EP4731754A2 - Serum-free methods for derivation of stem cell-derived retinal pigment epithelial cells and related cells, compositions, methods, and systems - Google Patents

Abstract

Provided herein are serum-free methods for the derivation of stem cell-derived retinal pigment epithelial cells (SC-RPE) and related cells, compositions, methods and systems that in several embodiments allow efficient, differentiation, isolation, and/or maturation of SC-RPE from a variety of different mammalian stem cells. In particular, provided herein serum free media related method and systems for maturation, enrichment and/or expansion of SC-RPE cells, as well as additional or alternative method and system for preparation of freezing stock, preparation of SC-RPE patch or cells suspension and related uses to transplant the SC-RPE in the sub-retinal space of an individual.

Description

Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT SERUM-FREE METHODS FOR DERIVATION OF STEM CELL-DERIVED RETINAL PIGMENT EPITHELIAL CELLS AND RELATED CELLS, COMPOSITIONS, METHODS, AND SYSTEMS CROSS REFERENCE TO RELATED APPLICATIONS [0001] The present application claims priority to U.S. Provisional Application No.63/510,095, entitled “Serum-Free Methods for the Derivation of Stem Cell-Derived Retinal Pigment Epithelial Cells and Related Cells, Compositions, Methods, and Systems” filed on June 23, 2023, with docket number P2765-USP, the contents of which is incorporated by reference in its entirety. FIELD [0002] The present disclosure relates to cells which are stem cell-derived, and in particular to stem cell-derived retinal pigment epithelial cells. More particularly, the present disclosure relates to serum-free methods for the derivation of stem cell-derived retinal pigment epithelial cells and related cells, compositions, methods, and systems. BACKGROUND [0003] Stem cells are at the center of many efforts to develop a technology capable of offering efficient and effective methods for cell generation and regeneration as well as for treatment and/or prevention of a variety of conditions. [0004] In particular, stem cell technology has been applied to retinal pigment epithelium (RPE) to address RPE degeneration and loss of retinal pigment epithelial cells associated with a number of degenerative retinal diseases. [0005] Challenges however remain due to a generally low and/or variable yield of RPE derivation procedures, as well as to relatively high complexity which impact the efficiency of RPE derivation as would be understood by a skilled person. SUMMARY [0006] Provided herein are serum-free methods for the derivation of stem cell-derived retinal pigment epithelial cells (SC-RPE) and related cells, compositions, methods, and systems that in several embodiments allow efficient, differentiation, isolation, and/or maturation from a variety of different mammalian stem cells. [0007] In particular, according to a first aspect, serum-free SC-RPE methods and related cells, Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT compositions, methods and systems are based on the unexpected finding that stem cell line- specific combinations of serum-free cell culture differentiation media and cell culture substrates, can be used to provide an efficient SC-RPE differentiation in a stem cell line specific manner. [0008] Accordingly, a toolbox method and systems and related serum-free SC-RPE differentiation media are herein described, in which combinations of serum-free SC-RPE differentiation medium and SC-RPE cell culture substrate are selected depending on the starting stem cell line, to perform SC-RPE differentiation by generating precursor cells capable of obtaining a mature RPE phenotype in accordance with the present disclosure. [0009] In the SC-RPE differentiation method of the disclosure, the method comprises contacting stem cells of a starting stem cell line with an SC-RPE differentiation medium, and an SC-RPE cell culture substrate, the SC-RPE differentiation medium, and SC-RPE cell culture substrate selected depending on the starting stem cell line. In the SC-RPE differentiation method of the disclosure, the contacting is performed for a time and under conditions to obtain a differentiated SC-RPE cells. [0010] In the SC-RPE differentiation system of the disclosure, the system comprises at least one serum-free SC-RPE differentiation medium and at least one SC-RPE cell culture substrate. In SC-RPE differentiation system of the disclosure, the at least one cell culture medium and the at least one cell culture substrate are included for combined use to provide a cell culture environment (herein also indicated as support) configured to allow introduction and/or maintenance of stem cells cultured in the cell culture support to obtain a differentiated SC-RPE cells according to methods of the disclosure. [0011] Combinations of an SC-RPE differentiation medium and an SC-RPE cell culture substrate configured for the SC-RPE differentiation of a starting stem cells line can be identified by a method comprising contacting the starting stem cell line with one or more candidate combinations of an SC-RPE differentiation medium and an SC-RPE cell culture substrate, the contacting performed for a time and under condition allowing SC-RPE differentiation and selecting a candidate combination of the one or more candidate combinations which following the contacting results in SC-RPE differentiation of the starting stem cell line. [0012] According to a second aspect, the serum-free SC-RPE methods and related cells, compositions, methods, and systems are also based on the unexpected finding that the yield Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT and maturation of differentiated SC-RPE cells can be promoted and/or enhanced by a serum- free RPE Maturation Medium of the disclosure. [0013] The RPE Maturation Medium of the disclosure is a cell culture medium comprising a buffered physiological salt solution, vitamins, essential lipids, aerobic and anerobic energy sources, essential amino acids (EAA) and nonessential amino acids (NEAA), insulin, selenite and transferrin/Fe⁺³, progesterone, taurine, triiodothyronine (T3), corticosteroid, retinoid (technically included in the before mentioned vitamin class), and anti-oxidants in an effective amount to promote maturation of differentiated SC-RPE cells. [0014] The SC-RPE maturation method of the disclosure is a method to maturate a differentiated stem cell-derived retinal pigment epithelial (SC-RPE) cell, the method comprising contacting differentiated SC-RPE cells with a cell culture maturation medium of the present disclosure. For a time and under conditions to obtain a mature SC-RPE cell. [0015] According to a third aspect, the serum-free SC-RPE methods and related cells, compositions, methods, and systems are also based on the unexpected finding that mature SC- RPE can be identified by the presence of marker CD57, the presence of the marker CD104, and/or the absence of marker CD49b. [0016] Accordingly, an SC-RPE enrichment/purification method and system in accordance with the third aspect are described. [0017] The SC-RPE enrichment method of the disclosure is a method to enrich mature SC- RPE cells, the method comprising providing differentiated and/or mature stem cell-derived retinal pigment epithelial cells; and contacting the cells with a probe specific for a marker selected from CD57, CD104, and CD49b for a time and under condition to obtain cells stained for CD57, CD104, and/or CD49b markers, if any is present. The SC-RPE enrichment method of the disclosure also comprises sorting the stained cells to select the cells stained for CD57 and/or CD104 markers, and discard cells stained for CD49b marker, to obtain a population of cells enriched in mature SC-RPE cells. [0018] In addition or in the alternative to detection performed through marker selection in SC- RPE methods of the disclosure, the SC-RPE enrichment method provides for selection of mature SC-RPE based on detection of pigmentation of the differentiated and/or mature stem cells to be performed with one or more sorting devices configured to perform detection of pigmentation through absorption of light by melanin and/or scatter of light by melanosomes Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT present in the cells. In some embodiments selection of differentiated and/or mature stem cells through light absorption and/or light-scattering by the pigment of the cells can be performed in combination with selection based on marker selection as will be understood by a skilled person. [0019] The SC-RPE enrichment system of the disclosure is a system to enrich mature stem cell-derived retinal pigment epithelial (SC-RPE) cells, the system comprising combination of one or more probes specific for a marker selected from CD57, CD104, and/or CD49b, reagents for the detection of the marker, and/or stem cell-derived retinal pigment epithelial (SC-RPE) cells for combined use to stain differentiated and or mature SC-RPE cells in a method to obtain a population of cells enriched in SC-RPE cells. In addition or in the alternative to probes for detection of SC-RPE, the system can comprise cell sorting devices to perform selection based on pigmentation and obtain a population of cells enriched based on detection of pigmentation in addition or in the alternative to enrichment based on presence of one or more markers. [0020] According to a fourth aspect, the serum-free SC-RPE methods and related cells, compositions, methods, and systems are also based on the unexpected finding that an RPE Maintenance Medium of the present disclosure can be used to perform efficient expansion of SC-RPE cells that retain the capacity to obtain a mature RPE phenotype and function. [0021] The RPE Maintenance Medium of the disclosure comprises an RPE Maturation Medium of the disclosure supplemented with basic fibroblast growth factor (bFGF), a Rho- associated coiled-coil containing protein kinase (ROCK) inhibitor and/or transforming growth factor beta receptor (TGFBR/ALK5) kinase inhibitor, and dimethyl sulfoxide (DMSO) in an effective amount to promote expansion of SC-RPE cells that retain the capacity to obtain a mature RPE phenotype and function. In some embodiments, ROCK inhibitors can comprise Y-27632, Thiazovivin, or RevitaCell, and the TGFBR kinase inhibitors can comprise A-83-01, RepSox (ALK5-inibitor), SB431542, or SB525334. [0022] The SC-RPE expansion method of the disclosure is a method for expansion of stem cell-derived retinal pigment epithelial (SC-RPE) cells that retain the capacity to obtain a mature RPE phenotype and function, the method comprising providing mature stem cell-derived retinal pigment epithelial (SC-RPE) cells; and contacting the SC-RPE cells with the RPE Maintenance Medium of the disclosure on an SC-RPE substrate of the disclosure, the contacting for a time and under condition to obtain a target cell density of the SC-RPE cells. [0023] The SC-RPE expansion system of the disclosure is a system for expansion of stem cell- Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT derived retinal pigment epithelial (SC-RPE) cell, the system comprising the RPE Maintenance Medium, and an SC-RPE substrate of the disclosure for combined use in a method to perform primary expansion of the SC-RPE cells of the disclosure. [0024] According to further aspects of the disclosure, the methods, and systems and related medium according to any one of the first to the fourth aspect can be used in connection with methods, systems and compositions related to various stages of the SC-RPE derivation process. [0025] For example, according to a fifth aspect an SC-RPE frozen stock preparation method and system and SC-RPE obtainable or obtained thereby are described based on the use of the RPE Maintenance Medium of the disclosure. [0026] The SC-RPE frozen stock preparation method according to the fifth aspect is method to prepare frozen stock of stem cell-derived retinal pigment epithelial (SC-RPE) cells, the method comprising performing expansion of SC-RPE cells with the expansion method of the disclosure to obtain SC-RPE cells having a target cell density. The SC-RPE freezing stock preparation method further comprise harvesting the SC-RPE cells having the target cell density to obtain harvested SC-RPE cells, and freezing the harvested SC-RPE cells. [0027] The SC-RPE frozen stock preparation system according to the fifth aspect is a system for frozen stock preparation of stem cell-derived retinal pigment epithelial (SC-RPE) cells, the system comprising the RPE Maintenance Medium of the present disclosure, the SC-RPE substrate of the present disclosure, and freezing media for combined use in a method to provide frozen stock of the present disclosure. [0028] According to a sixth aspect an SC-RPE method and systems are described for the production of RPE cells on a physical support, and SC-RPE cells monolayers obtainable and/or obtained thereby (herein also SC-RPE Patch production method), which are based on the use of the RPE Maintenance Medium of the disclosure. The SC-RPE Patch production method according to the sixth aspect is directed to production of functional RPE cells on a physical support that can be selected in view of the use of the product obtained thereby (e.g., experimental study or clinical use). Accordingly, the support can be on a physical non- permeable, permeable, or porous support (such as TC plastic, 2D permeable film or membrane, 2D-porous support, 3-D membrane/matrix, porous or solid microcarriers, and additional identifiable by a skilled person) coated with or modified with a suitable RPE substrate as will Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT be understood by a skilled person. In some cases the patch production method can be used to repair a region of the retinal pigment epithelium where the RPE is dysfunctional, degenerated, or missing as will be understood by a skilled person. [0029] The SC-RPE Patch production method for the production of RPE cells on a physical support of the disclosure is a method to manufacture stem cell-derived retinal pigment epithelial (SC-RPE) cells on a physical support, the method comprising performing expansion of SC-RPE cells according to the method of the disclosure and to obtain SC-RPE cells having a target cell density. The SC-RPE method of the production of RPE cells on a physical support further comprises contacting the SC-RPE cells having the target cell density with a membrane support and the RPE Maturation Medium or an RPE Maintenance Medium of the disclosure to obtain a population of quiescent partially mature or mature SC-RPE cells monolayers. [0030] According to a seventh aspect, a method to manufacture an SC-RPE cell suspension is described, the method comprises obtaining the SC-RPE cell suspension from any one of the SC-RPE cells of the present disclosure following performing the SC-RPE enrichment method of the disclosure. [0031] According to an eighth aspect SC-RPE method and systems are described for transplanting an individual with SC-RPE monolayers on a flexible solid support, and/or the SC-RPE cells suspension obtainable and/or obtained with the methods and systems of the present disclosure (herein SC-RPE Transplantation method). The SC-RPE transplantation method comprises placing the SC-RPE monolayers on a flexible solid support, and/or the SC- RPE cells suspension of the disclosure in the subretinal space of the individual thus transplanting the individual with the of SC-RPE cells in accordance with methods of the of the disclosure. [0032] The SC-RPE systems are described for transplanting SC-RPE in an individual, comprises SC-RPE monolayers on a flexible solid support, and/or the SC-RPE cells suspension together with reagents and devices for performing the placing according to a method transplanting SC-RPE in an individual of the present disclosure. [0033] According to a ninth aspect a toolbox SC-RPE derivation method and system are described in which a user selects the steps and/or component in accordance with the starting stem cells and the experimental design. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [0034] The SC-RPE derivation method of the disclosure is a method to derive retinal pigment epithelial (RPE) cell from a starting stem cell (SC), comprising differentiating the stem cell (SC) to obtain differentiated stem cell-derived retinal pigment epithelial (SC-RPE) cells and maturating the differentiated SC-RPE cells to obtain mature SC-RPE cells. [0035] The SC-RPE derivation methods of the disclosure can optionally further comprise performing enrichment of mature SC-RPE stem cells, preparing a frozen stock of the SC-RPE, performing expansion of the SC-RPE, manufacturing SC-RPE cells for use in the SC-RPE Transplantation method and system of the disclosure and/or manufacturing SC-RPE cells on a physical support to obtain a population of quiescent and at least partially mature up to fully mature SC-RPE cells monolayers depending on the percentage of mature cells in the monolayer as will be understood by a skilled person upon reading of the present disclosure. [0036] In the SC-RPE derivation methods of the disclosure at least one of the differentiating, the maturating, the performing enrichment, the preparing a frozen stock, the performing expansion, and the preparation of SC-RPE suspensions or the preparation of SC-RPE patches is performed according to method of the present disclosure. [0037] The SC-RPE derivation system of the disclosure comprises at least one of the SC-RPE differentiation medium, the SC-RPE substrate, the RPE Maturation Medium, at least one probe specific for a marker selected from CD57, CD104, and CD49b, the RPE Maintenance Medium, at least one SC-RPE freezing medium, and at least one SC-RPE scaffold of the present disclosure. [0038] According to a tenth aspect SC-RPE cells are described obtainable or obtained by the SC-RPE differentiation method, the SC-RPE maturation method, the SC-RPE enrichment method, the SC-RPE expansion method, or the SC-RPE derivation of method of the disclosure. In particular SC-RPE cell populations obtained with the methods and systems of the disclosure can have an percentage purity level of SC-RPE differentiated cells and/or SC-RPE mature cells, in the obtained cell population increased with respect to SC-RPE cells obtained in outcome corresponding differentiation methods, maturation methods, enrichment methods expansion methods, and/or derivation methods as will be understood by a skilled person upon reading of the present disclosure. [0039] According to an eleventh aspect, any of the matured SCE-RPE cells herein described, Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT possibly obtained following maturation method, enrichment method, expansion method, derivation methods possibly following thawing from a frozen stock of the present disclosure can be used to treat and/or prevent retinal conditions in the individual. [0040] The serum-free differentiation methods and systems herein described allow to decrease the variability in differentiation efficiency known to be associated with stem cell differentiation in with particular reference to differentiation performed with serum-free protocols. In particular, serum free protocols can provide varying levels of RPE differentiation yields efficiency due for example to issues such as disaggregation of embryoid bodies (EBs) in protocols employing EBs, insufficient pigmentation and/or insufficient RPE epithelial properties as will be understood by a skilled person. [0041] In particular, the serum free differentiation methods and systems herein described allow one of skill to consistently perform differentiation of SC-RPE across a range of different starting stem cells to obtain yields of differentiated SC-RPE cells that can be higher than 50% as will be understood by a skilled person. [0042] [The serum-free RPE Maturation medium and related methods and systems herein described allow obtaining in several embodiments a population of mature SC-RPE cells having RPE marker genes expression and corresponding a quiescent state, an RPE morphology, and/or functionality comparable to functional RPE in vivo, with surprising increased in yields and a decreased variability among starting stem cells compared to existing serum free media methods and systems for maturation of RPE cells. [0043] In particular it is expected that, serum-free RPE Maturation medium and related methods and systems herein described allow in some embodiments to consistently obtain a population of mature SC-RPE cells having RPE marker genes expression and a corresponding quiescent state, an RPE morphology, and functionality comparable to functional RPE in vivo, in percentage equal to or greater than 40% of the starting differentiated cells in the cell population, and in preferred and most preferred embodiments herein described up to or greater than 95%, or up to or greater than 99% of the starting differentiated cells in the cell population. [0044] The serum-free RPE Maturation Medium and related methods and systems herein described also comprise standardized serum-free protocols which allow production of mature SC-RPE cells with reliable and reproducible results as will be understood by a skilled person Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT upon reading of the present disclosure. [0045] The serum-free RPE Enrichment methods and systems herein described also allow enrichment of a starting population of differentiated SC-RPE and/or mature RPE of at least 95% (mean = 98% +/- 1, n=22). [0046] The serum-free RPE Maintenance medium and related methods and systems herein described also allow efficient proliferation with growth rates comparable to that seen with serum supplemented medium and a greater than 50-fold increase in number of RPE that can obtain a quiescent monolayer, having RPE marker expression, morphology, and functionality comparable to RPE in vivo compared to serum-free medium without the additional components of RPE-MMM using standardized protocols, which is unexpected in view of variability, and technical complexity of existing serum-free media. [0047] The serum-free RPE Maintenance medium and related frozen stocks methods and systems herein described also allow compared to existing serum-free methods a consistent cryopreservation as well as decreased cellular stress and damage with corresponding increased quality of recovered cells following thawing and replating allowing for the attainment of quiescent RPE monolayers with up to and greater than 95% of the cells having RPE marker expression, morphology, and functionality comparable to RPE in vivo. [0048] The serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods, and systems can be used in several embodiments to allow for a substantial improvement in yield, purity, and quality, as well as a significant shortening of the time required to complete the process compared to existing procedures. [0049] In particular, the serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods, and systems can be used in some embodiments to obtain a final SC-RPE yield of about 15,000 SC-RPE per starting stem cell for cases with differentiation efficiencies >50%, a purity >97% expected, often >99% with top-producing stem cell lines, and quality (uniform pigmentation, characteristic RPE morphology and phenotype, RPE signature gene expression comparable to cultured fetal RPE, minimal evidence of mesenchymal RPE gene expression) compared to existing procedures while using a complete processing time of only about 90 day. [0050] In addition, the serum-free methods for the derivation of SC-RPE cells and related SC- Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT RPE cells, compositions, methods, and systems in embodiments comprising the SC-RPE differentiation methods and systems herein described, is expected to provide increased yields in connection with a greater number of stem cell lines than existing derivation methods employing a single differentiation method. [0051] The serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods, and systems can be used in some embodiments comprising enrichment methods of the disclosure, particularly when using cell sorter-based enrichment and standard 2-D culture methods, to allow a substantial reduction in labor and hence increased SC-RPE manufacture productivity in comparison to existing methods employing manual based enrichment or embryoid body culture. For example, it is expected that 1-2 persons working in a non-cGMP setting can produce sufficient frozen stocks of SC-RPE to generate as many as one trillion SC-RPE in about three months using stem cell lines and differentiation methods with differentiation efficiencies of 50% or greater, in the preferred embodiments, and with maximal SC-RPE expansion. [0052] Accordingly, the serum-free methods for the derivation of SC-RPE cells and related cells, compositions, methods, and systems can be used in several embodiments to achieve high yield RPE derivation from many different starting stem cell lines. In some embodiments when stem cell lines are used that have differentiation efficiencies greater than 50%, final yields in excess of 15,000 SC-RPE cells per single stem cell are possible. With stem cell lines that have lower differentiation efficiencies or when the user chooses to reduce the extent of culture expansion the yield will be reduced proportionally, as will be understood by a skilled person upon reading of the disclosure. [0053] The serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods and systems can be used in several embodiments to achieve large-scale SC-RPE production (>30 billion SC-RPE per 50 cm² starting differentiation culture, with differentiation efficiencies >50% and maximal SC-RPE expansion) in a single run in the time span of approximately four months using standard culture methods. In clinical applications where the common dosages range from 100,000 to 200,00 cells this equates to enough cells to produce over 150,000 doses. [0054] The serum-free methods for the derivation of SC-RPE cells and related cells, compositions, methods, and systems can be used in several embodiments to produce clinical Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT grade stem cell-derived mammalian cells since the components of the compositions used in the derivation of SC-RPE methods described herein are usable with xeno-free cGMP compliant reagents. [0055] The serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods and systems can be used in several embodiments to select an efficient protocol for a given starting stem cell from a collection of spontaneous and semi-directed differentiation protocols which employ the use of serum-free SC-RPE differentiation and/or serum-free RPE Maturation Medium of the disclosure. [0056] The serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods, and systems can be used in several embodiments for culture expansion and/or identification and/or testing of a drug product that promotes RPE maturation and limits persistent RPE-to-mesenchymal transition. [0057] The serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods, and systems can be used in several embodiments to create intermediate frozen SC-RPE stocks for on-demand production of final product. [0058] The serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods, and systems can be used in several embodiments to enact an automation compatible method for the isolation of pure populations of RPE cells from mixed cell populations based on the expression the cell surface antigens CD57 and/or CD104, lack of expression of CD49b, and/or light absorbance or light scattering of melanin/melanosomes. [0059] The serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods and systems can be used in several embodiments to provide cell-based therapeutic compliant pipeline for large-scale manufacture of high purity (^99%), high fidelity SC-RPE for use in the treatment of degenerative eye disorders involving RPE degeneration an/or loss of function. Fidelity refers to homogeneity of phenotype (pigmentation, shape, RPE gene expression relative to that of primary differentiated primary fetal RPE) and lack of or minimal expression of non-RPE genes or genes associated with RPE wound response as will be understood by a skilled person. [0060] The serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods, and systems herein described can be used in any field where efficient Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT production of SC-RPE and/or production of high-quality SC-RPE cells are desired. Exemplary applications comprise medical and in particular clinical applications related to treatment and/or prevention of retinal degenerative conditions such as macular degeneration. [0061] The serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods and systems herein described can also be used for the manufacture of SC-RPE capable of delivering specific biologics (gene products) when used with genetically engineered stem cells or when the SC-RPE are genetically engineered during the production process. [0062] The serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods and systems herein described can further be used in drug research and/or to develop diagnostic and/or therapeutic approaches and/or tools (such as diagnostic and/or therapeutic cellular agents), to counteract conditions involving the retinal epithelium, such as monitoring of therapeutic cell/agent efficacy and safety during developmental stages and clinical usage. [0063] Additional exemplary applications include uses of the serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods and systems herein described in several fields including basic biology research, applied biology, bio- engineering, bio-energy, medical research, medical diagnostics, therapeutics, and in additional fields identifiable by a skilled person upon reading of the present disclosure. [0064] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS [0065] The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with the detailed description and the examples, serve to explain the principles and implementations of the disclosure. [0066] Figure 1 shows a set of whole well incident illumination images of exemplary induced pluripotent (iPSC) and embryonic (ESC) stem cell lines following incubation with exemplary combinations of SC-RPE differentiation media and SC-RPE extracellular matrix and Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT subsequent incubation with to RPE Maturation Media (iPSC: #032411, #110211, and #120111; ESC: Shef1, H1, and H9). The iPSC lines were developed in-house from late passage donor fetal RPE cells using an RNA-based reprogramming method (ReproRNA™-OKSGM, STEMCELL Technologies, Inc.). The day that the image was collected, which corresponds to the day the culture was harvested for SC-RPE enrichment or discarded due to poor yields is indicated at the bottom of each image. Black image borders indicate cell line:media:substrate combinations that gave rise to acceptable quantities of pigmented SC-RPE to allow for production of substantial quantities of enriched SC-RPE. Each well image is a representative image selected from 3-6 replicate wells. [0067] Figure 2 shows the beneficial effect of sequential incubation with the differentiation media and the RPE Maturation Medium on SC-RPE differentiation. Top Row: Whole well incident light images of Shef1 differentiation cultures where the step of incubation of with the RPE Maturation Medium was omitted and the cells remained in the exemplary differentiation for the entire culture period after the initial switch to the differentiation media. Second Row: Whole well incident light images of Shef1 cells differentiated using the standard procedure where the cells were incubated with the exemplary differentiation medium from Day-3 through Day-30 and thereafter with RPE Maturation Medium. Third Row: Whole well incident light images of iPSC #110211 cultures where the switch to the exemplary differentiation media was omitted and the cells were only incubated with RPE Maturation Medium beginning at Day-3 of the differentiation process. Bottom Row: Whole well incident light images of iPSC #110211 cells that were differentiated using the standard procedure where the cells are incubated with the exemplary differentiation media from Day-3 through Day-30 and thereafter with RPE Maturation Medium. The day that the image was collected is indicated at the bottom of each image. [0068] Figure 3 shows that switching to RPE Maturation Medium after Day-31 of the differentiation protocol does not substantially improve the yield of pigmented SC-RPE. Whole well incident light images of cultures plated on the three exemplary substrates and switched from stem cell culture medium directly to RPE Maturation Medium at Day-3 (Top Row) or to CTS-KO-DMEM/20%CTS-KOSR Medium at Day-3 and then switched to RPE Maturation Medium at Day-31 or Day-59 (Middle Rows). The bottom row shows whole well incident light images of negative control cultures where the cells were switched to CTS-KO- DMEM/20%CTS-KOSR Medium at Day-3 and maintained in CTS-KO-DMEM/20%CTS- Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT KOSR Medium for the entire differentiation time course. The day that the image was collected is indicated at the bottom of each image. [0069] Figure 4 shows the substantial improvement in pigmented SC-RPE yield and homogeneity that can be achieved by passaging the cells prior to cell surface marker-based enrichment. Shef1 cells were plated on rhVTN-N or LAM521 coated plates and differentiated in CTS-KO-DMEM/20%CTS-KOSR Medium using the optional Activin A treatment and then switched to RPE Maturation Medium on Day-31. On Day-87 the two cultures were harvested by protease digestion and a portion of the cells were plated at 100,000 cell/cm² on LAM521 culture wells and maintained in RPE Maintenance Medium for 42 additional days. Top Row: Whole well incident images of the cultures immediately before and 42-days after passage. Bottom Four Rows: Brightfield images of the corresponding cultures at different magnification. Regions of the culture with pigmentation were selected for the collection of the 10-100X images of the pre-passage culture to evaluate the nature of the SC-RPE. Note the marked improvement in the proportion of pigmented cells and normal RPE morphologies after passage. [0070] Figure 5 shows results of experiments performed to evaluate how each of the RPE Maturation Medium components contribute to RPE phenotype maturation and pigmentation. Figure 5 Panel A shows 2×210X brightfield stitched images of one of three replicate wells in which Shef1-RPE produced using the CTS-KO-DMEM/20%CTS-KOSR:PC-VTN or X- VIVO™ 10/XF-B27:rhVTN-N exemplary differentiation and sorting protocols were grown in complete RPE Maturation Medium (RPE-MM) or RPE Maturation Medium missing one of the components as indicated. Figure 5 Panel B shows a diagram illustrating the quantitation of the amount of pigment for cells maintained in the complete RPE Maturation Medium (RPE-MM) and the n-1 formulations. The relative amount of pigmentation was determined based on the absorbance of light at 510 nm using a 3×3 multipoint scan of each well (mean +/- SEM, n=3). [0071] Figure 6 shows results of experiments demonstrating the superiority of RPE Maturation Medium to facilitate RPE maturation and pigmentation compared to the other media formulations. Figure 6 Panel A shows 2×210X brightfield stitched images of one of three replicate wells in which Shef1-RPE produced using the CTS-KO-DMEM/20%CTS- KOSR:PC-VTN (RPE-DM1 SC-RPE) or X-VIVO™ 10/XF-B27:rhVTN-N (RPE-DM2 SC- RPE) exemplary differentiation and sorting protocols grown in RPE Maturation Medium Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT (RPE-MM), CTS-KO-DMEM/20%CTS-KOSR (RPE-DM1), or X-VIVO™ 10/XF-B27 (RPE- DM2) or RPE-DM1, RPE-DM2, CTS-KO-DMEM (KO-DMEM), or CTS-KO-DMEM/F12 (KO-DMEM) with the addition of the RPE-MM non-base medium components (NEAA, N1, B27, taurine, and hydrocortisone). Figure 6 Panel B shows 20X brightfield images of the same cells maintained in RPE Maturation Medium, X-VIVO™ 10/XF-B27, and X-VIVO™ 10/XF- B27 supplemented with the RPE-MM non-base medium components (NEAA, N1, B27, taurine, and hydrocortisone). [0072] Figure 7 shows a schematic representation of an exemplary method of stem cell expansion and SC-RPE differentiation. [0073] Figure 8 shows a schematic representation of exemplary methods and system for SC- RPE differentiation of the present disclosure. [0074] Figure 9 shows a schematic representation of an exemplary method and system for SC-RPE Fluorescence Activated Cell Sorting (FACS) Enrichment of the present disclosure. [0075] Figure 10 shows three FACS scatter plots illustrating results of an exemplary SC-RPE gating strategy to sort SC-RPE. Pigmented SC-RPE are identified based on their relatively high side scatter (SSC) to forward scatter (FSC) ratio (Figure 10 left side diagram), positive expression of CD104 and CD57 (Figure 10 middle diagram), and no expression of CD49b (Figure 10 right side diagram). [0076] Figure 11 shows results of cell sorting based on pigmentation and cell surface markers on the enrichment in populations of SC-RPE. Figure 11 Panel A Top Row: shows results of cells identified based on forward and side light scattering (FSC & SSC) profiles and SC-RPE sorting based on expression of both CD104 and CD57 and lack of expression of CD49b. The far-right panel shows the light scatter profiles of the CD104 Positive/CD57 Positive/CD49b Negative cells. Figure 11 Panel A Bottom Row: shows results of cells sorting based on pigmentation and cell surface marker expression. Figure 11 Panel B shows diagrams illustrating SC-RPE Purity analysis based on the expression of the RPE marker gene product PMEL of cells sorted based on pigmentation and surface marker expression. [0077] Figures 12A and 12B show the results of experiments directed to detect the effect on pigmentation and the establishment and maintenance of RPE barrier function of basic FGF (bFGF) plus DMSO. Figure 12A shows a diagram illustrating the amount of pigmentation of Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Shef1-RPE manufactured using the CTS-KO-DMEM/CTS-20%KOSR:rhVTN-N differentiation protocol and RPE Maturation Medium with and without 2 ng/ml bFGF + 0.1% DMSO according to the standard protocol. In the diagram of Figure 12A the amount of pigmentation of the Shef1-RPE was determined by measuring the absorbance of 510 nm light using 3×3 multipoint scan (background subtracted absorption value, n=1) at the indicated timepoints. Figure 12B shows a diagram illustrating RPE barrier function of the cells of Figure 12A determined by measuring the transepithelial electrical resistance of the macroporous insert cultures (mean +/- SEM, n=3). [0078] Figure 13 shows results of experiments directed to measure effects of the addition of DMSO and bFGF to RPE Maturation Medium on SC-RPE pigmentation. Figure 13 Panel A shows representative 4X brightfield images of three replicate wells of SC-RPE cultured in RPE Maturation Medium supplemented with 0.1% DMSO (DMSO), 2 ng/mL bFGF (bFGF), or 0.1% DMSO + 2 ng/mL bFGF (DMSO bFGF) when plated at a density (4500 cell/cm²) at which they will not mature and pigment without additional treatment. Figure 13 Panel B show Whisker plots of the level of pigmentation detected in the of SC-RPE cultured of Figure 13 panel A. The middle two boxes indicate the middle data quartiles and the whiskers indicate the outer quartiles, the “X” denotes the mean, and any dots indicate outliers. The level of pigmentation was quantified by measuring the absorbance of 510 nm light using a 3×3 multipoint scan of each replicate well. [0079] Figures 14A to 14D show results of exemplary expansion by continual treatment of SC-RPE with varying combinations of 0.1% DMSO, 2 ng/mL bFGF, and 5 ^M Y-27632. Figure 14A shows a schematic illustration of a workflow for the expansion of a frozen stock vial of iPSC-RPE. Figure 14B shows representative 4X brightfield images of a replicate well of SC-RPE cultured at a density conducive for non-passaged SC-RPE maturation (80,000 cell/cm²) in each of the media (DMSO, DMSO+bFGF, DMSO+Y27632, DMSO+Y27632+bFGF) schematically reported in Figure 14A as a function of passage. The day post plating of imaging is indicated for each condition. Figure 14C shows a diagram illustrating the relative level of pigmentation at Day-34 plotted versus the cumulative number of total population doublings up to the point of each passage (mean +/- SEM, n=3). Figure 14D shows a table of calculated yields at a level of pigmentation equal to 90% of the level observed at Passage 1. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [0080] Figure 15 shows a schematic representation of an exemplary method and system for SC-RPE Primary Expansion method and system in combination with an exemplary method and system for Preparation of Frozen SC-RPE Stocks of the present disclosure. [0081] Figure 16 shows a schematic representation of an exemplary method and system for thawing of Frozen Stocks, in combination with an exemplary method and system for SC-RPE secondary Expansion and an exemplary method and system for SC-RPE Patch Production of the present disclosure. [0082] Figure 17 shows whole well incident light images for a set of different RPE differentiation and maturation methods obtained using two iPSC stem cell lines taken at the end of RPE maturation. The stem cells were plated on Matrigel coated culture wells and differentiated using a X-VIVO™ 10/SM1 semi-directed differentiation method and maturated with RPE-MM with different combinations of supplements. Top Row: iPSC #110211 maturated with RPE-MM supplemented with N1 and 5%FBS, SM1 Neurocult minus vitamin A (Vitamin A Minus), or SM1 Neurocult (Vitamin A Plus). Second Row: iPSC #110211 maturated with RPE-MM supplemented with N2 and 5%FBS, SM1 Neurocult minus vitamin A (Vitamin A Minus), or SM1 Neurocult (Vitamin A Plus). Third row: iPSC #082809 maturated with RPE-MM supplemented with N1 and 5%FBS, SM1 Neurocult minus vitamin A (Vitamin A Minus), or SM1 Neurocult (Vitamin A Plus). Bottom row: iPSC #110211 maturated with RPE-MM supplemented with N2 and 5%FBS, SM1 Neurocult minus vitamin A (Vitamin A Minus), or SM1 Neurocult (Vitamin A Plus). [0083] Figure 18 shows the analysis of RPE barrier function by measurement of the transepithelial electrical resistance (TEER). Fetal RPE (n=5) and enriched SC-RPE #1102111 (n=4). The SCRPE #110201 cells were obtained from the cultures shown in Figure 17. Cells were grown on microporous transwell membranes using RPE-MM:N1 with SM1 minus vitamin A (grey bars) or RPE-MM:N1 with SM1 plus vitamin A (black bars). For the SC-RPE the cells were grown in the media used for differentiation. At one-month the TEER for each culture set of cells and culture conditions was measured and the Ohm•cm² (mean +/- SEM) values were plotted. [0084] Figure 19 shows the average transcriptome profiles for 6 Enriched SC-RPE cell lines obtained by differentiation using a X-VIVO™ 10/SM1 semi-directed differentiation method and maturated with RPE-MM:N1 medium supplemented with SM1 with (Plus) and without Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT (Minus) vitamin A. After enrichment and a post-sort maturation period of one-month using their corresponding RPE-MM medium their transcriptome profiles were determined by RNA AmpliSeq and the log2 transformed Plus Vitamin A/Minus Vitamin A ratio values plotted after alphabetical sorting of the gene symbols. The gene expression for profiles for several gene sets are shown. Top Chart: All Expressed Genes. Second chart down: Top-500 RPE Genes, up- regulated genes associated with RPE maturation. Third chart down: RPE Signature Genes, a curated set of 90 RPE marker genes. Fourth chart down: Top-500 induced genes associated with reversible RPE wound response. Top-500 induced genes associated with persistent EMT. [0085] Figure 20 shows the log2 transformed Vitamin A Plus/Vitamin A Minus expression ratios for a select set of genes related to key RPE functions for SC-RPE differentiated and maintained using RPE-MM prepared using the B27-based supplement SM1 Neurocult with or without vitamin A (black bars) along with the Vitamin A Plus/Vitamin A Minus expression ratios obtained from fetal RPE cultured in the same two media (grey bars). [0086] Figure 21 shows the gene expression levels of a select set of disease-associated genes obtained from enriched SC-RPE that were derived and matured for one-month using RPE-MM prepared with the B27-based supplement SM1 Neurocult with (Minus) or without (Plus) vitamin A, along with the values obtained when the SC-RPE were switched and matured in their corresponding opposite RPE-MM medium (Plus to Minus, Minus to Plus). The gene expression values are the trimmed mean of the M-value normalized reads per million (mean +/- SEM, Minus and Plus, n=6, Plus to Minus and Minus to Plus, n=3). [0087] Figure 22 shows the vitamin A dose response curves for the expression of eight select genes obtained from fetal RPE matured for one month in RPE-MM prepared using a 2-fold dilution series (100-0%) of RPE-MM:SM1 Neurocult plus vitamin A diluted with RPE- MM:SM1 Neurocult minus vitamin A. Gene expression values are the trimmed mean of the M-value normalized reads per million as determined by RNA AmpliSeq. The 0% vitamin A values are plotted on the y-axis. [0088] Figures 23A and Figure 23B show a comparison of the gene expression profiles of SC-RPE contrasted to pluripotent iPSC and fetal RPE. Enriched SC-RPE were obtained from eight ESC-RPE differentiations depicted in Figure 1: Shef1:CTS-KO-DMEM/20%KOSR: PC- VTN, rhVTN-N, and LAM521; Shef1: X-VIVO™ 10/XF-B27:LAM521; Shef1: X-VIVO™ 10/XF-B27+Activin A:PC-VTN and rhVTN-N; H1: X-VIVO™ 10/XF-B27:LAM521; and Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT H9:CTS-KO-DMEM/20%KOSR: rhVTN-N. After enrichment, the SC-RPE along with primary fetal RPE (fRPE) cells were matured for one month using RPE-MM:N1:CTS-B27 and their transcriptome profiles were determined by RNA AmpliSeq. In particular in Figure 23A shows scatter plots are shown comparing the average gene expression profiles of the eight SC- RPE lines, four pluripotent stem cell lines and four primary cultured fetal RPE lines. Figure 23B shows scatter plots comparing the expression of a Top-500 up-regulated gene set associated with RPE maturation for iPSC and fetal RPE (top-left) and comparing each of the eight SC-RPE with fRPE. [0089] Figures 24A and Figure 24B shows the expression of RPE-signature genes and select RPE function genes for SC-RPE and fetal RPE. In particular Figure 24A shows expression levels of a curated gene set comprised of 90 RPE signature genes for all SC-RPE (black bars, mean, n=8) and all fRPE (grey bars, mean=4). The expression values are the trimmed mean of the M-values normalized reads per million. Figure 24B shows Average expression levels of a select set of RPE function genes in SC-RPE and fetal RPE. The gene expression levels are expressed relative to the average values obtained from four pluripotent iPSC line cultures. [0090] Figure 25 shows a schematic representation of a cell sorter usable in the methods and systems of the present disclosure. [0091] Figure 26 shows a schematic representation of devices configured to transplant RPE cells in sub-retinal space of an individual and usable in the methods and systems of the present disclosure. [0092] Figure 27 shows a chart depicting the results of examining the effect of the timing, split ratios, and supplementation of RPE-MM with bFGF and DMSO on corrected SC-RPE differentiation yields when an optional intermediate passage is performed during the maturation phase of SC-RPE differentiation and maturation. DETAILED DESCRIPTION [0093] Provided herein are serum-free methods for the derivation of stem cell-derived retinal pigment epithelial cells (SC-RPE) and related cells, compositions, methods, and systems that in several embodiments allow efficient, differentiation and/or maturation from a variety of different mammalian stem cells. [0094] The wording “stem cell” as used herein indicates an undifferentiated cell of a Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT multicellular organism which is capable of giving rise to indefinitely more cells of the same type, and from which certain other kinds of cells arise by differentiation [1]. In particular stem cells encompass undifferentiated or partially differentiated cells that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell [2]. Distinguished from progenitor cells, stem cells in the sense of the disclosure can be categorized as adult stem cells, embryonic stem cells and induced stem cells. [0095] The wording “adult stem cells” or “somatic stem cells” in the sense of the disclosure indicate undifferentiated cells, found throughout the body of an individual such as animals mammals and humans after development, that multiply by cell division to replenish dying cells and regenerate damaged tissues [3]. [0096] The wording “embryonic stem cells” or “ESCs” as used herein indicate pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage pre-implantation embryo. In particular, embryonic stem cells in the sense of the disclosure comprise human embryonic stem cells or hESC as well as ESC from other mammals. ESC can be obtained by isolating the inner cell mass (embryoblast) of an embryo stage 4–5 days post fertilization, when the blastocyst consists of 50–150 cells using methods such as immunosurgery, mechanical dissection, microdissection, laser dissection, or minimized trophoblast cell proliferation as will be understood by a skilled person [4], [5]. [0097] In some embodiments ESC in the sense of the disclosure are ESC derived using non- embryo-destructive methods. Methods for making ESC using non-embryo destructive methods are identifiable to a skilled person (see e.g. [6], [7], [8]). [0098] The wording “induced stem cells” or “iSC” in the sense of the disclosure indicates stem cells derived from somatic, reproductive, pluripotent, or other cell types by deliberate epigenetic reprogramming. They are classified as either totipotent (iTC), pluripotent (iPSC) or progenitor (multipotent – iMSC, also called an induced multipotent progenitor cell – iMPC) or unipotent – (iUSC) according to their developmental potential and degree of dedifferentiation. Progenitors are obtained by so-called direct reprogramming or directed differentiation are also called induced somatic stem cells as will be understood by a skilled person [9]. [0099] In particular, it is expected that suitable iSC can give rise to all three germ layer lineages (ectoderm, endoderm and mesoderm). Therefore iMSC/iMPC cells which have this property Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT are expected to be suitable iSC. Moreover, it is also expected that a some RPE progenitors do not need to go through the differentiation step and can go straight to RPE maturation medium. For those RPE progenitor cells cell sorting is expected to be optional. Also, it is expected that for iTC, an additional step would be required to first generate pluripotent cells. [00100] In some embodiments, the starting stem cells of the disclosure are from stem cell lines. In some embodiments, the starting cells are primary RPE as will be understood by a skilled person upon reading for the present disclosure. In particular, some aspects of the process can be used for the culture, enrichment, freezing, and/or expansion of primary RPE as will be understood by a skilled person. [00101] The wording “stem cell line” in the sense of the disclosure indicates a group of stem cells that is cultured in vitro and can be propagated indefinitely. Stem cell lines are derived from either animal or human tissues and come from one of three sources: embryonic stem cells, adult stem cells, or induced stem cells. Accordingly, stem cell line refers to a group of identical stem cells that can be grown and nurtured in a lab dish. A line originates from a single cell or group of cells of common genetic origin. All resulting cells in the line are replicates of the original cells and stem cell lines can be propagated in culture to produce large numbers of cells [10], [11]. Stem cell lines can become heterogenous at the genetic and/or epigenetic level over time due to culture related artifacts. Therefore, replicates of the original cells of a stem cell lines can be subjected to a genetic or epigenetic drift over time that might alter their propensity to give rise to each of the three different germ layer cells as will be understood by a skilled person [12] [13] [14] [15] [16]. [00102] In some embodiments herein described, serum- and xeno-free methods and systems and related composition are used to perform efficient derivation from starting stem cells to retinal pigment epithelial (RPE) cells. [00103] The wording “serum-free” as used herein indicates the absence of the use of whole serum, plasma, or complex undefined mixtures of blood-derived products. It does not exclude the use of the use of defined serum components (such as and not limited to, serum albumin, insulin, transferrin, vitronectin, or growth factors) derived from serum, plasma, blood, or recombinant gene expression technologies. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00104] The wording “xeno-free” as used herein indicates the use of protein components derived from the same species as the stem cell used for the derivation of RPE or derived by recombinant technologies and having the same amino acid sequence as that of the stem cell species. [00105] The wording “retinal pigment epithelial”, “retinal pigment epithelium”, or “RPE” as used herein indicates the pigmented cell layer of an eye of individual located outside the neurosensory retina firmly attached to the underlying choroid and overlying retinal visual cells or cells isolated from such cell layer. Key RPE characteristics are an epithelial morphology (monolayer of hexagonal shaped cells with apical/basal polarization and tight lateral junctions), expression of genes products involved in the retinoid cycle, phagocytosis of photoreceptors, polarized secretion of factors that support the function and maintenance of the retina and choroid, transport of metabolites and catabolites between the retina and choroid, and establishment of the retina:blood barrier, as will be understood by a skilled person. RPE in the sense of the disclosure can be subjected to various disfunction due to degeneration of the RPE which can results in conditions of the retina such as retinal degenerative diseases (RDD), including age-related macular degeneration (AMD), Stargardt’s macular dystrophy (SMD), Best’s disease (BEST), and retinitis pigmentosa (RP). RPE are also involved in diabetic retinopathy and in Gardner syndrome which is characterized by FAP (familial adenomatous polyps), osseous and soft tissue tumors, retinal pigment epithelium hypertrophy and impacted teeth. [17], [18]. [00106] The wording “derivation” as used herein indicates a process by which a stem cell loses pluripotency and specializes into a specific cell type. The derivation process comprises a differentiation and a maturation stage where the cell type is determined, and the cell phenotype acquired. In some contexts, not referring specifically to stem cells, it can also refer to as being isolated from. [00107] The term “differentiation” when referred to stem cells indicates the process of changing of a stem cell to a more specialized cell type, which involves a switch from proliferation to specialization. This involves a succession of alterations of gene expression, cell morphology, membrane potential, metabolic activity and signal responsiveness as will be understood by a skilled person [19]. [00108] Differentiation of stem cells can be a spontaneous differentiation or a directed Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT differentiation. [00109] The wording “spontaneous differentiation” in the sense of the disclosure indicates to a default differentiation process that occurs naturally upon removal of medium components that sustain the pluripotent state or switching to a medium that does not contain these components. In general, spontaneous differentiation relies on the presence or absence of signals derived from other cells in the culture of different germ layer origin and cell type to proceed. Accordingly, RPE can be derived from stem cells by removing factors that act to promote and maintain pluripotency, such as and not limited to TGFB1 and bFGF, in stem cell medium or by switching to a medium that does not have these factors. The yield of RPE from removal of factors is often < 20% and highly variable from line to line, presumably due to line specific preponderances to generate different ratios of ectoderm, endoderm, and mesoderm germ layer cells. Based on current understanding of RPE development, lines that generate substantial quantities of ectoderm cells with sufficiently low levels of mesoderm and endoderm cells to allow for neuroectoderm and eye field precursor cell differentiation, but not so low as to prevent their further development into RPE progenitors would be expected to result in the best yields. Increased yields can be obtained by screening multiple lines using a specific protocol to find a line with a desired yield. Alternatively, the initial culture conditions can be altered to affect the germ layer distribution. [00110] The wording “directed differentiation” in the sense of the disclosure indicates the addition of components, such as small molecules or biologics, to the medium that promote differentiation; usually in conjunction with the removal of components that promote the maintenance of the pluripotent state. Accordingly, directed differentiation of a starting stem cell can be performed by using various biologics and small molecules to direct the differentiation toward RPE. In the case of RPE differentiation, directed differentiation protocols typically use media that favor ectoderm growth and inhibitors that block mesoderm- derived signals that promote ectodermal-to-epidermal differentiation and medium that support neuroectoderm cells, followed later by addition of mesoderm/endoderm derived factors that promote RPE progenitor development. In some instances, directed differentiation is associated with increased complexity of the procedure and/or requires a selection or adjustments of a specific differentiation method for optimal RPE differentiation from different stem cell lines. [00111] The wording “semi-directed differentiation” in the sense of the disclosure refers to Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT differentiation methods that rely primarily on spontaneous differentiation processes for cell fate differentiation and employ the use of small molecules or biologics to direct a single or limited subset of differentiation steps. Semi-directed differentiation methods for a given cell type typically have complexity intermediate to that of spontaneous and directed differentiation methods. [00112] Differentiation of SC- RPE cells is marked by the appearance of intracellular golden- brown to black pigment, and preferably also by the SC-RPE cell morphology (polygonal shape and/or formation of monolayer with tight junctions) as will be understood by a skilled person. The appearance of pigment in differentiated SC-RPE cells can be detected with various methods such as i) macroscopic observation on a suitable background (e.g. on a white background) using indirect illumination (see Example 12 and Figure 1), ii) observation using brightfield microscopy, and/or iii) image analysis or by scanning light absorbance readings at wavelength between 480-600 nm to quantitively detect pigmented culture area, and additional methods as will be understood by a skilled person upon reading of the present disclosure. Differentiated SC-RPE cells in the sense of the disclosure comprise partially differentiated SC- RPE which are marked by qualitatively detectable pigment and fully differentiated SC-RPE which comprise detectable pigment in increased quantity compared to the qualitative detectable pigment of the partially differentiated SC-RPE. Accordingly, specific quantitative detection threshold to distinguish between partially differentiated SC-RPE cells and fully differentiated SC-RPE cells can be set in connection to a specific imager configuration, specific image collection settings, and specific image analysis software. [00113] The term “maturation” as used herein with respect to stem cells indicates the process of cells maturing and specializing, and eventually losing their ability to divide and become new cells. Pluripotent stem cells have the ability to become any cell in the human body. These cells form other cells, and through development, and maturation become specialized to a specific task [20]. [00114] The term “maturation” as used herein with respect to RPE or SC-RPE refers to any step in the process where a proliferating cell with a determined RPE cell fate establishes a confluent monolayer, becomes quiescent, acquires a characteristic RPE morphology, increases expression of RPE marker genes, decreases expression of genes associated with proliferation and wound repair, develops melanosomes, and acquires the characteristic functionality of Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT native RPE in vivo [21]. [00115] The term “passage” as used herein with respect to the culturing of cells refers to the collecting of cells from one culture device and transferring the cells to a new culture device. For adherent cell culture this generally entails the use of cell dissociation agents or mechanical means to remove the cells from the culture surface and generate a suspension of single cells and/or cell clumps. The resulting suspensions are then seeded or plated in the new culture device such as, a culture container (dish, flask microplate, roller bottle) or bioreactor; typically at a lower density than they were at in the originating culture. When they are plated at the same or higher density the process can be referred to as “replating”. [00116] The term “confluence” as used herein with respect the culturing of cells refers to the complete (100%) coverage of the culture surface with cells with exception of any areas where cells are absent due to plating artifacts, substrate defects, or incidental injury as evident by clear regions absent of any cells, often with clearly defined borders or defined areas where cells appear to have been removed or damaged by scrapping or excessively forceful pipetting. [00117] The term “near confluence” as used herein with respect to the culturing of cells refers to the approximately 24-hour period preceding the time that they are estimated to become confluent; in other words, the day before confluence is estimated to occur based on known or observed doubling rate of the cell line in the cell culture on a selected substrate. For example, one of skill can follow culture growth on a daily basis and based on this observation estimate the culture doubling rate. With that knowledge reasonable predictions of whether confluence occurs in the next 24 hours can be made. For example, if the culture doubles every day, then near confluence would correspond to having confluence of 50-99%. If desired, one can validate the prediction by continuing to grow the culture with the lowest cell density and determine its state of confluence the next day. [00118] The term “just confluence” as used herein with respect to the culturing of cells refers to anytime within the 24- to 32-hour period after confluence occurs. Identification of just confluence can be performed by daily monitoring of the cultures and detection of just confluent cells starting from the time when the cells are first observed to be confluent. [00119] With respect to mature SC-RPE, it is generally accepted that the SC-RPE should be qualitatively indistinct from primary RPE in that, they should express all key RPE genes and Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT few non-RPE genes as will be understood by a skilled person. Accordingly, maturation can be detected by example in the form of gene expression analysis where SC-RPE detected genes are compared to genes detected in fetal RPE. [00120] Accordingly, a skilled person will understand that “differentiation” in methods of the disclosure refer to the case of a cell type changing to another cell type (i.e. cell type determination) and the term “maturation” in methods of the disclosure refer to the change in phenotype that occurs when a determined RPE cell becomes confluent, exits the cell cycle, and obtains a prototypical functional phenotype. The term “dedifferentiation” describes the process where RPE undergo an “irreversible switch to a mesenchymal phenotype due to accumulative rounds of cell division or suboptimal culture conditions”. These cells can be called RPE- derived mesenchymal cells or mesenchymal-RPE. Whenever RPE are in a state of proliferation but still have the potential to turn into mature RPE they are generally referred as RPE unless otherwise indicated. [00121] In some embodiments, herein described serum-free methods are described and related cells compositions methods and systems that allow to obtain an efficient SC-RPE differentiation in a stem cell line specific manner. [00122] In those embodiments, methods and systems of the disclosure, an alternative approach is used based on the unexpected finding that the differentiation media and substrate act as a functional unit to alter RPE cell fate determination and different cell lines have unique preference profiles across a collection of different media and substrate combinations as can be most easily observed after a process of SC-RPE maturation that enables and/or enhances SC- RPE pigmentation or by using immunohistology methods to identify immature SC-RPE prior to maturation. [00123] In particular, the differentiation methods and system of the present disclosure are based, in part, on the unexpected finding that, some cells have preferences for specific media:substrate combinations where for a certain cell line some media:substrate combinations yield more SC-RPE than do other media:substrate combinations and where for another cell line other media:substrate combinations yield more SC-RPE. [00124] For example, for cell lines H1, H9 Shef1, iPSC #. 110211, and iPSC #032411, differentiation performed on exemplary media:substrate combinations provided little to no SC- Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT RPE yield, or yields well in excess of 50%, in a cell line specific manner (see Example 13 and Figure 1). Additionally, for those cell lines and media:substrate combinations, it has been surprisingly found that the cells lines have varying degrees of media:substrate dependency. In particular, it has been found that one line gave SC-RPE yields greater than about 50% on 66% of the tested media:substrate pairs, and another line gave only yields of greater than 50% in 11% of the same media:substrate combinations (see Example 13 and Figure 1). [00125] Consequently, the methods and systems for SC-RPE cell differentiation described herein can be used by the skilled artisan to select a stem cell line and corresponding preferred differentiation medium:substrate combination to obtain an efficient generation of a population of partially differentiated SC-RPE, presumptive RPE progenitors, and immature SC-RPE across a greater number of stem cell lines than is possible using a single differentiation medium and substrate. [00126] Accordingly, a “toolbox” method and systems and related serum-free SC-RPE differentiation media are herein described, wherein the term “toolbox” indicates a method where the specific steps and reagents used are selected by the user in view of the starting material and the result desired, as well as experimental design. [00127] In particular, in method and systems of the disclosure and related serum-free SC-RPE differentiation media herein described combinations of serum-free SC-RPE differentiation medium and SC-RPE cell culture substrate are selected depending on the starting stem cell line, to perform SC-RPE differentiation in accordance with the present disclosure. [00128] The wording “culture medium” or “medium” as used herein in connection with cell culture indicates a liquid or semi-solid (e.g., gel) designed to support the survival, growth, and cellular functions of the referenced cell. Accordingly, culture media typically comprise a carbon source such as glucose, water, various salts, a source of amino acids and nitrogen as well as any other component required for the survival growth and cellular function of the cultured cells. Cell culture media can also contain pure protein additives like growth factors as well as vitamins, cholesterol, amino acids, and fatty acids as well as other components from various sources as will be understood by a skilled person. Culture media can comprise components from natural animal sources (such as tissue extracts and biological fluids like plasma, serum, and amniotic fluid) as well as from artificial or synthetic sources. [00129] Accordingly, culture media can be categorized as media from natural sources and Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT synthetic or artificial media which can contain nutrients, vitamins, salts, oxygen and carbon dioxide gas phases, blood serum proteins, carbohydrates and cofactors in various combinations depending on the cultured cell [22]. Cell culture media can also be categorized as serum containing media and serum-free media. Bovine calf serum, newborn calf serum, fetal bovine serum, horse serum and human serum are used in cell culture to provide hormones, growth factors, protective protease inhibitors, chelators or carriers for nutrients, essential nutrients, components that promote cell adherence, as well as to bind and neutralize toxins. Serum-free media or ‘defined culture media’, are media which lack serum but have defined quantities of growth factors, lipoproteins and other proteins normally found in serum. Stem cells can be typically cultured in media containing serum or serum-free media with specific growth factors and additives which either promote stem cell self-renewal or promote differentiation into specialized cell types. The factor bFGF (basic fibroblast growth factor) is added to regulate stem cell-renewal for human embryonic stem cells (ESCs), induced pluripotent stem cells and neural stem cells. Leukemia Inhibitory Factor (LIF) inhibits differentiation and promotes the expansion of mouse but not human ESCs. Culture medium used for the propagation of human stem cells most typically includes the addition of transforming growth factor beta 1 (TGFB1) or nodal to maintain pluripotency. Retinoic acid, ascorbic acid, hormones and DNA demethylating agents, intracellular cAMP elevating IBMX, growth factors, or small molecule regulators of different extracellular or intracellular pathways can induce stem cells to differentiate down defined pathways [23]. [00130] A “serum free SC-RPE differentiation medium” in the sense of the disclosure is a cell culture medium which lacks serum but have a combination of components configured to allow for the establishment of an effective ratio of ectoderm, endoderm, and mesoderm progenitor cells within the stem cell culture. The optimal ratio can vary depending on stem cell line and culture substrate, but in general it should skew towards the production of ectodermal cells yet with sufficient numbers of endoderm and mesoderm cells to provide the proper cues to enable the natural development of RPE. Aspects of the differentiation medium that affect cell growth rates or the relative length of time in different stages of the cell cycle such as the concentration or composition of the carbon energy sources, essential and non-essential nutrients (amino acids, lipids, and vitamins), metal salts, trace minerals, growth factors or those that might affect cell signaling, such as divalent cations, pH or specific amino acids can all act alone or in concert as will be understood by a skilled person. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00131] The term “cell culture substrate” or “substrate” as used herein indicates a material on which a cell or organism grows or is attached. Exemplary cell culture substrate comprise extracellular matrices composed mainly of proteins which are configured to facilitate attachment of cells through binding to specific cell surface matrix receptors and to provide chemical cues that affect or guide the behavior of cells interactive with each matrix. Cell substrates are commonly used to coat the inert physical supports that the cells are grown on such as plastic cultureware, porous or nonporous films or membranes, or porous or non-porous microcarriers. Additional exemplary substrate is the basement membrane, also referred to as basal laminae, which comprise extracellular sheets of proteins that surround tissues, providing structural support, a filtration function, and a surface for cell attachment, migration, and differentiation. In some instances, substrates can be used directly to produce 2- or 3- dimensional supports on which the cells may be grown on or in [24]. [00132] An “SC-RPE cell culture substrate” in the sense of the disclosure is a substance configured to support stem cell attachment and growth and allow SC-RPE differentiation of stem cells in presence of a serum-free SC-RPE differentiation medium of the disclosure. In particular, the substrate facilitates the establishment of the optimal ratio of ectodermal, endodermal and mesodermal progenitor cells (ectoderm greater than endoderm and mesoderm) to allow for the natural development of RPE as will be understood by a skilled person upon reading of the present disclosure. Two-dimensional culture indicates the process of growth of cells on solid surface on which cells are seeded onto and that cannot be penetrated by the cells. Exemplary SC-RPE cell culture substrate which can be used in methods and systems herein described comprise laminin 521, such as truncated or full-length vitronectin, laminin 111, laminin 211, laminin 221, laminin 332, laminin 411, laminin 511, and soluble basement membrane matrix extract such as Matrigel^TM and additional SC-RPE cells culture substrate identifiable by a skilled person. [00133] In embodiments herein described a “serum-free SC-RPE differentiation medium” refers to a cell culture medium comprising a defined combination of essential components (e.g. serum albumin, insulin, transferrin, selenium), salts (Na, K, Ca, Mg, Cl, SO₄, PO₄ of composition similar to serum), pH buffer(s), carbohydrate energy source(s), vitamins, amino acids, lipids and trace minerals) that support cell growth and absent of factors or agents that maintain pluripotency such as bFGF, TGFB1, or nodal. For optimal efficiency of spontaneous SC-RPE differentiation, the differentiation medium in conjunction with a specific cell Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT substrate, results in the preferential production of ectoderm germ layer cells in the immediate days following its addition to allow for subsequent efficient production of neuroectoderm cells, yet with sufficient quantities of mesoderm and endoderm germ layer cells to later direct the production of RPE progenitors. In addition, the differentiation medium supports the maintenance and growth of RPE progenitors. As each stem cell line is unique due to different genetics and culture history, the preferred medium for a given substrate according to the present disclosure is typically identified empirically, as it is expected that the medium that works best with one cell line may or may not work well with another cell line. [00134] Accordingly in some preferred embodiments assessing different differentiation media or modified media formulations is performed by carrying out a complete RPE differentiation process of a candidate differentiation medium on tested RPE cells with methods identifiable by a skilled person upon reading of the present disclosure, determining the final efficiency of SC-RPE differentiation based on detected relative proportion of pigmented cells or the production of RPE gene products such as the highly expressed and secreted protein, pigment epithelial-derived factor (PEDF) according to methods identifiable by a skilled person; and selecting the tested RPE cells having a final efficiency above a set threshold indicative of a target efficiency based on the experimental design. However, for faster or larger scale screening of differentiation media it is possible to monitor performance by determining the absolute quantity and percentage of desired cell types which can be performed by detection of germ layer markers (e.g. SOX17, GATA4, T, NODAL, HAND1, CDX2) in the days following transition to the differentiation medium and neuroectoderm/eye field/RPE markers (e.g. PAX6, RAX, LHX2, MITF, SOX9, SOX10) in the days and weeks following the switch to differentiation medium using appropriate methods. [00135] In some embodiments, an SC-RPE differentiation media herein described is from the RPE Differentiation Medium 1 (RPE-DM1) group of media of the disclosure and comprises the commercially available base medium KnockOut™ DMEM supplemented with KnockOut™ Serum Replacement Medium, GlutaMAX™, nonessential amino acid supplement, ^-mercaptoethanol, and normocin. A distinguishing feature of RPE-DM1 is the use of KnockOut™ Serum Replacement (KOSR) as the source of additional supplements required for cell growth and maintenance. [00136] KnockOut™ DMEM (KO-DMEM) is a basal medium based on the formulation of DMEM and optimized for growth of undifferentiated embryonic and induced pluripotent stem Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT cells. DMEM (Dulbecco’s Modified Eagle Medium) is a widely used basal medium for supporting the growth of many different mammalian cells. The composition of standard DMEM is reported in the following Table 1. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Table 1 DMEM composition [25]. [00137] DMEM is unique from other media as it contains 4 times the concentration of amino acids and vitamins than the original Eagle’s Minimal Essential Medium. DMEM was originally formulated with low glucose (1 g/L) and sodium pyruvate, but is often used with higher glucose levels, with or without sodium pyruvate. The KO-DMEM used in the preferred formulation of RPE-DM1 of this disclosure is based on the high glucose plus pyruvate DMEM formulation. DMEM contains no proteins, lipids, or growth factors. Therefore, DMEM requires supplementation, commonly with 10% Fetal Bovine Serum (FBS). DMEM uses a sodium bicarbonate buffer system (3.7 g/L), and therefore requires a 5–10% CO2 environment to maintain physiological pH [26]. [00138] In KnockOut™ DMEM, which contains no L-glutamine, the osmolarity of original DMEM is optimized to approximate that of mouse embryonic tissue (~320 mOsm/L). High glucose plus pyruvate DMEM, which KnockOut™ DMEM is based on, has a calculated osmolarity of approximately 360 mOsm/L. It is expected that the osmolarity of KnockOut™ DMEM has been adjusted with respect to DMEM by reducing the amount of sodium chloride and/or the amount of sodium bicarbonate in the original DMEM formulation by about 40 mOsm/L (see Table 1), as these are two major contributors to the final osmolarity of the medium. Alternatively, the osmolarity of DMEM may be reduced by reduction of the concentration of all medium components (except glucose and pyruvate whose concentration in KO-DMEM are stated) by about 10%. KnockOut™ DMEM has been used in literature in the Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT culture of stem cells. (see e.g. [27], [28], [29]). [00139] KnockOut™ DMEM or the cell therapy manufacturing grade CTS™ KnockOut™ DMEM with phenol red and without glutamine is available by the manufacturer ThermoFisher with product code: Gibco™ 10829018 and A1286101^ [30] at the filing date of the present disclosure. [00140] KnockOut™ Serum Replacement (KOSR) is a FBS-free formulation designed to replace FBS in feeder-dependent ESC and iPSC cultures. It has been cited in more than 2,000 publications on various PSC-related applications. The preferred composition and expected usable concentration ranges, formulation methods, and guidelines for its use are reported in patent publication WO1998030679A1 [31] incorporated herein by reference in its entirety. Based on this publication it is expected that KnockOut™ Serum Replacement is comprised of the proteins lipid-rich albumin (AlbuMAX), insulin, and holo-transferrin; the amino acids glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L- hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine; the vitamins and antioxidants thiamine, L-Ascorbic acid 2-phosphate, reduced glutathione; and the trace elements Ag+, Al⁺³, Ba⁺², Cd⁺², Co⁺², Cr⁺³, Ge⁺⁴, Se⁺⁴, Br⁻, I⁻, F⁻, Mn⁺², Si⁺⁴, V⁺⁵, Mo⁺⁶, Ni⁺², Rb⁺, Sn⁺², Zr⁺⁴. For ease of reference, the preferred concentration of each of these components is provided in Table 2. KnockOut™ Serum Replacement is commercially available and sold by manufacturer ThermoFisher with catalog numbers 10828010 for 100 mL and 10828028 for 500 mL or 12618013 for the CTS™ KnockOut™ SR XenoFree Medium which is the cell therapy manufacturing grade equivalent. [32] at the filing date of the present disclosure. ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^{^}^ ^^ ^^ ^^^^^^^^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^ ^^ ^^ ^^ ^^^^^^^^ ^^^^^ ^^ ^^ ^^ ^^^^^^ ^^^!" ^^^ #^^#^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ $^%^^^"^ ^^^&^ ^^ ^^ ^^ ^^^^^'^^^^"^ ^^&^^ ^^ ^^ ^^ ^^^%^ ^^%^ ^^^^"^ ^()^ ^^ ^^ ^^ ^^^^^^"^^^^"^ ^*^^^^ ^^ ^^ ^^ ^^^"'^^^^^^"^ (#)^ ^^ ^^ ^^ ^^+^"^%^^^^^^^"^ ^#&^^^ ^^ Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^{^}^ ^^ ^^ ^^ ^^+ ^ ^^ ^^ ^^ ^^," ^^"^ ^(^)^ ^^ ^^ ^^ ^^^^ "^^^^"^ ^&^(^^ ^^ ^^ ^^ ^^^ %^'^^^^^^ ^^*^^ ^^ ^^ ^^ ^^^% ^^^^"^ ^^)^ ^^ ^^ ^^ ^^-^^^^"^ ^#^&(^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^ ^^ ^^ ^^ ^^ ^^^^^^ ^^^^^^^^^^^^^^^^^^'"^ ^^^&^ ^^ ^^ ^^ ^^^^^^^"^ ^^^&^ ^^ ^^ ^^ $^^'^'^^^^"^^ "^^^"^^^ )^^*^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^./0)^1^.²3^ #^##*)#^ ^^ ^^ ^^ ^^4^)^5^(^^0^1^^⁾²3^ #^#^^^^ ^^ ^^ ^^ 6^^4^^^0^^^^16^^{^2}3^ #^#)^*^ ^^ ^^ ^^ 4^,0^^5^&^^0^14^^{^2}3^ #^^#^^ ^^ ^^ ^^ 4^4^^^5^^^0^14^^{^2}3^ #^#(^(^ ^^ ^^ ^^ 4 ^^,0^^)^5^^^0^14 ⁾²3^ #^##^)^^ ^^ ^^ ^^ $"0^^1$"^{^2}3^ #^#^&^^ ^^ ^^ ^^ /^_^,"0₎^1,"^{^2}3^ #^^^*^ ^^ ^^ ^^ ^_^,"0₎^1,"^{^2}3^ #^^**^ ^^ ^^ ^^ 76 ^16⁸3^ #^##*#^^ ^^ ^^ ^^ 7^^1^⁸3^ #^##*^^^ ^^ ^^ ^^ ^^4^_^^5^^^_^0^1^^^{^2}3^ #^#^#^^ ^^ ^^ ^^ /^9^19⁸3^ #^^^(^ ^^ ^^ ^^ /^^,^0)^5^^^^0^1,^^{^2}3^ )^*^^ ^^ ^^ ^^ /^-0)^1-^*23^ #^#^^^^ ^^ ^^ ^^ ^/^^^(^^^0^^^5^^^^0^1^^⁽²3^ #^#^&*^ ^^ ^^ ^^ /^,0^^5^(^^0^1/^^{^2}3^ #^##)&^^ ^^ ^^ ^^ ^^4^^1^^²3^ #^#*^^^ ^^ ^^ ^^ ,^4^^^1,^^{^2}3^ #^##^*&^ ^^ ^^ ^^ : 04^^^5^&^^0^1: ^{^2}3^ #^#&^^^ ^^ + "!" "^^*#;^,'^^<^! ^^^+^'"^'^ ^^ =0^^^&#)#(^^^^^ ^^ ^^{^} ^^"^^^'^>"^"^"^"^'^^^^^^^^^^'"^^^^^^ ^^<"'^^^0'^" ^^^^'^!^ ^^^ ^^%^^"^^^^^'^'^'"^^ [00141] The KnockOut Serum Replacement composition reported in Table 2 can be used in methods and systems herein described as Embryonic Stem Cell Serum Replacement (ESCSR) as will be understood by a skilled person upon review of the present disclosure. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00142] GlutaMAX™ supplement is an improved cell culture supplement that can be used as a direct substitute for L-glutamine in cell culture media. GlutaMAX™ Supplement is an alternative to L-glutamine, with increased stability that improves cell health [33]. GlutaMAX™ Supplement is suitable for both adherent and suspension culture of mammalian cells, with no adaptation required. GlutaMAX™ Supplement is offered as a 200 mM solution (100X) of L- alanyl-L-glutamine dipeptide in 0.85% NaCl. GlutaMAX™ Supplement is also included in a variety of media formulations. Compared to L-glutamine, GlutaMAX™ Supplement minimizes toxic ammonia build-up, improves cell viability and growth, and remains stable across a wide range of temperatures. GlutaMAX™ is commercially available from manufacturer ThermoFisher with catalog numbers 35050061 for 100 mL or 3505007920×100 mL or A1286001 for 100 mL of the clinical manufacture grade equivalent. Alternatively, the solid L-alanyl-L-glutamine dipeptide can be obtained from multiple alternative vendors and used as would be clear to a skilled person based on the above general description at the filing date of the present disclosure. [00143] In its most preferred embodiment, about 500 mL of RPE-DM1 Medium is formulated by combining 389 mL of CTS-KnockOut™ DMEM, 100 mL of CTS-KnockOut™ Serum Replacement, 5 mL of 200 mM CTS-GlutaMAX™ solution, 5 mL of 100X MEM Non- essential Amino Acids solution (100 mM alanine, 100 mM asparagine, 100 mM aspartic acid, 100 mM glutamic acid, 100 mM glycine, 100 mM proline, and 100 mM serine; ThermoFisher Scientific catalog numbers 11140050 or 11140076), and 909 ^L of 55 mM ^-mercaptoethanol (ThermoFisher Scientific catalog number 21985023). In addition, in applications where their use is allowed antibiotic or antimycotics such as penicillin streptomyocin, and normocin can be included in the medium, but their use is optional, and they are not believed to substantially impact RPE differentiation when used at their recommended concentrations. The concentration of the RPE-DM1 Medium components including the most preferred and the least preferred concentrations, are listed in Table 3. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00144] In each of Set-1, Set-2, Set-3, Set-4 and Set-5 reported in Table 3, each concentration within the indicated range for a component of the RPE-DM1 composition can be combined with each concentration within the indicated range of another component of the RPE-DM1 composition. Therefore, for example in Set 4 SnCl2 [Sn²⁺] can be comprised at a concentration of 0.000179 um in combination with ZrOCl₂ [Zr⁴⁺] at a concentration of 0.016 uM and in combination with any other concentrations comprised within each range of the other components as will be understood by a skilled person, providing that the osmolarity remains within the prescribed ranges. [00145] For xeno-free applications of the method the protein components of the media should be derived from the same species as the stem cell line. Alternatively, nonprotein-based substitutes can be employed as will be understood by a skilled person. [00146] Variations in the exemplified RPE-DM1 Medium of similar composition are expected to give rise to RPE differentiation. For example, alternative medium formulations can be made by changing the percentage of the KnockOut™ Serum Replacement from the most preferred concentration of 20% to within the range of 15 to 20% (preferred), 10 to 25% (less preferred), or 5 to 30% (least preferred). Additionally, in some cases lipid-rich serum albumin or transferrin substitutes can be used in place of or in addition to these two protein components as will be understood by a skilled person. For example, in some embodiments, lipid-rich Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT albumin is substituted with its lipid components, and in particular with lysophosphatidic acid and sphingosine-1-phosphate, either by themselves or in combination with lipid-depleted (Fraction V) albumin [34]. In some embodiments, transferrin can be substituted with a complex of a lipophilic iron chelator, such as pyridoxal isonicotinoyl hydrazone, and ferric iron [35]. Similarly, the concentration of the nonessential amino acids, GlutaMAX™ and ^- mercaptoethanol may be altered by adjusting their relative ratios of their solutions and glutamine can be used as a direct 1:1 substitute for GlutaMAX™ (see Table 3 for allowable concentration ranges, see any one of sets 1 to 5). Other base medium such as, DMEM, KnockOut™ DMEM:F12, DMEM:F12 mixtures, ^-MEM, RPMI 1640, and Iscove’s Modified Dulbecco’s Medium and their variations can be substituted for KnockOut™ DMEM providing that the concentration of the individual medium components and final medium osmolarity remain in the indicated ranges in Table 3 (see any one of sets 1 to 5). The concentration of the medium components can be calculated based on the published alternative base medium formulations and the preferred composition of Knockout Serum Replacement (International Patent application PCT/US1998/000467 Publication No. WO1998030679A1, incorporated herein by reference in its entirety) [31] used as an Embryonic Stem Cell Serum Replacement (ESCSR) supplement. RPE-DM1 formulation variants are denoted by their base medium and percent volume of KnockOut^TM Serum Replacement with additional changes to the other components (NEAA, GlutaMAX™, ^ME, or KOSR composition) addended (e.g., KO- DMEM/20%KOSR, CTS-KO-DMEM/20%KOSR, KO-DMEM/15%KOSR, CTS-KO- DMEM/20%KOSR/Glutamine, KO-DMEM:F12/20%KOSR). Media formulations that meet the requirements listed in Table 3 are expected to support stem cell differentiation to some degree, although differences in yield and substrate preference between media for a given stem cell line are expected. Their utility for derivation of RPE from specific stem cell lines can be assessed by monitoring stem cell survival and growth as well as the expression of germline, retinal progenitor, and RPE signature genes after transition to the differentiation medium using simple experimentation as would be understood by a skilled person in the field. [00147] A representative example of an SC-RPE differentiation media formed by the most preferred formulation and allowable concentration ranges of RPE-DM1 Medium (Table 3) in accordance with the present disclosure and related method of preparation is reported in Example 2. [00148] Providing that the individual components and final medium osmolarity remain in the Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ranges listed in Table 3, in some embodiments RPE-DM1 Medium formulations not possible by simple combination of commercially available base medium and KnockOut™ Serum Replacement can be made using custom base medium formulations, diluted or concentrated base medium, alteration of the concentration of the individual components in base medium by supplementation or subtraction of individual medium components, blended mixtures of base medium, or modified KnockOut Serum Replacement as described in International Patent application PCT/US1998/000467 Publication No. WO1998030679A1[31]. [00149] In general, changes of the composition are expected to affect media:substrate preference and/or RPE yield as will be understood by a skilled person upon reading of the present disclosure. The rate of growth is expected to affect a stem cells propensity into the different germ layer cell types, either directly by changing the relative and absolute time in various stages of the cell cycle or by affecting the cell density and timing of events in that are cell density dependent. Furthermore, different stem cell lines have different inherent growth rates; possibly due to common genetic variation which is another prime determinant of differentiation potential [36]. Hence, changes to the medium composition that would be expected to alter growth rates such as altering the concentration and ratio of the two primary carbon energy sources (glucose and pyruvate), the concentration of the amino acids, substantial reduction in the concentration of the vitamins, changes to the pH (carbonate/CO2 and HEPES), the inorganic salt concentrations and relative ratios, and the concentration of the protein components (insulin, lipid rich serum albumin, and transferrin), and redox potential would have an expected effect on cell growth that would be clear to a skilled person in the field and could be tested by simple measurement of growth rates in the alternative medium formulations. In addition, some medium components may have effects on RPE differentiation that are not mediated by effects on cell growth. For example, it is expected that at least in some cases calcium signaling plays an essential role in ectoderm-to-neuroectoderm differentiation and calcium plays an essential role in cell:substrate and cell:cell adhesion. As such, altering the calcium concentration is expected to affect the yield and possibly the substrate preference in a stem cell line dependent fashion. The suitability of alternative medium in accordance with the present disclosure is preferably ultimately validated experimentally using several different stem cell lines with different propensities as would be understood by skilled person in the field. [00150] In some embodiments, an SC-RPE differentiation media of the disclosure is a medium comprising X-VIVO™ 10 Medium supplemented one or more of the following supplements: Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT insulin, transferrin, sodium selenite, progesterone, putrescine, serum albumin, catalase, superoxide dismutase, corticosterone or hydrocortisone, d-galactose, ethanolamine, glutathione (reduced), L-carnitine, linoleic acid, linolenic acid, triiodo-I-thyronine (T3), DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A (retinyl acetate, retinol acetate) along with the optional ingredient biotin and lipoic acid. The medium resulting from the combination of X-VIVO™ 10 medium and the listed supplements is herein referred to a RPE Differentiation Medium 2 (RPE-DM2). The distinguishing feature of media classified as being RPE-DM2 is the use of the complete medium X-VIVO^TM 10 (or medium of similar composition and functionality) that is preferably further augmented with a B27-based supplement. [00151] The X-VIVO™ 10 Medium is a commercially available serum-free Hematopoietic Cell Medium, with L-glutamine, and with or without gentamicin and phenol red that was designed to support the generation of LAK cells in a serum-free environment. X-VIVO™ 10 Media formulations are designed to support slower growing, less mature cells such as hematopoietic stem cells and natural killer cells. X-VIVO^TM 10 also supports the generation of lymphokine activated killer (LAK) cells and peripheral blood lymphocytes (PBL). Key applications for the use of X-VIVO™ 10 medium are: proliferation of peripheral blood lymphocytes, proliferation of tumor infiltrating lymphocytes, cryopreservation of human tissue, cultivation of human monocytes and macrophages, cultivation of dendritic cells, cultivation of CAR-T cells, and cultivation of hematopoetic stem cells. X-VIVO™ 10 Media is available from Lonza Biosciences as a 1X liquid in three different formulations: research grade X-VIVO^TM 10, which contains L-glutamine, gentamicin, and phenol red (catalog number BE04-380Q), cGMP grade TheraPeak X-VIVO^TM 10, which contains L-glutamine and without gentamicin, or phenol red and uses native human transferrin (catalog number BEBP04-743Q), and cGMP grade TheraPeak X-VIVO^TM 10, which contains L-glutamine and without gentamicin, or phenol red and uses recombinant human transferrin [catalog number (BEBP02- 055Q) [37]. [00152] X-VIVO^TM 10 is a medium formulation comprised of a base medium, human serum albumin, human insulin, human transferrin, and possibly unknown additional supplements such as essential lipids and antioxidants. Iscove’s Modified Dulbecco’s Medium (IMDM), RPMI 1640, and DMEM are the most common base medium (in decreasing order) used in serum supplemented hematopoietic cell culture media [38] and IMDM is known to be the base medium used in two other commercially available serum-free media that are used for the same Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT applications as X-VIVO^TM 10 (StemSpan Media family SFEM, SFEMII, H3000, and ACF from STEMCELL Technologies and QBSF-60 from Quality Biological). It is therefore expected that IMDM and possibly RPMI 1640 and DMEM can be comprised within or equivalent to the base medium of X-VIVO^TM. [00153] The additional supplements are a collection of compounds commonly used in neuronal culture. The supplement mixture is a defined yet complex mixture of antioxidant enzymes, proteins, vitamins, and fatty acids that are combined in optimized ratios to support neuronal survival in culture. [00154] In some embodiments of the disclosure RPE-DM2 is prepared using the base culture media Iscove's Modified Dulbecco's Medium (IMDM) and/or RPMI 1640. In particular, RPE- DM2 can be prepared using IMDM, liquid, with phenol red (#12440053, 500 mL ThermoFisher), and RPMI 1640 liquid formulation, with glutamine, with phenol red (#11875093, 500 mL, ThermoFisher), The composition of preferred formulations of IMDM and RPMI 1640 is reported in Table 4 below. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00155] Other formulations of IMDM are also available (such as powder, with GlutaMax, or Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT with phenol red that can also be used as well as would be understood by a skilled person. [00156] Other formulations of RPMI 1640 are also available (such as powder, or with GlutaMax, or with HEPES, or with phenol red etc.) that can also be used as well as would be understood by a skilled person. [00157] In some embodiments RPE-DM2 is prepared by blending the IMDM and RPMI 1640 in various proportions to obtain unique RPE-DM2 base media formulations in the sense of the present disclosure. In particular, the resulting general formulation of an RPE-DM2 medium is reported in Table 4a below. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00158] In the RPE-DM1 formulation of Table 4a, each concentration within the indicated range for a component of the RPE-DM1 composition can be combined with each concentration within the indicated range of another component of the RPE-DM1 composition within the indicate osmolarity. Due to the substantial differences due to the use of HEPES and increased amounts of glucose in RPMI 1640 and the compensatory differences in inorganic salt composition (primarily NaCl and NaHCO³), when comprising a media using the individual components the medium resulting from the combination of various components at any one of Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT the concentration within the indicated ranges, it is preferred that the medium has an osmolarity about 290-325 mOsm/L, and is preferably 300-310 mOsm/L by adjusting the amount of NaCl accordingly. In addition for CaCl₂ and Ca(NO3)₂ the two should be considered together and when using Ca(NO3)₂ it is preferred that the amount of CaCl₂ be reduced. The same principle holds for mono- and dibasic sodium phosphate. [00159] In some embodiments, RPE-DM2 medium can be provided by combining IMDM:RPMI1640 in various proportions as will be understood by a skilled person. In particular, in some embodiments the base medium of RPE-DM2 can be provided by mixing fixed proportions of each media (IMDM:RPMI = (10:0), (8:2), (6:4), (4:6), (2:8), and (0:10)) with ranges between adjoining the fixed mixtures IMDM:RPMI (10:0) to (8:2), (8:2) to (6:4), (6:4) to (4:6), (4:6) to (2:8), and (2:8) to (0:10). The related composition including ranges of possible concentrations of each component, comprised between the fixed dilution ratios is reported in Tables 4b to 4f below. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00160] In each of the RPE-DM2 media reported in Tables 4a to 4f, each concentration within the indicated range for a component of the RPE-DM2 composition can be combined with each Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT concentration within the indicated range of another component of the RPE-DM2 composition providing that the osmolarity remains in the specified range, as will be understood by a skilled person. [00161] In some embodiments a custom RPE-DM2 medium can be prepared based on the concentration ranges of the individual components provided in Tables 4a to 4f with the proviso that the calculated osmolarity of the custom formulation is 290-330 mOsm/L, preferably 300- 320, and more preferably 300-310. This can be generally achieved by adjusting the sodium chloride concentration to a value in the range of 113-139 mM as necessary. [00162] In some embodiments of RPE-DM2 medium of the disclosure, IMDM, RPMI 1640, or the IMDM:RPMI blended media of any one of Tables 4a to 4f can then be supplemented with insulin, selenite, transferrin, ethanolamine, and albumin (collectively known as ITSE+A) to generate a minimal RPE-DM2 (IMDM-ITSE+A, RPMI-ITSE+A, or IMDM:RPMI- ITSE+A). [00163] In some embodiments of RPE-DM2 medium of the disclosure, xeno-free ITSE+A can be obtained as a 100X cocktail from InVitra (catalog number 777ITS092). ITSE+A is comprised of 1.00 g/L recombinant human insulin, 0.55 g/L recombinant human transferrin, 0.00067 g/L sodium selenite, 0.20 g/L ethanolamine, and 20.00 g/L recombinant human albumin. [00164] In some embodiments of RPE-DM2 medium of the disclosure, ITSE+A used at the preferable strength of 1X, less preferably at 0.25-0.5X, 0.5-0.75X, 0.75-1X, 1-1.5X, 1.5-2.0X, or 2-3X. To gain further control of the concentration of individual components ITSE+A they can be obtained individually, or as sub-cocktails (ITS and ITSE) and custom formulations can be prepared where the concentration of one or more of the individual components is adjusted within the forementioned range. [00165] In some embodiments, an SC-RPE differentiation media RPE-DM2 of the disclosure is supplemented with a B27-based supplement. The original serum-free neuronal culture supplement composition, B27, was developed by Dr. Gregory Brewer and colleagues and is described in [39] and [40] and a modified B27-based composition, named NS21, is described in [41]. Typically, these supplements are prepared as concentrated stocks which are then added to the base medium. The formulations of B27 and NS21 at 1X strength based on an online Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT published protocol [42] and the original publications are provided in Table 5. Suitable B27- based 50X liquid stock solutions are commercially available from ThermoFisher Gibco (B- 27™ Supplement (50X), serum free [catalog numbers 17504001 and 17504044]; B-27™ Plus Supplement (50X), [catalog number A3582801]; B-27™ Supplement, XenoFree, [catalog number A1486701], CTS™ B-27™ Supplement XenoFree (50X) [catalog number A5047501]) and STEMCELL Technologies (Neurocult SM1, catalog number 05711). NS21- based medium supplements can be obtained from Millipore Sigma (N21 Medium Supplement (50X), catalog number SCM081) and R & D Systems (N21-MAX Media Supplement (50X), catalog number AR008). These commercial stocks have the same composition of compounds listed in Table 5, but the exact formulations are proprietary and the source (e.g., species, recombinant) and concentration of some of the components may vary from the original published versions and between different commercial products. ^^^^^^*^^+^,^{^}^^^^^-^^^^{^}^^^^^^^^^^^^.^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^/^^^^^^^^ ^^ ^^ ^^ .^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ?^^ ^^ ^^ ^^^^^^^^^ ^^ ^^ ^^ ^^ 6^>^^"^," ^^^^^^^^^^@^9 ^^'^^^^-^ )^^(^ ^^ ^^ ^^ ^^^^^^^@^^^>^^"^ #^(^&^ ^^ ^^ ^^ ^^^^^^ ^^^!" ^^@^^^^^^^ #^#(^*^ ^^ ^^ ^^ 4^'^^^^"@^^^>^^"^ #^#^#^^ ^^ ^^ ^^ ,^^" ^^^^"^A^^^^'^^"@^^^>^^"^ #^#^(^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^0^^^^^1^^^^^^^^^ ^^ ^^ ^^ ^^ $^^'^'^^^^"^^ "^^^"^^^ )^^*^ ^^ ^^ ^^ ,^^^^^^,"^"^^'"^ #^#^^*^{^}@^#^#&)#^{^}^ ^^ ^^ ^^ A^^^^^^^^^^^^^^" ^^^^^"'^'"^ ^^^^^ ^^ ^^ ^^ A^^^^^^^^^^^^^^" ^^^ ^^^#^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ 2^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ B'^^^^^^^^^"^ ^(^^^ ^^ ^^ ^^ ^^4^ ^^'^^"^^ ^#^^^ ^^ ^^ ^^ ^^^^^"^^^^^^^^ )^*^^ ^^ ^^ ^^ ^^^^^"^^^^^^^^^ )^*^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^3^^^^^^^^ ^^ ^^ ^^ ^^ A^6^^'^^^ ^#^^^{^}^ ^^ ^^ ^^ ^"'^^^^^^^"'^'"^ #^)#^^ ^^ ^^ ^^ ^"'^^^^^ #^)^^^{^}^ ^^ ^^ ^^ 4^ '^^^^'" ^^"^ #^#*^^^ ^^ Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^ ^^ + ^."^'" ^^"^ #^#^##^ ^^ ^^ ^^ ^ ^^^^^^^^'^% ^^^^"^^^)^^ #^##^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^4^^^ ^^ ^^ ^^ ^^ A^$^^^^'^^"^ &)^)^ ^^ ^^ ^^ ^^^^^^^^^^^^ #^^^&^{^}^ ^^ ^^ ^^ +^' "^^^^"^ ^##^ ^^ ^^A" ^>"^^! ^^^6 "C" ^"'^^^^@^^^^)1)^3^D^6 "C" ^^^^^4^'^^^@^^^&^1^#3^^ ^^ ^^4^"^^"'^^^^@^^##&1^^3^{^} ^ ^ ^^E^^F^"^6^^^4^^^"^' ^'^^^^ ^^ ^^ ^^E^^F^"^/,^^^4^^^"^' ^'^^^^ ^^ ^^ [00166] In some embodiments the RPE-DM2 supplemented with B27 is a medium comprising X-VIVO™ 10 Medium. In some embodiments the RPE-DM2 supplemented with B27 is a medium comprising a composition of any one of Tables 4a to 4f as will be understood by a skilled person, upon reading of the present disclosure. [00167] In preferred embodiment of RPE-DM2 medium of the disclosure, ITSE+A supplemented RPE-DM2 media are further supplemented with any of the B27-type supplements of the disclosure at a strength of 1X, less preferably 0.25-0.5X, 0.5-0.75X, 0.75- 1X, 1-1.5X, 1.5-2X, 1.5-2X, and 2-3X. [00168] In preferred embodiments of RPE-DM2 medium of the disclosure, the B27-type supplements are with vitamin A. [00169] In some embodiments of RPE-DM2 medium of the disclosure, the B27-type supplements are without vitamin A. [00170] In some embodiments of RPE-DM2 medium of the disclosure, since all of the components of ITSE+A are also provided by the B27 supplements, supplementation of IMDM, RPMI 1640, or IMDM:RPMI blends with ITSE+A any one or more of ITSE+A’s individual components can be omitted and the base media can be then supplemented with B27. In this case it is expected that higher strengths of the B27 supplements can be required to achieve a desired outcome. [00171] In some embodiments of RPE-DM2, supplementation with ITSE+A can be omitted, and the base media can be supplemented with a B27-base supplement alone. [00172] A skilled person will understand that the specific IMDM, RPMI, or IMDM:RPMI Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT based RPE-DM2 differentiation media can be evaluated first by assessing the differentiation cultures in the immediate day or two following the switch from stem cell media to RPE-DM2 to insure that there is not excessive loss of cells ( roughly >20%). In the following days the onset of expression of eye-field transcription factors (such as PAX6, OTX2, LHX2, and RAX can be evaluated. [00173] Expression of transcription factor can be performed for example with quantitative PCR, immunohistochemistry, or other equivalent methods as would be understood by a skilled person. In the following weeks the expression of the pigmented cell transcription factor MITF, RPE genes such as TYR and PMEL can be determined and at later times near the time of or after initiating RPE maturation successful outcomes can be quantified based on the percentage of pigmented culture area. In most preferred embodiments all these measures be done relative to a cell and differentiation method known to work. In the alternative, since a feature of the prototype RPE-DM2 based media X-VIVO 10 is that it supports at least some degree of RPE maturation it may be possible to prescreen novel formulations of RPE-DM2 to determine if they also support some degree of RPE maturation. It is expected that RPE-DM2 formulations that support the maturation of RPE would be good candidates for SC-RPE differentiation. This analysis can be carried out using previously obtained SC-RPE or with primary RPE cells. [00174] In a most preferred embodiment RPE-DM2 is prepared by adding the appropriate amount of any of the commercially available or custom made 50X B27-based supplement stocks prepared according to the composition provided in Table 5 to one of the three X- VIVO^TM 10 formulations to result in a 1X concentration of the supplements in medium. In addition, in applications where their use is allowed antibiotic or antimycotics such as penicillin, streptomyocin, and normocin can be included in the medium, but their use is optional and they are not believed to substantially impact RPE differentiation when used at their recommended concentrations. In some embodiments of RPE-DM2 the concentration of the additional B27- based supplements can be 0.25-3X, more preferably between 0.5-2X, and even more preferably between 0.75-1.25X. [00175] In some embodiments the concentration of the B27-based supplements can be in the range of 0-0.25X. This is generally less preferred as the yields of RPE are typically reduced, but for some lines it may be advantageous. The identification of optimum RPE-DM2 medium formulations can be determined by simple experimentation to determine their effect on germ Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT layer bias and RPE differentiation as would be understood by a skilled person upon reading of the present disclosure. [00176] A representative example of one of the most preferred embodiments of RPE-DM2 Medium formulation of the disclosure (TheraPeak X-VIVO^TM 10 with recombinant transferrin + 1X B-27™ Supplement, XenoFree) and related method of preparation is reported in Example 3. Variations in the exemplified X-VIVO^TM 10 + XF-B27 RPE-DM2 Medium are expected as will be understood by a skilled person upon reading of the present disclosure. For example, other grades of B27 or other B27-based supplement formulations such as NeuroCult SM1 (StemCell Technologies), N21 (Sigma/Millipore), N21-Max (R & D Systems) can be substituted directly for B27 as can custom made preparations of B27 and N21 based on the formulations provided in Table 5, thus providing alternative RPE-DM2 media such as X- VIVO^TM 10/B-27, X-VIVO^TM 10/B-27+, X-VIVO^TM 10/CTS-B27, X-VIVO^TM 10/NeuroCultSM1, X-VIVO^TM /N21, X-VIVO^TM 10/N21-Max, X-VIVO^TM 10/custom B27, and X-VIVO^TM 10/custom N21 and where the term X-VIVO^TM 10 denotes any X-VIVO^TM 10 of similar composition to the previously stated X-VIVO^TM 10 formulation. [00177] The choice of which of the most preferred RPE-DM2 variants to use is largely dependent on the intended use of the cells. To produce SC-RPE that are intended to be used as a clinical therapeutic the media and supplements should preferably be xeno- and animal-free and certified for the manufacture of a cell-based clinical therapeutic. For purely research purposes any of the media and supplement choices are suitable, but non-clinical grade materials are generally less costly and easier to source. However, the source or specific derivation of B27 can affect media:substrate germline bias profile in a cell line-dependent manner. For example, it has been observed that some stem lines give higher yields of SC-RPE with research grade B27, whereas others give better yields with clinical grade B27. Hence, once a media and B27- based supplement version or source is selected for a given cell line it should not be changed. [00178] As previously stated, in some embodiments, B27-based supplements can be omitted in its entirety, albeit with the possible reduction of the yields of SC-RPE. As such, in some embodiments of RPE-DM2, custom B27 or N21 formulations with varying concentrations of the individual components within the ranges that result from the previously described RPE- DM2 compositions in which the concentration of the individual components of the[00166] and the concentrations listed in Table 4 or missing one or more of the components can be used. For Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT example, since biotin and lipoic acid are unique to B27 and N21 respectively, yet B27- and N21-based supplements can be used interchangeably, it follows that biotin and lipoic acid supplementation may not be essential. Similarly, since serum albumin, insulin, transferrin, and selenite are also present in X-VIVO^TM 10, it would be expected that additional supplementation with these three components may not be essential or that their concentration in the supplement mixture may be reduced. In addition, the source of the components may be altered from what is used in the original supplement formulations. For example, lipid rich serum albumin can be substituted for lipid poor albumin or iron chelators can be substituted for transferrin. As each stem cell line is unique and there is no method to predict how a given cell line may behave, assessment of the utility of custom B27-derived formulations should be determined by simple experimentation using the stem cell lines of interest, as will be understood by a skilled person upon reading of the present disclosure. [00179] In addition to X-VIVO^TM 10, it is expected that other serum-free medium commonly used for the culture of hematopoietic cells can be used as a serum-free base medium for the formulation of RPE-DM2 according to the fore described guidelines. Examples of suitable commercially available alternative serum-free base medium include, StemSpan^TM-SFEM, StemSpan^TM-SFEM, StemSpan^TM-H3000, and StemSpan^TM-ACF from STEMCELL Technologies; AIM V^TM, CTS AIM V^TM and StemPro^TM-34 SFM from ThermoFisher Gibco; and QBSF-60 from Quality Biological. Alternatively, custom serum-free media that use IMDM, RPMI 1640, DMEM:Ham’s F12 mixtures, or DMEM as described in US Patents 5,945,337 and 6,224,860B1 can be used. Finally, it is expected that the base media, IMDM, RPMI1640, DMEM, KnockOut^TM DMEM, KnockOut^TM DMEM:F12, and DMEM:F12 mixtures each supplemented with insulin, transferrin, and selenium, and albumin (lipid-rich or -free) (ITSA) can be substituted for X-VIVO^TM 10. Each of these X-VIVO^TM 10 alternatives can then be combined with a B27-based supplement to create a RPE-DM2 medium variant as would be understood by skilled person up reading this disclosure. Alternative RPE-DM2 formulations are denoted by their base medium and type of B27-based supplement. When the final concentration of the B27 supplement is not 1X that is also indicated (e.g., X-VIVO^TM 10/CTS-B27, X-VIVO^TM 10/2X-CTS-B27, X-VIVO^TM 10/N21-MAX, X-VIVO^TM 10/NC- SM1, StemSpan-ACF/CTS-B27, IMDM/CustomB27, RPMI/CustomB27, and IMDM/ITSA/CustomB27, etc.). While the use of these alternative serum-free base media is expected to support SC-RPE differentiation, they are also expected to have unique cell line Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT specific germ layer/substrate profiles. As with all SC-RPE differentiation media of the disclosure, the suitability of these alternatives for a given cell line can be determined experimentally as would be understood by skilled person in the field and upon reading of the present disclosure. [00180] In some embodiments, the various SC-RPE differentiation media of the disclosure are further supplemented with Activin A (herein also RPE-DM1+ActivinA and RPE- DM2+ActivinA). Embodiments that employ the use of Activin A are considered to be semi- directed differentiation methods. [00181] The wording “Activin A” as used herein indicates the dimeric protein product of the inhibin, beta A gene (INHBA) gene identifiable by a skilled person. Activin A is a member of the transforming growth factor beta (TGFB) superfamily and plays a role in the regulation of multiple cellular functions including, but not limited to, cell proliferation, differentiation, wound responses, apoptosis, and metabolism. In vivo, Activin A produced by neighboring extraocular mesenchyme promotes the development of RPE from retinal/RPE precursors. For the case of in vitro spontaneous SC-RPE differentiation, it is believed that efficient generation of RPE requires a sufficiently low percentage of mesenchymal germ layer to allow for ectoderm to neuroectoderm differentiation, but enough mesenchymal cells to subsequently promote ocular neuroectoderm to RPE differentiation. Supplementation of the differentiation medium with Activin A after the period of ocular neuroectoderm differentiation is expected to increase the yield of SC-RPE when there are insufficient mesenchymal cells to promote that transition by natural means and embodiments that employ the use of Activin A are considered to semi-directed differentiation methods. [00182] In some embodiments of the differentiation methods of this disclosure RPE- DM1+ActivinA and RPE-DM2+ActivinA can be prepared freshly on the day of use by adding Activin A to any of the various embodiments of RPE-DM1 or RPE-DM2 medium to a final concentration of 140 ng/mL (most preferred), 70-210 ng/mL (preferred), or 20-280 ng/mL (less preferred). Lower concentrations than indicated are not expected to increase the efficiency of SC-RPE differentiation and higher concentrations than indicated, can possibly result in adverse effects due to increase binding to non-Activin A TGF^ superfamily receptors. For stem cell lines where the addition of Activin A to the differentiation medium is beneficial there is typically an earlier onset of pigmentation, an increase in the number of pigmented cells, an Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT apparent intensity pigmentation, and an increase in the expression of RPE signature genes. [00183] A representative example of a preferred embodiment of the RPE-DM2+ActivinA SC- RPE differentiation medium of the disclosure and related method of preparation is reported in Example 4. Variations in the exemplified RPE-DM2+ActivinA and RPE-DM1+ActivinA SC- RPE differentiation media are expected as will be understood by a skilled person upon reading of the present disclosure. For example, lower dosages of Activin A are expected to result in a decrease in RPE differentiation. Significantly higher dosages can result in poor yields due to an increased potential for cross-reactivity with non-Activin A receptors. Other members of the TGFB superfamily that are expected to be able to substitute for Activin A, such as but not limited to, are TGFB1, GDF11, and GDF8. Addition of a ROCK inhibitor (such as Y-27632) is expected to augment the activity of Activin A as is further supplementation of the medium with nicotinamide. [00184] Addition of Activin A is expected to result in a functional SC-RPE differentiation medium for all possible embodiments of RPE-DM1 and RPE-DM2, as will be understood by a skilled person upon reading of the present disclosure, but the benefit of activin supplementation will vary depending on the specific cell line/substrate/differentiation medium combination. The decision as to whether use an Activin A supplemented SC-RPE differentiation medium is generally determined empirically for a given cell line/substrate/medium combination by quantifying the SC-RPE yield as would be understood by skilled worker upon reading of the disclosure. Alternatively, the potential for its use can be assessed by determining the relative amounts of mesenchymal cells in the culture after the period of neuroectoderm differentiation (about one week) when not using Activin A. It would be expected that cell line/substrate/medium combinations that have low levels of mesenchymal cells compared to cultures that are known to have efficient SC-RPE differentiation without the use of Activin A supplementation might benefit from the use of Activin A. In the SC-RPE differentiation methods of the disclosure the Activin A supplemented SC-RPE differentiation media of the disclosure can be used in combination with the matching SC-RPE differentiation medium without Activin A. Typically, the period of its use is between day-6 and -14 of the RPE differentiation phase of the protocol after which the Activin A is medium is replaced with differentiation medium without Activin A. In some instances, the start of its use can be adjusted depending on the growth rate of the stem cells and timing of the switch from stem cell medium to SC-RPE differentiation medium (RPE-DM1 or RPE-DM2) and the cells can continue to be Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT maintained in RPE-DM+ActivinA until the RPE Maturation phase of the protocol. [00185] An SC-RPE cell culture substrate is a substance configured to support stem cell attachment and growth and allow SC-RPE differentiation of stem cells in the presence of a serum-free SC-RPE differentiation medium of the disclosure. SC-RPE cell culture substrate of the disclosure are substrates configured to effect cell division times, alter cell morphology, and differentially effect gene expression through their specific receptors to provide an optimum ratio of three germ layer precursors. More specifically, in combination with an RPE-DM an optimum substrate should favor the production of ectoderm cells yet allow for the production of sufficient mesoderm and endoderm cells to provide the necessary signals for the development of ocular ectoderm and RPE progenitors. Different cell lines can have different substrate preferences and the preference can vary in an RPE-DM dependent manner. [00186] In some embodiments, SC-RPE substrates are comprised of extracellular matrix or basement membrane proteins, derivatives, or mimics that allow for the attachment of stem cells by binding to specific receptors on the stem cell surface. Substrates with documented history of promoting the development of ectoderm-derived cell types are preferred. At an effective concentration, candidate substrates should support the attachment of essentially all viable stem cells. For a given cell line and RPE-DM combination, prospective substrates can be evaluated by comparing germ layer and progenitor cell profiles during the early stages of differentiation to that of cell line/RPE-DM/substrate concentration with known history of promoting SC-RPE differentiation. Alternatively, a substrate can be evaluated by its ability to generate SC-RPE based on the appearance of pigment and the expression of RPE signature genes. [00187] In some embodiments, SC-RPE substrates comprise laminin 521, recombinant truncated vitronectin, or full-length plasma vitronectin (see e.g., Examples 5 and 6 and also e.g., Example 9, Example 19 and Example 21). [00188] The wording laminin 521 as used here indicates a heterotrimeric protein comprised of ^5, ^2, and ^1 laminin subunits which are coded by the laminin family genes LAMA5, LAMB2, and LAMC1, respectively. [00189] The wording recombinant truncated vitronectin as used here indicates a a recombinant protein having a vitronectin portion limited to, and possibly consisting of, amino acid residues 62-478 of the protein coded by the vitronectin gene, VTN. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00190] The wording plasma vitronectin as used here indicates the mature vitronectin protein coded by the vitronectin gene that results after cleavage of the signal peptide and that has been isolated from serum. [00191] The wording full-length vitronectin as used here indicates the mature vitronectin protein coded by the vitronectin gene that results after cleavage of the signal peptide. Full- length vitronectin can be derived from natural sources such as plasma or recombinant methodologies. [00192] Substrates that can also be used as SC-RPE differentiation substrate comprise soluble basement membrane matrix extract purified from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma cell line (commercially available as Matrigel, Gentex, and Cultrex BME), Collagen IV, or other members of the laminin protein family (e.g., LAM 111 (^1^1^1), LAM211 (^2^1^1), LAM221 (^2^2^1), LAM332 (^3^3^2), LAM411 (^4^1^1), and LAM511 (^5^1^1)). The synthetic substrate coating, Synthemax, which is a short synthetic vitronectin- derived peptide containing the arginine-glycine-asparagine amino acid domain that mediates binding to cellular integrin receptors has also been used for the derivation of RPE from stem cells and can also be used as a substrate. Similar laminin-derived peptides or fragments of laminin that retain laminin receptor binding activity can also be used as a substrate. When any substrates produced by recombinant methodologies are used can have amino acid sequence modifications so long as those modifications do not prevent binding to their specific receptors as will be understood by a skilled person. Custom combinations of any one of these substrates can also be used. While typically used for the derivation of endo- and mesodermal cells, the proteins collagen type 1 and fibronectin can be suitable substrates for stem cell lines that produce very low levels of these germ layer cells and fail to produce significant quantities of RPE using the previously described substrates. [00193] The SC-RPE differentiation media and SC-RPE differentiation substates herein described can be used in the SC-RPE differentiation methods of the disclosure. In particular, in embodiments herein described the method comprises contacting stem cells growing on an SC-RPE substrate with one of the SC-RPE differentiation media. The optimal substrate- differentiation combination is stem cell line dependent. In the SC-RPE differentiation method of the disclosure, the contacting is performed for a time and under conditions to obtain a differentiated SC-RPE cells. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00194] In some embodiments, the stem cells are from an embryonic stem cell line, which can be a hESC. In some of those embodiments the stem cells can be from an ESC line selected from H1 (WA01) or H9 (WA09). In some of those embodiments the stem cells can be from an ESC line selected from Shef3, and Shef6. H1 and H9 are available from WiCell Research Institute and Shef3 and Shef6 can be obtained from the University of Sheffield Centre for Stem Cell Biology. [00195] In some embodiments, the stem cells are from an iPSC stem cell line, which can be a human iPSC. In some of those embodiments the stem cells comprise stem cells from an iPSC line selected from CW70207, CW70066, CW70203, CW70316 which are from the Blinding Eye Disease Control Group of the California Institute for Regenerative Disease Repository and are available through FUJIFILM Cellular Dynamics. [00196] In some embodiments, the stem cells are expected to be from human adult stem cells derived from bone marrow, umbilical cord blood, or adipose tissue all available from the American Type Culture Collection (ATCC). [00197] In some embodiments, the stem cells are from the stem cell line Shef1, a human embryonic cell line derived by the University of Sheffield Centre for Stem Cell Biology. [00198] In some embodiments of the SC-RPE differentiation method of the disclosure, starting stem cells are cultured in appropriate stem cell culture media in cultureware comprising one or more of the SC-RPE substrate of the disclosure. [00199] The term “cultureware” in the sense of the disclosure indicates any device for aseptic cell culture that contains the cell culture medium, provides a surface that can be coated with the SC-RPE substrate on which cells can be grown on, and allows for gas exchange. Examples of suitable cultureware are tissue culture dishes, flasks, multiwell plates, or bioreactors. The surface on which the cells grow upon can be solid, porous, membranes, permeable films, or microcarriers. Most commonly the cultureware are plastic multiwell plates or tissue culture flasks and the support is the solid bottom surface of the plate or flask. [00200] In embodiments of the SC-RPE differentiation method of the disclosure, stem cells can be thawed and plated on proper cultureware coated with an SC-RPE substrate of the disclosure (see e.g., the protocol of Example 7). Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00201] In embodiments of the SC-RPE differentiation method of the disclosure, stem cells can be propagated and/or cultured on SC-RPE differentiation substrate coated cultureware using standard methodologies that support the maintenance of a nondifferentiated state in serum-free conditions. In general, the medium should at a minimum be comprised of a physiological salt solution, essential and non-essential amino acids, essential vitamins, carbon energy source and growth factors that maintain pluripotency such as TGF^1 and bFGF. Examples of suitable medium are E8 [43] or TeSR1-based media such as mTeSR1, mTeSR-Plus, and TeSR-AOF [44] [45]. Other media such as the KnockOut™ Serum replacement-DMEM or DMEM/F12 media supplemented with bFGF and TGF^1 can be used. The substrates must enable efficient stem cell attachment to the culture surface and can also play a role in maintenance of pluripotency. Examples of suitable substrates are rh-VTN-N, laminin 521, plasma vitronectin, recombinant derived full-length vitronectin, and soluble basement membrane extracts (e.g., Matrigel). Passage of the stem cells is done at subconfluence using mechanical, calcium chelator-based and/or protease-based cell dissociation reagents to detach the cells from the substrate and create single cells and/or cell clumps and cells are plated using a dilution factor of about 1:6 in stem cell medium with or without inhibitors of epithelial-to-mesenchymal transition (see e.g., culture medium of Example 1 and procedure of Example 5). While any serum-free media that supports stem cell growth and maintains pluripotency is expected to work, stem cells will have different growth rates and phenotype which can affect the substrate/RPE-DM preference and SC-RPE yield and may require adjustments to the differentiation method such as altering the plating density or the timing of the media switches. [00202] In some embodiments of the SC-RPE differentiation method of the disclosure, cultureware can be precoated with one or more of the SC-RPE differentiation substrate of the disclosure (e.g., by the manufacturer). In some embodiments, the method can further include the optional additional step of substrate coating which can be performed in accordance with the manufacturer’s recommended concentrations and conditions for the selected substrates (see e.g., Example 6). [00203] In some embodiments the method can further comprise screening a plurality of combinations of candidate SC-RPE differentiation medium and SC-RPE cell culture substrate by contacting the plurality of combinations with the starting stem cell, the contacting performed to select a combination of SC-RPE differentiation medium and SC-RPE cell culture substrate configured to provide differentiated SC-RPE cells from the starting stem cell, thus providing Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT the selected SC-RPE differentiation medium and SC-RPE cell culture substrate. [00204] In the representative embodiments of the Examples section and related differentiation screening protocols, cell cultureware was coated with 1 ^g/cm² of Cultrex Human Vitronectin, CTS-rhVTN-N, or Biolaminin 521 CTG. Adjusting the concentration of SC-RPE differentiation substrate is a variable in the method that can be altered and is expected to influence outcomes by affecting cell shape (flattening), rates of proliferation, and gene expression as will be understood by a skilled person. Such an adjustment of the concentration SC-RPE differentiation substrate can also affect the extent that cells remain attached to the dish early in the process when medium changes occur and later when the cells have differentiated. The optimal substrate concentration varies among cell lines and can be determined once the best candidate medium:substrate combination is determined as will be understood by a skilled person upon reading of the disclosure. The minimum possible substrate concentration is that which allows for attachment of at least 75% of viable stem cells. The optimal concentration can be further assessed by monitoring the expression of germ layer, neuroectoderm, ocular ectoderm and RPE marker gene expression at the appropriate times of differentiation. [00205] In some embodiments, propagating and/or culturing the starting stem cells can be performed for a time period of 1 to 5 days or up to 6-7 days for slow growing cultures. The optimal timing will vary depending on the viability of the plated cells, the intrinsic growth rate of the specific cell line, stem cell medium, and substrate as will be understood by a skilled person. For the purpose of simple propagation of pluripotent stem cells the determination of the appropriate timing is based on the degree of cell confluence which should preferably be in the range of 60-90% (see e.g., the protocol of Example 8). For the rare, particularly slow growing culture, times of longer than 5 days can be necessary to achieve the desired degree of confluence and desired number of cells. Alternatively, to achieve the desired number of cells the seeding density can be increased to keep the passage times in line with the preferred timing, more individual cultures can be used, or additional passages can be performed to achieve the desired number of cells In preferred embodiments, passaging of stem cells, if any is performed, is performed before the stem cells reach confluence to minimize premature differentiation in particular with reference to stem cell lines that have a propensity to lose pluripotency during routine propagation, as would be understood by a skilled person [00206] In some embodiments of the SC-RPE differentiation method of the disclosure, stem Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT cell culture can be performed under feeder-free conditions using Essential 8 Medium (Example 7) and truncated vitronectin (rh-VTN-N) coated cultureware (Example 6). Other standard feeder-free stem cell media such as mTESR and cultureware coatings such as laminin (LAM111, LAM211, LAM221, LAM332, and LAM411, LAM511, and LAM521), full-length vitronectin, or Matrigel can also be used. In an exemplary procedure stem cells are generally passaged as a mixture of single cells and cell clumps of roughly 2-20 cells per clump using EDTA based solutions (Versene), but enzymatic methods such as protease digestion (e.g., TrypLE™, Accutase™) or physical methods such as scraping and trituration can be used as well (Example 8). Transient addition of the ROCK inhibitors (e.g., Y-27632, thiazovivin, RevitaCell^TM Supplement) to the medium can also be used at the time of passage to promote survival and limit the loss of pluripotency and is preferred as will be understood by a skilled person, but their use is not essential in particular, when the suspension contains minimal numbers of single cells (Example 8). Because the choice of stem cell culture medium and substrate coating effects growth rate and other properties it will likely effect the final differentiation results. Cell lines with slow to normal growth rates are expected to benefit from the use of Essential 8^TM medium, whereas faster growing cell lines are expected to benefit from the use of mTeSR^TM based media which typically result in a lower rate of growth. [00207] In embodiments the of the SC-RPE differentiation method of the disclosure, the stem cells are contacted with an SC-RPE differentiation media in combination with an SC-RPE differentiation substrate (see e.g., the protocols of Example 9, Example 10, Example 19, Example 21, and Example 23). [00208] In some embodiments, the SC-RPE differentiation media can be one of the variants of the KnockOut^TM Serum Replacement-based RPE-DM1 formulated with different base media and amounts of KnockOut^TM Serum Replacement: CTS-KO-DMEM/5-30%KOSR, KO- DMEM/5-30%KOSR, DMEM/10-30%KOSR, CTS-KO-DMEM:F12/5-30%KOSR, KO- DMEM:F12/5-30%KOSR, DMEM:F12/5-30%KOSR, ^MEM/5-30%KOSR, RPMI1640/5- 20%KOSR, and IMDM:/5-20%KOSR where the KOSR is certified for clinical manufacturing (CTS grade), research grade, or ESCSR as provided in Table 2 of the present disclosure. [00209] In some embodiments, the SC-RPE differentiation media can be one of the X-VIVO™ 10-based RPE-DM2 variants formulated with one of the B27-derived supplements at 0X to 2X strength: X-VIVO™ 10/CTS-B27, X-VIVO™ 10/XF-B27, X-VIVO™ 10/B27-Plus, X- VIVO™ 10/NC-SM1, X-VIVO™ 10/cGMP-N21-MAX, and X-VIVO™ 10/N21-MAX where Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT X-VIVO™ 10 is any of the three different formulations of X-VIVO™ 10 or an SC-RPE differentiation media where the supplement is made from the individual components of B27 as provided as described in Table 5. [00210] In some embodiments, the SC-RPE differentiation media RPE-DM1 and RPE-DM2 variants described in the present disclosure, can be supplemented with Activin A. [00211] In some embodiments, wherein spontaneous differentiation of the starting stem cells is desired the SC-RPE differentiation media can be an RPE-DM1 variant formulated with different base media and amounts of KnockOut^TM Serum Replacement selected from CTS-KO-DMEM/5-30%KOSR, KO- DMEM/5-30%KOSR, DMEM/10-30%KOSR, CTS-KO-DMEM:F12/5-30%KOSR, KO- DMEM:F12/5-30%KOSR, DMEM:F12/5-30%KOSR, ^MEM/5-30%KOSR, RPMI1640/5- 20%KOSR, and IMDM:/5-20%KOSR where KOSR is certified for clinical manufacturing (CTS grade),research grade, or ESCSR as provided in Table 2 of the present disclosure. an RPE-DM2 variant formulated with one of the B27-derived supplements at 0X to 2X strength selected from X-VIVO™ 10/CTS-B27, X-VIVO™ 10/XF-B27, X-VIVO™ 10/B27- Plus, X-VIVO™ 10/NC-SM1, X-VIVO™ 10/cGMP-N21-MAX, and X-VIVO™ 10/N21- MAX where X-VIVO™ 10 is any of the three different formulations of X-VIVO™ 10 and where the supplement is made from the individual components of B27 as provided as described in Table 5. [00212] In some embodiments, wherein semi-directed differentiation of the starting stem cells is desired the SC-RPE differentiation media can be an RPE-DM1 variant formulated with different base media and amounts of KnockOut^TM Serum Replacement selected from CTS-KO-DMEM/5-30%KOSR, KO- DMEM/5-30%KOSR, DMEM/10-30%KOSR, CTS-KO-DMEM:F12/5-30%KOSR, KO- DMEM:F12/5-30%KOSR, DMEM:F12/5-30%KOSR, ^MEM/5-30%KOSR, RPMI1640/5- 20%KOSR, and IMDM:/5-20%KOSR where KOSR is certified for clinical manufacturing (CTS grade),research grade, or ESCSR as provided in Table 2 of the present disclosure that is further supplemented with Activn A. an RPE-DM2 variant formulated with one of the B27-derived supplements at 0X to 2X strength selected from: X-VIVO™ 10/CTS-B27, X-VIVO™ 10/XF-B27, X-VIVO™ 10/B27- Plus, X-VIVO™ 10/NC-SM1, X-VIVO™ 10/cGMP-N21-MAX, and X-VIVO™ 10/N21- MAX where X-VIVO™ 10 is any of the three different formulations of X-VIVO™ 10 or an Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT RPE-DM2 variant where the supplement is made from the individual components of B27 as provided as described in Table 5 that is further supplemented with Activn A. [00213] In particular, in some embodiments, RPE-DM1 formulated with CTS-KnockOut^TM DMEM and CTS-20% KnockOut^TM Serum Replacement (CTS-KO-DMEM/20%CTS-KOSR) and RPE-DM2 formulated with cGMP grade TheraPeak X-VIVO^TM 10, without gentamicin or phenol red and 1X XenoFree-B27 (TP-X-VIVO™ 10/XF-B27) are used for spontaneous differentiation and RPE-DM2 formulated with cGMP grade TheraPeak X-VIVO^TM 10 without gentamicin or phenol red and 1X XenoFree-B27 supplemented with Activin A (X-VIVO™ 10/XF-B27+Activin A) are used as a simple semi-directed differentiation media. In some of those embodiments, these three media are then used in conjunction with three different substrates, preferably full-length human serum vitronectin (hsVTN), recombinant human truncated vitronectin (rhVTN-N), and recombinant human laminin 521 (LAM521) in all possible combinations: CTS-KO-DMEM/20%CTS-KOSR:hsVTN, CTS-KO- DMEM/20%CTS-KOSR:rh-VTN-N, CTS-KO-DMEM/20%CTS-KOSR:LAM521, TP-X- VIVO™ 10/XF-B27: hsVTN, TP-X-VIVO™ 10/XF-B27:rhVTN-N, TP-X-VIVO™ 10/XF- B27:LAM521, TP-X-VIVO™ 10/XF-B27+ActivinA: hsVTN, TP-X-VIVO™ 10/XF- B27+ActivinA:rhVTN-N, TP-X-VIVO™ 10/XF-B27+ActivinA:LAM521. Each of these media were then contacted with the embryonic stem cell lines Shef1, H1, H9 and the induced pluripotent stem cell lines iPSC #032411, iPSC #110211, and iPSC #120111. [00214] The use of other pluripotent stem cell lines and other combinations of medium and substrate using the media formulations such as any one of an RPE-DM1 variant formulated with different base media and amounts of KnockOut^TM Serum Replacement selected from CTS-KO-DMEM/5-30%KOSR, KO- DMEM/5-30%KOSR, DMEM/10-30%KOSR, CTS-KO-DMEM:F12/5-30%KOSR, KO- DMEM:F12/5-30%KOSR, DMEM:F12/5-30%KOSR, ^MEM/5-30%KOSR, RPMI1640/5- 20%KOSR, and IMDM:/5-20%KOSR where KOSR is commercially available CTS or research grade or made from the individual components of KOSR as described in Table 2 of the present disclosure, or an RPE-DM2 variant formulated with one of the B27-derived supplements at 0X to 2X strength selected from: X-VIVO™ 10/CTS-B27, X-VIVO™ 10/XF-B27, X-VIVO™ 10/B27- Plus, X-VIVO™ 10/NC-SM1, X-VIVO™ 10/cGMP-N21-MAX, and X-VIVO™ 10/N21- MAX where X-VIVO™ 10 is any one of the three different X-VIVO™ 10 formulations or Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT where the supplement is or made from the individual components of B27 as described in Table 5 of the present disclosure in combination with alternative substrates selected from Vitronectin, CTS-rhVTN-N, or Biolaminin 521 are possible as would be understood by a skilled person. [00215] Additional combinations of an SC-RPE differentiation medium and an SC-RPE cell culture substrate configured for the SC-RPE differentiation of a starting stem cells line can be identified by contacting the starting stem cell line with one or more candidate combinations of an SC-RPE differentiation medium and an SC-RPE cell culture substrate, the contacting performed for a time and under condition allowing SC-RPE differentiation and selecting a candidate combination of the one or more candidate combinations which following the contacting results in SC-RPE differentiation of the starting stem cell line. [00216] For “spontaneous differentiation” stem cells are generally contacted with differentiation medium 2-10 days (for example, 2-5 days, 3-4 days, 2-6 days, 3-6 days, 2-8 days, or 3-8 days) after plating in stem cell medium, at which time the majority of the replicate cultures are typically 60-90% confluent with some replicates possibly being of lesser confluence and/or some replicates being in the range of 90% to just confluent. Depending on the inherent cell line growth rate, choice of stem cell medium, substrate choice, plating density, and post-plating survival the timing of the first contact with differentiation medium can be extended to 1-7 days after plating in stem cell medium. The cells are contacted with differentiation medium for a total of about 11 to 56 days, more preferably 21-42 days, and most preferably 28-35 days. Longer contact times in differentiation medium are possible, but minimal to no significant increase in the yield of SC-RPE is expected. At the end of the differentiation medium contact period the cultures are contacted without passage with RPE Maturation Medium. [00217] For “semi-directed differentiation stem cells are generally contacted with differentiation medium 2-10 days (for example, 2-5 days, 3-4 days, 2-6 days, 3-6 days, 2-8 days, or 3-8 days) after plating in stem cell medium, at which time the majority of the replicate cultures are typically 60-90% confluent with some replicates possibly being of lesser confluence and/or some replicates being in the range of 90% to just confluent. Depending on the inherent cell line growth rate, choice of stem cell medium, substrate choice, of plating density, and post-plating survival the timing of first contact with differentiation medium can be extended to 1-7 days after plating. The cells are contacted with differentiation medium for Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT a total of 2-6 days (preferably 3 days) after which they are contacted with the corresponding differentiation medium supplemented with Activin A, for 4-28 days, more preferably 6-21 days, and most preferably 8 days. At the end of the Activin A treatment the cells are contacted with differentiation medium without Activin A for 0-60 days, more preferably 7-35 days, most preferably 14-21 days, after which time the differentiation medium without Activin A are contacted without passage with RPE Maturation Medium. [00218] For both the spontaneous and semi-directed differentiation methods best results with respect to final yields the contacting with RPE Maturation Medium is carried out until the percentage of mature SC-RPE in the culture plateaus (generally 30-60 days). Cultures can be maintained in RPE Maturation Medium beyond this point; however, the yield can decrease due to difficulties in dissociating cultures for downstream processing. [00219] The efficacy of the differentiation medium:substrate combinations for a given cell line can be assessed by determining the relative expression level of germ layer, neuroectoderm, retinal/RPE progenitors (e.g. by detection of germ layer markers (e.g. SOX17, GATA4, T, NODAL, HAND1, CDX2)) and retinal/RPE progenitors (e.g. (LHX2, OTX2, RAX, PAX6, MITF, SOX9, SOX10) in the days following transition to the differentiation medium and RPE marker genes in the days and weeks following the switch to RPE Maturation Medium (e.g. by detection of genes associated with pigmentation (e.g. PMEL, TYR, TYRP1, DCT), retinoid cycle (e.g. LRAT, RPE65, ALDH1A3, RBP1, RDH5, RDH10), RPE:RPE cell adhesion (e.g. TJP1, CDH19, CLDN19), ion channel (e.g. BEST1, TRPM1, TRPM3), or phagocytosis (e.g. MERTK, ITGAV)) as a function of time as would be understood by a skilled worker in the field. Alternatively, the differentiation process can be taken to its conclusion and RPE yield can be assessed based on the percentage of pigmented cells or the relative concentration of PEDF secreted into the medium can be monitored in real-time. Conditions that give rise to greater than 50% pigmented cells would be regarded as highly successful. Lower efficiencies of differentiation down to approximately 10% can also result in substantial quantities of final product, albeit with a corresponding reduction in final yield. [00220] Accordingly in those embodiments, the differentiation protocols can be used with candidate combinations to act as screening kit. Using each candidate media:substrate combination on a cell of interest leads to selection of the combination that better provides for the desired results. As an outcome of this method six different stem cell lines have been identified, 3 human embryonic stem cell lines discussed in the Examples section (see e.g., Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Examples 9, 10, and 12) and 3 additional human iPSC lines. H1 and H9 cells can be obtained from the WiCell Research Institute, Madison, WI, USA. The Shef1 cell line was isolated by the University of Sheffield Centre for Stem Cell Biology. The iPSC lines were produced by the inventors of this disclosure from primary fetal RPE obtained from three different donors. The fetal RPE were passaged until they became persistently mesenchymal, after which the cells were then reprogrammed using ReproRNA (STEMCELL Technologies) and iPSC were isolated based on the expression of Tra-1-60. The iPSC lines are available from the inventors of this disclosure. [00221] In some embodiments, following identification and selection of candidate combination of choice, it is possible to modify the additional variables such as plating density, clump size, and the timing of contacting as well as media switches to provide a further customized protocol for the selected combination of SC-RPE differentiation medium and an SC-RPE cell culture substrate in connection with the specific starting stem cells according to embodiments of the methods herein described. [00222] In some embodiments of the differentiation method, in particular those using rhVTN- N and laminin 521 as the substrate, the expanded stem cells are most preferably seeded at a plating density of approximately 20,000 cell/cm² of coated substrate surface area, preferably 15,000 to 25,000 cell/cm², less preferably 10,000 cell/cm² to 40,00 cell/cm², and least preferably, 5,000 to 60,000 cell/cm². The preferred concentration of 20,000 cell/cm² is recommended starting density for any cell line, but the optimal density can vary depending on the particular line and choice of stem cell medium. In general, media:substrate:cell line combinations that result in larger cells and less compact colonies or faster growth rates, can benefit from lower seeding densities. Additionally, it is expected that the density will affect the preferred timing of the switch to RPE differentiation medium which is reflected by the extent of confluence. Determination of the optimal plating density and its associated timing of the contacting with differentiation medium for a cell line can be determined experimentally based on the expression of cell type markers at different time points during the differentiation process, and/or based on the accumulation of pigmentation after SC-RPE maturation. On this basis one can then select the condition that provides the highest yield of SC-RPE according to the experimental design, as would be understood by a skilled worker upon reading of this disclosure. [00223] In some embodiments of the differentiation method, in particular those using full- Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT length vitronectin as the substrate, the expanded stem cells are most preferably seeded at a plating density of approximately 40,000 cell/cm² of coated substrate surface area, preferably 30,000 to 50,000 cell/cm², less preferably 20,000 cell/cm² to 60,00 cell/cm², and least preferably, 10,000 to 70,000 cell/cm². In general, cells plated on full-length vitronectin are smaller and less flattened than when plated on rhVTN-N or laminin 521; hence the optimum density is often higher. The preferred concentration of 40,000 cell/cm² is a preferred starting density for any cell line, but the optimal density is expected to vary depending on the particular line and choice of stem cell medium. Consideration for the choice of cell density on VTN coated cultureware and methods of evaluation are as described in the present disclosure. [00224] In all embodiments of the differentiation protocol, stem cells are plated as a mixture of single cells and clumps of varying cell numbers. While the relationship between clump sizes is not clearly understood, in many instances the pattern of pigmentation that is observed after differentiation suggest that SC-RPE develop at the borders of colonies or where different cell masses contact. Since smaller clumps sizes should result in an increase in the colony circumference:area ratio and a greater extent of colony:colony contacts it follows that they should result in a greater efficiency of RPE differentiation. It further follows that single cells are expected to give the best yields of RPE. However, stem cells can be sensitive single cell passage, and even in the presence of compounds such as ROCK inhibitors that act to promote stem cell survival after single cell passage there can be variable extents of survival. Therefore, as compromise a clump size distribution is in the range of 1-10 cells per clump and is a preferred starting point. Once a preferred differentiation medium:substrate combination is identified for specific cell line, further optimization of SC-RPE yield can be performed by varying the range of clump sizes. Further note, that clump size and plating density are expected to be inter-connected, hence a 2-dimensional matrix experimental design can assist in determining the optimal clump size and plating density combination by assessing the relative efficiency of RPE differentiation-based cell type specific marker gene expression as would be understood by skilled person upon reading this disclosure. [00225] In some embodiments the stem cells are plated in stem cell medium containing the ROCK inhibitor, Y-27632, at a concentration of 10 ^M (most preferred), 7.5-12.5 ^M (preferred), 5-15 ^M less preferred, or 2.5-25 ^M. Thiazovivin, when used at a 5-fold lower concentration range or RevitaCell Supplement (ThermoFisher Scientific, catalog numbers A2644501 and A4238401) used in accordance with the manufactures instructions can be Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT substituted for Y-27632. In certain embodiments, other ROCK1/ROCK2 inhibitors (e.g., GSK429286A, RKI-1477, Ripasudil, Netrasudil, Y-39983, Fasudil, Hydroxyfasudil, H-1152, Azaindole 1, WAY-624704) can be substituted for Y-27632. Guidance as to an appropriate concentration for an alternative ROCK inhibitor can be obtained by comparing the Ki values for ROCK1 and ROCK2 to that of Y-27632 and empirical testing using single cell stem cell passage to determine the minimal dosage required to enable cell survival without inhibiting growth rates. [00226] In some embodiments, the contacting of the starting stem cell line with one or more candidate combinations of an SC-RPE differentiation medium and an SC-RPE cell culture substrate, is performed for a time and under conditions to provide differentiated SC-RPE cells. [00227] In particular in some embodiments of the “spontaneous” differentiation protocol cells are generally contacted with differentiation medium 2-5 days after plating in stem cell medium, at which time the majority of the replicate cultures are typically 60-90% confluent with some replicates possibly being of lesser confluence and/or being in the range of 90% to just confluent, for a total of about 11 to 56 days, more preferably 21-42 days, and most preferably 28-35 days, with the optimal time being determined by the one that gives rise to the highest level of expression of RPE marker genes or the highest percentage of cells expressing RPE marker genes in the shortest length of time. Alternatively, the optimal period of contact with differentiation medium can be determined based on the percentage of pigmented cells in the culture after transitioning the culture to RPE Maturation Medium and a sufficient period of culture (> 2 weeks) to allow for visualization of pigmentation. Contact times beyond 56 days are possible however, with little to no benefit in final yields of SC-RPE. [00228] In all embodiments of the SC-RPE differentiation protocol cultures are fed with sufficient volumes of medium as necessary to maintain the composition of the medium. This is typically every 1-2 days with 2-3 mL of medium per 10 cm² of culture surface area and a roughly 90% media exchange depending on the protocol stage, the medium choice, and extent of medium acidification. Yellowish medium when using media containing phenol, a measured pH below 7.0. or cell death not associated with changes in type of media (e.g., stem cell medium to SC-RPE differentiation medium) is indicative of a need to feed the cells more frequently or increase the feeding volume. More frequent than necessary feeding schedules or greater than recommended volumes are possible, but those protocol changes are expected to affect Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT outcomes due to dilution of secreted factors that regulate the differentiation process as will be understood by a skilled person. [00229] In some embodiments, during the period of stem cell growth post-plating in stem cell medium (e.g., Essential 8^TM or mTeSR^TM1) cells are fed every day until the cells reach 60-90% confluence with 2 mL/10 cm² of stem cell medium. When media that employ the use of stabilized bFGF and with increased pH buffering (e.g., Essential 8^TM Flex or mTeSR^TM Plus) are employed, a feeding frequency of every other day can be used. Increasing the frequency and/or volume of feeding can also provide higher nutrient levels beneficial to the cells during this period of rapid growth as will be understood by a skilled person. [00230] In “spontaneous” or “semi-directed” differentiation embodiments, which employ the use of RPE-DM1, and in particular those that use RPE-DM1 formulated with KnockOut^TM DMEM, the preferred protocol is that cells be fed daily with 2 mL/10 cm² at all times during its use. On occasion, a feeding frequency of every other day is possible especially in the later stages of differentiation. In this case it is preferred that the volume be increased by 50-100%. [00231] In “spontaneous” or “semi-directed” differentiation embodiments which employ the use of RPE-DM2, the cultures are preferably fed daily with 2 mL/10 cm² for the first feedings after the switch from stem cell medium to RPE-DM2. Thereafter, in embodiments that use RPE-DM2 formulated with X-VIVO™ 10 the cells can be fed every other day with 2 mL/10 cm² of RPE-DM2. The use of RPE-DM2 formulated with other complete or base media can require more frequent feeding depending on its pH buffering capacity as will be understood by a skilled person. [00232] In embodiments of the disclosure, the media, substrate, cultureware and/or other reagents used to perform the SC-RPE differentiation method of the disclosure can be comprised within corresponding SC-RPE differentiation system of the disclosure. [00233] The SC-RPE differentiation system of the disclosure comprises at least one serum-free SC-RPE differentiation medium and at least one SC-RPE cell culture support coated or uncoated with a substrate of the present disclosure In the SC-RPE differentiation system of the disclosure, the at least one cell culture medium and the at least one cell culture substrate in combination with an appropriate cultureware or device are included for combined use to provide a cell culture system configured to allow introduction and/or maintenance of stem cells culture within the system to obtain a differentiated SC-RPE cells according to methods of the Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT disclosure. [00234] In some embodiments, SC-RPE differentiation methods and system of the disclosure allow efficient SC differentiation which can be readily scaled up for large-scale cGMP manufacturing when cGMP compliant reagents are employed. [00235] In some embodiments, SC-RPE differentiation methods and system of the disclosure allow efficient SC differentiation in a time period of ranging from 2 to 8 weeks with the preferred methods requiring a total of 4-5 weeks. [00236] In some embodiments, SC-RPE differentiation methods and system of the disclosure provide a toolbox approach that allows differentiation of different stem cell lines. [00237] In some embodiments, SC-RPE differentiation methods and system of the disclosure provide simplified approach minimizing requirement of multiple manipulations or steps. [00238] In embodiments of the disclosure SC-RPE differentiated cells can be matured using an RPE Maturation Medium of the disclosure (herein RPE-MM), which has been unexpectedly found to promote and/or enhance yield and maturation of differentiated SC-RPE cells. [00239] In some embodiments, the ability of the RPE Maturation Medium of the disclosure to promote and/or enhance yield and maturation of differentiated SC-RPE cell can occur even when the differentiation medium gives rise to minimal amounts of discernable RPE. [00240] In some embodiments, in addition to supporting the establishment of a mature RPE phenotype, the RPE Maturation Medium of the disclosure appears to also play a role in earlier stages of SC-RPE development with at least some stem cell line:differentiation medium:substrate combinations, based on the observation that passaging during the time of contacting with RPE Maturation Medium can result in significant increases in SC-RPE differentiation efficiencies when cultures are passaged at later times (about 2-weeks) during the period of maturation compared to passaging after just a few days (3-days, Example 46 and Figure 27). [00241] In some embodiments, the SC-RPE Maturation Medium allows for improved isolation of SC-RPE cells based on the presence of pigmentation, by enabling or enhancing pigment accumulation and melanosome maturation. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00242] The RPE Maturation Medium of the disclosure is a cell culture maturation medium comprising a buffered balanced salt solution, essential amino acids and nonessential amino acids, vitamins, hormones, essential components for lipid metabolism, carbon energy sources, serum albumin, insulin, transferrin, selenite, and antioxidants in an effective amount to promote maturation of differentiated SC-RPE cells. [00243] In some embodiments, the RPE Maturation Medium of this disclosure uses a common serum-free medium formulation comprised of a base medium supplemented with the components of B27 as will be understood by a skilled person upon reading of the present disclosure. [00244] In some embodiments of the disclosure RPE-MM can further be supplemented by additional nonessential amino acids (NEAA), insulin, transferrin, selenite, putrescine, progesterone, and hydrocortisone to obtain a RPE Maturation Medium of the disclosure according to additional embodiments of the disclosure. [00245] In some embodiments of the disclosure insulin, transferrin, selenite, putrescine, and progesterone supplementation is obtained using a N1-based supplement mixture. [00246] In embodiments of the RPE-MM of the disclosure, compositions of the RPE-MM can be made by modifying the amount of related base medium, B27 components and/or by adding further components in accordance with the indications of the present disclosure as will be understood by a skilled person. [00247] The effects of the changes in composition on the effect of the resulting RPE-MM of the disclosure can be monitored through detection of pigmentation and/or expression of marker genes associated with mature RPE phenotype (see Example 43 and Figures 24A and 24B). [00248] In outcome of the detection of pigmentation it has been surprisingly found that in embodiments of RPE-MM herein described, neither taurine nor additional supplementation with non-essential amino acids (NEAA), N1-based supplement mixture, or hydrocortisone, by themselves, is essential, to obtain mature RPE with typical RPE morphology. However, when maturation competent SC-RPE are plated at suboptimal density, such as 40,000 cell/cm², omission of any one of them individually results in an a 50-60% reduction in the percentage of pigmented SC-RPE and cells with a mesenchymal phenotype. (See Example 18 and Figure 5). Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00249] In particular, it will be understood by a skilled person upon reading of the present disclosure that, when culturing minimally expanded SC-RPE under optimal conditions, supplementation with additional hydrocortisone is not necessary, with no overt differences in SC-RPE quality being observed because of its omission. However, its addition can have some benefits for RPE expansion due to its ability to minimize wound response. Using serum-based RPE medium it has been found that it can be eliminated in its entirety with no obvious overt effects on pigmentation or cell morphology. [00250] It has also surprisingly been found that in preferred embodiments the RPE Maturation Medium, that removal of retinol and/or retinol acetate (collectively known as vitamin A) through the of use of B27 supplements without vitamin A results in significant changes in the expression of many RPE genes; yet the differentiation efficiencies are unchanged and the obtained SC-RPE have a normal pigment epithelial morphology and retain at least some RPE function. It has further been found that by varying the concentration of the vitamin A in RPE- MM by using B27 with and without vitamin A the expression the vitamin A-responsive genes can be altered in a dose-dependent and reversible manner. [00251] In particular, the inventors have unexpectedly found that in embodiments of RPE-MM of the disclosure the presence/absence and concentrations of Vitamin A are a result effective variable. Presence of Vitamin A is not essential to obtain high yields of differentiated, readily identifiable, pigmented SC-RPE that express RPE-signature genes or for the culture RPE that retain at least some RPE function, as based on the formation tight junctions. (See Example 42, Figures 17 and 18). However, in the absence of vitamin A RPE express highly reduced levels of a several key RPE genes relating to regulation of RPE gene expression and function (see Example 42, Figure 19 and 20). In addition, in the absence of vitamin A there is an elevated expression of genes associated with wound response. The expression levels of the genes can be regulated in a dose-dependent manner and the effect on gene expression is reversable. Accordingly, embodiments of the RPE-MM comprising vitamin A are preferred. [00252] In more preferred embodiments, the RPE-MM of the disclosure can comprise an amount of vitamin A selected in view of the features of the mature SC-RPE to be obtained as will be understood by a skilled person upon reading of the present disclosure. [00253] Accordingly, in some embodiments of the RPE-MM of the disclosure the B27 supplement is a commercially available B27 supplement without vitamin A or is made from Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT the individual components of B27 or NS21 without retinol or retinol acetate, to which retinol and/or retinol acetate can be added to achieve a desired concentration of vitamin A (e.g. all- trans-retinol, catalog number 95144; retinol acetate, also referred to as retinyl acetate, catalog number R7882 Sigma-Aldrich) to achieve the desired RPE phenotype as can be assessed using gene expression profiling or in the alternative, quantitative PCR to determine the expression level of LRAT, EYA2, RAX, and SPP. [00254] In some embodiments of the RPE-MM of the disclosure when vitamin A is provided from B27, at the highest concentration of vitamin A provided by B27 when used at its preferred 1X concentration, the expression levels for some of the RPE genes has been observed not to have reached their maximum based on the vitamin A dose-response analysis. This data supports the conclusion, that the quality of SC-RPE obtained using the serum-free RPE Maturation Medium can be advantageously improved by providing more vitamin A. Accordingly, in some preferred embodiments of the RPE-MM of the disclosure comprise B27 supplement, the concentration of vitamin A can be further increased by increasing the concentration of the B27 supplement in RPE Minimal Medium, using the B27-derived NS21 supplement which has twice the concentration of vitamin A, or preferably by further supplementation of RPE Maturation medium with retinol and/or retinyl acetate. The effect of altering the concentration of vitamin A and the selection of a desired concentration vitamin A can be determined using gene expression profiling or in the alternative, quantitative PCR to determine the expression level of LRAT, EYA2, RAX, and SPP1 as will be understood by a skilled person. Exemplary techniques for detection of gene expression comprise quantitative Real-Time PCR (qRT-PCR) RNA Sequencing (RNA-Seq), Microarray analysis involves hybridizing cDNA to a chip containing probes for thousands of genes in situ hybridization, immunohistochemistry and additional techniques identifiable by a skilled person. [00255] In some embodiments, the desired phenotype is obtained at a concentration of vitamin A lower than what is provided by B27 supplement with vitamin A. In those embodiments, the concentration of vitamin A in RPE-MM can be reduced by first diluting B27 supplement with vitamin using a B27 supplement without vitamin A (NeuroCult™ SM1 Without Vitamin A (05731 STEMCELL Technologies) B-27 Supplement Minus Vitamin A (A1370701, ThermoFisher), CTS B-27 Supplement Xeno-Free Minus Vitamin A (A5047601, ThermoFisher), or N21-MAX Vitamin A Free Media Supplement (AR012 R & D Systems) prior to preparing the base media to obtain the desired concentration. In the alternative, RPE- Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT MM with and without vitamin A can be prepared with the respective B27 supplements and the media can be mixed to achieve the desired concentration. [00256] In some embodiments of the RPE-MM of the disclosure, where it is desired to further increase B27 concentration, but not alter the vitamin A concentration any of the B27 supplements without vitamin A can be added and then adjusting the total concentration of Vitamin A to the desired concentration, preferably by adding a supplement from a same supplier (e.g. SM1 Neurocult Minus Vitamin A added to SM1 Neurocult (without vitamin A). [00257] All-trans-retinol is processed by the RPE cells during the visual (retinoid) cycle to regenerate the photosensitive visual pigment 11-cis-retinal. In addition to being essential to vision, dysregulation of the retinoid cycle has been linked to a number of retinal and/or RPE diseases and disorders. [00258] Beyond the effects of Vitamin A addition with regard to obtaining high quality SC- RPE the ability to regulate the RPE phenotype by manipulating the related concentration, RPE obtained with RPE-MM comprising vitamin A concentration have specific useful applications, such as providing disease models for both basic research or drug screening and drug development. [00259] In some embodiments, the RPE maturation medium of the disclosure can comprise ^- MEM as base medium supplemented with additional nonessential amino acids (NEAA), N1 or N2 supplement, taurine, hydrocortisone, and a B27-based supplement. [00260] The term “^-MEM” or “alpha-MEM” as used herein indicates Eagle’s Minimum Essential Medium, Alpha Modification suitable for cell culture with L-glutamine and sodium bicarbonate, and without ribonucleosides and deoxyribonucleosides. [00261] The Minimum Essential Medium (MEM), developed by Harry Eagle, is one of the most widely used of all synthetic cell culture media. Early attempts to cultivate normal mammalian fibroblasts and certain subtypes of HeLa cells revealed they had specific nutritional requirements that could not be met by Eagle’s Basal Medium (BME). Subsequent studies using these and other cells in culture indicated additions to BME could be made to aid growth of a wider variety of fastidious cells. MEM, which incorporates these modifications, includes higher concentrations of amino acids so the medium more closely approximates the composition of cultured mammalian cells. Optional supplementation of non-essential amino Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT acids to the formulations that incorporate either Hanks’ or Earle’s salts has broadened the usefulness of this medium. [00262] The Alpha modification of MEM with Earle’s Balanced Salts, commonly referred to as alpha-MEM, contains non-essential amino acids, sodium pyruvate, and additional vitamins. These modifications were first described by Stanners for use in growing hybrid mouse and hamster cells. The preferred formulation of this disclosure is without the deoxyribonucleosides and ribonucleosides originally used in Stanners’ studies[46], [47]. [00263] The Minimum Essential Medium Eagle (MEM) Alpha Modifications without nucleosides is commercially available medium in powder and liquid form from Corning Life Sciences (Product Numbers: 50-012-PC, powder and 15-012, liquid). The powdered form is provided without sodium bicarbonate, which must be added separately. The liquid form is provided without glutamine, which must be added separately. The formulation, concentration of the components and specification of the powder form (50-012-PC) is provided in Table 6 below. [00264] Table 6. ^MEM Formulation and Specification ^{^^ ^^ ^^} ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ .^^^^^5^^^6^^^^^^^^^^^^^7*%8%^^8^.^ ^^ ^^ ^^ ^^ ^^ ^592^ ^:^ ^^^ ^^ ^^ ^^ ^^^5^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ /^4^^ (&##^ *&^^^ ^^()*^^ ^^ ^^ ^^ ^^ /^^40)^^^^^^^ ^^##^ &^^#^ ^(^&&^ ^^ ^^ ^^ ^^ 74^^ ^##^ ^^^(^ *)(*^ ^^ ^^ ^^ ^^ /^^^+0^5^^0^ ^^#^ ^)&^#^ ^#^*^ ^^ ^^ ^^ ^^ 4^4^^^ ^##^ ^^^^#^ ^&#^^ ^^ ^^ ^^ ^^ ^.,0^^ ^^^^^ ^^#^^^ &^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^"^ ^*^ &^^^^ ^&^^ ^^ ^^ ^^ ^^ ^^^ .^^^^"5^4^^ ^^(^^^ ^^#^^^ (##^ ^^ ^^ ^^ ^^ ^^^^^^ ^.^^"5^^0^ *#^ ^*#^^^ )))^ ^^ ^^ ^^ ^^ ^^^^^^ '^^^^^^^^ )#^ ^))^^^ ^^*^ ^^ ^^ ^^ ^^ ^^4%^'"^^"5^4^5^^0^ ^##^ ^^*^(^ *(^^ ^^ ^^ ^^ ^^ ^^4%^'^^"5^^4^^ )^^^^ )^)^^^ ^^^(^ ^^ ^^ ^^ ^^ ^^$^^'^^^^^^^^^^ ^*^ ^^^^^^ *^#^ ^^ ^^ ^^ ^^ ^^$^^'^^^^"^ ^^^^ ^^(^^^ ^^^&^ ^^ ^^ ^^ ^^ $^%^^^"^ *#^ ^*^^^ (((^ ^^ ^^ Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Table 6. ^MEM Formulation and Specification ^^ ^^ ^^ ^_^ ^{^^ ^^ ^^ ^^} _{^^ ^^ ^^} ^^ ^^ ^^ .^^^^^5^^^6^^^^^^^^^^^^^7*%8%^^8^.^ ^^ ^^ ^^ ^^ ^^ ^^^^^'^^^^"5^4^5^^0^ ^^^^^ ^#^^(^ ^##^ ^^ ^^ ^^ ^^ ^^^^^^"^^^^"^ *^^*^ ^)^^^^ ^##^ ^^ ^^ ^^ ^^ ^^^"^^^^"^ *^^*^ ^)^^^^ ^##^ ^^ ^^ ^^ ^^ ^^^%^^^"5^4^^ ^^^*^ ^&^^^^ )^^^ ^^ ^^ ^^ ^^ ^^^"'^^^^^^"^ ^*^ ^^^^^^ ^#^^ ^^ ^^ ^^ ^^ ^^+^"^%^^^^^^^"^ )^^*^ ^(*^^^ ^^^^ ^^ ^^ ^^ ^^ ^^+ ^^^^"^ ^#^ ^^*^^^ )^^^ ^^ ^^ ^^ ^^ ^^," ^^"^ ^*^ ^#*^^^ ^)&^ ^^ ^^ ^^ ^^ ^^^^ "^^^^"^ ^^^(^ ^^^^^^ ^##^ ^^ ^^ ^^ ^^ ^^^ %^'^^^^^^ ^#^ ^#^^^^ ^^^#^ ^^ ^^ ^^ ^^ ^^^% ^^^^"5^/^5^^_^0^ *^^^^ ^()^^^ ^^^^ ^^ ^^ ^^ ^^ ^^-^^^^"^ ^(^&^ ^^^^^^ )^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^ ^^^^^^^^^ *#^ ^^(^^^ ^&^^ ^^ ^^ ^^ ^^ 6^^'^^^ #^^^ ^^^^)^ #^^#^^ ^^ ^^ ^^ ^^ 4^^^^^"^^^^^ ^^"^ ^^ ^)^^(^ ^^^(^ ^^ ^^ ^^ ^^ 9^^^^^^^^^^ ^^ ^^^^^^ ^^^^^ ^^ ^^ ^^ ^^ ^^^^^^^'^^^ ^^ ^&#^^^ ^^^^#^ ^^ ^^ ^^ ^^ /^^^'^^^^^^"^ ^^ ^^^^^^ &^^^^ ^^ ^^ ^^ ^^ A^4^^^^^^^^^^'^'^"^^'"^ ^^ ^^(^*^ ^^^#^ ^^ ^^ ^^ ^^ +% ^^^^^^"5^4^^ ^^ ^#*^(^ ^^&(^ ^^ ^^ ^^ ^^ ^^^^!^^>^^^ #^^^ )^(^^^ #^^((^ ^^ ^^ ^^ ^^ ^^^^^^^"5^4^^ ^^ ))^^)^ ^^^(^ ^^ ^^ ^^ ^^ -^'^^^^^6^^^ ^^)(^ ^)**^^^ ^^##^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^4^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ A^$^^^^^"^ ^###^ ^&#^^^ ***^^ ^^ ^^ ^^ ^^ ,^^^^^^^% ^>^'"^ ^^#^ ^^#^#^ ^###^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^ #^^^ ^#(^)^ #^^(^^ ^^ ^^ ^^ ^^ +^"^^^^^"^5/^^ ^#^ )^(^^^ ^(^(^ ^^ ^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^^^^_^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ,^"^^!^^^'^^^^ ^^ ^^ ^^ ^^^C^'^^^'^/^^40₎^ ^^ )^*^2^#^*^ ^^ ^^ ^^ ^^ ^^^C^'^^/^^40)^G^^'^^^^^" ^^^40^^ ^^#^2^#^*^ ^^ ^^ ^^ ^^ 0^^^^^^^'%^C^'^^/^^40)^ ^^ ^^#^2^)#^^0^^H<.^^^0^ ^^ ^^ ^^ ^^ B^^^'^^^^^ ^^ I^#^^*^BEH^^^G^9=^ ^^ ^^ ^^ ^^ ^%^^^^^^^^^ ^^ /".^'^>"^ ^^ ^^ ^^ ^^ 4"^^^$ ^C'^^^-B^0@^+^7@^/$^^#&^^ +^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00265] In some embodiments the alpha-MEM formulation Table 6 can comprise one or more of the nucleosides (adenosine, cytidine, guanosine, adenosine, thymidine, uridine, 2’- deoxyadenosine, 2’-deoxycytidine, 2’-deoxyadenosine, each at a concentration of about 40 ^M. [00266] Alpha-MEM of Table 6 without nucleosides is also available in powder form from ThermoFisher (catalog number: 12000) and Sigma (catalog number: M0894) and in liquid form from ThermoFisher (catalog number: 12561), Sigma (catalog number: M0200) and STEMCELL Technologies (catalog number: 36453). However, all of these products have slight differences in formulation compared to the Corning ^-MEM formulation. ThermoFisher powdered ^-MEM has 23% lower amounts of L-cystine, 17% higher amounts of tyrosine, and it uses pyridoxal•HCl as opposed to pyridoxine•HCl as its source of vitamin B6. ThermoFisher liquid medium has the additional difference that the amount of L-arginine is reduced by 17%. By comparison powdered and liquid Sigma ^-MEM has 25% lower amounts of CaCl₂, 12% lower amounts of ascorbic acid, 2-fold greater amounts of panthothenate, 25% higher amounts of pyruvate, and it uses pyridoxal•HCl as opposed to pyridoxine•HCl as its source of vitamin B6. STEMCELL Technologies medium has 25% lower amounts of CaCl₂, 23% lower amounts of L-cystine, 15% higher amounts of NaH2PO4, and it uses pyridoxal•HCl as opposed to pyridoxine•HCl as its source of vitamin B6. For example, the ThermoFisher powdered and liquid formulations have been found to support RPE maturation with minimal demonstrable differences based on the observance of pigmentation and morphology compared to the Corning ^-MEM formulation. While it is expected that the ^-MEM formulations from other suppliers will support RPE maturation, when using alternative sources of ^-MEM, the concentration of the components with reduced amounts compared to Corning® ^-MEM can be increased by supplementation. Formulations of ^-MEM with nucleosides are advantageous in cGMP application because they allow use of commercially available phenol-free media to provide the components of the RPE-MM of the disclosure. [00267] In embodiments of the disclosure, other base cell culture media of formulation similar to alpha-MEM are expected to be usable in providing a base medium for RPE-MM of the disclosure, but in some cases commonly used media such as DMEM or DMEM:F12 (50:50) have been shown not to be able to substitute for alpha-MEM. (Example 18 Figure 6). [00268] In some embodiments, the alpha-MEM of Table 6 is supplemented with a B27 Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT supplement made from the individual components of B27 or NS21 without retinol or retinol acetate. [00269] In some embodiments, the alpha-MEM of Table 6 is supplemented by NeuroCult™ SM1 Without Vitamin A (05731 STEMCELL Technologies) B-27 Supplement Minus Vitamin A is (A1370701, ThermoFisher), CTS B-27 Supplement XenoFreeMinus Vitamin A (A5047601, ThermoFisher), or N21-MAX Vitamin A Free Media Supplement (AR012 R & D Systems). [00270] In some embodiments, the alpha-MEM of Table 6 is supplemented with retinol and/or retinol acetate (e.g. all-trans-retinol, catalog number 95144; retinol acetate, also referred to as retinyl acetate, catalog number R7882 Sigma-Aldrich). [00271] In some embodiments, the alpha-MEM of Table 6 is supplemented with B27 supplement made from the individual components of B27 or NS21 and retinol and/or retinol acetate (e.g. all-trans-retinol, catalog number 95144; retinol acetate, also referred to as retinyl acetate, catalog number R7882 Sigma-Aldrich).are added to achieve a desired vitamin A concentration. [00272] In some embodiments, the alpha-MEM of Table 6 is supplemented by NeuroCult™ SM1 Without Vitamin A, B-27 Supplement Minus Vitamin A, CTS B-27 Supplement XenoFreeMinus Vitamin A, or N21-MAX Vitamin A Free Media Supplement and retinol and/ retinol acetate are added to achieve a desired concentration of vitamin A. [00273] In some embodiments, the alpha-MEM of Table 6 is supplemented with any the B27 supplements with vitamin A of the disclosure is further supplemented with by NeuroCult™ SM1 without Vitamin A (05731 STEMCELL Technologies) B-27 Supplement Minus Vitamin A is (A1370701, ThermoFisher), CTS B-27 Supplement XenoFreeMinus Vitamin A (A5047601, ThermoFisher), or N21-MAX Vitamin A Free Media Supplement (AR012 R & D Systems). [00274] In some embodiments, the alpha-MEM of Table 6 is supplemented with any the B27 supplements with vitamin A of the disclosure and retinol and/ retinol acetate are added to achieve a desired concentration of vitamin A. [00275] In some embodiments of the RPE-MM of the disclosure, ^-MEM is supplemented with Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT additional nonessential amino acids (NEAA).100X MEM Non-essential Amino Acids solution (10 mM alanine, 10 mM asparagine, 10 mM aspartic acid, 10 mM glutamic acid, 10 mM glycine, 10 mM proline, and 10 mM serine) is available from ThermoFisher Scientific (catalog numbers 11140050 or 11140076). In some embodiments of RPE-MM, the NEAA solution is added at a final strength of 1X (most preferred), 0.75 to 1.25X (preferred), 0.5 to 1.5X (less preferred), 0.25 to 2X (least preferred). While supplementation with additional NEAA has been associated with improved RPE phenotype on average (see e.g., Example 18 and Figure 5), it is not essential for RPE cells to pigment and obtain a normal RPE morphology. The addition of supplemental NEAAs is considered to be optional; although, some reduction in RPE quality is expected. Strengths higher than 2X is expected to be possible, but no further improvement in RPE phenotype and function is expected. [00276] In some embodiments of the RPE-MM of the disclosure, ^-MEM is supplemented with B27- or NS21-based supplements which were derived from the defined serum-free medium, B18, of Brewer et al., 1989 [40] which was developed as a supplement to facilitate neuronal cell culture. B27 and NS21 provide the additional components of serum albumin, insulin, transferrin, selenite, putrescine, progesterone, galactose, catalase, superoxide dismutase, D,L- alpha-tocopherol, D,L-alpha-tocopherol acetate, reduced glutathione, ethanolamine, linoleic acid, linolenic acid, carnitine, triiodo-I-thyronine (T3) and vitamin A (retinol acetate (B27 and NS21) and retinol (NS21)) [39], [41]. B27 also includes biotin and NS21 also includes lipoic acid, both of which are also present in ^-MEM. Typically, these supplements are prepared as concentrated stocks which are then added to the base medium. The formulations of B27 and NS21 at 1X strength based on an online published protocol [42] and the original publications are provided in Table 5. 50X stock solutions of the supplements can be prepared using the reagent lists and instructions of the publications. Alternatively, suitable B27-based 50X liquid stock solutions are commercially available from ThermoFisher Gibco (B-27™ Supplement (50X), serum free [catalog numbers 17504001 and 17504044]; B-27™ Plus Supplement (50X), [catalog number A3582801]; B-27™ Supplement, XenoFree, [catalog number A1486701]; CTS™ B-27™ Supplement XenoFree (50X) [catalog number A5047501]); and STEMCELL Technologies (NeuroCult^TM SM1, catalog number 05711). NS21-based medium supplements can be obtained from Millipore Sigma-Aldrich (N21 Medium Supplement (50X), catalog number SCM081) and R & D Systems (N21-MAX Media Supplement (50X), catalog number AR008). These commercial stocks have the same composition of compounds listed in Table Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT 5, but the source (e.g., species, recombinant) and concentration of some of the components can vary from the original published versions and between different commercial products from that listed in Table 5. B-27™ Supplement (50X), serum free; B-27™ Supplement, XenoFree; CTS™ B-27™ Supplement XenoFree (50X); NeuroCultTM SM1; and N21-MAX Media Supplement (50X) have all found to be interchangeable when formulating RPE-MM, albeit with some differences in differentiation media:substrate preferences and yields. Based on this observation it is expected that RPE-MM formulated based on the individual B27/N21 components and concentrations in Table 5 will yield a functional RPE-MM. [00277] In some embodiments of RPE-MM, 50X B27-based supplements are most preferably used at a final concentration of 1X, preferably at 0.75 to 1.25X, less preferably at 0.5 to 1.5X, and least preferably at 0.25 to 2X. [00278] In some embodiments of the RPE-MM of the disclosure, ^-MEM is supplemented with N1 supplement [48]. The N1 supplement is a 100X solution comprised of human insulin (0.5 mg/mL), partially iron saturated human transferrin (0.5 mg/mL), sodium selenite (0.5 ^g/mL), putrescine (1.6 mg/mL), and progesterone (0.73 ^g/mL) in Earle’s Balanced Salt Solution. It is typically used at 1X strength. Insulin, transferrin, and selenite are referred to as IST and are required for serum-free cell culture. Insulin, transferrin, selenite, and putrescine are also added as individual media additives in the serum supplemented RPE medium of Hu and Bok [49,]. All five components of N1 are also present in B27 supplement. The observation that the omission of addition of N1 supplements to RPE-MM results in diminished RPE pigmentation (see Example 18 and Figure 5) suggests that the concentration of at least one of these components in either supplement is limiting for RPE maturation. In context of the changes in RPE energy metabolism with maturation, insulin is expected to be possibly critical. N1 Medium Supplement (100×) can be obtained from Millipore Sigma-Aldrich (Catalog N6530) or alternatively it can be made from the individual components which are available from numerous suppliers. [00279] In some embodiments, the RPE-MM of the disclosure uses N-2 supplement as an alternative to N1 supplement. N2 differs from N1 with respect to the concentration of some of the components and the use of holo-transferrin instead of partially iron saturated transferrin. It is also available in grades suitable for clinical manufacturing. N-2 supplement is a 100X solution comprised of human insulin (0.5 mg/mL), holo-human transferrin (10 mg/mL), Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT sodium selenite (0.52 ^g/mL), putrescine (1.611 mg/mL), and progesterone (0.63 ^g/mL) in water available from Thermofisher Scientific (N-2 Supplement (100X) Catalog 17502001 and 17502048, CTS™ N-2 Supplement. (100X) Catalog A1370701 or A13707-01). In some embodiments, N1 and N2 have been found to be interchangeable with respect to supporting RPE maturation with minimal to no overt differences as evident based on pigmentation and morphology (see Example 42 and Figure 17). [00280] In some embodiments, the RPE-MM of the disclosure uses High Insulin N-2 (HI-N-2) supplement as an alternative to N1 supplement. High Insulin N-2 supplement has been shown to provide optimal conditions for neural stem cell expansion [51] and differs from normal N-2 in that the concentration of insulin in the 100X supplement stock solution is 2.5 mg/L compared to 0.5 mg/L. High Insulin N-2 is available from R & D Systems (N-2 Plus Media Supplement (100X, N-2+), catalog number AR003 with bovine insulin; N-2-MAX, catalog number AR009, with recombinant human insulin and human transferrin; and GMP N-2 MAX Media Supplement (100X), Animal-free, catalog number AR016, with recombinant human insulin and recombinant human transferrin. [00281] 100X N1 supplement and its related 100X N-2, and 100X HI-N-2 supplements are most preferably used at final concentration of 1X, preferably 0.75 to 1.25X, less preferably 0.5 to 1.5X, and least preferably 0.25 to 2.0X. When RPE-MM is formulated using greater than 1X B27-based supplements, which contain all of the components of N1, N-2, or HI-N-2 at a similar concentration, the amount of the N1-related supplement can be reduced accordingly. [00282] N1 type supplementation can also be achieved by custom formulation using the individual components and concentration ranges of the preceding described commercial preparations of N1, N-2, and HI-N-2 as would be understood by skilled person. The individual components or mixtures of subsets of the components (e.g., IST) suitable for cell culture are available from numerous vendors. Custom formulated N1-type supplements are expected to work in the same concentration range as the commercially available supplements. [00283] In the RPE-MM of the disclosure ^-MEM can be supplemented with taurine . Taurine is not a component of alpha-MEM. Taurine is the most abundant amino acid in the retina and the retina has the highest concentration of taurine in the body. Much of that taurine is delivered to the retina from the vasculature via active transport by the RPE. Furthermore, in highly aerobic cells such as the RPE, taurine is concentrated in mitochondria where it reduces Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT superoxide generation and in the RPE and photoreceptor it also serves to sequester retinaldehydes, which are toxic intermediates of the visual cycle. Given the apparent importance of taurine in retina/RPE physiology, taurine may play a role in normal RPE maturation and function in addition to providing further protection against oxidative stress. [00284] RPE-MM medium is most preferably supplemented with taurine to final concentration of 2.0 mM (0.25 g/L), preferably 1.5 to 2.5 mM, less preferably, 0.5 to 3 mM, least preferably 0 to 4 mM. Taurine inclusion in the medium is not essential for the production of pigmented RPE, however its addition increases the percentage of mature pigmented RPE in a given culture when cells are plated at lower than optimal seeding density (75,000-150,000 cell/cm²) (see Example 18 and Figure 5). Concentrations greater than 4 mM are expected to be possible, but no further improvement in RPE quality is expected. Taurine suitable for cell culture can be obtained from Millipore Sigma-Aldrich (catalog number T8691). Other high quality reagent grades, such as high purity molecular biology grade, which are available from numerous vendors would be expected to substitute for cell culture grade (USP) when the intended use of the SC-RPE is for non-clinical purposes. [00285] In the RPE-MM of the disclosure ^-MEM is supplemented with hydrocortisone. Hydrocortisone an anti-inflammatory gluco- and mineral-corticoid that can affect cellular differentiation and has been shown to promote the upregulation of genes involved epithelial polarization, establishment of lateral tight junctions, and downregulates genes involved in epithelial-to-mesenchymal transition (EMT). [00286] When culturing minimally expanded SC-RPE under optimal conditions, supplementation of RPE-Minimal Medium with additional hydrocortisone is not necessary, with no overt differences in SC-RPE quality being observed because of its omission. However, its addition can have some benefits for RPE expansion due to its ability to minimize wound response. Using serum-based RPE medium it has been found that hydrocortisone can be eliminated in its entirety with no obvious overt affects. [00287] On that basis, hydrocortisone can, in some embodiments, be a desirable RPE medium additive, even if a non-essential one, for high quality RPE (see Example 18 and Figure 5). Small molecule inhibitors of EMT such as ROCK inhibitors or TGFBR kinase inhibitors have a similar, but more potent effect, although they do so through a different mechanism. Note that B27 supplement contains corticosterone which is the prodrug form of hydrocortisone, Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT suggesting that the concentration of corticosterone in B27 is insufficient on its own when SC- RPE are plated and suboptimal seeding densities(generally <75,000 cell/cm²). [00288] RPE-MM is most preferably supplemented with hydrocortisone to a final concentration of 20 ^g/L (55.2 nM), preferably 15 to 25 ^g/L, less preferably 10 to 30 ^g/L, and least preferably 0 to 40 ^g/L using water soluble, cyclodextrin encapsulated hydrocortisone (Millipore Sigma-Aldrich, catalog number H0396). Concentrations greater than 40 ^g/L are expected to be possible but are not expected to result in further improvements in RPE phenotype and function. It is expected that other forms of hydrocortisone such as the sodium salt of hydrocortisone 21-hemisucinate (e.g., STEMCELL Technologies, catalog numbers 74142 or 74144), hydrocortisone stock solutions (e.g., STEMCELL Technologies, catalog number 07925 or 07926), hydrocortisone solubilized in ethanol (Millipore Sigma-Aldrich, catalog number H0888) can be used as an alternative. It is also expected the corticosterone (Millipore Sigma Aldrich, catalog number C2505) could be substituted for hydrocortisone; however, slightly higher concentrations are expected to be possibly required due to a lower anti-inflammatory potential. [00289] In some embodiments of the disclosure RPE-MM can be the formulation RPE-MM which comprises the base medium ^-MEM supplemented with additional non-essential amino acids (glycine, alanine, asparagine, aspartate, glutamate, proline, and serine), insulin, transferrin, selenite, putrescine, progesterone, serum albumin, galactose, catalase, superoxide dismutase, D,L-alpha-tocopherol, D,L-alpha-tocopherol acetate, glutathione, ethanolamine, linoleic acid, linolenic acid, biotin, carnitine, vitamin A, taurine, triiodothyronine, corticosterone, and hydrocortisone, wherein the components in italics are expected to be non- essential but to possibly result in more homogenous pigmentation and epithelial morphology. [00290] In the most preferred embodiment, the RPE Maturation Medium (RPE-MM) can be prepared as follows: 10.08 g of powdered medium ^-MEM medium and 2.2 g of sodium bicarbonate in tissue culture grade H₂O to a final volume of 950 mL. For each 950 mL of dissolved ^-MEM medium 10 mL of 100X penicillin/streptomycin solution, 1 mL of normocin solution, 10 mL of N1 Supplement, 10 mL of 100X non-essential amino acids solution (NEAA), 20 mL of CTS-B27 Supplement, 0.25 g taurine, and 8 ^L of 2.5 mg/ml hydrocortisone are added and the complete medium is filter sterilized (see Example 11). [00291] The concentration of all components of the most preferred formulation of RPE-MM Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT and allowable (general) concentration ranges of RPE-MM buffered balanced salt solution, essential amino acids and nonessential amino acids, vitamins, hormones, essential components for lipid metabolism, carbon energy sources, serum albumin, insulin, transferrin, selenite, and antioxidants in an effective amount to promote maturation of differentiated SC-RPE cells are provided in Tables 7A to 7E. For xeno-free culture the protein components should be from the same species as the stem cell line. It is expected that lipid-rich albumin, such as and not limited to albumin purified from serum using non-denaturing chromatography methods or the commercially available AlbuMAX, can be substituted for Serum Albumin, Fraction V, which is lipid-poor, at the concentrations indicated in Table 7A to 7E. When using lipid poor albumin, some benefits such as improved growth and RPE phenotype, and function can be expected by the addition the lipids found in lipid rich albumin associated [34]. Table 7A. RPE Maturation Medium ^^{^} ^^^'^ ^^ ^^ + "!" "^^ ^^ ^^^^^^ ^ ^!"^ ^^ ^^ ^?^^^^ ^^ ^^ ^^^5^^^^^^^^^^^ ^ ^ ^^'^^^/^²^ ^^)^&^^ ^^ ^^'^^^7²^ *)(*^ ^^ ^^'^^^4^^{2^}^ ^&#^^ ^^ ^^'^^^^.^{2^}^ &^^^ ^^ ^^'^^^^^^{2^}^ ^^^ ^^ ^^'^^^9"^{2^}^ ^^^ ^^ ^^'^^^9"²⁾^ ^^^ ^^ ^^'^^^4^^{2^}^ ^^^ ^^ ^^'^^^:^^{2^}^ ^^^ ^^ ^^'^^^4^⁸^ ^^^)#^^ ^^ ^^'^^^+0_^ ⁸⁾^ ^#^*^ ^^ ^^'^^^,0^^{8^}^ &^^^ ^^ ^^'^^^/#)⁸^ ^^^ ^^ ^^'^^^,"0₎ ^{8^^}^ #^^^^^ ^^ ^^ ^^ ^^ /^4^^ ^^()*^^ ^^ /^^40₎^ ^(^&&^ ^^ 74^^ *)(*^ ^^ /^^_^+0_^^ ^#^*^ ^^ 4^4^^^ ^&#^^ ^^ ^.,0_^^ &^^^ ^^ /^_^,"0₎ ^{8^^}^ #^^^^^ ^^ Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Table 7A. RPE Maturation Medium ^^{^} ^^^'^ ^^ ^^ + "!" "^^ ^^ ^^^^^^ ^ ^!"^ ^^ ^^ ^?^^^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^ ^ ^ ^^^^^^^^"^ )&^^ ^^ ^^^ .^^^^"^ (##^ ^^ ^^^^^^ ^.^^"^ ^))^ ^^ ^^^^^^ '^^^^^^^^ )^*^ ^^ ^^4%^'"^^"^ *(^^ ^^ ^^4%^'^^"^ ^##^ ^^ ^^$^^'^^^^^^^^^^ (^#^ ^^ ^^$^^'^^^^"^ ^^^&^ ^^ $^%^^^"^ ^((^ ^^ ^^^^^'^^^^"^ ^##^ ^^ ^^^^^^"^^^^"^ ^##^ ^^ ^^^"^^^^"^ ^##^ ^^ ^^^%^^^"^ )^^^ ^^ ^^^"'^^^^^^"^ ^#^^ ^^ ^^+^"^%^^^^^^^"^ ^^^^ ^^ ^^+ ^^^^"^ ^^^^ ^^ ^^," ^^"^ ))&^ ^^ ^^^ ^^"^ ^###^ ^^ ^^^^ "^^^^"^ ^##^ ^^ ^^^ %^'^^^^^^ ^^^ ^^ ^^^% ^^^^"^ ^^^^ ^^ ^^-^^^^"^ )^^^ ^^ ^^ ^^ ^^ 2^^^^^^^^^^^^^^^^ ^ ^ 4^^^^^"^^^^^ ^^"^ ^^^(^ ^^ B'^^^^^^^^^"^ ^(^^^ ^^ ^^^^^^^'^^^ ^^^^^ ^^ ^^4^ ^^'^^"^^ ^#^^^ ^^ ^^^^^"^^^^^^^^ )^*^^ ^^ ^^^^^"^^^^^^^^^ )^*^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^ -^^^^^^^^^ ^ ^ Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Table 7A. RPE Maturation Medium ^^{^} ^^^'^ ^^ ^^ + "!" "^^ ^^ ^^^^^^ ^ ^!"^ ^^ ^^ ^?^^^^ ^^ ^^ ^^^^ ^^^^^^^^^ ^^ 6^^'^^^ ^#^(^ ^^ A^4^^^^^^^^^^'^'^"^^'"^^{^}^ ^^^#^ ^^ 9^^^^^^^^^^ ^^^^^ ^^ ^%^^^^^'^^^"^ ^^^ ^^ /^^^'^^^^^^"^ &^^^^ ^^ +^' "^^^^"^ ^^^^ ^^ +% ^^^^^^"^^{^}^ ^^&(^ ^^ ^"'^^^^^ #^ ^^ ^"'^^^^^^^"'^'"^ #^)#^#^ ^^ ^^^^!^^>^^^ #^^((^ ^^ ^^^^^^^"^ ^^^(^ ^^ -^'^^^^^6^^^ ^^##^ ^^ ^^ ^^ ^^ ^^ ^^ 3^^^^^^^ ^ ^ 4^ '^^^^'" ^^"H^%^ ^^^ '^^^^"^ #^^^^^^ ^^ + ^."^'" ^^"^ #^#^)^^ ^^ ^ ^^^^^^^^'^% ^^^^"^^^)^^ #^##)#^ ^^ ^^ ^^ ^^ .^^^^^^^^^^5;^^^^^^^^ ^ ^ A^$^^^^'^^"^ &)^)###^ ^^ A^$^^^^^"^ ***^^ ^^ +% ^>^^^^^^^^ ^###^ ^^ 0^^^^^^"'^'"^ ^^^ ^^ ^^ ^^ ^^ ^^^^^^^^^ ^ ^ ," ^^^^^^^^^^^ )^^(^ ^^ ^^^^^^^^ ^^*(^ ^^ ^ ^^^!" ^^^ #^^^*^ ^^ 4^'^^^^"^ #^#^#^^ ^^ ,^^" ^^^^"^A^^^^'^^"^ #^#^(^^ ^^ ^^ ^^ ^^ <^^^^^^^^^^^^ ^^^1^^5^^59^^^^^0^^^^^^ ^ ^ $^^'^'^^^^"^^ "^^^"^^^ )^^*^ ^^ A^^^^^^^^^^^^^^" ^^^^^"'^'"^ ^^^^^ ^^ A^^^^^^^^^^^^^^" ^^^ ^^^#^ ^^ ^^^^^^^^^^^^ #^^(^^ ^^ ^^ ^^ ^^ Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Table 7A. RPE Maturation Medium ^{^^} ^^^'^ ^^ ^^ + "!" "^^ ^^ ^^^^^^ ^ ^!"^ ^^ ^^ ^?^^^^ ^^ ^^ -^^^^^^^^^^ ^ ^ ^^"^^^^^"^ ^^^ ^^ 4%'^^^^"^ ^^^ ^^ $^^^^^^^"^ ^^^ ^^ ^^%^^^^^"^ ^^^ ^^ E ^^^^"^^^^%^ ^^^^^ ^^^ ^^ ^J^A"^^%^^"^^^^^"^ ^^^ ^^ ^J^A"^^%^%'^^^^"^ ^^^ ^^ ^J^A"^^%.^^^^^^^"^ ^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^3^ ^^^^^^^^^&^^^^^^^^'^^ ^ ^ +^"^^^^ "^^ ^(^(^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^^&^^^^^^^^'^ ^ /^ ^^^^^^ *#^^##^?.H^^^ +"^^^^^^^^^ ^##^EH^^^ ^^ ,' "^'^^%^^^^^ ^##^?.H^^^ ^^ A^+^^'^^'^"^^^^^^^^5K4^^^^^^ ^"^^^^^'^'^'"^^!^ ^A^4^^^^^^^ ^^^'^'^"^^'"^^ ^^ ^^ +% ^^^^^^^^^^^^"^^^^^'^'^'"^^ !^ ^+% ^^^^^^"^ ^^ ^^ ^^^^^^,+^^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ + "!" "^^ ^^^^^^ ^ ^^ ^#$^ %^ ^^&^ ^^ ^^ ^?^^^^ ^^ ^^^5^^^^^^^^^^^ ^ ^ ^ ^ ^^'^^^/^²^ ^^ ^^^#^*^ ^^ ^^(&^)^ ^^'^^^7²^ ^^ ^^^^^ ^^ *^^)^ ^^'^^^4^^{2^}^ ^^ ^*^^^ ^^ ^&^#^ ^^'^^^^.^{2^}^ ^^ ^^^^ ^^ &^&^ ^^'^^^^^^{2^}^ ^^ #^ ^^ #^###^^*^ Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^^^^^,+^^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ + "!" "^^ ^^^^^^ ^ ^^ ^#$^ %^ ^^&^ ^^ ^^ ^?^^^^ ^^ ^^'^^^9"^{2^}^ ^^ #^ ^^ #^)^)^ ^^'^^^9"²⁾^ ^^ #^ ^^ #^#(^^ ^^'^^^4^^{2^}^ ^^ #^ ^^ #^##^)#^ ^^'^^^:^^{2^}^ ^^ #^ ^^ #^)^^^ ^^'^^^4^⁸^ ^^ ^^^)*(^ ^^ ^^^&^&^ ^^'^^^+0_^ ⁸⁾^ ^^ ^^#^ ^^ ^#^(^ ^^'^^^,0^^{8^}^ ^^ ^#)^ ^^ &^&^ ^^'^^^/#₎ ⁸^ ^^ #^ ^^ #^^&*^ ^^'^^^,"0)^{8^^}^ ^^ #^#^^^^ ^^ #^^*^^ ^^ ^^ ^^ ^^ ^^ /^4^^ ^^ ^^^#^^^ ^^ ^^&(&(^ /^^40)^ ^^ ^*^#)^ ^^ ^&)(^^ 74^^ ^^ ^^^^^ ^^ *^^)^ /^^^+0^^ ^^ ^^#^ ^^ ^#^(^ 4^4^^^ ^^ ^*^*^ ^^ ^&)&^ ^.,0_^^ ^^ ^^^^ ^^ &^&^ /^^,"0)^{^}^ ^^ #^#^^^^ ^^ #^^*^^ 9"^/0)^)^ ^^ #^ ^^ #^ 9",0_^^ ^^ #^ ^^ #^ :^,0^^ ^^ #^ ^^ #^ 4^,0_^^ ^^ #^ ^^ #^ ^^4^^^ ^^ #^ ^^ #^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^ ^ ^ ^ ^ ^^^^^^^^"^ ^^ )*(^ ^^ ^#(^ ^^^ .^^^^"^ ^^ *(*^ ^^ (^^^ ^^^^^^ ^.^^"^ ^^ ^#&^ ^^ ^*&^ ^^^^^^ '^^^^^^^^ ^^ )##^ ^^ )*#^ ^^4%^'"^^"^ ^^ ^(&^ ^^ *&^^ ^^4%^'^^"^ ^^ &^^#^ ^^ ^^#^ ^^$^^'^^^^^^^^^^ ^^ *&*^ ^^ ()*^ ^^$^^'^^^^"^ ^^ ^^(^^ ^^ ^^^#^ $^%^^^"^ ^^ ^^^^ ^^ ^^^^ ^^^^^'^^^^"^ ^^ ^&*^ ^^ ^#^^ ^^^^^^"^^^^"^ ^^ )^^^ ^^ ^^^^ ^^^"^^^^"^ ^^ )^^^ ^^ ^^^^ ^^^%^^^"^ ^^ )^#^ ^^ ^*^^ ^^^"'^^^^^^"^ ^^ ^&^^^ ^^ ^^)^ ^^+^"^%^^^^^^^"^ ^^ ^^^^ ^^ ^^^^ ^^+ ^^^^"^ ^^ ^^^^ ^^ ^^^^ ^^," ^^"^ ^^ )^)^ ^^ )()^ Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^^^^^,+^^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ + "!" "^^ ^^^^^^ ^ ^^ ^#$^ %^ ^^&^ ^^ ^^ ^?^^^^ ^^ ^^^ ^^"^ ^^ ^*##^ ^^ ^^*#^ ^^^^ "^^^^"^ ^^ )^^^ ^^ ^^(^ ^^^ %^'^^^^^^ ^^ ^(^^^ ^^ *^^^^ ^^^% ^^^^"^ ^^ ^^^^ ^^ ^^^^ ^^-^^^^"^ ^^ )^^^ ^^ ^^^^ ^^ ^^ ^^ ^^ ^^ 2^^^^^^^^^^^^^^^^ ^ ^ ^ ^ 4^^^^^"^^^^^ ^^"^ ^^ ^^#)^ ^^ ^^^^^ B'^^^^^^^^^"^ ^^ ^^^)^ ^^ ^#^*^ ^^^^^^^'^^^ ^^ ^#^^^ ^^ ^*^^^ ^^4^ ^^'^^"^^ ^^ ^^*&^ ^^ ^^^(^ ^^^^^"^^^^^^^^ ^^ ^^(&^ ^^ ^^*#^ ^^^^^"^^^^^^^^^ ^^ ^^(^^ ^^ ^^^^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^ -^^^^^^^^^ ^ ^ ^ ^ ^^^^ ^^^^^^^^^ ^^ ^^)^ ^^ ^^#^ 6^^'^^^ ^^ ^^^(^ ^^ ^)^^^ A^4^^^^^^^^^^'^'^"^^'"^^{^}^ ^^ ^^#(^ ^^ )^^^^ 9^^^^^^^^^^ ^^ ^^^^^ ^^ )^*#^ ^%^^^^^'^^^"^ ^^ #^ ^^ ^^)^^ /^^^'^^^^^^"^ ^^ &^#^^ ^^ ^^^^^ +^' "^^^^"^ ^^ ^*#^ ^^ ^^^^ +% ^^^^^^"^^{^}^ ^^ ^^^&^ ^^ ^^#(^ ^"'^^^^^ ^^ #^ ^^ #^)^^^ ^"'^^^^^^^"'^'"^ ^^ #^^^&^ ^^ #^)&#^ ^^^^!^^>^^^ ^^ #^^(^^ ^^ #^)^^^ ^^^^^^^"^ ^^ ^^^^^ ^^ ^^)(^ -^'^^^^^6^^^ ^^ #^^&*^ ^^ ^^#^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ 3^^^^^^^ ^ ^ ^ ^ 4^ '^^^^'" ^^"H^%^ ^^^ '^^^^"^ ^^ #^#&^^^ ^^ #^^^^^^ + ^."^'" ^^"^ ^^ #^#)^^^ ^^ #^#*^#^ ^ ^^^^^^^^'^% ^^^^"^^^)^^ ^^ #^##^^)^ ^^ #^##)^^^ ^^ ^^ ^^ ^^ ^^ .^^^^^^^^^^5;^^^^^^^^ ^ ^ ^ ^ A^$^^^^'^^"^ ^^ (^^*^ ^^ ^#^^ A^$^^^^^"^ ^^ *^^&^ ^^ ^^(^^ +% ^>^^^^^^^^ ^^ &^*^ ^^ ^#&^^ 0^^^^^^"'^'"^ ^^ #^ ^^ ^&)^ Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^^^^^,+^^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ + "!" "^^ ^^^^^^ ^ ^^ ^#$^ %^ ^^&^ ^^ ^^ ^?^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^ ^ ^ ^ ^ ," ^^^^^^^^^^^ ^^ ^&^^^ ^^ ^^^#^ ^^^^^^^^ ^^ ^^^^^ ^^ )^^(^ ^ ^^^!" ^^^ ^^ #^#^^#^ ^^ #^^)^^ 4^'^^^^"^ ^^ #^##^&#^ ^^ #^#^)#^ ,^^" ^^^^"^A^^^^'^^"^ ^^ #^#*^^^ ^^ #^#^(^^ ^^ ^^ ^^ ^^ ^^ <^^^^^^^^^^^^ ^^^1^^5^^59^^^^^0^^^^^^ ^ ^ ^ ^ $^^'^'^^^^"^^ "^^^"^^^ ^^ ^^^^^ ^^ ^^#(^ A^^^^^^^^^^^^^^" ^^^^^"'^'"^ ^^ ^^*^^ ^^ ^^(*^ A^^^^^^^^^^^^^^" ^^^ ^^ ^^&#^ ^^ )^##^ ^^^^^^^^^^^^ ^^ #^^*^^ ^^ ^^^^^^ ^^ ^^ ^^ ^^ ^^ -^^^^^^^^^^ ^ ^ ^ ^ ^^"^^^^^"^ ^^ #^ ^^ ^^^)^ 4%'^^^^"^ ^^ #^ ^^ ^)^(^ $^^^^^^^"^ ^^ #^ ^^ ^^^^^ ^^%^^^^^"^ ^^ #^ ^^ ^)^(^ E ^^^^"^^^^%^ ^^^^^ ^^ #^ ^^ ^)^*^ ^J^A"^^%^^"^^^^^"^ ^^ #^ ^^ ^)^^^ ^J^A"^^%^%'^^^^"^ ^^ #^ ^^ ^)^&^ ^J^A"^^%.^^^^^^^"^ ^^ #^ ^^ ^^^)^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^3^ ^^^^^^^^^&^^^^^^^^'^^ ^ ^ ^ ^ +^"^^^^ "^^ ^^ ^#^^^ ^^ ^^^&^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^^&^^^^^^^^'^ ^ ^ ^ /^ ^^^^^^ ^^ ^#^ ^^ ^^##^?.H^^^ +"^^^^^^^^^ ^^ ^#^ ^^ ^^##^EH^^^ ,' "^'^^%^^^^^ ^^ ^#^ ^^ ^^##^?.H^^^ A^+^^'^^'^"^^^^^^^^5K4^^^^^^ ^"^^^^^'^'^'"^^!^ ^A^4^^^^^^^ ^^^'^'^"^^'"^^ ^^ ^^ ^^ ^^ +% ^^^^^^^^^^^^"^^^^^'^'^'"^^ !^ ^+% ^^^^^^"^ ^^ ^^ ^^ Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^^^^^,.^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^"^^^+ "!" "^^ ^^^^^^ ^ ^#&^ %^ ^^$^ ^^ ^?^^^^ ^^ ^^^5^^^^^^^^^^^ ^ ^ ^ ^^'^^^/^²^ ^)&^(&^ ^^ ^^^^(^^ ^^'^^^7²^ ^(^^^ ^^ **&#^ ^^'^^^4^^{2^}^ ^)^^^ ^^ ^&^(^ ^^'^^^^.^{2^}^ ^))^ ^^ &^^^ ^^'^^^^^^{2^}^ #^ ^^ #^###^^^^ ^^'^^^9"^{2^}^ #^ ^^ #^^^^^ ^^'^^^9"²⁾^ #^ ^^ #^^^)^ ^^'^^^4^^{2^}^ #^ ^^ #^##^*^^ ^^'^^^:^^{2^}^ #^ ^^ #^^^&^ ^^'^^^4^⁸^ ^^^^^#^ ^^ ^)^)^^^ ^^'^^^+0^⁸⁾^ ^(*^ ^^ ^^)^^ ^^'^^^,0_^ ^{8^}^ *^*^ ^^ &^^^ ^^'^^^/#)⁸^ #^ ^^ #^)^#^ ^^'^^^,"0)^{8^^}^ #^#(#^^ ^^ #^^&^^ ^^ ^^ ^^ ^^ /^4^^ ^^^^)#^ ^^ ^^^#^)^ /^^40₎^ ^*^^&^ ^^ )#*^#^ 74^^ ^(^^^ ^^ **&#^ /^^^+0^^ ^(*^ ^^ ^^)^^ 4^4^_^^ ^)&^^ ^^ ^&^^^ ^.,0^^ ^))^ ^^ &^^^ /^^,"0)^{^}^ #^#(#^^ ^^ #^^&^^ 9"^/0₎^₎^ #^ ^^ #^^^)^ 9",0^^ #^ ^^ #^^^^^ :^,0_^^ #^ ^^ #^^^&^ 4^,0^^ #^ ^^ #^##^*^^ ^^4^^^ #^ ^^ #^###^^^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^ ^ ^ ^ ^^^^^^^^"^ ))^^ ^^ ^)^^ ^^^ .^^^^"^ *)^^ ^^ (^^^ ^^^^^^ ^.^^"^ )&)^ ^^ ^&)^ ^^^^^^ '^^^^^^^^ ^^*^ ^^ )^*^ ^^4%^'"^^"^ )(^^ ^^ *^^^ ^^4%^'^^"^ (^^)^ ^^ ^^#^ ^^$^^'^^^^^^^^^^ *(#^ ^^ ((#^ ^^$^^'^^^^"^ ^^^^^ ^^ ^*^^^ $^%^^^"^ ^^(^ ^^ &^(^ ^^^^^'^^^^"^ ^(^^ ^^ ^#&^ ^^^^^^"^^^^"^ )&^^ ^^ ^&^^ Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^^^^^,.^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^"^^^+ "!" "^^ ^^^^^^ ^ ^#&^ %^ ^^$^ ^^ ^?^^^^ ^^ ^^^"^^^^"^ )&^^ ^^ ^^^^ ^^^%^^^"^ )&^^ ^^ *#^^ ^^^"'^^^^^^"^ ^(^&^ ^^ ^^*^ ^^+^"^%^^^^^^^"^ ^&&^ ^^ ^^^^ ^^+ ^^^^"^ )^^^ ^^ ^^^^ ^^," ^^"^ ^&&^ ^^ )&&^ ^^^ ^^"^ ^###^ ^^ ^*##^ ^^^^ "^^^^"^ )&*^ ^^ ^^)^ ^^^ %^'^^^^^^ ^^^^^ ^^ *)^)^ ^^^% ^^^^"^ ^^#^ ^^ ^^^^ ^^-^^^^"^ )&^^ ^^ ^^^^ ^^ ^^ ^^ ^^ 2^^^^^^^^^^^^^^^^ ^ ^ ^ 4^^^^^"^^^^^ ^^"^ (^^#^ ^^ )*^(^ B'^^^^^^^^^"^ &^^#^ ^^ ^^^(^ ^^^^^^^'^^^ ^#^^^ ^^ ^#^^^ ^^4^ ^^'^^"^^ *^#*^ ^^ ^*^^^ ^^^^^"^^^^^^^^ ^^^^^ ^^ *^^)^ ^^^^^"^^^^^^^^^ ^^&#^ ^^ *^)^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^ -^^^^^^^^^ ^ ^ ^ ^^^^ ^^^^^^^^^ ^^^^ ^^ ^^*^ 6^^'^^^ *^)^^ ^^ ^*^^^ A^4^^^^^^^^^^'^'^"^^'"^^{^}^ ^^#^^ ^^ ^^^)^ 9^^^^^^^^^^ ^^^&^ ^^ ^^^)^ ^%^^^^^'^^^"^ #^ ^^ &^^^^ /^^^'^^^^^^"^ ^^&^^ ^^ ^*^(^ +^' "^^^^"^ ^##^ ^^ ^^^^ +% ^^^^^^"^^{^}^ ^^^^^ ^^ ^^^(^ ^"'^^^^^ #^ ^^ #^^)(^ ^"'^^^^^^^"'^'"^ #^^*^^ ^^ #^^*(^ ^^^^!^^>^^^ #^^(^ ^^ #^*^^ ^^^^^^^"^ ^^&*^ ^^ *^^*^ -^'^^^^^6^^^ #^^((^ ^^ ^^#^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ 3^^^^^^^ ^ ^ ^ 4^ '^^^^'" ^^"H^%^ ^^^ '^^^^"^ #^#*(^^ ^^ #^^(^)^ + ^."^'" ^^"^ #^#^^(^ ^^ #^#(^&^ ^ ^^^^^^^^'^% ^^^^"^^^)^^ #^##^^^^ ^^ #^##^^(^ Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^^^^^,.^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^"^^^+ "!" "^^ ^^^^^^ ^ ^#&^ %^ ^^$^ ^^ ^?^^^^ ^^ ^^ ^^ ^^ ^^ .^^^^^^^^^^5;^^^^^^^^ ^ ^ ^ A^$^^^^'^^"^ ^^^^^ ^^ ^^*^ A^$^^^^^"^ *)^*^ ^^ ^^^^^^ +% ^>^^^^^^^^ (*#^ ^^ ^^^*^ 0^^^^^^"'^'"^ #^ ^^ *((^ ^^ ^^ ^^ ^^ ^^^^^^^^^ ^ ^ ^ ," ^^^^^^^^^^^ ^&^&^ ^^ *(^^^ ^^^^^^^^ #^^^^^ ^^ *^)*^ ^ ^^^!" ^^^ #^#(^^^ ^^ ^^)*^ 4^'^^^^"^ #^##*^#^ ^^ #^#^*(^ ,^^" ^^^^"^A^^^^'^^"^ #^#)&*^ ^^ #^^^*^^ ^^ ^^ ^^ ^^ <^^^^^^^^^^^^ ^^^1^^5^^59^^^^^0^^^^^^ ^ ^ ^ $^^'^'^^^^"^^ "^^^"^^^ ^^()^ ^^ ^^&&^ A^^^^^^^^^^^^^^" ^^^^^"'^'"^ ^^#(^ ^^ )^^&^ A^^^^^^^^^^^^^^" ^^^ ^^^#^ ^^ )^(#^ ^^^^^^^^^^^^ #^^))^ ^^ ^^^^^ ^^ ^^ ^^ ^^ -^^^^^^^^^^ ^ ^ ^ ^^"^^^^^"^ #^ ^^ ^^^^^ 4%'^^^^"^ #^ ^^ ^^^^^ $^^^^^^^"^ #^ ^^ ^)^)^ ^^%^^^^^"^ #^ ^^ ^^^^^ E ^^^^"^^^^%^ ^^^^^ #^ ^^ ^^^#^ ^J^A"^^%^^"^^^^^"^ #^ ^^ ^(^)^ ^J^A"^^%^%'^^^^"^ #^ ^^ ^^^*^ ^J^A"^^%.^^^^^^^"^ #^ ^^ ^^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^3^ ^^^^^^^^^&^^^^^^^^'^^ ^ ^ ^ +^"^^^^ "^^ ^)^^^ ^^ ^^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^^&^^^^^^^^'^ ^ ^ ^ /^ ^^^^^^ ^#^ ^^^ ^^##^?.H^^^ +"^^^^^^^^^ ^#^ ^^^ ^^##^EH^^^ ,' "^'^^%^^^^^ ^#^ ^^^ ^^##^?.H^^^ A^+^^'^^'^"^^^^^^^^5K4^^^^^^ ^^ ^^ ^^ Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^^^^^,.^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^"^^^+ "!" "^^ ^^^^^^ ^ ^#&^ %^ ^^$^ ^^ ^?^^^^ ^^ ^"^^^^^'^'^'"^^!^ ^A^4^^^^^^^ ^^^'^'^"^^'"^^ +% ^^^^^^^^^^^^"^^^^^'^'^'"^^ !^ ^+% ^^^^^^"^ ^^ ^^ ^^^^^^,!^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^"^^'^+ "!" "^^ ^^^^^^ ^ ^&^^ %^ ^"^^ ^^ ^?^^^^ ^^ ^^^5^^^^^^^^^^^ ^ ^ ^ ^^'^^^/^²^ ^)*^(^^ ^^ ^*^(**^ ^^'^^^7²^ ^^^(^ ^^ *(&^^ ^^'^^^4^^{2^}^ ^^&^^ ^^ ^^^^^ ^^'^^^^.^{2^}^ (^^^ ^^ &(#^ ^^'^^^^^^{2^}^ #^ ^^ #^###)^^^ ^^'^^^9"^{2^}^ #^ ^^ ^^^^^ ^^'^^^9"²⁾^ #^ ^^ #^^&*^ ^^'^^^4^^{2^}^ #^ ^^ #^##)&^^ ^^'^^^:^^{2^}^ #^ ^^ ^^^^^ ^^'^^^4^⁸^ ^^&^(*^ ^^ ^)^^^#^ ^^'^^^+0^⁸⁾^ ^^^^ ^^ ^^^^^ ^^'^^^,0_^ ^{8^}^ ^&^^ ^^ &(#^ ^^'^^^/#)⁸^ #^ ^^ #^***^ ^^'^^^,"0₎ ^{8^^}^ #^#)#^^ ^^ #^^^*^ ^^ ^^ ^^ ^^ /^4^^ ^#^^^*^ ^^ ^^))^#^ /^^40)^ ^^^))^ ^^ )^^^*^ 74^^ ^^^(^ ^^ *(&^^ /^^_^+0_^^ ^^^^ ^^ ^^^^^ 4^4^^^ ^^&#^ ^^ ^^^#^ ^.,0^^ (^^^ ^^ &(#^ /^_^,"0₎ ^{^}^ #^#)#^^ ^^ #^^^*^ 9"^/0)^)^ #^ ^^ #^^&*^ 9",0_^^ #^ ^^ ^^^^^ :^,0_^^ #^ ^^ ^^^^^ 4^,0^^ #^ ^^ #^##)&^^ ^^4^_^^ #^ ^^ #^###)^^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^ ^ ^ ^ Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^^^^^,!^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^"^^'^+ "!" "^^ ^^^^^^ ^ ^&^^ %^ ^"^^ ^^ ^?^^^^ ^^ ^^^^^^^^"^ )#(^ ^^ ^&^^ ^^^ .^^^^"^ ^^(^ ^^ (^)^ ^^^^^^ ^.^^"^ )*&^ ^^ *))^ ^^^^^^ '^^^^^^^^ ^*#^ ^^ ^^*^ ^^4%^'"^^"^ ^((^ ^^ (#^^ ^^4%^'^^"^ ^(^(^ ^^ ^)#^ ^^$^^'^^^^^^^^^^ *)*^ ^^ ^^#^ ^^$^^'^^^^"^ ^&&^^ ^^ ^&^)^ $^%^^^"^ (^^^ ^^ &((^ ^^^^^'^^^^"^ ^*^^ ^^ ^^^^ ^^^^^^"^^^^"^ )^*^ ^^ *^*^ ^^^"^^^^"^ )^*^ ^^ *^^^ ^^^%^^^"^ )^*^ ^^ *(^^ ^^^"'^^^^^^"^ ^*^ ^^ ^)^^ ^^+^"^%^^^^^^^"^ ^&^^ ^^ ^(*^ ^^+ ^^^^"^ )^^^ ^^ *^^^ ^^," ^^"^ ^()^ ^^ ^)&^ ^^^ ^^"^ *##^ ^^ ^^*#^ ^^^^ "^^^^"^ )^^^ ^^ *)^^ ^^^ %^'^^^^^^ ^^^^^ ^^ **^^^ ^^^% ^^^^"^ ^&(^ ^^ ^()^ ^^-^^^^"^ )^)^ ^^ *^^^ ^^ ^^ ^^ ^^ 2^^^^^^^^^^^^^^^^ ^ ^ ^ 4^^^^^"^^^^^ ^^"^ (^^(^ ^^ ^^^&^ B'^^^^^^^^^"^ ^^^#^ ^^ )^^&^ ^^^^^^^'^^^ ^#^*^ ^^ **^#^ ^^4^ ^^'^^"^^ ^^*)^ ^^ ^#^^^ ^^^^^"^^^^^^^^ #^&^)^ ^^ ^^^*^ ^^^^^"^^^^^^^^^ #^&^^*^ ^^ ^^^&^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^ -^^^^^^^^^ ^ ^ ^ ^^^^ ^^^^^^^^^ ^^^#^ ^^ )#^^ 6^^'^^^ ^^((^ ^^ ^#^&^ A^4^^^^^^^^^^'^'^"^^'"^^{^}^ ^^^&^ ^^ *^^*^ 9^^^^^^^^^^ ^^^^^ ^^ *^^^^ ^%^^^^^'^^^"^ #^ ^^ ^)^^^ /^^^'^^^^^^"^ ^^^)^ ^^ ^^^^^ +^' "^^^^"^ ^^^&^ ^^ ^##^ +% ^^^^^^"^^{^}^ ^^()^ ^^ ^^^*^ Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^^^^^,!^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^"^^'^+ "!" "^^ ^^^^^^ ^ ^&^^ %^ ^"^^ ^^ ^?^^^^ ^^ ^"'^^^^^ #^ ^^ #^*^^^ ^"'^^^^^^^"'^'"^ #^#^(#^ ^^ #^(#&^ ^^^^!^^>^^^ #^^*^ ^^ #^(^^^ ^^^^^^^"^ ^^&#^ ^^ ^^^^^ -^'^^^^^6^^^ #^^*^ ^^ ^^#(^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ 3^^^^^^^ ^ ^ ^ 4^ '^^^^'" ^^"H^%^ ^^^ '^^^^"^ #^#^&^^ ^^ #^^^*&^ + ^."^'" ^^"^ #^#^#&^ ^^ #^#&#^^ ^ ^^^^^^^^'^% ^^^^"^^^)^^ #^###^^)^ ^^ #^##*^^^ ^^ ^^ ^^ ^^ .^^^^^^^^^^5;^^^^^^^^ ^ ^ ^ A^$^^^^'^^"^ ^#^&^ ^^ ^(^^ A^$^^^^^"^ *^^^^ ^^ ^((^^^ +% ^>^^^^^^^^ ^^*^ ^^ ^^(^^ 0^^^^^^"'^'"^ #^ ^^ &^&^ ^^ ^^ ^^ ^^ ^^^^^^^^^ ^ ^ ^ ," ^^^^^^^^^^^ ^^^#^ ^^ ^*^^^ ^^^^^^^^ #^)^#^ ^^ ^#^#^ ^ ^^^!" ^^^ #^#)^)^ ^^ ^^(^^ 4^'^^^^"^ #^##^(#^ ^^ #^#^#&^ ,^^" ^^^^"^A^^^^'^^"^ #^#^^^^ ^^ #^^*)&^ ^^ ^^ ^^ ^^ <^^^^^^^^^^^^ ^^^1^^5^^59^^^^^0^^^^^^ ^ ^ ^ $^^'^'^^^^"^^ "^^^"^^^ #^&^^*^ ^^ (^*#^ A^^^^^^^^^^^^^^" ^^^^^"'^'"^ #^*)##^ ^^ ^^^^^ A^^^^^^^^^^^^^^" ^^^ #^(###^ ^^ ^^&#^ ^^^^^^^^^^^^ #^^^*^ ^^ ^^)^^ ^^ ^^ ^^ ^^ -^^^^^^^^^^ ^ ^ ^ ^^"^^^^^"^ #^ ^^ )^^^^ 4%'^^^^"^ #^ ^^ ^^^^^ $^^^^^^^"^ #^ ^^ )*^)^ ^^%^^^^^"^ #^ ^^ ^^^)^ E ^^^^"^^^^%^ ^^^^^ #^ ^^ ^^^#^ ^J^A"^^%^^"^^^^^"^ #^ ^^ )^^&^ ^J^A"^^%^%'^^^^"^ #^ ^^ ^^^^^ ^J^A"^^%.^^^^^^^"^ #^ ^^ )^^^^ Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^^^^^,!^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^"^^'^+ "!" "^^ ^^^^^^ ^ ^&^^ %^ ^"^^ ^^ ^?^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^3^ ^^^^^^^^^&^^^^^^^^'^^ ^ ^ ^ +^"^^^^ "^^ ^^)^ ^^ )#^)^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^^&^^^^^^^^'^ ^ ^ ^ /^ ^^^^^^ ^#^ ^^^ ^^##^?.H^^^ +"^^^^^^^^^ ^#^ ^^^ ^^##^EH^^^ ,' "^'^^%^^^^^ ^#^ ^^^ ^^##^?.H^^^ A^+^^'^^'^"^^^^^^^^5K4^^^^^^ ^"^^^^^'^'^'"^^!^ ^A^4^^^^^^^ ^^^'^'^"^^'"^^ ^^ ^^ ^^ +% ^^^^^^^^^^^^"^^^^^'^'^'"^^ !^ ^+% ^^^^^^"^ ^^ ^^ ^^^^^^,^^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^^/^ ^^ ^^;^ ^^^^^^ ^ !'(^ %^ ^(&^ ^^ ^?^^^^ ^^ ^^^5^^^^^^^^^^^ ^ ^ ^ ^^'^^^/^²^ ^)^###^ ^^ ^*(###^ ^^'^^^7²^ )&^#^ ^^ *&##^ ^^'^^^4^^{2^}^ ^###^ ^^ ^^*#^ ^^'^^^^.^{2^}^ (*#^ ^^ &&#^ ^^'^^^^^^{2^}^ #^ ^^ #^###(##^ ^^'^^^9"^{2^}^ #^ ^^ ^^^^^ ^^'^^^9"²⁾^ #^ ^^ #^^*#^ ^^'^^^4^^{2^}^ #^ ^^ #^##(##^ ^^'^^^:^^{2^}^ #^ ^^ ^^##^ ^^'^^^4^⁸^ ^^**##^ ^^ ^)&###^ ^^'^^^+0_^ ⁸⁾^ ^##^ ^^ ^)##^ ^^'^^^,0^^{8^}^ )&#^ ^^ &&*^ ^^'^^^/#)⁸^ #^ ^^ #^^*#^ ^^'^^^,"0₎ ^{8^^}^ #^#^##^ ^^ #^))#^ ^^ ^^ ^^ ^^ /^4^^ ^#^###^ ^^ ^^(###^ Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^^^^^,^^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^^/^ ^^ ^^;^ ^^^^^^ ^ !'(^ %^ ^(&^ ^^ ^?^^^^ ^^ /^^40₎^ ^^^##^ ^^ )*###^ 74^^ )&^#^ ^^ *&##^ /^^_^+0_^^ ^^(^ ^^ ^)##^ 4^4^^^ ^^#^ ^^ ^^*#^ ^.,0^^ (*#^ ^^ &&#^ /^_^,"0₎ ^{^}^ #^#^##^ ^^ #^)^(^ 9"^/0)^)^ #^ ^^ #^^*#^ 9",0_^^ #^ ^^ ^^##^ :^,0^^ #^ ^^ ^^##^ 4^,0^^ #^ ^^ #^##(##^ ^^4^_^^ #^ ^^ #^###(##^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^ ^ ^ ^ ^^^^^^^^"^ ^^^^^ ^^ *^#^ ^^^ .^^^^"^ ^##^ ^^ &##^ ^^^^^^ ^.^^"^ ^*^#^ ^^ (##^ ^^^^^^ '^^^^^^^^ ^*^#^ ^^ ^*#^ ^^4%^'"^^"^ ^(#^ ^^ (^#^ ^^4%^'^^"^ ^^^#^ ^^ ^^#^ ^^$^^'^^^^^^^^^^ ^*^#^ ^^ &^#^ ^^$^^'^^^^"^ ^)^#^ ^^ )#^#^ $^%^^^"^ ^)#^ ^^ ^###^ ^^^^^'^^^^"^ ^)^^ ^^ ^^(^ ^^^^^^"^^^^"^ ^(#^ ^^ *^#^ ^^^"^^^^"^ ^(#^ ^^ *^#^ ^^^%^^^"^ ^(#^ ^^ (^#^ ^^^"'^^^^^^"^ ^#^#^ ^^ ^*#^ ^^+^"^%^^^^^^^"^ ^)#^ ^^ ^^#^ ^^+ ^^^^"^ &#^#^ ^^ (##^ ^^," ^^"^ ^)^^ ^^ ^(#^ ^^^ ^^"^ #^ ^^ )###^ ^^^^ "^^^^"^ ^()^ ^^ *^#^ ^^^ %^'^^^^^^ ^#^#^ ^^ (*^#^ ^^^% ^^^^"^ ^)#^ ^^ ^^#^ ^^-^^^^"^ ^(#^ ^^ *^#^ ^^ ^^ ^^ ^^ 2^^^^^^^^^^^^^^^^ ^ ^ ^ 4^^^^^"^^^^^ ^^"^ ^^^#^ ^^ (^^#^ B'^^^^^^^^^"^ ^^^^^ ^^ ^)^(^ ^^^^^^^'^^^ ^^)^ ^^ ^#^#^ ^^4^ ^^'^^"^^ #^####^ ^^ ^^^#^ Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^^^^^,^^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^^/^ ^^ ^^;^ ^^^^^^ ^ !'(^ %^ ^(&^ ^^ ^?^^^^ ^^ ^^^^^"^^^^^^^^ #^*&^^ ^^ ^^^#^ ^^^^^"^^^^^^^^^ #^*^^^^ ^^ ^^**^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^ -^^^^^^^^^ ^ ^ ^ ^^^^ ^^^^^^^^^ ^*^#^ ^^ ^##^ 6^^'^^^ #^^#^^ ^^ ^^^#^ A^4^^^^^^^^^^'^'^"^^'"^^{^}^ ^^^#^ ^^ (^^#^ 9^^^^^^^^^^ ^^*#^ ^^ ^^^#^ ^%^^^^^'^^^"^ #^ ^^ ^&^#^ /^^^'^^^^^^"^ *^^#^ ^^ ^)^#^ +^' "^^^^"^ ))^#^ ^^ *^#^ +% ^^^^^^"^^{^}^ )^^#^ ^^ ^^^#^ ^"'^^^^^ #^ ^^ #^^##^ ^"'^^^^^^^"'^'"^ #^ ^^ #^&^#^ ^^^^!^^>^^^ #^^^*^ ^^ #^^^#^ ^^^^^^^"^ ^^##^ ^^ &^*#^ -^'^^^^^6^^^ #^^*#^ ^^ ^^)#^ ^^ ^^ ^^ ^^ ^^ 3^^^^^^^ ^ ^ ^ 4^ '^^^^'" ^^"H^%^ ^^^ '^^^^"^ #^####^ ^^ #^)^#^ + ^."^'" ^^"^ #^##^^#^ ^^ #^^^#^ ^ ^^^^^^^^'^% ^^^^"^^^)^^ #^###^^#^ ^^ #^##^^#^ ^^ ^^ ^^ ^^ .^^^^^^^^^^5;^^^^^^^^ ^ ^ ^ A^$^^^^'^^"^ #^####^ ^^ ^^^^ A^$^^^^^"^ *###^ ^^ ^#^##^ +% ^>^^^^^^^^ )##^ ^^ ^)*#^ 0^^^^^^"'^'"^ #^ ^^ ^)*#^ ^^ ^^ ^^ ^^ ^^^^^^^^^ ^ ^ ^ ," ^^^^^^^^^^^ (^^#^ ^^ ^##^ ^^^^^^^^ #^^(#^ ^^ ^^^#^ ^ ^^^!" ^^^ #^#^^#^ ^^ )^*#^ 4^'^^^^"^ #^ ^^ #^#^&#^ ,^^" ^^^^"^A^^^^'^^"^ #^ ^^ #^^^##^ ^^ ^^ ^^ ^^ <^^^^^^^^^^^^ ^^^1^^5^^59^^^^^0^^^^^^ ^ ^ ^ $^^'^'^^^^"^^ "^^^"^^^ #^ ^^ &^^#^ Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^^^^^,^^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^^/^ ^^ ^^;^ ^^^^^^ ^ !'(^ %^ ^(&^ ^^ ^?^^^^ ^^ A^^^^^^^^^^^^^^" ^^^^^"'^'"^ #^ ^^ *^(#^ A^^^^^^^^^^^^^^" ^^^ #^ ^^ (^^#^ ^^^^^^^^^^^^ #^^*#^ ^^ ^^*#^ ^^ ^^ ^^ ^^ -^^^^^^^^^^ ^ ^ ^ ^^"^^^^^"^ #^ ^^ *#^#^ 4%'^^^^"^ #^ ^^ **^#^ $^^^^^^^"^ #^ ^^ ^^^#^ ^^%^^^^^"^ #^ ^^ **^#^ E ^^^^"^^^^%^ ^^^^^ #^ ^^ *^^*^ ^J^A"^^%^^"^^^^^"^ #^ ^^ *)^#^ ^J^A"^^%^%'^^^^"^ #^ ^^ **^*^ ^J^A"^^%.^^^^^^^"^ #^ ^^ *#^#^ ^^ ^^ ^^ ^^ ^^ ^3^ ^^^^^^^^^&^^^^^^^^'^^ ^ ^ ^ +^"^^^^ "^^ #^ ^^ )*^#^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^^&^^^^^^^^'^ ^ ^ ^ /^ ^^^^^^ #^ ^^ ^^##^?.H^^^ +"^^^^^^^^^ #^ ^^ ^^##^EH^^^ ,' "^'^^%^^^^^ #^ ^^ ^^##^?.H^^^ A^+^^'^^'^"^^^^^^^^5K4^^^^^^ ^"^^^^^'^'^'"^^!^ ^A^4^^^^^^^ ^^^'^'^"^^'"^^ ^^ ^^ ^^ +% ^^^^^^^^^^^^"^^^^^'^'^'"^^ !^ ^+% ^^^^^^"^ [00292] In each of the RPE-MM reported in Tables 7A to 7E each concentration within the indicated range for a component can be combined with each concentration within the indicated range of another component with the proviso that the osmolarity of the media remains within the prescribed range, as will be understood by a skilled person. [00293] Variations in the composition of Table 7A to 7E are expected to be functional when performed within the configuration of the RPE Maturation Medium of the disclosure. For example, ascorbic acid is an essential nutrient and hence it is expected to be an essential component of SC-RPE Medium. Low glucose plus pyruvate is expected to be beneficial by Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT forcing the RPE to become more dependent of aerobic energy production, which is their predominant mode of energy production in vivo and results in production of oxaloacetate. Data supporting this expectation comprise the finding that using low glucose DMEM plus pyruvate as the base medium for RPE-MM results in improved epithelial morphology compared to high glucose DMEM; while, neither DMEM nor pyruvate support RPE pigmentation. Oxaloacetate, is also expected to be possibly used as an alternative to pyruvate or in addition to pyruvate. [49]^{^}Use of pyruvate or oxalacetate is considered optional. Additional supplementation of RPE Maturation Medium with zinc, copper, or manganese ions is expected to yield slight improvements in RPE phenotype even if none of those metals are essential. Hypoxanthine is a common cell culture medium supplement that can promote cell growth it also is not found in alpha-MEM and its addition is optional. [00294] From the experiment shown in Example 18 and Figures 5 and 6, it has been determined that each of the base media additions and the base media itself contribute to RPE phenotype maturation. However, except in the cases of the base media and B27 the effects of the individual media components on their own was merely quantitative and while they each result in improved RPE phenotype, they are not by themselves essential. From a comparison of the various base media, an examination of the constituents of the N1 and B27 supplements, the role of the individual components in cell function, and a knowledge of the unique aspects of RPE metabolism and function some conclusions as to the essential media requirements for RPE maturation are expected. [00295] Accordingly in some embodiments of the RPE Maturation Medium of the disclosure, variations based on modification of the B27 components can be made. Serum albumin is a nonspecific carrier protein for hydrophobic steroids and fatty acids which is expected to be essential for RPE maturation. Galactose is both an energy source and a necessary substrate for protein glycosylation, which is not considered to be essential for RPE maturation but can be advantageous during periods of rapid growth and development. In certain embodiments, galactose is omitted during long term maintenance of RPE in culture. Inclusion of catalase, superoxide dismutase, D,L-alpha-tocopherol, D,L-alpha-tocopherol acetate, glutathione, and taurine all play a role in protecting against oxidative stress. Individually, none of these are expected to be essential, but inclusion of one or more of these is expected to improve RPE quality in culture and is expected to be essential for RPE maturation. Ethanolamine, linoleic acid, linolenic acid, biotin, and carnitine all participate in fatty acid and lipid biosynthesis and Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT are all expected to be essential but for carnitine which is expected to be conditionally essential. It is expected that concentration of some of the components related to lipid biosynthesis can be reduced by the use of lipid-rich serum albumin. [00296] Vitamin A is non-essential for RPE differentiation, pigmentation, and epithelial morphology, but its inclusion is considered to be beneficial based on an observed effect on visual cycle and complement pathway gene expression as well as subtle improvements to morphology (See Example 42 and Figures 20, 21, and 22). [00297] Hydrocortisone and possibly corticosterone are non-essential and are expected not to be necessary for culture of minimally expanded cultures of RPE, but are expected to result in improved RPE quality, especially in instances where the cells have undergone significant cell doublings during the course of culture expansion. Moreover, hydrocortisone and corticosterone are expected to be interchangeable, albeit with some changes to the concentration to account for differences in potency. Progesterone has been shown to regulate calcium signaling in RPE and to up-regulate vascular endothelial growth factor by RPE. Hence its inclusion is predicted to be beneficial for the production of quality RPE. [00298] In some embodiments of RPE-MM, the complete B27-based supplement is replaced with a modified B27-based supplement with altered concentration of specific components or missing one or more of the complete B27-based supplement components. This can be achieved by custom formulation based on the guidelines for production of these supplements in the previously cited protocols and publications relating to B27 and N21 and referencing the concentration ranges listed in Tables 7A to 7E as would be understood by a skilled person. B27-based supplements without antioxidants, vitamin A, and insulin are also available from ThermoFisher Scientific and STEMCELL Technologies. [00299] In some embodiments of the RPE maturation medium of the disclosure, variations based on the use of any one of a N1-type 100X supplements (e.g., N1, N-2, CTS-N-2, N-2- Plus, N-2-MAX, GMP-N-2-MAX, Custom N1) and any one of a B27 50X supplement equivalent or derivative, (e.g., B27, B27 Plus (B27+), B27 XenoFree (XF-B27), CTS-B27 XenoFree (CTS-B27) NeuroCult SM1, NS21, N21-MAX, GMP-N21-MAX, and Custom B27) in any combination with the most preferred ^-MEM base medium and additional maturation medium supplements (NEAA, taurine, and hydrocortisone) can be used to provide a RPE Maturation Medium (RPE-MM). The resulting different RPE-MM variants can be denoted Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT using the naming convention, RPE-MM:“N1”/“B27”, where RPE-MM denotes the use of one of the commercially available powdered ^-MEM without nucleosides formulations and the most preferred concentrations of the additional maturation medium supplements, “N1” denotes the type of N1 supplement, and “B27” denotes the type of B27 supplement at the preferred strength of 1X. For example, when using N1 supplement and B27 XenoFree, the medium would be labeled RPE-MM:N1/XF-B27. It is expected that when used at 1X strength the concentration of the components derived from commercial B27-based supplements will fall within the concentration ranges in Tables 7A to 7C. When alternative amounts of N1 or B27 supplements are used they can be noted by parenthetical amendment of the concentration to the supplement names, (e.g., RPE-MM:CTS-N-2(0.5X)/CTS-B27(1.5X)). When using strengths other than 1X of either N1 or B27 compensatory changes in the amounts of the other supplement is expected to possibly be necessary or desirable to stay within the acceptable concentration ranges due to the shared components in the two supplements. [00300] In some embodiments of the RPE Maturation Medium of the disclosure can be made based on the expectation that since the components of N1 and hydrocortisone are all contained within B27, their addition can be omitted by increasing the amount of B27 Supplement in an RPE Maturation Medium. In some embodiments of the SC-RPE maturation medium of the disclosure, the maturation medium does not comprise antibiotics. [00301] In some embodiments the formulation of RPE-MM can be advantageously described from a manufacturing standpoint as the result of a combination of an alpha-MEM medium and a B27 supplement which can be variated to provide different version of the RPE-MM of the disclosure. [00302] Additionally, embodiments where the RPE-MM are derived from alpha-MEM are known or expected to provide a high percentage of pigmented cells with a mature RPE phenotype and morphology (generally> 95%, often >99 %) Additionally, RPE-MM are derived from alpha-MEM enables or enhances SC-RPE enrichment based on pigmentation non-manual automation capable cell sorting as will be understood by a skilled person. [00303] In those embodiments a set of core components derived from the combination of alpha- MEM and B27 is reported in Table 8 below Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00304] The core components of the RPE-MM of Table 8, can be used to obtain maturation of RPE cell or be variated to provide additional embodiments of buffered balanced salt solution, essential amino acids and nonessential amino acids, vitamins, hormones, essential components for lipid metabolism, carbon energy sources, serum albumin, insulin, transferrin, selenite, and antioxidants in an effective amount to promote maturation of differentiated SC-RPE cells. As will be understood by a skilled person. [00305] In some embodiments, the core set of components of RPE-MM of Table 8 can be variated in accordance with the indications of the present disclosure to optimize performance of the RPE-MM on the RPE cells to be monitored through detection of pigmentation and/or preferably expression of marker genes of RPE maturation as will be understood by a skilled person. [00306] In those embodiments the variations can be provided in terms of variations of base component alpha-MEM and B27 inclusive of modification of the related components and additional supplementation with advantage in related manufacturability as will be understood by a skilled person. [00307] For example in preferred embodiments of RPE Maturation Medium of Table 8 obtained by combining alpha-MEM at 100% as described in Table 6, can be variated to include alpha-MEM in an amount of less than 100%, for example by using alpha-MEM at 97-100%, 94-97%, 90-94%, or 85-90% of the concentration reported in Table 6. [00308] In some embodiments, when using powdered alpha-MEM, the RPE Maturation Medium of Table 8 can be variated, by providing powdered alpha-MEM at 100-103%, 103- 106%, or 106-110% of its normal concentration reported in Table 6. [00309] In preferred embodiments, the RPE Maturation Medium of Table 8 can be variated by Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT combining alpha-MEM in any variation herein described with a B27-based supplement with vitamin A such as the B27 formulation of Table 5 is used at 1X, less preferably 0.25-0.5X, 0.5-1X, 1-1.5X, 1.5-2.X, or 2-3X. [00310] In some embodiments, the RPE Maturation Medium of Table 8 can be variated by combining alpha-MEM in any variation herein described with the B27-based supplement without vitamin A, preferably used at 1X, less preferably 0.25-0.5X, 0.5-1X, 1-1.5X, 1.5-2.X, or 2-3X. [00311] In some preferred embodiments, the RPE Maturation Medium of Table 8 can be variated by combining alpha-MEM in any variation herein described with B27 in any variation herein described and with taurine used at preferred concentrations of 2 mM, less preferably at 0-2 mM, 0.5-1 mM, 1-1.5 mM, 1.5-2 mM, 2-2.5 mM, 2.5-3 mM or 3-4 mM. [00312] The preferred embodiment the RPE Maturation Medium of Table 8 can be variated by combining alpha-MEM in any variation herein described with B27 in any variation herein described, and further with the supplemental N1-based cocktail concentration at 1X, less preferably 0-0.25X, 0.25-0.5X, 0.5-1X, 1-1.5X, 1.5-2X. In some of those embodiments the N1- based cocktail is made using the individual constituents (insulin, apo- or holo-transferrin, selenite, progesterone, and putrescine) at any of the concentrations found in N1, N2, or High Insulin N-2. These custom blended N1-based cocktails are used at 1X, less preferably 0-0.25X, 0.25-0.5X, 0.5-1X, 1-1.5X, 1.5-2.X. [00313] In some embodiments the RPE Maturation Medium of Table 8 can be variated by combining alpha-MEM in any variation herein described with B27 in any variation herein described with N1-based cocktail, missing one or more of the individual components and the included components are used at any of the concentrations found in N1, N2, or High Insulin N-2. These custom blended N1-based cocktails are used at 1X, less preferably 0-0.25X, 0.25- 0.5X, 0.5-1X, 1-1.5X, 1.5-2.X. [00314] In preferred embodiments the RPE Maturation Medium of Table 8 can be variated by combining alpha-MEM in any variation herein described with B27 in any variation herein described, and further supplementing the medium with a cocktail of NEAA (alanine, asparagine, aspartic acid, glutamic acid, glycine, proline, and serine) to increase the concentration of the non-essential amino acids in the medium by100 ^M, less preferably 0-25 Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^M, 25-50 ^M, 50-100 ^M, 100-150 ^M, 150-250 ^M, or 250-300 ^M. In some of those embodiments of the RPE Maturation Medium is supplemented with a cocktail of NEAA missing one or more of alanine, asparagine, aspartic acid, glutamic acid, glycine, proline, and serine to increase the concentration of the non-essential amino acids in the medium by100 ^M, less preferably 0-25 ^M, 25-50 ^M, 50-100 ^M, 100-150 ^M, 150-250 ^M, or 250-300 ^M. [00315] In some embodiments of the RPE Maturation Medium of Table 8 can be variated by combining alpha-MEM in any variation herein described with B27 in any variation herein described. [00316] In preferred embodiments the RPE Maturation Medium of Table 8 can be variated by combining alpha-MEM in any variation herein described with B27 in any variation herein described further supplemented with hydrocortisone at a concentration is 50 nM, less preferably 0-10 nM, 10-20 nM, 30-40 nM, 30-60 nM, 60-80 nM, or 80-100 nM, 100-150 nM, or 150-200 nM. [00317] In embodiments wherein the RPE Maturation Medium of Table 8 is variated , the outcome of reducing or increasing the concentration of alpha-MEM from its preferred strength is a reduction in the percentage of pigmented cells beginning about 1-2 weeks after becoming confluent a decrease in the level of RPE marker genes as provided elsewhere in the disclosure. In the extreme slow growth and cell death would be expected. The same is true for the expected outcomes of altering the amount of the B27. [00318] For taurine, and additional supplementation of NEAA, N1, and hydrocortisone complete removal of any one of them individually is expected to reduce the percentage of pigmented cells in the culture by as much as 50% or more depending on the seeding densities (<40,000 cell/cm²) and the passage history of the cells. When more than one of these is removed it is expected that the effect would be combinatorial, with respect to RPE phenotype, but the cells are expected to propagate, albeit at a possibly reduced rate. When all four are eliminated it is still expected that the cells will grow. When culturing cells with higher concentrations of the supplements some improvement in culture phenotype can be expected, but at the high end of the concentrations some toxicity and accompanied loss or RPE might be observed. [00319] The outcome of variating the concentrations of the RPE Maturation Medium of Table 7 using the supplements at non-preferred concentrations or amounts can most simply be Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT achieved by comparing to cells cultured in the preferred embodiment. Either by eye to qualitatively assess phenotype and pigment, by absorbance to quantitatively measure pigmentation, by qPCR using RPE marker probes, by quantification of PEDF secretion into the culture medium, or more preferably by gene expression profiling to fully assess the cell phenotype as would be understood by a skilled person upon reading of the disclosure. [00320] In preferred embodiments of the disclosure a variated RPE Maturation Medium of Table 8 can be supplemented with vitamin A via B27- or NS21-based supplements that contain retinyl acetate (B27) or retinyl acetate and retinol (NS21). When the supplements are used at their preferred strength of 1X, retinyl acetate is provided to the medium at a final concentration of about 300 nM and retinol is provided at a final concentration of 350 nM. Since vitamin A is a potent regulator of RPE gene expression, when it is desired to reduce the amount of the B27 or NS21 supplement, addition of retinyl acetate and/or retinol should be considered and when the amount of these supplements are increased use of a B27 supplement without Vitamin A to increase the B27 concentration should be considered. Since NS21 has effectively twice the concentration of vitamin A compared to B27 some changes in gene expression are expected between the two supplements. [00321] In additional preferred embodiments a variated RPE Maturation of Table 8 can comprise 1. Alpha-MEM based on the formulation in (Table 6, at 90-92%, 92-94%, 94-96%, 96- 98%, 98-100%, 100%, or up to 110%) which allows for use of liquid formulations when necessary or convenient (see e.g. cGMP embodiments) or a. In any of the available formulations b. With or without nucleosides 2. B27/NS21(based on the formulation in Table 5), with possible variations herein described a. Plus/Minus Vitamin A (B27/NS21 formulations are available commercially with and without retinol/retinyl acetate in both research and cGMP grade), b. Plus/Minus Insulin (B27/NS21 formulations are available commercially with and without insulin in both research and cGMP grade) allows for adjustment of insulin (available in both research and cGMP grade) concentration, possible alternative to using N1 supplement to provide further control over process. When RPE is matured it is expected that being able to alter the insulin Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT concentration can allow an improved control of RPE maturation and phenotype at least in some embodiments. c. Plus/Minus Antioxidants (available commercially without^DL alpha-tocopherol acetate, DL alpha-tocopherol, superoxide dismutase, catalase, and glutathione) The presence of the antioxidants provided by B27 supplement is not expected to be essential in for obtaining SC-RPE but there addition may lead to improved RPE health. Omission of antioxidants can reduce medium complexity which is beneficial in clinical manufacture and omission of antioxidants are of particular relevance with embodiments entailing oxidative stress studies, d. Plus/Minus Triiodo-I-thyronine (T3) T3 has been implicated in RPE maturation proliferation and migration and is expected to regulate genes related to RPE function. The use of T3-minus B27 is expected to allow for further control to obtain a desired RPE phenotype. [00322] The concentration of all components of the most preferred formulation of RPE-MM and allowable (general) concentration ranges of RPE-MM obtained by combination of Alpha- MEM and B27 of Table 8 variated and supplemented in accordance with the present disclosure is reported in Tables 8a to 8f. For xeno-free culture the protein components should be from the same species as the stem cell line. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00323] In each of the RPE-MM media reported in Tables 8a to 8h, each concentration within the indicated range for a core component of the RPE-MM can be combined with each concentration within the indicated range of another core component, of the composition, with the provision that the osmolarity also remains in the specified range as will be understood by a skilled person. For example when choosing a high glucose and high bicarbonate concentration, one would need to compensate by choosing a lower concentration of NaCl to maintain the osmolarity in the prescribed range. [00324] In preferred embodiments of the disclosure the RPE media are prepared using powdered medium. This is done for two main reasons. First it allows for the ability to make different media formulations comprised of varying volumes of different liquid supplements and maintain the desired concentrations of all components. Secondly, it allows for the possibility of preparing more concentrated base media, the reason for which is that cultures subject greater evaporation, such as the outer wells in a multi-well plate or cultures in incubator with poor humidity control have been observed to have increased pigmentation and higher percentages of pigmented cells. By using powder media one can make more concentrated media with higher osmolarity if desired by adding more medium powder or reducing the amount of water used to dissolve it, as will be understood by a skilled person. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00325] In some embodiments, any one of the RPE Maturation Media of the disclosure use a powder form of ^-MEM without nucleosides. [00326] In some embodiments, any one of the RPE Maturation Media of the disclosure can use a liquid form of ^-MEM. In some of those embodiments the addition of sodium bicarbonate can be omitted and glutamine is expected to possibly be needed to be added, depending on the specific medium formulation. When using liquid media some slight reduction in the quality of RPE phenotype can be expected due to dilution of the base medium components by the RPE- MM liquid supplements (RPE-MM) as will be understood by a skilled person. [00327] When using glutamine-free base medium GlutaMAX (alanyl-L-glutamine dipeptide) can be directly substituted for glutamine. [00328] The use of antibiotics or antimycotics is not essential to the function of RPE-MM. In some embodiments, where the use of antibiotics and/or antimycotics is not desirable (e.g., the production of SC-RPE for clinical use) the addition of penicillin, streptomycin, and/or normocin can be omitted. [00329] Any media formulation where the concentration of the individual components, the inorganic ions, and osmolarity falls within the ranges provided in Tables 7A to 7E and more preferably in Tables 8A to 8g are all considered to be RPE Maturation Media. For example, while some components by themselves, such as additional NEAAs, taurine and hydrocortisone, can be omitted with modest reduction in RPE quality, it is expected that reduction of the concentration of such components in combination can result in a failure of the RPE to mature. The efficacy of alternative RPE-MM formulations can be assessed by simple experimentation as would be understood by skilled person in the field and upon reading of this disclosure. Using SC-RPE produced by the methods of this disclosure, primary RPE, or SC-RPE obtained from commercial sources the various RPE-MM formulations can be evaluated for their ability to promote RPE maturation by quantitative measurement of pigment accumulation, RPE gene expression, transepithelial electrical resistance, and photoreceptor outer segment phagocytosis; as well as by qualitative assessment of RPE morphology. The results obtained with the alternative media can be compared to those obtained using the most preferred formulation of RPE-MM. [00330] For simple deviations of the formulation the medium name is modified to indicate the Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT change by simple amendment of the name with a suitable qualifier (e.g., RPE-MM, taurine- free; RPE-MM, hydrocortisone-free; RPE-MM, without additional NEAA; RPE-MM, without Pen/Strep/Normocin; RPE-MM, low calcium; RPE-MM, liquid; RPE-MM, with nucleosides; RPE-MM, without vitamin A; RPE-MM, without anti-oxidants; RPE-MM, high insulin; RPE- MM, without Vitamin E; RPE-MM, with Fe³⁺; etc.). When using alternative commercially available sources of ^-MEM with slight variations to the most preferred formulation (Table 6), the media name is amended to indicate the vendor and/or product identification. For alternative media where many changes are made, such as the case of the use of alternative base media such as DMEM:F12 mixtures or custom base media that meet the criteria of Tables 7A to 7E and more preferably in Tables 8A to 8g, they can be identified using a numbering scheme, i.e., RPE-MM1, RPE-MM2, RPE-MM3, and so on) and referencing a separate detailed description of the multiple changes. [00331] In some embodiments, any one of the RPE Maturation Media of the disclosure can be used in the SC-RPE Maturation Method of the disclosure which is a method comprising contacting differentiated SC-RPE cells with a cell culture maturation medium of the present disclosure for a time and under conditions to obtain mature stem cell-derived retinal pigment epithelial (SC-RPE) cells. [00332] In some embodiments of the SC-RPE maturation method of the disclosure coupled with the “spontaneous” differentiation method where the contacting of the cells with RPE differentiation medium begins 4 to 5 days after the initial plating, the contacting with RPE- MM is performed beginning between about 7 to 60 days after the initial plating; most preferably on about Day- 32, preferably between 21 and 37 days, less preferably between, 14 and 49 days, and least preferably between 7 and 60 days. After Day-37 there is typically no increase in mature SC-RPE yield and after Day-60 there can be a decrease in yield due to decreased isolation efficiency. Initial contact times substantially prior to 30 days are expected to be associated with decreased yields of RPE. (See Figures 1, 2, and 3). For embodiments where the contacting with RPE differentiation medium begins prior to or after 4-5 days after stem cell plating the timing and ranges of contacting with RPE-MM can be changed accordingly. [00333] In some embodiments of the SC-RPE maturation method of the disclosure coupled with the “semi-directed” differentiation method, the contacting with RPE-MM follows the same guidelines as the differentiation protocol without Activin A. In embodiments of the “semi-directed” differentiation protocol where the contacting with RPE differentiation medium Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT starts 4-5 days after first plating the stem cells and the last day of contacting with Actin A occurs on Days-15 or -16 this translates to a range of times to begin the contacting with RPE- MM between about 15 and 60 days after the initial plating, with the most preferable time being about Day-32, preferably between 21 and 37 days, and less preferably between 15 and 49 days, and least preferably between 15 and 60 days. For contact times beginning after Day-37 there is typically no increase in mature SC-RPE yield and after Day-60 there can be a decrease in yield due to decreased isolation efficiency. Initial contact times substantially prior to 30 days are expected to be associated with decreased yields of RPE. For embodiments where the contacting with RPE differentiation medium begins prior to or after 4-5 days after stem cell plating the timing and ranges of contacting with RPE-MM can be changed accordingly. For embodiments where the contacting with Activin A ends before Day-14 or continues after Day- 15 after stem cell plating the same guidelines for the initiation of contacting with RPE-MM apply with the proviso that the contacting cannot start until the after the last day of contacting with Activin A in RPE differentiation medium. (See Figures 1 and 2). [00334] In a preferred embodiment the length of SC-RPE maturation for a given cell line:medium:substrate combination is determined empirically by monitoring the extent of pigmented cell accumulation and PEDF secretion as will be understood by a skilled person. [00335] In embodiments of the SC-RPE maturation method of the disclosure coupled with the “spontaneous” or “semi-directed” differentiation method, the length of the period of contacting with RPE-MM is best determined empirically by regular assessment of the extent of pigment accumulation. Alternatively, it is expected that the extent of RPE maturation can be determined by measuring the amount pigment epithelium derived factor (PEDF) in the cell culture medium at the time of the regular culture feedings. PEDF is a highly abundant mature RPE secreted protein product of the SERPINF1 gene. Detection of pigmentation and PEDF secretion can be performed with methods known to a skilled person. In particular, pigmentation accumulation can most readily be assessed by collecting photographs of the cultures on a white background using incident light illumination or by collecting images using a common document scanner as function of time in culture and visually comparing the resulting images to determine when the extent of pigmentation in the culture reaches a plateau. Additional methods such as automated image analysis of culture images, measurement of light absorption to quantify pigmentation, as well as use AI based high resolution image analysis to quantify the degree of pigment accumulation are also envisioned as will be understood by a skilled person. The amount of Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT PEDF secreted into the medium over a prescribed period of time as a function of the RPE maturation time course can be determined using standard analytical methods, such as using commercially available enzyme-linked immunosorbent assays (ELISA). When the amount of PEDF secretion per unit time plateaus minimal increase in SC-RPE yield is expected with increased RPE maturation time. [00336] The optimal length of contact time in RPE-MM to result in maximal yields of SC-RPE is generally in the range of 30-60 days. Shorter times are expected to result in correspondingly reduced yields of SC-RPE and contact times greater than 90 days can result in decreased yields due to increasing difficulty in the physical isolation of the SC-RPE due to tight junction formation. Shorter contact times often being optimal for media of the RPE-DM2 family and protocols employing Activin-directed differentiation. Longer contact times are often required for media of the RPE-DM1 family. In addition, cell lines that appear to have different rates of RPE maturation have been observed. In several embodiments, with different cell lines, contacting with RPE Maturation Medium beginning at about Day-30 after stem cell plating and ending between Days-60 and -90 from the start of the SC-RPE differentiation method of the disclosure gave optimal results as defined as the time required for the percentage of pigmented cells to plateau. [00337] In some embodiments of the SC-RPE maturation method of the disclosure, the frequency of feeding during the contacting can be based on the extent of medium acidification. With normal volumes of culture medium (about 3 mL/10 cm²) this is typically at least every 3 days for the first week or two after the medium switch. Thereafter feeding twice a week (every 3-4 days) is possible. [00338] In some embodiments of the SC-RPE maturation method of the disclosure, the contacting is performed on differentiated SC-RPE differentiated cells obtained with one of the SC-RPE differentiation methods of the disclosure (see e.g. protocols of Examples 12, 13, 16, 19, 20, 21, and 24). [00339] In particular, in some embodiments of the SC-RPE maturation method of the disclosure, following completion of the SC-RPE differentiation method of the disclosure the differentiation media are replaced with one of the RPE Maturation Medium herein described. In particular the differentiation media can be replaced with one of the RPE Maturation media prepared combining any one of a N1-type 100X supplements and any one of a B27 50X supplement equivalent or derivative, in any combination with the most preferred ^-MEM base Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT medium and additional maturation medium supplements. such as the media specifically described in [00299]. [00340] In some of those embodiments the replacement of the SC-RPE differentiation medium of the disclosure with the RPE Maturation Medium of the disclosure can be performed after about 30 days from the beginning of the SC-RPE differentiation method. The selection of 30 days as being the default timing of the beginning of the SC-RPE maturation method was based on the empirical finding that this is the general time when the first hints of pigmentation are often observed in differentiation media that have the capacity to support some degree of RPE maturation on their own (i.e., X-VIVO™ 10-based RPE-DM2). Interestingly, switching directly to RPE-MM from stem cell medium results in the emergence of numerous foci of pigmentation in the first few weeks of culture, but the foci fail to enlarge with time; suggesting that some presumptive RPE form quite quickly in the process, but RPE-MM inhibits the differentiation of additional RPE precursors when applied at these early stages of RPE differentiation. In the case of some cell line/substrate/media combinations hints of RPE pigmentation prior to 30 days in RPE-differentiation media have also been noted. It follows that in these cases earlier beginning contact times with RPE-MM is expected to be possible and would thus allow for shortening of the method. Furthermore, the observation that major improvements in yield are not seen after Day-30 would imply the earlier times are likely possible. That said, with respect to final RPE yield there is no downside to waiting to day-30 to initiate the beginning of the RPE other than the increase in time and costs. In addition to using the first onset of pigmentation as an indicator of the time to initiate contact with RPE- MM, it is expected that similar indication could be obtained by monitoring the onset and extent of expression RPE precursor marker gene, such as MITF, LHX2, and OTX2 and chose the timing of timing of the initiation or RPE-MM contact accordingly. [00341] In some embodiments, in addition to the actual combinations of media and substrates, another dominant determinant of SC-RPE yield appears to be density, colony size, and extent of confluence of the stem cell colonies at the time of switching from stem cell medium to the RPE determination medium based on the observed patterns SC-RPE differentiation, as will be understood by a skilled person. Preferred conditions vary depending on the specific cell line:medium:substrate combination as will be understood by a skilled person upon reading of the present disclosure. Changes or adjustments to the protocol, as would be understood by skilled person, that affect the global and local cell density are expected to alter SC-RPE Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT differentiation efficiency. For example, cell line:media:substrate combinations that result in substantial pigmentation in regions known to often have lower initial cell densities or on the side wall of the culture dish or flask are expected to possibly benefit from lower density plating or switching to the differentiation medium at earlier times, whereas those that have more prominent pigmentation in the center of the well or outer third of the well can benefit from higher density plating or initiating the switch to differentiation medium at a later time. [00342] In some embodiments of the SC-RPE maturation method of the disclosure, the cells will spontaneously detach as sheets depending on cell line:medium:substrate combinations (For example, see Example 13 and Figure 1, culture H9:X-VIVO™ /XF-B27+ActivinA:PC- VTN for one of several cases of a partially detached sheet). In those embodiments, while it is possible to continue to maintain the cultures as floating sheets the yield of RPE is often poor, even when there is a significant amount of presumptive RPE. In this case it is possible to rescue the culture at the time of detachment by dissociating the sheets using proteases (e.g., trypsin, TrypLE™, Accutase™, or Accumax™) as would be understood by a skilled person, and culturing the resulting cell suspensions on a substrate of choice (e.g., laminin or vitronectin, Matrigel, etc.) using RPE Maturation Medium. In some embodiments some cell lines give rise to 2-dimensional tissue like sheets of cells after RPE maturation that are highly resistant to dissociation and hence reduced the yield of SC-RPE at the time of enrichment. In this case the protocol can be adjusted by passaging the cells at the time of switching to RPE-MM or preferably in the days following the switch. Alternatively, it is possible to routinely proactively passage the cells at the time of the switch to RPE-MM or in the days following the switch with any or all cell lines and embodiments of the RPE differentiation method. Improved efficiencies of SC-RPE derivation have been associated with increased times of contact with RPE-MM prior to passage, supporting the conclusion that contact with RPE-MM prior to differentiation culture dissociation contributes to the generation RPE-progenitors or presumptive RPE as well as RPE maturation. While routinely proactively passage the cells can allow for the use of differentiation medium:substrate combinations where substrate detachment or lower than desired differentiation yield is an issue, it does come at the expenses of increased materials costs, an increase in the time required for SC-RPE derivation, and increases the amount of material that must be processed for enrichment of the SC-RPE (see e.g. Example 17 and Figure 4). [00343] In some embodiments of the present disclosure, SC-RPE enrichment methods and Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT systems are described to obtain an enriched population of mature SC-RPE cells, based on detecting the presence of markers CD57, the presence of CD104, and/or the absence of marker CD49b (see e.g., Example 26, 27, 28, and 29). [00344] In some embodiments of the of the SC-RPE enrichment methods and systems a population of mature SC-RPE cells, is obtained based on detecting the presence of markers CD57, the presence of CD104, and/or the absence of marker CD49b and the presence of pigmentation (see e.g., Example 26, 27, 28, and 29). [00345] The terms “detect” or “detection” as used herein indicates the determination of the existence, presence or fact of a target in a limited portion of space, including but not limited to a sample, a reaction mixture, a molecular complex and a substrate. The “detect” or “detection” as used herein can comprise determination of chemical and/or biological properties of the target, including but not limited to ability to interact, and in particular bind, other compounds, ability to activate another compound and additional properties identifiable by a skilled person upon reading of the present disclosure. The detection can be quantitative or qualitative. A detection is “qualitative” when it refers, relates to, or involves identification of a quality or kind of the target or signal in terms of relative abundance to another target or signal, which is not quantified, such as presence or absence. A detection is “quantitative” when it refers, relates to, or involves the measurement of quantity or amount of the target or signal (also referred as quantitation), which includes but is not limited to any analysis designed to determine the amounts or proportions of the target or signal. A quantitative detection in the sense of the disclosure comprises detection performed semi-quantitatively, such as, above/below a certain amount of nucleic acid molecules as will be understood by a skilled person. [00346] The wording “CD57” (Cluster of Differentiation 57) as used herein indicates an antigen also known as HNK1 (human natural killer-1) or LEU7. It is expressed as a carbohydrate epitope that contains a sulfoglucuronyl residue in several adhesion molecules of the nervous system. The B3GAT1 gene codes for an enzyme that plays a key role in a glucuronyl transfer reaction during the biosynthesis of the carbohydrate CD57 epitope. These enzymes exhibit strict acceptor specificity, recognizing nonreducing terminal sugars and their anomeric linkages. [52]. [00347] The wording “CD104” (Cluster of Differentiation 104), as used herein indicates the gene product of integrin beta 4 (ITGB4). When associated with integrin alpha 6, the two Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT integrin subunits combine to act as laminin receptor. [00348] The wording “CD49b” (Cluster of Differentiation 49b), as used herein indicates the protein product of the integrin alpha 2 gene (ITGA2). When associated with integrin beta 1 (ITGB1), the two integrin subunits combine to form a functional receptor that binds to laminin, collagen, thrombospondin, E-cadherin, and tenascin. [00349] In some embodiments of the of the SC-RPE methods an enriched population of mature SC-RPE cells is obtained based on the presence of pigmentation alone as determined by the absorption of light by melanin and/or the scatter of light by melanosomes [00350] The word “pigmentation” as used herein indicates a colored material that is completely or nearly insoluble in water which is found within cells. In particular, pigmentation of a mature SC-RPE generally indicates the material within the pigment granules of SC-RPE cells which comprise melanin as will be understood by a skilled person. These pigmented granular cell organelles are also referred to as melanosomes. In immature SC-RPE, some diffuse and slight darkening of cells can often be observed in the absence of visibly discernable pigment granules using light microscopy. Melanin has a broad light absorption spectral profile with peak absorption at 320 nm that decreases by 50% by about 400 nm and then decreases to near zero by about 750 nm. Because of the broad spectral absorbance in the visual light spectrum pigmented cells appear golden brown to black under white light illumination depending on its concentration. In cells melanin can readily be detected and quantified by measuring light absorption in the range of 350-650 nm, a spectral range where few other cellular molecules absorb light. Best signal to noise ratios will typically be achieved using light wavelengths in 350-550 nm range. In addition, melanosomes can also be detected based on light scattering. When light is shined on a cell some of the light will be deflected or scattered at narrow angles (forward scatter, FSC), some will be scattered to the side (side scatter, SSC), and some will be reflected or scattered backwards (back scatter, BSC). In the case of cells the amount of FSC is mostly indicative of cell size whereas the amount of SSC and BSC are indicative of the amount of intracellular granularity (melanosomes, mitochondria, lysosomes, vesicles, vacuoles, etc.) Due to the relationship between particle size and scatter, light scatter analysis of cells is often done with laser light wavelengths from 405 to 488 nm, but wavelengths outside of this range can be useful depending on cell and particle size. While side and back light scatter are not exclusive to melanosomes, well pigmented cells can readily be distinguished from most, but Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT not all, non-pigmented cells of similar size based on their relatively high SSC:FSC or BSC:FSC ratios due to highly effective light scatter by melanosomes and the amount of melanosomes per SC-RPE. [00351] Melanin has a broad light absorption profile and pigmented cells can be readily detected and distinguished from non-pigmented cells using light wavelengths in the range of 350-550 nm light since non-pigmented cells have minimal light absorbance in this range. In some embodiments of the SC-RPE enrichment method of the disclosure pigmented cells are detected using a cell sorter configured with a 488 nm light absorption detector. Guidance for determining the appropriate sorting thresholds (or gates) can be obtained by determining the light absorption values of known non-pigmented cells, such as stem cells, fibroblasts, human embryonic kidney cells, or non-RPE in the culture as identified based on their CD49b, CD104, and/or CD57 staining profile. Alternatively, as would be understood by a person skilled in the field of cell sorting, in many cases it is possible to identify pigmented cells using scatter or contour plots where the light absorption values are plotted versus one of the light scatter values (forward, side, or back) and the pigment cells can identified as clear population of cells with the highest absorbance values and set gates to select that population. For best results, the gate selection should be confirmed by sorting a small number of cells and determining the percentage of observable pigment cells by microscopy and cell counting. Depending on the results the gates can be adjusted until the desired purity and yield is obtained. [00352] The scatter of light by melanosomes is a function of melanosome size and the light wavelength. In general, laser wavelengths suitable for the detection of cells and intracellular particles such as melanosomes using light scatter detection are in the range of 405-488 nm, with 488 nm being the most commonly used. In some embodiments of the SC-RPE enrichment method of the disclosure pigmented cells are identified using a cell sorter configured with 488 nm laser light side scatter and forward scatter detectors and the values for both parameters for each cell is plotted using a scatter, contour, or ratio plot. Forward scatter signal intensity is proportional to cell size and side scatter is proportional to the amount of intracellular particles, like mitochondria, lysosomes, and melanosomes. Typically, a clear group of cells (comprised largely of pigmented cells) with relatively high side scatter to forward scatter signal intensity ratios compared to a group of cells (comprised largely of non-pigmented and minimally pigmented cells) with a lower side scatter to forward scatter ratio can readily be identified. As would be understood by a person skilled in cell sorting, appropriate side and forward scatter Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT gates can be set to effectively select the group of cells with high side versus forward scatter ratios. Further, guidance for determining the appropriate sorting thresholds (or gates) can be obtained by identifying non-RPE in the culture based on their CD49b, CD104, and/or CD57 staining profile. For best results, the gate selection should be confirmed by sorting a small number of cells and determining the percentage of observable pigment cells by microscopy and cell counting. Depending on the results the gates can be adjusted until the desired purity and yield is obtained. [00353] The SC-RPE enrichment method entails the use of a device to separate SC-RPE from non-RPE cells herein also indicated as cell sorters. A cell sorter in the sense of the disclosure has a detector capable of measuring the parameter of interest (fluorescence, light scatter, and/or light absorbance) and has the capability to then separate the desired cell from the unwanted cells. Typically flow cytometer-based instruments are used, but imaging-based devices, such as laser microdissection instruments, can be used to enrich SC-RPE as well. In preferred embodiments of the SC-RPE enrichment method of the disclosure the sorting device is a fluorescence activated cell sorter (FACS). For sorting based on marker staining of all three markers the FACS should have light scatter detectors capable of identifying single cells (forward and side and/or back scatter) and it must be capable of 3-color sorting using fluorescence detectors. For sorting based light absorption by melanin, the sorter must have a detector capable of measuring light absorption at wavelength within the 350-650 nm range and preferably in the range of 350-550 nm, more preferably 405-550 nm, even more preferably 405-488 nm, and most preferably 488 nm. Typically the light source is a laser, but light emitting diodes, band pass filtered light, or spectrometer-type light sources and detectors are possible. In addition, it is preferred that the light absorption enabled sorter is also capable of detecting single cells based on light scatter. For sorting based on the light scattering properties of melanosomes, the sorter must have a detector to measure cell size (typically forward light scatter) and a side and/or back light scatter detector to assess the degree of cell granularity (i.e. melanosome content). The scatter detectors use laser light in the range of 405-650 nm (more preferably 405-550 nm, even more preferably 405-488 nm, and most preferably 488 nm). For sorting based on all parameters it is preferred that a sorter capable of all detection mode is used, but a combination of multiple sorters can be used. For SC-RPE enrichment for the purpose of preparing material for clinical use it is desirable the sorter uses a closed fluidics system and preferably a uses a disposable, self-contained sorting chamber or cartridge An exemplary cell Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT sorter in the sense of the disclosure is described in Example 44 and schematically shown in Figure 25. [00354] In some embodiments of the SC-RPE enrichment method of the disclosure pigmented cells are enriched using a FACS configured with 405-650 nm (more preferably 405-550, even more preferably 405-488 nm, and most preferably 488 nm) laser light side scatter and forward scatter detectors and the values for both parameters for each cell is plotted using a dot, contour, or ratio plot. Typically, a clear group of cells with relatively high side scatter to forward scatter signal intensity ratios (comprised largely of pigmented cells) can readily be distinguished from a group of cells with a lower side scatter to forward scatter ratio (comprised largely of non- pigmented and minimally pigmented cells). As would be understood by a person skilled in cell sorting, appropriate side and forward scatter gates can be set to effectively select the group of cells with high side versus forward scatter ratios. Further, guidance for determining the appropriate sorting thresholds (or gates) can be obtained by identifying non-RPE in the culture based on their CD49b, CD104, and/or CD57 staining profile. For best results, the gate selection should be confirmed by sorting a small number of cells and determining the percentage of observable pigment cells by microscopy and cell counting. Depending on the results the gates can be adjusted until the desired purity and yield is obtained. Alternatively, on suitably configure cell sorters images of the sorted cells can be collected in real-time and the pigmented cell purity can be calculated (also in real-time) using machine learning or artificial intelligence based image analysis. Additional cell sorting methods and devices can be identified by a skilled person. [00355] In some embodiments of the SC-RPE enrichment method of the disclosure pigmented cells are enriched using a FACS most configured most preferably with a 488 nm light absorption detector (preferably 405-488 nm, less preferably 405-550 nm, and least preferably 350-650 nm) and pigmented SC-RPE cells are identified based on their relatively light absorption compared to non-pigmented cells. Guidance for determining the appropriate sorting thresholds (or gates) can be obtained by determining the light absorption values of known non- pigmented cells, such as stem cells, fibroblasts, human embryonic kidney cells, or non-RPE in the culture as identified based on their CD49b, CD104, and/or CD57 staining profile. Alternatively, as would be understood by a person skilled in the field of cell sorting, in many cases it is possible to identify pigmented cells using scatter or contour plots where the light absorption values are plotted versus one of the light scatter values (forward, side, or back) and Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT the pigment cells are then identified as a clear population of cells with similar light scatter to each other and the highest absorbance values compared to the other cells, Sorting gates can then be to select pigmented SC-RPE population. Further guidance for gate selection can be got based on known non-pigmented cells as previously described. For best results, the gate selection should be confirmed by sorting a small number of cells and determining the percentage of observable pigment cells by microscopy and cell counting. Depending on the results the gates can be adjusted until the desired purity and yield is obtained. Alternatively, on suitably configure cell sorters images of the sorted cells can be collected in real-time and the pigmented cell purity can be calculated (also in real-time) using machine learning or artificial intelligence based image analysis. Additional cell sorting methods and devices can be identified by a skilled person. [00356] In some embodiments of the SC-RPE enrichment method of the disclosure SC-RPE are cells are enriched based on their CD49b, CD104, and/or CD57 staining profile using a FACS configured for the detection pigmentation by light absorption and/or light scatter. After identifying single cells based on light scatter, as would be understood by a skilled person, CD49b negative, CD104 positive and/or CD57 positive cells are collected. Guidance for selecting gates can be gotten using negative isotype control antibodies and by comparing the staining profiles of the pigmented and non-pigmented populations as well as by examining dot plots of all possible pairwise marker combination for both the pigmented and non-pigmented cell populations, as would be understood by skilled person. [00357] It has been found that mature pigmented SC-RPE cells have a specific pattern of CD57, CD104 and CD49b marker expression and that extracellular epitopes of these markers can be recognized by specific probes which survive the process of cell dissociation required for enrichment. In particular they are marked by the presence of CD57 and CD104, and the absence of CD49b. This is consistent with published gene expression profiling of differentiated and mesenchymal RPE where it has been shown that the expression of B3GAT1 (CD57) and ITGB4 CD104) increase as RPE obtain a mature phenotype and that there is minimal expression of ITGA2 (CD49b) in differentiated RPE and elevated expression in mesenchymal RPE [21]. The inventors have found that the proteins expressed by B3GAT1 (CD57), ITGB4 (CD104), and ITGA2 (CD49b) are expressed in sufficient levels for detection, do survive the extensive protease digestion required to generate single cells, and therefore can be used as marker. It has also been found that some nonpigmented cells, which have the same marker Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT profile, in most cases can give rise to mature pigmented RPE upon subsequent culture. Cells in the enriched immature RPE population that fail to pigment upon further culture can be cells in the early stage of dedifferentiation to being a mesenchymal RPE, non-RPE with slightly overlapping marker staining profile or aberrant SC-RPE that failed to differentiate properly. [00358] Accordingly, in some embodiments detection of mature SC-RPE cells can be performed as part of an SC-RPE enrichment method of the disclosure to enrich for a population of mature SC-RPE by selecting pigment cells with an RPE marker profile and to enrich for immature SC-RPE cells by selecting nonpigmented cells with a RPE-marker profile using non- manual methods that are less prone to user bias and skill and that can be readily automated. Enrichment based on RPE marker profile alone will result in the enrichment of a mixed population of mature and immature SC-RPE, many or most of which will mature on further culture. Enriched populations including immature SC-RPE will most always have lower purity but can be useful for some studies and can be cultured and resorted including pigmentation as a selection parameter to obtain more mature SC-RPE cells than in case where the SC-RPE immature cells comprising with an RPE marker profile were simply discarded. [00359] The method comprises providing differentiated stem cell-derived retinal pigment epithelial (SC-RPE) cells and staining the differentiated SC-RPE cells with a probe specific for a marker selected from CD57, CD104, and/or CD49b to obtain stained differentiated SC-RPE cells stained for CD57 and/or CD104, and not stained for CD49b markers (see e.g. Examples 27 and 28). [00360] The SC-RPE enrichment method of the disclosure further comprises sorting the stained SC-RPE cells to select the differentiated SC-RPE cells stained for CD57 and/or CD104 markers, and/or to discard cells stained for the CD49b marker, to obtain a population of SC- RPE cells enriched in mature SC-RPE cells. [00361] In preferred embodiments of the SC-RPE enrichment method, the cells are sorted based on their marker staining profile and the presence of pigment relative to known or identifiable nonpigmented cells as a skilled person would understand upon reading of the disclosure. [00362] In some embodiments of the SC enrichment, the cells can be isolated based on the presence of pigment alone. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00363] The wording “staining” as used herein in connection with cell detection indicates a series of techniques that can be used to improve or enable detection such as optical detection of the cells as will be understood by a skilled person. In particular, the word staining as used herein indicates techniques that can be used to enable or improve detection of living cells; in particular living SC-RPE cells following contacting with a probe specific for any one of the CD57, CD104, and/or CD49b markers as will be understood by a skilled person. [00364] The term “probe” as described herein indicates a molecule capable of specifically detecting a target molecule such as one of the markers herein described. The wording “specific”, “specifically” or “specificity” as used herein with reference to the binding of a first molecule to second molecule refers to the recognition, contact and formation of a stable complex between the first molecule and the second molecule, with substantially less to no recognition, contact and formation of a stable complex between each of the first molecule and with other molecules that can be present. Exemplary specific bindings are antibody-antigen interaction, cellular receptor-ligand interactions, polynucleotide hybridization, enzyme substrate interactions, and additional interactions identifiable by a skilled person. [00365] In some embodiments, staining can be performed with probes specific for any one of the CD57, CD104, and/or CD49b such as antibodies, and or aptamers with the further requirement that the probe must recognize an epitope in the markers that survives the procedure to obtain single cells such as protease digestion during the process of producing single cells after SC-RPE differentiation and maturation, or any other culture or tissue containing SC-RPE or RPE. (e.g. Examples 27 and 28). [00366] Depending on the selected mode of cell isolation, the probe can be coupled to any molecule that allows for sorting of the cells with appropriate staining pattern such as a fluorescent molecule, a paramagnetic bead or particle, a solid support, or a ligand or receptor. [00367] In some embodiments the probe specific for CD57, CD104, and CD49b comprise anti- CD104-PE (Clone: REA236, Miltenyi Biotec), anti-CD57-PE/Vio770 (Clone: REA739, Miltenyi Biotec) and anti-CD49b-APC (Clone: REA188, Miltenyi Biotec) antibodies. [00368] The wording “sorting” as used herein in connection with cells, indicates the process through which a particular cell type is separated from others contained in a sample based on its physical or biological properties, such as size, morphological parameters, viability and both Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT extracellular and intracellular protein expression [53]. [00369] Examples of methods to perform cell sorting in the sense of the disclosure comprise Fluorescence Activated Cell Sorting (FACS), magnetic cell sorting, or solid phase affinity selection as will be understood by a skilled person [53]. [00370] In some embodiments, the sorting can be performed based on a relative high side scatter (SSC) to forward scatter (FSC) ratio to identify melanosome containing pigmented cells and negative CD49b expression, positive CD104, and positive CD57 expression (see e.g., Example 29). [00371] In some embodiments, light absorbance-based sorting and marker-based enrichment of SC-RPE cells can be performed by detecting light absorbance from a target cell culture and selecting cells of the target cell culture having absorbance of light between 350-650 nm (more preferably between 405-550 nm, even more preferably 405-488, and most preferrable 488 nm) as compared to known nonpigmented negative control cells or identifiable nonpigmented cells in the population and by negative CD49b expression, positive CD104, and/or positive CD57 expression. In some embodiments, the method can further comprise calibrating a light absorption threshold setting by sorting a small number of test cells, quantifying the percentage of pigmented cells in the sorted population, and then adjusting the threshold setting as necessary to achieve a desired yield and pigmented cells purity. Alternatively, on a suitably configured cell sorter, images of the sorted cells can be collected in real-time and the pigmented cell purity can be calculated (also in real-time) using machine learning or artificial intelligence based image analysis as will be understood by a skilled person. Additional cell sorting methods and devices can be identified by a skilled person. [00372] In some embodiments, the sorting can be performed by selecting a population with a relative high absorbance of light within the range of 350-650 nm and with a relative high side scatter (SSC) to forward scatter (FSC) ratio, as compared to known nonpigmented cells, to identify pigmented cells and with negative CD49b expression, positive CD104, and/or positive CD57 expression. [00373] In some embodiments, the sorting can be performed based on a relative high side scatter (SSC) to forward scatter (FSC) ratio, as compared to known nonpigmented cells, to identify melanosome containing pigmented cells. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00374] In some embodiments, the sorting can be performed by selecting a population with a relative high absorbance of light in the range of 350-650 nm, as compared to known nonpigmented cells, to identify pigmented cells. [00375] In some embodiments, the sorting can be performed based by selecting a population with a relative high absorbance of light in the range of 350-650 nm, as compared to known nonpigmented cells, to identify pigmented cells using microfluidics, microchip-based FACS. [00376] Methods to perform cell sorting in the sense of the disclosure further comprise affinity selection of SC-RPE cells. For example, a suspension of RPE cells can be first stained with an anti-CD49b antibody coupled to a paramagnetic particle and CD49b positive cells can be removed by magnetic selection. The resulting CD49b negative cells can then be stained with anti-CD104 antibodies coupled to a paramagnetic particle and with biotin conjugated anti- CD57 antibody. CD104 positive cells can then be captured by magnetic selection and after release the CD57 positive cells can be collected using a streptavidin coupled solid support. Other similar strategies are possible as would be understood by a person skilled in the field. [00377] The homogeneous cell population obtained after sorting, which is optionally subjected to post-sorting recovery period (see e.g. Example 30), can be used for a variety of applications including research, diagnosis, and therapy as will be understood by a skilled person [53]. [00378] In embodiments of the enrichment method of the disclosure positive selection can be performed based on the cell surface markers CD57 and/or CD104, which have been determined to be expressed on mature SC-RPE, and/or negative selection based on the absence of CD49b, which has been determined to be expressed on RPE that have undergone epithelial-to- mesenchymal transition and presumably by some non-SC-RPE. [00379] In embodiments of the enrichment method of the disclosure, each of the positive detection of any one cell surface markers CD57 and/or CD104, and negative detection of CD49b can be used to select mature SC-RPE cells. Preferably selection is performed following multiple detection of at least two of CD57, CD104, and CD49b. More preferably detection of all three markers is performed. [00380] Accordingly, in some embodiments the staining is performed to label cells presenting CD57 and CD104 and the selected cells are SC-RPE cells positive for at least one and preferably both of CD57 and CD104. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00381] In some embodiments the staining is performed to label cells presenting CD57 and CD49b and the selected cells are SC-RPE cells positive for CD57, SC-RPE cells negative for CD49b and preferably SC-RPE cells each positive for CD57 and negative for and CD49b. [00382] In some embodiments the staining is performed to label cells presenting CD104 and CD49b and the selected cells are SC-RPE cells positive for CD104, SC-RPE cells negative for CD49b and preferably SC-RPE cells each positive for CD104 and negative for and CD49b. [00383] In some embodiments the staining is performed to label cells presenting CD57, CD104 and CD49b and the selected cells are SC-RPE cells positive for CD57, SC-RPE cells positive for CD104, and/or SC-RPE cells negative for CD49b. In preferred embodiments, the selected cells are SC-RPE cells are each positive for at least one and more preferably both of CD57 and CD104 or positive for CD104 and negative for and CD49b, or positive for CD57 and negative for CD49b. In most preferred embodiments, the selected cells are SC-RPE cells are each positive for both of CD57 and CD104 and negative for CD49b. [00384] In embodiments of the enrichment method and system of the present disclosure while it is possible to achieve a large degree of enrichment based on a single cell surface marker alone, better levels of enrichment are achieved using multiple markers as will be understood by a skilled person upon reading of the present disclosure. [00385] In addition, the enrichment method of the present disclosure can further comprise detecting RPE pigmentation of the SC-RPE cells based on light scattering by melanosome and/or or absorbance of light by melanosomes and melanin and selecting the SC-RPE cells having pigmentation associated with all or a subset of the mature SC-RPE markers (see e.g. Example 29). [00386] In some embodiments, sorting of cells can be performed by FACS based on multiple criteria. In those embodiments, the choice of fluorescent labels will be dependent on the specifications of the cell sorter, but as much as possible dyes with high quantum yield and fluorescent intensity should be used to best discriminate labeled from unlabeled cells. [00387] When sorting by FACS based on multiple criteria (more than one of pigmentation, CD57, CD104 and CD49b) the order of gating or gating protocol is irrelevant. However, the proper selection of the gate locations or windows can be aided by comparing the fluorescent labeling patterns of the pigmented and nonpigmented cell populations using all possible Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT pairwise marker comparisons (i.e., CD57 vs. CD104, CD57 vs CD49b, and CD104 vs CD49b) to achieve the best level of purity and yield. [00388] In some embodiments of the SC-RPE enrichment method and related system of the disclosure, the sorting is preferably based on detection of CD57, CD104 and CD49b as well as based on the detection of pigmentation of the SC-RPE cells to increase the final purity of mature SC-RPE cells obtained by the method. [00389] In those preferred embodiments, the sorting of SC-RPE cells in the enrichment method of the disclosure, can further comprise sorting SC-RPE cells having a detectable pigmentation to further select mature SC-RPE cells based on presence or absence of pigmentation. In those embodiments, detecting SC-RPE cells having a detectable pigmentation can be performed after marker staining and the pigment-based sorting can be done in conjunction with sorting based on presence or absence of markers CD57, CD104 and/or CD49b. Alternatively, the pigmentation-based sorting can be done prior to staining. The resulting enriched population of pigmented cells are then stained follow by a round of sorting based on the presence or absence of markers CD57, CD104 and/or CD49b in conjunction with or without a second round of pigment sorting. [00390] In those preferred embodiments, the enrichment method and systems of the disclosure can be performed by a combination of any method to perform marker-based selection with a cell sorting method based on absorbance of light by melanin and/or side-scatter of light by melanosomes to discriminate pigmented RPE from non-pigmented cells as will be understood by a skilled person upon reading of the present disclosure. For detecting absorbance wavelengths from 350 to 650 nm are possible, but the best signal to noise ratios are expected in the range from at least 400 nm, preferably from 405 to 550 nm. The same general guidance regarding wavelength is true for use for detection of melanosome by determination of side- scatter (see e.g., Example 29). [00391] Accordingly in some embodiments, the detection of pigmented mature SC-RPE can be performed by measuring light absorbance readings at wavelength of at least 400 nm, preferably between 480-600 nm to quantitively detect pigmented culture area. ^ [00392] In some embodiments, detection of pigmented mature SC-RPE can be performed by by light scattering, preferably by detecting relative light side scatter to forward scatter ratio Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT following directing a light beam towards the cell culture are. [00393] In some embodiments of the SC-RPE enrichment method and related system of the disclosure, sorting can be performed using a subset of markers to yield satisfactory results in cases of cell lines that have high differentiation efficiencies, where yield is a priority over purity, or where it is desirable to reduce cost and complexity as will be understood by a skilled person upon reading of the disclosure. [00394] In some of the embodiments where the use antibodies or probes is not desired and the cells are enriched based on physical properties and the presence of melanin or melanosomes alone, improvements in purity can be expected by staining a portion of the total population for one or more of the markers CD49b, CD104, and CD57 and use the resulting staining profile to aid in the selection of the optimal scatter and pigment sorting gates. The resulting gates can then be used to sort the reserved unstained portion of the cell population. A similar strategy can be used when only a subset of marker probes is used for the actual sorting process. [00395] In some embodiments sorting using multiple sort modalities can be combined to result in shortened sorting processing times. As an example, cells can be stained with an anti-CD57- paramagnetic microbead (Miltenyi BioTec catalog number 130-092-073), anti-CD104-PE, and anti-CD49b/APC. After capture and release of the CD57 positive cells by magnetic selection, the resulting CD57 positive cells can be further enriched by FACS based on, positive CD104 staining, negative CD49b staining, and/or pigmentation. Similarly, unwanted CD49b positive cells can be removed first by magnetic bead separation and the remaining cells can be sorted by FACS based on CD57, CD104, and/or pigmentation. Furthermore, when selection of CD57 or CD104 cells is carried out by methods such as paramagnetic bead selection, the cells can be dual stained with fluorescent labeled antibodies and paramagnetic beads directed at the same antigen. In this way, a bulk separation of CD57 or CD104 positive cells is possible while still allowing for isolation based quantitative levels of expression of all three markers and pigmentation. This can be especially advantageous with populations of cells with heterogenous expression of the RPE specific markers and allows for both decreased sorting times and enrichment of an SC-RPE population with enhanced homogeneity and purity. [00396] In addition, or in the alternative, in some preferred embodiments, the enrichment method and systems of the disclosure, can be performed in combination with detection and separation of pigmented cells based on the light scattering or absorbance property of Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT melanosomes or melanin. [00397] In particular, in some embodiments, SC-RPE with the desired phenotype can then then isolated using fluorescence activated cell sorting (FACS, see e.g., Example 29). [00398] In addition, in some embodiments the enrichment method and systems of the disclosure can be performed in combination with staining with viable or dead cell dyes or reagents to allow for the selection of viable SC-RPE. [00399] In some embodiments of the SC-RPE enrichment method and related system of the disclosure, additional consideration for selection of the set of markers and/or pigmentation to be used for the staining and sorting steps, comprise the observation that nonpigmented cells that have the proper CD antigen expression profile can in some cases yield substantial number of cells that will give rise to pigmented SC-RPE upon re-culture, even though such a result is expected to generally lead to lesser purity due to the preponderance of non-SC-RPE in this population and overlapping labeling profiles. A skilled person will thus be able to identify the proper set of markers to use in specific application based on the specific starting cells, and experimental design. [00400] In some embodiments of the enrichment methods and systems of the disclosure the staining and sorting can be preceded by a cell harvesting in particular in embodiments where the starting SC-RPE cells are SC-RPE differentiated cells of the disclosure (see e.g., Example 27). The harvesting is preferably performed at a time when the increase in the number pigmented cells and their degree of pigmentation begins to plateau. This time will vary depending on the specific stem cell line:media:substrate combination, but it is typically between 60-90 days after the initial plating for differentiation. Enrichment of SC-RPE at times prior to the plateau in the percentage of pigmented cells in the culture will result in correspondingly lower yields due to decreased numbers of SC-RPE, reduced or heterogeneous levels of marker expression, and reduced levels of pigmentation. After the plateau of pigmentation is reached no further increases in yields or lasting improvement in RPE phenotype are expected. Enrichment at times up to differentiation day 120 are generally acceptable, but with increasing time in culture decreased yields can result due to increasing difficulty in preparing single cell suspensions. [00401] In some embodiments of the enrichment methods of the disclosure and systems and Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT related systems, the method comprises a cell harvesting step, the cell harvesting can be performed by dissociating the cultures by protease digestion using standard cell culture methods appropriate for the chosen protease. Preferred protease employed in this embodiment of the enrichment method comprise trypsin, TrypLE™, Accutase™ and Accumax™. Additional protease such as papain or dispase are expected to be possibly used. The proteases can be used alone, in combination, or sequentially. The preferred method is to use sequential digestion with trypsin or TrypLE™ in conjunction with Accutase™ or Accumax™. Beyond being able to dissociate the cultures into viable single cells that can survive the rigors of the enrichment process, the primary requirement directing the choice of protease is that it does not destroy the epitope recognized by the detection reagent, as will be understood by a skilled person. [00402] In some embodiments of the enrichment methods of the disclosure and systems and related systems, the method comprises a post-sort recovery step, based on the observation that after sorting the cells can be used directly, but higher quality and yields are generally obtained if one allows them to recover and reenter the cell cycle before downstream use. In those embodiments, the post recovery step is preferably performed by allowing cells to grow to near or just confluence. Shorter periods of recovery are acceptable but will be associated with reduced yields. Substantially longer periods of time that allow for the cells to establish a mature phenotype generally result in difficulties with subsequent harvesting of the cells and are therefore less preferred embodiments. [00403] In some embodiments of the enrichment method post-sort recovery step, cells are plated on substrate coated cultureware, at a preferrable density of about 75,000 to 150,000 cell/cm². Lower plating density down to about 10,000 cell/cm² are possible and can result in higher final yields, but at an increased risk of reduced RPE quality due to epithelial-to- mesenchymal transition (EMT). Higher densities up to about 250,000 are possible but will reduce the final yield of SC-RPE. [00404] The preferred substrate for the post-sort recovery is laminin 521 at a recommended amount of about 0.5-1 ^g/cm² of culture surface area. Lower concentrations can be used but can be associated with reduced cell attachment. Higher concentrations are expected to work as well, but no improvement in RPE quality is expected. [00405] In some embodiments of the post-sort recovery step, other substrates that support RPE Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT attachment and growth can be substituted for laminin 521, such as but not limited to, truncated or full-length vitronectin, laminin 111, laminin 211, laminin 221, laminin 332, laminin 411, laminin 511, and soluble basement membrane matrix extract such as Matrigel^TM. It is recommended that these substrates are used about 0.5-1 ^g/cm² of culture surface area or according to the manufacture’s guidelines. [00406] In the preferred embodiments of the post-sort recovery step, the culture medium is RPE Maintenance Medium (RPE-MMM). [00407] The wording RPE-Maintenance medium (RPE-MMM) as used herein indicates RPE Maturation Medium supplemented with one or more of three additional additives that when used together unexpectedly act synergistically to allow for the production of increased numbers of differentiation competent SC-RPE cells (not dedifferentiated) by cell division (also referred to as cell proliferation, expansion, or propagation) via multiple modes of action. An RPE Maintenance Medium in the sense of the disclosure is thus a RPE Maturation Medium further configured to enhance cell survival and cell attachment after culture harvest using cell dissociation reagents and subsequent re-culture (passage), to facilitate and enhance cell-cycle reentry of quiescent mature SC-RPE when passaging, and to delay the onset of and/or reverse persistent proliferation-dependent epithelial-to-mesenchymal transition (EMT, RPE dedifferentiation) whenever cells are being expanded. RPE Maintenance Medium has other beneficial effects as well. [00408] Accordingly, an RPE-Maintenance Medium in the sense of the disclosure is configured to enhances the rate of SC-RPE proliferation (thus shortening culture times to achieve a desired cell number or density), to improve SC-RPE phenotype homogeneity within a population, and to enhances the SC-RPE functionality. [00409] The RPE-Maintenance Medium is a preferred medium for all SC-RPE post-enrichment culture of the disclosure. RPE-Maintenance Medium is particularly preferred for post-sort recovery to enhance mature SC-RPE survival and cell-cycle reentry, after thawing of frozen cells to promote survival and presumably limit freeze/thaw induced injury, when SC-RPE are plated at moderate to low densities <40,000 cell/cm² for the purpose of achieving mature SC- RPE, or when greater numbers of cells are desired than can be achieved for a particular population SC-RPE in RPE Maturation Medium, which will vary depending on prior culture history of the particular population. When all of the effects of RPE-MMM SC-RPE are not Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT needed or desired, as would be understood by a skilled person upon reading of the disclosure, variations of RPE-MMM missing one or two of the additional factors or RPE-MM can be used. [00410] In some embodiments of the disclosure, the RPE Maintenance Medium is RPE Maturation Medium supplemented with Y-27632, bFGF and 0.1% DMSO. The addition of Y- 27632 promotes substrate attachment, facilitates cell cycle re-entry, and inhibits the onset of persistent EMT. Basic FGF facilitates cell cycle re-entry, increases growth rates, and in combination with Y-27632 results in further delay in the onset of persistent EMT. By itself bFGF does not delay the onset of passage-associated persistent EMT; however, it does act to enhance the development of at least some aspects of the mature RPE phenotype. DMSO acts in conjunction with bFGF to result in a more homogeneous RPE phenotype. By itself DMSO is not known to have any beneficial effects. (Examples 30, 31, and 32, Figures 13 and 14.) [00411] In some embodiments of RPE-MMM, Y-27632 is most preferably at a concentration of 5 ^M, preferably 5-10 ^M, less preferably 2.5-15 ^M, even less preferably 1.5-20 ^M, and least preferably 1-25 ^M. At concentrations below 1.5 ^M there is a marked decrease in efficacy and over 20 ^M there is a decrease in growth rates, a decrease in pigmentation, and cytotoxic effects becomes evident. [00412] In some embodiments of RPE-MMM the concentration of bFGF is most preferably 4 ng/mL, preferably 2-8 ng/mL, less preferably 2-10 ng/mL, even less preferably 0.5-12 ng/mL, furtherly less preferable 1-20 ng/mL, and least preferably 0.5-50 ng/mL. At concentrations below 0.5 ng/mL there is a substantial reduction in efficacy and above 12 ng/mL there is a reduction in pigment levels and there appears to be a reduction in epithelial morphology. [00413] In some embodiments of RPE-MMM, the concentration of DMSO is most preferably 0.1%, preferably 0.05-0.2%, less preferably 0.05-0.3% ng/mL and least preferably, 0.025- 0.5%. [00414] In some embodiments of the post-sort recovery step Y-27632 is omitted from the medium (RPE-MMM, without Y-27632) any time after about 1 day. For transient Y-27632 medium supplementation of 4 days or less, the most preferred concentration is 10 ^M, preferably 5-10 ^M, less preferably 2.5-15 ^M, even less preferably 1.5-20 ^M, and least preferably 1-25 ^M. [00415] In some embodiments of the post-sort recovery, SC-RPE cells are preferably allowed Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT to recover in RPE-MMM until they are near to just confluent. With typical post-sort viability (about 50% or greater) and preferred post-sort plating densities (75,000-150,000 cell/cm²) confluence generally occurs 4-7 days after plating. Shorter times of recovery down to about 2 days are possible. This will reduce the potential for proliferation dependent EMT but will result in a corresponding decrease in total yield. Longer times up to 2 weeks result in slight increases in yield, but with a slight to moderate increase in difficulty in harvesting the cells. Periods of recovery beyond 2 weeks result in substantial increase in difficulty in harvesting due to tight junction formation, RPE maturation, and exit from the cell cycle; and while possible, are not recommended for the purpose of recovery after sorting. [00416] The SC-RPE cell enrichment method and related system are easily scalable, require minimal technical expertise, can be automated, and can achieve purity levels greater than 98% in their preferred embodiments. [00417] In embodiments where the selection can be done based on quantitative measurement of surface marker expression and levels of pigmentation, the SC-RPE cell enrichment method and related system allow a user to further discriminate between high fidelity SC-RPE and SC- RPE of lesser quality based on robust expression of the two RPE markers and the presence of pigmentation; hence, allowing for further improvement in overall product quality by removal of outlier SC-RPE that have substantially reduced or elevated levels of SC-RPE marker expression compared to the population average. [00418] The SC-RPE cell enrichment method and related system is not limited to the enrichment of RPE derived culture from stem cells. It is also applicable to the enrichment of primary RPE cells from retinal pigment epithelium tissue, from cultured primary RPE, or from RPE derived from directed reprogramming of somatic cells. [00419] The SC-RPE Enrichment System of the disclosure is a system to enrich mature stem cell-derived retinal pigment epithelial (SC-RPE) cells, the system comprising at least one labeled probe specific for a marker selected from CD57, CD104, and/or CD49b, in combination with at least another probe specific for another for marker selected from CD57, CD104, and/or CD49b, a device configured to select pigmented cells based on light scatter and/or light absorption, reagents to perform the detection reaction and/or an RPE cell for combined use to stain differentiated SC-RPE cells in a method to obtain a population of SC-RPE cells enriched in mature SC-RPE cells. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00420] The terms “label” and “labeled probe” as used herein refer to a molecule capable of detection while preserving the viability of cells, including but not limited to, fluorophores, chemiluminescent dyes, chromophores, enzymes, enzymes substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, nanoparticles, metal solids, ligands (such as biotin, avidin, streptavidin or haptens) and the like. The term “fluorophore” refers to a substance or a portion thereof which is capable of exhibiting fluorescence in a detectable image. As a consequence, the wording “labeling signal” as used herein indicates the signal emitted from the label that allows detection of the label, including but not limited to radioactivity, fluorescence, chemiluminescence, production of a compound in outcome of an enzymatic reaction and the like. [00421] In embodiments of the SC-RPE enrichment system of the disclosure the system can comprise any combination of probes, such as recombinant antibody clones, monoclonal antibodies, polyclonal antibodies, antibody fragments and/or aptamers directed against the named antigens, provided the methods of culture dissociation does not destroy the antigen, and in particular the specific marker epitope recognized by the probes. Any fluorescent labels can be used depending on the specification of the sorting method after the staining such as FACS. Furthermore, non-antibody-based reagents that detect the named CD antigens (e.g., nucleic acid aptamers, peptide aptamers, synthetic peptides), or live cell nucleic acid stains that detect their expression could be used. Other physical means of enriching the SC-RPE based on their expression of the CD antigens include any of the affinity capture methods such as magnetic bead-based separation can be used as well. [00422] For example, a complete RPE enrichment kit can comprise single cell dissociation reagents and solutions, probes for each of the three markers coupled to an appropriate label based on the physical enrichment method of choice (e.g., FACS, magnetic capture, or affinity selection), staining solutions, substrate (e.g. laminin or vitronectin), live/dead cell staining reagents, and the components to constitute RPE-MMM. [00423] In some embodiments the serum-free SC-RPE methods and related cells, compositions, methods, and systems also comprise an RPE maintenance medium of the present disclosure that can be used to perform efficient expansion of immature and mature SC-RPE cells. [00424] The RPE Maintenance Medium of the disclosure comprises an RPE Maturation Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Medium of the disclosure (RPE-MM) supplemented with basic fibroblast growth factor (bFGF), a protein kinase inhibitor, and dimethyl sulfoxide (DMSO) in an effective amount to promote expansion of SC-RPE cells while maintaining their ability to mature, herein RPE- MMM. (See e.g. Examples 31 and 32). [00425] In some embodiments of the post-sort recovery step the culture medium is RPE-MMM, without Y-27632. In this case some reduction in cell attachment and survival can be expected as well as an increased potential for EMT. [00426] In some embodiments of the post-sort recovery step the culture medium is RPE-MMM, without bFGF. In this case, slower growth rates and a greater potential for EMT is expected. [00427] In some embodiments of the post-sort recovery step, the medium is RPE-MMM, without DMSO. It is expected that omission of DMSO from RPE-MMM can result in increased SC-RPE phenotype heterogeneity. [00428] In some embodiments of the post-sort recovery step the medium is RPE-MM, with Y- 27632, and Y-27632 is most preferably at a concentration of 5 ^M, preferably 5-10 ^M, less preferably 2.5-15 ^M, even less preferably 1.5-20 ^M, and least preferably 1-25 ^M. In this case, slow growth rates and a greater potential for EMT are expected. [00429] In some embodiments of the post-sort recovery step the medium is RPE-MM, with bFGF, and bFGF is most preferably 4 ng/mL, preferably 2-8 ng/mL, less preferably 2-10 ng/mL, even less preferably 0.5-12 ng/mL, furtherly less preferable 1-20 ng/mL, and least preferably 0.5-50 ng/mL. In this case reduced cell attachment, survival, and population homogeneity are expected. [00430] In some embodiments of the post-sort recovery step the medium is RPE-MM, with DMSO, with DMSO most preferably at a concentration of 0.1%, preferably 0.05-0.2%, less preferably 0.05-0.3% ng/mL and least preferably, 0.025-0.5%. [00431] In some embodiments of the post-sort recovery step the medium is RPE-MM. In this case a substantial reduction in post-sort recovery can be expected, especially in the cases of SC-RPE enrichment of differentiation cultures older than 90 days. [00432] Cultures are fed every 1-3 days by replacement of the expended medium with fresh Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT medium, with 2 days being the preferred feeding interval when using a volume of medium of about 1 mL of medium per 3 cm² of culture surface area. [00433] In some embodiments the configuration of the RPE-MMM of the disclosure also includes reagent that promote RPE survival and growth after selection, preservation of their capacity to obtain a prototypical RPE phenotype, and that inhibit passage associated EMT. In a preferred embodiment this entails the use of the additives Rho-kinase (ROCK) inhibitor Y- 27632, bFGF, and DMSO to RPE Maturation Medium. Other ROCK inhibitors such as thiazovivin can substitute for Y-27632. Thiazovin, (~ 2 ^M) can substitute for Y-27632 but its use should be limited to the first day or two of culture after SC-RPE enrichment. Additionally, it is expected that other inhibitors of RPE-to-mesenchymal transition such as the TGF^ receptor (ALK5) kinase inhibitors RepSox (~100 nM), A-83-01 (~100 nM), or SB-431542 (~1 ^M) can be used with similar effect either as an alternative to or in addition to Y-27632. Beyond the use of Y-27632 or thiazovivin for the first day post-enrichment the use of the media additives are not absolutely essential. However, in cases were maximal expansion of cultures are desired to produce large quantities of cells or due to less than optimal differentiation efficiencies their use can vastly improve the quality of the final product. [00434] The SC-RPE expansion method of the disclosure is a method for expansion of stem cell-derived retinal pigment epithelial (SC-RPE) cells, the method comprising providing stem cell-derived retinal pigment epithelial (SC-RPE) cells; and contacting the SC-RPE cells with the RPE Maintenance Medium of the disclosure on an SC-RPE substrate of the disclosure, the contacting for a time and under condition to obtain a target cell density of the SC-RPE cells. The objective of this step is to both increase the number of cells and to obtain a cell population that is in an active state of proliferation while maintaining the potential to achieve a prototypical RPE phenotype upon reaching confluence by reducing or minimizing potential changes due to proliferation dependent EMT or freezing-induced wounding that can lead to changes in RPE quality. In its most preferred embodiment, the expansion method of the disclosure employs the use of RPE Maintenance Medium (RPE-MMM) to preserve the potential for RPE to acquire a prototypical RPE phenotype, minimize culture expansion induced wound responses, and delay the onset of proliferation dependent EMT. [00435] In some embodiments the SC-RPE expansion method of the disclosure can be performed on SC-RPE cells after recovery from SC-RPE enrichment either to generate Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT additional cells or obtain cells in state suitable for preparation of frozen stocks of SC-RPE. [00436] In some embodiments the SC-RPE expansion method of the disclosure can be performed using frozen SC-RPE stocks. [00437] In some embodiments of the SC-RPE expansion method of the disclosure it can be performed in serial fashion using previously passaged cells to generate increased numbers of cells. [00438] In some embodiments, the SC-RPE expansion method of the disclosure can be performed by culturing of enriched SC-RPE on any of the SC-RPE substrates of the disclosure coated cultureware in the above described RPE Maintenance Medium (RPE-MMM) comprising the addition of basic fibroblast growth factor (bFGF), a rho-associated coiled-coil containing protein kinase (ROCK) inhibitor, and dimethyl sulfoxide (DMSO) which facilitate RPE growth while suppressing EMT (see e.g., the protocol of Example 35 and 40). [00439] In some embodiments, the SC-RPE expansion method of the disclosure can be performed by culturing of enriched SC-RPE on laminin or vitronectin coated cultureware in the above described RPE Maintenance Medium RPE-MMM comprising the addition of basic fibroblast growth factor (bFGF), a rho-associated coiled-coil containing protein kinase (ROCK) inhibitor, and dimethyl sulfoxide (DMSO) which facilitate RPE growth while suppressing EMT (see e.g., the protocol of Example 35 and 40). [00440] In some embodiments, the SC-RPE expansion method of the disclosure can be performed by culturing of enriched SC-RPE on laminin 521, recombinant truncated vitronectin, or full-length plasma vitronectin in the above described RPE Maintenance Medium (RPE-MMM) comprising the addition of basic fibroblast growth factor (bFGF), a rho- associated coiled-coil containing protein kinase (ROCK) inhibitor, and dimethyl sulfoxide (DMSO) which facilitate RPE growth while suppressing EMT (see e.g., the protocol of Example 35 and 40). In some embodiments, the SC-RPE expansion method of the disclosure can be performed by culturing of enriched SC-RPE on laminin 511, laminin 521, laminin 111, laminin 211, laminin 221, laminin 332, and laminin 411 in the above described RPE Maintenance Medium RPE-MMM comprising the addition of basic fibroblast growth factor (bFGF), a rho-associated coiled-coil containing protein kinase (ROCK) inhibitor, and dimethyl sulfoxide (DMSO) which facilitate RPE growth while suppressing EMT (see e.g., the protocol Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT of Example 35 and 40). [00441] In some embodiments the RPE-MMM comprises the ROCK inhibitor Y-27632, which suppresses trauma- and proliferation-induced EMT and in doing so acts to preserve the capacity of the SC-RPE to obtain their prototypical phenotype after culture passage and freezing. Basic FGF promotes SC-RPE proliferation and by itself bFGF promotes SC-RPE maturation when the cells are plated at low density, but it does not prevent the onset of passage-dependent EMT. However, in combination with Y-27632 it acts synergistically to further suppress passage induced EMT and thus allows for additional increases in yield. Dimethyl sulfoxide has no overt benefits by itself, but in conjunction with bFGF its use leads to and in particular acts synergistically to allow for more uniform RPE phenotype. Best results are obtained when all three media additives are used together, but some benefits are achieved with bFGF or a ROCK inhibitor alone (see e.g. the results of the experiments of Examples 31 and 32, Figures 13 and 14). [00442] In some embodiments, of the SC-RPE expansion method of the disclosure after reaching a desired cell density, the SC-RPE cells are harvested, replated in fresh RPE Maturation Medium plus bFGF, ROCK inhibitor, and DMSO (RPE-MMM) and a substrate. When the cultures reach the desired density, the cells can be harvested by protease digestion and frozen directly using one of several commercially available freezing media. Alternatively, if a greater number of cells are desired for preparation of frozen stocks the culture can be expanded further by replating and growth in RPE-MMM and a substrate. After reaching the desired density the cells can then be harvested and frozen. Optimal passaging and freezing efficiencies are achieved when the cells are harvested prior to establishing intercellular tight junctions and significant RPE phenotype maturation (< 1-2 weeks). (See e.g., the protocol of Examples 36 and 37). [00443] In some embodiments the SC-RPE expansion method of the disclosure are performed for Primary Expansion and/or Preparation of Frozen SC-RPE Stocks (see Examples 35 and 36). The goal of Primary Expansion is to expand the SC-RPE cultures to increase yield and to prepare intermediate frozen stocks of SC-RPE for later use while maintaining their ability to obtain a mature RPE phenotype. To accomplish this goal this process incorporates the use of a ROCK inhibitor to prevent passaged-induced EMT and bFGF/DMSO to promote RPE growth and the potential to differentiate. The other key aspect of this process which aids in successful Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT freezing is to harvest the cells prior to them fully exiting the cell cycle, beginning to acquire a mature RPE phenotype, and establishing tight junctions. As a general guideline optimal results are expected when the cells are harvested between 1 day prior to the day that the cells are estimated to reach confluence (near confluent) and 1 day after reaching confluence (just confluent), with harvesting at near confluence being preferable. Since SC-RPE have a doubling time of about one day, near confluent cultures would be expected to be in the range of 60-99% confluency. Just confluent cultures can easily be identified by daily observation of the cultures. Somewhat longer culture times than recommended between passage and before freezing are possible as are the omission of the media additives; however, expect to have increased difficulty in harvesting cells and/or reduced viability after harvest, reduced recovery after thawing, and an increased percentage of maturation incompetent mesenchymal RPE. In particular, early harvesting is expected to result in lower yields due to a reduction in the number of cells in the culture. As a general rule it is best to harvest cells for freezing when the cells are at about 80% confluence. [00444] In some embodiments of the SC-RPE expansion method of the disclosure performed for Primary Expansion, the method can comprise harvesting of SC-RPE. In those embodiments the harvesting can be performed by protease (e.g., TrypLE™ or trypsin) digestion using standard methods in keeping with the choice of protease. There is no requirement for the protease other than it results in a viable cell suspension and preferably results in cells capable of attaching to the substrates/supports of choice. In embodiments where the harvesting is performed following the SC-RPE enrichment method of the disclosure the harvesting can be performed after 2-14 days of culture post-sorting, in particular for the purpose of culture expansion. Typically, the cells can be harvested at 7 days, at which time the post-sort recovery cultures are typically confluent but have not begun to establish tight junctions, and obtain adequate post-harvest viability (> 90%). In some embodiments, it is possible to have much longer times of post-sort recovery before harvest (e.g.1-3 months). In those embodiments, no improvement is expected in the product as a result and beyond 2 weeks of culture the use of RPE Maturation Medium supplemented with a ROCK inhibitor (e.g., RPE-MM + Y-27632) as the recovery medium is preferred to reduce the extent of tight junction formation, while still inhibiting wound response and the onset of EMT. In addition, long post-sort recovery periods can require more extensive protease digestion or the use of multiple proteases to achieve efficient monolayer dissociation. (See e.g., the protocol of Example 36). Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00445] In some embodiments of the SC-RPE expansion method of the disclosure performed for Primary Expansion, the harvesting is followed by performing a primary expansion of the harvested cells. In those embodiments the method can comprise removing or neutralizing the protease, and plating the cells on a substrate (e.g., laminin, vitronectin, Matrigel^TM, or other SC-RPE substrate of the disclosure) coated cultureware using a passage ratio of about 1:8, contacting the cells with RPE Maintenance Medium of the disclosure such as RPE-MMM comprising Y-27632, bFGF, and DMSO to result in a seeding density of 20,000 to 60,000 cell/cm² (e.g., at a seeding density of 40,000 cell/cm²) when starting with a near to just confluent culture (see e.g., the protocol of Examples 34 and 35). Lower plating densities down to at least with 5000 cell/cm² are possible and will result in greater expansion ratios, albeit at some increased risk for an increase in the prevalence of mesenchymal RPE. Higher plating densities are associated with a decrease in the expansion ratio. [00446] In some embodiments of the SC-RPE expansion method of the disclosure performed for Primary Expansion, the RPE-MMM of the disclosure can be used to provide cell expansion, and in particular, in some of those embodiments for a large expansion to further maintain RPE quality. [00447] In some embodiments of the SC-RPE expansion method of the disclosure the culture medium is RPE-MMM, without Y-27632; RPE-MMM, without bFGF; RPE-MMM, without DMSO; RPE-MM, with Y-27632; RPE-MM, with bFGF; RPE-MM, with DMSO; or RPE- MM with potential impacts on RPE quality as would be understood by skilled person upon reading of this disclosure. [00448] In some embodiments of the SC-RPE expansion method the cells can be cultured transiently for one or more days in RPE-MMM; any of the RPE-MMM variants; or RPE-MM, with Y-27632, bFGF, or DMSO and then switched to one of the other variants of RPE-MMM or RPE-MM. In some instances, this can be advantageous in cases where it is desirable to produce cells that are cultured in medium free of one or more of the RPE-MMM supplements. In cases where transient contact with Y-27632 is employed, the use of higher concentrations of Y-27632 is most preferred (e.g., 10 ^M). In some embodiments the method and systems of the disclosure comprise the combined use of bFGF and DMSO. [00449] In some embodiments of the SC-RPE expansion method of the disclosure performed for Primary Expansion, the method comprises feeding the cells as required in view of the Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT experimental design with any one of the RPE-MMM media and/or the RPE-MM media of the disclosure; generally every 1-3 days using a volume of medium equivalent to 2-3 mL/10 cm² of culture surface area as will be understood by a skilled person upon reading this disclosure (see e.g., the protocol of Example 35). [00450] In some embodiments, one can just use the ROCK inhibitor for 1-2 days post plating or the entire culture period, while expecting a slightly reduced yield due to the absence of bFGF. The bottom line is that the three medium additives provide insurance against EMT and will result in a more consistent product. [00451] In some embodiments of the SC-RPE expansion method of the disclosure, the method can be performed for additional, serial expansion of SC-RPE to generate greater final yields of maturation competent SC-RPE by addressing the main factor that limits SC-RPE yield, which is the limited ability of RPE to proliferate without undergoing irreversible EMT. Most of the time, labor, and costs of producing SC-RPE goes into the derivation and enrichment phases of their production. Increasing the extent that the cells can be expanded post-freezing can have a dramatic effect on increasing final yields, with minimal increases in production time and total cost. From frozen stocks of minimally expanded SC-RPE one can obtain roughly 7 further cell doublings prior to loss of the ability of the cells to establish a prototypical mature RPE phenotype when using RPE-MMM compared to RPE-MM. (See e.g., the protocol of Example 40 and experiments of Example 32.) [00452] To achieve further expansion while maintaining the ability to obtain a mature and functional phenotype, frozen stocks are thawed and plated at low to moderate density (about 5000 to 20,000 cell/cm²) in RPE Maintenance Medium (e.g. comprising RPE-MM of the disclosure bFGF, ROCK inhibitor, and DMSO) on an SC-RPE substrate of the disclosure (such as laminin or vitronectin) coated cultureware (see e.g., the protocol of Examples 39 and 40 and experiments of Example 32). Lower plating densities are possible and can result in greater final yields but with a potential increase in the number of mesenchymal RPE, while high densities can result in a slight increase in RPE quality with a corresponding reduction in total yield. [00453] Prior to the establishment of tight junctions, the cells can be harvested and used for production of the final product, or they can be expanded further. When all three media additives are used in concert roughly six additional populations doublings can be achieved compared to Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT using RPE Maturation Medium alone. This equates to a 72.5-fold increase in total yield compared to using RPE Maturation Medium alone and an approximately 180-fold overall expansion post-thaw. (See e.g., the protocol of Example 41 and experiments of Example 32.) [00454] After the desired amount of culture expansion, the SC-RPE can then be used for production of a clinical product or for experimental purposes in the RPE Maintenance Medium. Depending on the extent of culture expansion the RPE Maturation Medium can be used in place of the RPE Maintenance Medium of the disclosure, even if RPE Maintenance Medium can be beneficial, in experimental designs where lesser yields are needed, or in cases where the addition of the additional supplements to RPE-MM are unwanted due to the intended use of the cells (e.g. for the use in experiments investigating RPE wound response or EMT). In the RPE Maintenance Medium the combination of bFGF and DMSO, promotes uniform differentiation, bFGF promotes the establishment of tight junctions and the ROCK inhibitor can act as insurance by preventing EMT onset as will be understood by a skilled person. (See e.g., the protocol of Example 41 and experiments of Examples 31 and 32). [00455] In embodiments herein described SC-RPE expansion system for expansion of stem cell-derived retinal pigment epithelial (SC-RPE) cell, comprises at least one RPE Maintenance Medium, and at least one SC-RPE substrate of the disclosure for combined use in a method to perform primary expansion of the SC-RPE cells of the disclosure. [00456] In some embodiments additional methods can be performed using the RPE Maturation Medium and/or the RPE Maintenance Medium of the disclosure. [00457] For example, an SC-RPE frozen stock preparation method and system and SC-RPE obtainable or obtained thereby are described based on the use of the RPE Maintenance Medium of the disclosure. The SC-RPE frozen stock preparation method comprises harvesting the SC- RPE cells having the target cell density following SC-RPE cell expansion of the disclosure to obtain harvested SC-RPE cells; and freezing the harvested SC-RPE cells. (See e.g., the protocol of Examples 36 and 37). [00458] In embodiments of the SC-RPE frozen stock preparation method, the pre-freeze harvesting can be performed when the cells reach near or just confluence (e.g., 2-4 days depending on plating density) for freezing by protease digestion and standard methods. Shorter times with result in lower yield, but it will decrease the number of cells to freeze. Longer times Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT will increase yield, but can result in poorer recovery after thawing. If the cells are allowed to become significantly mature as evident based on the accumulation of pigmentation, there will be a marked decrease in cell survival after thawing. After harvesting, performed for example with protease, cells can be collected by any suitable method such as centrifugation. (See e.g., the protocol of Example 36.) [00459] In embodiments of the SC-RPE frozen stock preparation method , the method can then comprise freezing the harvested cells, In particular, SC-RPE can be typically frozen at a concentration of 0.5-5 or 0.5 to 20 × 10⁶ cell/ml in volumes of 0.5 mL or greater. The choice of the cell concentration and aliquot size should be determined based on the number of cells required in the downstream applications. (See e.g., the protocol of Example 37). [00460] The SC-RPE frozen stock preparation method, the method can further comprise resuspending the harvested and collected cells in an appropriate volume of freezing medium and transfer desired size aliquots to an appropriate size cryovial or bag. During the aliquoting process it is best to maintain the cells at 4^{^} C until they are frozen. Serum-free freezing agents that are known to work are CryoStor CS10, CryoStor CS5, and CryoStor CS2 (Biolife Solutions); Synth-A-Freeze^TM and CTS-Synth-A-Freeze^TM (ThermoFisher); and mFreSR^TM (STEMCELL Technologies). If serum-free culture is not needed 10% DMSO in serum is also suitable. [00461] In the SC-RPE frozen stock preparation method, the method can also comprise after the aliquoting process is complete, freezing the cells using standard controlled rate freezing (approximately -1^{^} C/min) to a final set point of roughly -80^{^} C. At the completion of the freezing process the frozen cells can be transferred to -125 to -200^{^} C for long term storage. [00462] The SC-RPE frozen stock preparation system comprises a combination of the RPE Maintenance Medium of the present disclosure, the SC-RPE substrate of the present disclosure and freezing media for combined use in a method to provide frozen stock of the present disclosure, as will be understood by a skilled person. [00463] In some embodiments of the disclosure, the obtained SC-RPE can be used to produce a suspension of SC-RPE at a desired target density in a solution suitable for in vivo transplantation. The SC-RPE used to produce the suspensions can be post-sort enriched SC- RPE, post-sort recovered SC-RPE, expanded SC-RPE, post-thaw SC-RPE, and frozen SC- Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT RPE. The SC-RPE cells used to obtain the transplantation SC-RPE suspensions can be in any state of maturation ranging from nonpigmented proliferating RPE to fully mature quiescent SC-RPE. Examples of suitable suspension SC-RPE transplantation solutions are simple buffered saline solutions, buffered saline solutions with additional additives that act to maintain SC-RPE viability, and complex cell or tissue storage media. In addition, the transplantation solution can include additives that enhance SC-RPE survival and/or function after transplantation. The collective process of preparing SC-RPE suspensions for transplantation is herein referred to as the Suspension SC-RPE Transplantation method. [00464] In some embodiments of the Suspension SC-RPE Transplantation method SC-RPE suspensions for transplantation are prepared using mature pigmented SC-RPE obtained from any of the embodiments of the SC-RPE enrichment method. Use of SC-RPE enriched on both marker staining and pigmentation are expected to give suspensions with the highest SC-RPE purity. If it is desired that the use of marker stain be avoided due to concerns with the clinical compatibility or to avoid the high cost of acquiring marker probes certified for clinical use the SC-RPE can be enriched based on pigmentation, preferably using both light absorption and light scatter. One disadvantage of using SC-RPE directly post-enrichment is that the recovered cells often have significant quantities of debris and dead cells. This can be alleviated at least in part by using dead cell stains to select against dead cells either in the initial enrichment or in a second sequential enrichment providing that the dead cell staining is compatible with transplantation, as would be understood by skilled person. After collection the purity can be determined based on the percentage of viable pigmented cells. Enriched SC-RPE that meet a desired purity criteria can be then washed with saline solution using standard cell culture procedures, the number and percentage of viable can be quantified, and the enriched SC-RPE can be suspended at the desired target density in the chosen transplantation suspension solution. [00465] In some embodiments of the Suspension SC-RPE Transplantation method SC-RPE suspensions are prepared using adherent cultures of SC-RPE of varying degrees of maturity. The adherent SC-RPE can be obtained from post-sort recovery SC-RPE cultures, post-thaw recovery cultures, and expansion SC-RPE cultures grown for appropriate lengths of time to achieve the desired degree of maturity. The stage of maturity can be determined based on the expression of known immature and mature RPE markers [21]. as would be understood by a skilled person. After about 1-week post-confluence maturity can be assessed by light absorbance or brightfield microscopy image analysis. Other measures of RPE phenotype and Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT function, such as measurement of the transepithelial electrical resistance are possible as well. When the desired level of maturity is achieved the adherent SC-RPE cells can be harvested with a cell monolayer dissociation reagent (such as and not limited to TrypLE, trypsin or AccuMax) in accordance with the methods of the disclosure. After washing the cells with saline solution and determination of the number and percentage of viable cells, the collected SC-RPE can be suspended at the desired target density in the chosen transplantation suspension solution. [00466] In some embodiments of the Suspension SC-RPE Transplantation method immature SC-RPE suspensions are prepared using frozen SC-RPE. Frozen SC-RPE can be rapidly thawed in accordance with the recommendation of the cryopreservation solution manufacturer and the resulting SC-RPE suspension can be used directly without any further processing or they can be washed with saline, and after determination of the viable cell number and percentage they can be suspended at the desired target density in the choose transplantation solution. For using frozen SC-RPE directly without washing they should be frozen at the desired target density in a cryopreservation that is suitable for in vivo transplantation. CryoStor CS2 and CryoStor CS5 and are two examples of cryopreservation solutions expected to be suitable for in vivo use and can effectively be used to produce frozen stocks of SC-RPE with post-thaw viabilities of at least 80% and 90%. For direct transplantation of SC-RPE in cryopreservation solution it is preferable to determine the post thaw viable cell density and percentage to determine the actual viable cell dose or to allow for adjustment of the injected volume to deliver a constant viable cell dose. [00467] In some embodiments, the RPE Maturation Medium and/or the SC-RPE Maintenance medium of the disclosure can be used in an SC-RPE Patch production method and systems to obtain mature SC-RPE cells monolayers for the purpose of repairing degenerative RPE-based ocular disorders or scientific study. [00468] The SC-RPE Patch production method of the disclosure is a method to perform the manufacture of SC-RPE monolayers on a flexible solid support, the method comprising performing expansion of SC-RPE cells according to the method of the disclosure to obtain SC- RPE cells having a target cell density. The SC-RPE Patch production method further comprises contacting the SC-RPE cells having the target cell density with an SC-RPE substrate of the disclosure coated membrane support and the RPE Maturation or Maintenance Medium of the disclosure to obtain a population of quiescent and at least partially differentiated SC-RPE cells Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT monolayers on the membrane support. (See e.g., protocols of Examples 39 to 41.) [00469] In some embodiments the SC-RPE Patch production method, comprises thawing of frozen stocks, performing Secondary Expansion and Patch production by contacting the SC- RPE cells having the target cell density with a membrane support and the RPE Maturation or RPE Maintenance Medium of the disclosure. The goals of this process are to increase yields by culture expansion using conditions that minimize freezing and passage related EMT and to promote acquisition of a mature functional RPE monolayer after plating on a microporous membrane. This is best accomplished by culturing the cells in RPE Maintenance Medium. It is possible to omit all or some of the additives and obtained SC-RPE; however, there is a risk of increased occurrence of non-pigmented cells with poor morphology and expect to have reduced barrier function and lowered and less homogeneous expression of at least some RPE genes (see e.g. the protocols of Example 39 to 41 and experiments of Examples 31 and 32). [00470] In some embodiments, the SC-RPE Patch production method thawing and recovering of frozen stock comprises thawing a stock vial or container of cells in accordance with standard procedures for thawing cells. Generally, this entails rapid thawing, dilution of the thawed cells with culture medium, and collection of the cells by centrifugation (see e.g., the protocol of Example 39). [00471] In some embodiments the SC-RPE Patch production method thawing and recovering of frozen stock further comprises resuspending the cells in the RPE Maintenance Medium or in RPE Maturation Medium of the disclosure. The use of ROCK inhibitor in the RPE-MMM will reduce freezing related EMT and bFGF+DMSO will aid in rapid growth and further protection against proliferation dependent EMT. Maximal protection is achieved when all three additives are used in combination in an RPE-MMM of the disclosure. Additionally, some measure of protection against freezing related EMT can be achieved by only using ROCK inhibitors for the first day of culture after freezing. In that case, thiazovivin can be used as an alternative to Y-27632. Other ROCK inhibitors are expected to have a similar effect as well (see e.g. the protocol of Example 39 and experiments of Example 32). [00472] In some embodiments the SC-RPE Patch production method thawing and recovering of frozen stock also comprises plating the cells in an appropriate size cultureware coated with an SC-RPE substrate of the disclosure (e.g., laminin, vitronectin, or Matrigel^TM) at a plating density of roughly 6500 cell/cm². Higher density plating is acceptable but lower yields of cells Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT will result. Substantially lower densities will result in the potential for increased passage related EMT, especially in the case of omission of the RPE-XM media additives. (See e.g., the protocol of Example 39.) [00473] In some embodiments the SC-RPE Patch production method thawing and recovering of frozen stock comprises feeding the cells with RPE Maturation Medium (see e.g., the protocol of Example 39). [00474] In some embodiments of the SC-RPE Patch production method of the disclosure, it is possible to omit a post-freeze recovery and expansion step if greater numbers of cells are not necessary. This will decrease the potential for passage related EMT, as well as the quality of the resulting cultures due to some extent of post thaw cell death as will be understood by a skilled person. [00475] In some embodiments the SC-RPE Patch production method of the disclosure comprises harvesting the recovered cells. In those embodiments, when the cells reach near or just confluence, generally 3-4 days, the harvesting the cells can be performed by protease digestion using standard cell culture methods. Suitable proteases include TrypLE™, trypsin, Accutase™, and Accumax™. Other proteases are expected to be acceptable as long as the cells remain viable and capable of attachment. Shorter or longer times in culture before harvest are possible. Shorter times will result in reduced yield, whereas longer times in culture will increase the difficulty of harvesting cells due to cell:cell tight junction formation. After several days post confluence there is minimal cell growth and no further increases in yield will be achieved. In general, up to 7 days of post-thaw recovery will yield satisfactory results. (See e.g., the protocol of Example 40.) [00476] In some embodiments the SC-RPE Patch production method of the disclosure can further comprise recovering the harvested cells and exchanging the cells into the medium of choice (typically one of the RPE Maintenance Medium of the disclosure) for plating for experimentation or patch production. Alternatively, the cells can be expanded by additional passaging if even larger numbers of cells are desired. However, in this case it is recommend that a test run be carried out to verify that they are capable establishing mature RPE monolayers with minimal presence of mesenchymal RPE. (See e.g., the protocol of Example 40.) [00477] In some embodiments the SC-RPE Patch production method of the disclosure can Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT further comprise performing patch production of SC-RPE monolayers for implantation the cells. In those embodiments cells are plated on a membrane support of choice and are typically maintained for a sufficient period of time such that they become largely quiescent and at least partially differentiated prior to final release (see e.g., the protocol of Example 41 and experiments of Example 31.) [00478] In order to produce SC-RPE Patches, the method can comprise seeding the cells onto membrane supports coated with a substrate that supports RPE attachment and maturation (e.g., laminin 521, full-length vitronectin, or rhVTN-N) in RPE Maintenance Medium (RPE Maturation Medium + Y-27632, 2 ng/ml bFGF, and 0.1% DMSO) such that the initial density of cells on the membrane will be at a preferred density of about 100,000 cell/cm² (25,000- 250,000 cells/cm²). The total number of cells and plating volume will be dependent on the culture device and the relative surface area of the membrane in the culture device. Lower seeding densities can result in a decrease in culture uniformity and larger cell size. Higher densities will result in a slight increase in cell density at maturity. [00479] In some embodiments the SC-RPE Patch production method of the disclosure can further comprise feeding the cultures as necessary with RPE-Maintenance Medium (RPE Maturation Medium + 5 ^M Y-27632, 0.1% DMSO, and 2 ng/ml bFGF). Typically, this is every 2 days for the first week, and every 3-4 days thereafter using a volume of medium equivalent to about 3 mL/10 cm² of culture surface area. Additionally, after 2-3 weeks the addition of Y-27632, DMSO and/or bFGF to the medium can be omitted. Removal of bFGF/DMSO will result in a slight increase in the level of pigmentation. However, the establishment and maintenance of RPE:RPE tight junctions as well as the expression of genes associated with the RPE phenotypy are enhanced by Y-27632 and/or bFGF and their removal will result in reduced RPE barrier function over time until it stabilizes (see e.g. Example 31 and Figure 12). Other variations of RPE-MMM or RPE-MM can be used as well. However, additional variations are expected to result in a decrease in the quality and function of the SC- RPE as would be understood by a skilled person upon reading of this disclosure. Those variations however are expected to maintain a quality level suitable for use cases where the inclusion of Y-27632, DMSO, and/or bFGF is not desired. [00480] In some embodiments, SC-RPE method and systems are described for preparing a suspension of SC-RPE cells obtainable and/or obtained with the methods and systems of the Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT present disclosure that can be used for transplantation of the SC-RPE in an individual (SC-RPE suspension preparation method). [00481] The SC-RPE suspension preparation method comprises providing a suspension of mature SC-RPE cells of the disclosure, preferably following performing the SC-RPE enrichment method of the disclosure. [00482] In some of embodiments of the SC-RPE suspension preparation method of the disclosure providing a suspension of mature SC-RPE cells comprises performing expansion of mature SC-RPE cells according to any one of the methods of the disclosure, preferably after performing an SC-RPE Enrichment method of the present disclosure to obtain a suspension of mature SC-RPE cells having a target cell density. In those embodiments, placing the mature SC-RPE cells in a target ocular area of the individual is performed by placing the SC-RPE cells having the target cell density. [00483] In some of embodiments of the SC-RPE suspension preparation method of the disclosure providing a suspension of mature SC-RPE cells comprises contacting an adherent culture of differentiated and/or mature SC-RPE cells with one or more proteases (e.g. trypsin, collagenase, dispase and/or pronase), preferably after performing an SC-RPE Enrichment method on the adherent culture of differentiated and/or mature SC-RPE cells, and harvesting the cells following protease digestion in accordance with the methods of the disclosure in order to obtain a suspension of differentiated and/or mature SC-RPE cells. In those embodiments, when the obtained suspension of differentiated and/or mature SC-RPE cells SC-RPE comprises differentiated SC-RPE cells, the providing a suspension of mature SC-RPE cells further comprises contacting the obtained suspension of differentiated and/or mature SC-RPE cells SC-RPE comprising differentiated SC-RPE cells with the SC-RPE Maturation Medium and/or the SC-RPE Maintenance Medium of the disclosure, to provide the suspension of mature SC- RPE cells of the disclosure to be placed in the target ocular area of the individual. [00484] In some of embodiments of the SC-RPE suspension preparation method of the disclosure providing a suspension of mature SC-RPE cells comprises thawing a SC-RPE Frozen Stock of the present disclosure, and preferably performing expansion of thawed mature SC-RPE cells according to any one of the SC-RPE Expansion methods of the disclosure, to obtain a suspension of mature SC-RPE cells having a target cell density. In those embodiments, providing a suspension of mature SC-RPE cells can further comprise performing the SC-RPE Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Enrichment method of the present disclosure on the thawed mature SC-RPE cells, possibly before performing an SC-RPE Expansion method of the disclosure. [00485] In some of embodiments of the SC-RPE suspension preparation method, obtaining a suspension of SC-RPE can be performed using cells obtained from any step or time in the process after the SC-RPE Enrichment according to any one of the embodiments of the present disclosure. [00486] In some embodiments of the SC-RPE suspension preparation method when obtaining SC-RPE cells from periods of adherent culture after SC-RPE enrichment the cells can be, subconfluent and immature, confluent but immature, or fully mature provided that in each case the cells maintain the capacity to reestablish a functional RPE monolayer upon re-culture using appropriate culture conditions. [00487] In some embodiments, of the SC-RPE suspension preparation method, the method can further comprise preparing an SC-RPE cell suspension at a desired target density in a solution appropriate for use in transplantation. In some embodiments, the preparing can result in SC- RPE cells in a buffered saline or a cell storage solution configured to enhance cell survival that is also compatible with direct transplantation. Alternatively, the method can further comprise an intermediate step of preparing a suspension of SC-RPE in any solution configured to enhance cell survival during storage prior to transplantation and then transferring the SC-RPE cells to a more suitable solution for direct use in transplantation (e.g. balanced saline solution) at the time of use. In some embodiments, the cell suspension media can include additional components expected to promote cell survival and/or SC-RPE function after transplantation (Y-27632 for example). When using frozen stocks the cells can be used directly after thawing, provided they are frozen in cryopreservation suitable for direct in vivo use. Alternatively, cells can be thawed and transferred to suitable solution in accordance with the present disclosure. [00488] In some embodiments the SC-RPE monolayers are on a flexible solid support, and/or the SC-RPE cells in suspension obtainable and/or obtained with method of the disclosure can be used to treat and/or prevent retinal conditions in the individual. [00489] The term “condition” as used herein indicates a physical status of the body of an individual (as a whole or as one or more of its parts), that does not conform to a standard physical status associated with a state of complete physical, mental and social well-being for Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT the individual. Conditions herein described include but are not limited to disorders and diseases wherein the term “disorder” indicates a condition of the living individual that is associated to a functional abnormality of the body or of any of its parts, and the term “disease” indicates a condition of the living individual that impairs normal functioning of the body or of any of its parts and is typically manifested by distinguishing signs and symptoms. [00490] Retinal conditions in the sense of the disclosure refers to conditions involving and affecting the retina of an individual. Exemplary retinal conditions comprise various forms of Age-Related Macular Degeneration (AMD), such as Dry AMD, Wet AMD, Stargardt Disease, Retinitis Pigmentosa, Inherited Retinal Diseases: and additional conditions identifiable by a skilled person. [00491] The term “treatment” as used herein indicates any activity that is part of a medical care for, or deals with, a condition, medically or surgically. [00492] The term “prevention” as used herein indicates any activity which reduces the burden of mortality or morbidity from a condition in an individual. This takes place at primary, secondary and tertiary prevention levels, wherein: a) primary prevention avoids the development of a disease; b) secondary prevention activities are aimed at early disease treatment, thereby increasing opportunities for interventions to prevent progression of the disease and emergence of symptoms; and c) tertiary prevention reduces the negative impact of an already established disease by restoring function and reducing disease-related complications. [00493] In some embodiments the SC-RPE monolayers are on a flexible solid support, and/or the SC-RPE cells in suspension obtainable and/or obtained with method of the disclosure can be transplanted into an individual with an SC-RPE transplantation method of the present disclosure for research purpose or as part of a method to treat and/or prevent a retinal condition in the individual. [00494] In particular, the SC-RPE transplantation method comprises placing the SC-RPE monolayers on a flexible solid support, and/or the SC-RPE cells suspension of the disclosure in the subretinal space of the individual and placing the mature SC-RPE cells of the disclosure in the subretinal space of the individual thus transplanting the individual with the suspension of SC-RPE cells of the disclosure. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00495] In an SC-RPE Transplantation methods of the disclosure RPE cells can be transplanted as a cell suspension or as a monolayer. For example, human RPE stem cell-derived RPE (hRPESC-RPE) monolayers have been successfully transplanted under the macula in non- human primates, showing integration with the host retina and maintaining photoreceptor function without adverse effects like epithelial-mesenchymal transition or gliosis. Also, an SC- RPE suspension can be injected in the subretinal space as will be understood by a skilled person. [00496] Devices that can be used to perform SC-RPE transplantation methods can comprise a traphine and surgical kit, a Microfluidic and Actuator-Based Device, an Extracellular Matrix- Scaffold-Supported Transplantation Device, an intravenous Cannula for RPE Strips, and additional devices identifiable by a skilled person (see e.g. Example 45 and Figure 26). [00497] In some embodiments, at least one of the SC-RPE differentiation method, the SC-RPE maturation method of the disclosure, the SC-RPE enrichment method, the SC-RPE expansion method, the SC-RPE preparation of frozen stock method and the SC-RPE Patch production method can be used in a toolbox SC-RPE Derivation Method of the disclosure in which a user selects the steps and/or component in accordance with the starting stem cells and the experimental design. [00498] In embodiments herein described, the SC-RPE derivation methods comprises differentiating the stem cell (SC) to obtain differentiated stem cell-derived retinal pigment epithelial (SC-RPE) cells, maturating the differentiated SC-RPE cells to obtain mature SC-RPE cells, performing enrichment of mature SC-RPE stem cells, preparing a frozen stock of the SC- RPE, performing expansion of the SC-RPE, and/or production SC-RPE Patches to obtain a population of quiescent, at least partially mature SC-RPE cells monolayers on a membrane support. [00499] In the SC-RPE derivation methods of the disclosure at least one of the differentiating, the maturating, the performing enrichment, the preparing a frozen stock, the performing expansion is performed with a method of the present disclosure. [00500] In some embodiments, the SC-RPE derivation method of the disclosure encompasses - a collection of spontaneous and semi-directed differentiation protocols all of which employ the use of a novel xeno- and serum-free RPE medium; Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT - an automation compatible method for the isolation of pure populations of mature RPE cells from mixed cell populations based on any of the following properties: expression of the cell surface antigens CD57 and/or CD104, lack of expression of CD49b, and light absorbance or light scattering of melanin/melanosomes; - a method for the creation of intermediate frozen SC-RPE stocks for on-demand production of final product; - a method for culture expansion and production of final drug product that promotes RPE maturation and limits RPE-to-mesenchymal transition. [00501] Major features of this pipeline in many embodiments are - the utilization of the SC-RPE differentiation medium, and more notably the RPE Maturation Medium and SC-RPE-Maintenance Medium throughout the pipeline to both increase differentiation efficiencies and maintain the RPE phenotype in culture, - the efficient isolation of SC-RPE mature cells through the SC-RPE enrichment method, and - the combinatorial use of basic fibroblast growth factor, Rho-associated, coiled-coil containing protein kinase inhibitor in RPE Maintenance Medium, and dimethyl sulfoxide to suppress persistent proliferation-dependent epithelial-to-mesenchymal transitions during culture expansion. [00502] Together these features can allow for a substantial improvements in yield, purity, and quality, as well as a significant shortening of the time required to complete the process compared to many existing procedures. [00503] The SC-RPE derivation system of the disclosure comprises at least one of the SC-RPE differentiation system, the SC-RPE maturation system, the SC-RPE enrichment system, the SC-RPE maintenance system, the SC-RPE frozen stock preparation system, and the SC-RPE Patch production system of the present disclosure. [00504] In several embodiments, the SC-RPE derivation method of the disclosure allows multiple variations of the general method that allow for identification of an optimal method for a given stem cell line. [00505] In some preferred embodiments, the derivation method comprises Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT i) differentiating stem cells (SC) by contacting an RPE-DM1 differentiation medium with the stem cells on a SC-RPE cell culture substrate, preferably comprising or consisting of recombinant human truncated vitronectin, preferably for a differentiating time of 26-33 (e.g., about 28 or about 30) days to obtain differentiated stem cell-derived retinal pigment epithelial (SC-RPE) cells; ii) maturating the differentiated SC-RPE cells by contacting the differentiated SC-RPE cells with an RPE-MM medium of the present disclosure, preferably for a maturating time of one to two weeks to obtain matured SC-RPE cells; optionally wherein the method further comprises iii) performing protease digestion of the matured SC-EP cells to obtain protease digested matured SC-RPE cells; optionally wherein the method further comprises iv) replating the protease digested matured SC-RPE cells at split ratio of about 1:2 on recombinant human truncated vitronectin coated cultureware; optionally wherein the method further comprises v) culturing the replated protease digested matured SC-RPE cells by contacting the replated protease digested matured SC-RPE cells with RPE-MM for about 1 month or until the percentage of pigmented cells in the culture begins reach plateau to obtain a culture of matured SC-RPE. [00506] In preferred embodiments the stem cells are human pluripotent stem cells, and in particular embodiments the stem cells are human pluripotent stem cells (e.g., Shef1). [00507] In preferred embodiments the recombinant human truncated vitronectin in any one of steps i) to v) is CTS-Vitronectin (VTN-N) recombinant human protein, truncated. [00508] In preferred embodiments the RPE-DM1 medium in step i) can be CTS-KO- DMEM/20%CTS-KOSR or KO-DMEM/20%KOSR. [00509] In preferred embodiments, the RPE-MM in any one of steps ii) to v) can be liquid alpha MEM, without phenol red and with nucleosides, 1X CTS-B27 XenoFree, 1X CTS-N2, 2 mM taurine, 100 ^M NEAA, and 55 nM hydrocortisone. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00510] In some embodiments, the method further comprises, before the differentiating step, i), seeding the stem cells on a substrate (e.g., recombinant human truncated vitronectin) coated cultureware in complete CTS-Essential 8 medium, preferably for a seeding time of about 2 to 5 days, to provide the stem cells on a SC-RPE cell culture substrate (e.g., comprising recombinant human truncated vitronectin). [00511] In some embodiments, the CTS-Essential 8 medium is preferably supplemented with 5-10 ^M Y-27632 for the first 24-48 hours). [00512] In preferred embodiments of the derivation method, each of step i) to v) is performed in any possible combination one with the other. [00513] In some preferred embodiments the matured SC-RPE cells obtained in outcome of step ii), the protease digested matured SC-RPE cells obtained in outcome of step iii) the replated protease digested matured SC-RPE cells obtained in outcome of step iv) and/or the culture of matured SC-RPE obtained in outcome of step v) can be enriched using any one of the methods of the present disclosure. [00514] In some embodiments, enrichment can be performed by vi) contacting the matured SC-RPE cells obtained in step ii), the protease digested matured SC-RPE cells obtained in step iii) , the replated protease digested matured SC-RPE cells obtained in step iv), the culture of matured SC-RPE obtained in step v), and/or the mature SC-RPE cells sorted in step vii) with a probe specific for a marker selected from CD57, CD104, and/or CD49b to obtain stained mature SC-RPE cells stained for CD57, CD104, and/or CD49b markers; and sorting the stained SC-RPE cells to select the mature SC-RPE cells stained for CD57 and/or CD104 markers, and discard cells stained for CD49b marker, to obtain a population of SC-RPE cells enriched in mature SC-RPE cells. [00515] In addition, or in the alternative, in preferred embodiments the method can further comprise Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT vii) detecting pigmentation in the matured SC-RPE cells obtained in step ii), the protease digested matured SC-RPE cells obtained in step iii), the replated protease digested matured SC-RPE cells obtained in step iv) and/or the culture of matured SC-RPE obtained in outcome of step v), and/or the stained SC-RPE cells sorted in outcome of step vi) to identify pigmented mature SC-RPE cells, preferably by measuring light absorbance readings at wavelength of at least 400 and more preferably from 480-600 nm to quantitively detect pigmented culture area and sorting the mature SC-RPE cell populations to select pigmented mature SC-RPE cells and discard mature SC-RPE cells with no detected pigmentation, to obtain a population of SC-RPE cells enriched in mature SC-RPE cells. [00516] In some embodiments, the sorting step can be performed in a buffer, such as a MACS GMP PBS/MgCl2 buffer comprising the protease (e.g., tytonase) and optionally human serum albumin (HSA) and/or heparin. [00517] In preferred embodiments, the enriched SC-RPE can then undergo a post-sort recovery in embodiments of the enrichment method which in addition to steps vi) and/or vii) can further comprise viii) seeding the enriched mature SC-RPE cells on an SC-RPE substrate according to the disclosure, preferably recombinant human truncated vitronectin (CTS-Vitronectin (VTN-N) recombinant human protein, truncated) coated cultureware and also ix) contacting the seeded enriched mature SC-RPE cells with the recovery medium selected from an RPE-MMM or RPE-MM according to the disclosure, preferably for about 1 week to obtain recovered enriched mature SC-RPE. [00518] In some preferred embodiments of step ix), the recovery medium is liquid alpha-MEM, without phenol red and with nucleosides, 1X CTS-B27 XenoFree, 1X CTS-N2, 2 mM taurine, 100 ^M NEAA, and 55 nM hydrocortisone, 5 ^M Y-27632, 2-4 ng/mL bFGF, and 0.1% DMSO. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00519] In some preferred embodiments, derivation methods comprising steps viii) and ix) further comprise steps for the preparation of intermediate SC-RPE frozen stock x) harvesting recovered enriched mature SC-RPE by protease digestion of the recovered enriched mature SC-RPE cells to obtain harvested enriched mature SC-RPE and xi) preparing frozen vials of the harvested enriched mature SC-RPE cells at a desired density and by freezing in cryopreservation medium (preferably CryoStor® CS10, CryoStor® CS5, or CryoStor® CS2). [00520] In some preferred embodiments, preparation of intermediate SC-RPE frozen stock can also be performed by xii) harvesting recovered enriched mature SC-RPE by protease digestion of the recovered enriched mature SC-RPE cells to obtain harvested enriched mature SC-RPE xiii) seeding the harvested enriched mature SC-RPE onto a cultureware coated with an SC- RPE substrate in the sense of the disclosure, harvested enriched mature SC-RPE with an RPE-MMM or RPE-MM according to the disclosure to obtain expanded enriched mature SC-RPE, xiv) harvesting recovered enriched mature SC-RPE by protease digestion of the expanded enriched mature SC-RPE cells to obtain harvested enriched mature SC-RPE and xv) preparing frozen vials of the harvested enriched mature SC-RPE cells at a desired density by freezing in cryopreservation medium (preferably CryoStor® CS10, CryoStor® CS5, or CryoStor® CS2). [00521] In some preferred embodiments of step vii), ix) , x) and xi) the recombinant human truncated vitronectin is CTS-Vitronectin (VTN-N) recombinant human protein, truncated. [00522] In some preferred embodiments the seeding of step xii) is performed until the seeded enriched mature SC-RPE are near confluence, for example, for a culture expansion time of about 3-10 days (e.g., 3-4 days). [00523] In some preferred embodiments, methods of the disclosure can further comprise a method for preparation of a Frozen SC-RPE Drug Product from SC-RPE mature cells of the disclosure. The method can comprise xvi) thawing a vial of the intermediate frozen SC-RPE stock and further expanding SC-RPE Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT by seeding the cells with on cultureware coated with any one of the SC-RPE substrate in the sense of the disclosure, using an RPE-MMM or RPE-MM in the sense of the disclosure and xvii) harvesting the SC-RPE by using protease and frozen vials of cells at a desired density and amount are prepared by freezing in cryopreservation medium (preferably CryoStor® CS2, CryoStor® CS5, or CryoStor® CS10). [00524] Preferably in the method for preparation of a Frozen SC-RPE Drug Product from SC- RPE mature cells of the disclosure, the SC-RPE substrate in step xv) is a recombinant human truncated vitronectin (CTS-Vitronectin (VTN-N) recombinant human protein, truncated) [00525] .Preferably the seeding of step xv) is performed with liquid alpha MEM, without phenol red and with nucleosides, 1X CTS-B27 XenoFree, 1X CTS-N2, 2 mM taurine, 100 ^M NEAA, and 55 nM hydrocortisone, 5 ^M Y-27632, 2-4 ng/mL bFGF, and 0.1% DMSO). [00526] Preferably the harvesting of steps vi) can be performed when the cultures reach near confluence, which can, in some embodiments, be obtained within about 3-10 days (e.g., about 3-4 days). [00527] In several embodiments, the SC-RPE derivation method of the disclosure uses a xeno- and serum-free RPE medium that promotes efficient SC-RPE derivation and is comprised of reagents that are commercially available or can be readily sourced in cGMP-grade. [00528] The efficiency or yield of the SC-RPE production after maturation can most easily be determined by assessing the percentage of cells in the culture that are pigmented using macroscopic whole culture imaging. The degree of pigmentation achieved is sufficiently high such that it can readily be identified by direct observation and eyeball estimates generally suffice to assess the success of SC-RPE derivation for different cell line/differentiation/media- substrate combinations. For more quantitative analysis automated image analysis can be used by empirical adjustment of detection threshold settings such that the pigmented regions of the culture are clearly identified, as would be understood by a skilled person. [00529] In some embodiments, the yield can be determined by detecting the SC-RPE cells with a degree of pigmentation of the culture of about 50% or higher. In some of those embodiments it is expected that such a degree corresponds to overall yields stated in the disclosure for the most preferred embodiments for all subsequent steps. For some cell lines the yield can be Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT determined by detecting the SC-RPE cells with a degree of pigmentation of the culture of about >80%, depending on the intended use of the SC-RPE cells as will be understood by a skilled person. [00530] In some embodiments, the production of RPE during throughout the derivation process can most readily be non-destructively detected by the appearance of intracellular golden-brown to black pigment upon observation using brightfield microscopy or by macroscopic observation on a suitable background (e.g., white background) using indirect illumination (see Figure 1). The only other pigmented cells in mammals are melanocytes, iris pigmented epithelial cells, and some catecholaminergic neurons such as pigmented dopaminergic cells of the substantia nigra, none of which are produced in significant number using methods and conditions described here within. The earliest that pigment can be observed is around two weeks after the switch from stem cell medium to the differentiation medium, but most often substantial quantities are not seen until after the switch to RPE Maturation Medium. Typically, the pigment will first appear in a few cells and then “spread out” and intensify over the course of 1-2 months. In principle, the relative amount of pigmentation and percentage of pigmented culture area can be quantified by image analysis or by scanning light absorbance readings at wavelength between 480-600 nm; however this can be challenging due the common formation of cell multilayers which negatively affect optics. In practice manual estimates of the percentage of pigment cells generally suffice. Alternatively, a sacrificial well of cells can be harvested by protease digestion and the percentage of pigmented cells can be estimated using flow cytometry based on the property of increased side scatter (see Figure 7) or light absorption due to the presence of melanosomes or melanin. In addition, the amount and maturity of RPE can be non-destructively assessed by quantification of RPE specific factors secreted into the culture medium using immunoassays. Most notable in this regard is pigment epithelium-derived factor (PEDF) which is the product of the SERPINF1 gene and is one of the most abundantly expressed genes in mature RPE. Furthermore, RPE can be identified and quantified based on the expression of RPE genes coding for proteins involved in carrying out unique RPE function such as regulation of RPE specific gene expression (LHX2, OTX2, RAX, PAX6, MITF), pigmentation (PMEL, TYR, TYRP1, DCT), retinoid cycle (LRAT, RPE65, ALDH1A3, RBP1, RDH5, RDH10), RPE:RPE cell adhesion (TJP1, CDH19, CLDN19), ion channel (BEST1, TRPM1, TRPM3), or phagocytosis (MERTK, ITGAV). When detected using antibodies and immunocytochemistry or flow cytometry, both the relative number of RPE and Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT overall expression level of markers can be quantified. When detected using gene-based methods, such as quantitative PCR or RNA sequencing, relative amounts of expression can be determined. In general, differentiation yields more than 25% can give reasonable yields of SC- RPE when the optimal culture conditions described here within are used for isolation and expansion, but higher yields are always preferable. When determinations of relative amounts of SC-RPE are made using methods that determine average culture expression levels, higher levels are preferable and comparison to a standard such as differentiated primary RPE or previously isolated pure SC-RPE (both of which are available from commercial sources) can be helpful. [00531] In several embodiments, the SC-RPE derivation method of the disclosure allows completing the process of SC-RPE derivation in less than 90 days. [00532] In several embodiments, the SC-RPE derivation method of the disclosure requires at most three different medium switches with no other intermediate manipulations to complete the process. [00533] In several embodiments, the SC-RPE derivation method of the disclosure generates highly pigmented SC-RPE with a mature phenotype. [00534] In several embodiments, the SC-RPE derivation method of the disclosure allows use of standard 2-dimensional cell culture methods. [00535] In several embodiments, the SC-RPE derivation method of the disclosure provides a complete manufacturing pipeline for the large-scale production of clinical-grade stem cell- derived retinal pigment epithelial (SC-RPE) cells from a variety of stem cell lines. [00536] In several embodiments, the SC-RPE derivation method of the disclosure allows an improved cell-based therapeutic compliant pipeline for large-scale manufacture of high purity, high fidelity SC-RPE for use in the treatment of degenerative eye disorders involving RPE degeneration or loss of function. At current scale of 20-40 million starting stem cells this process can produce enough SC-RPE for over 14,000 doses (100,000 cells) in a single run in the time span of approximately four months with no secondary expansion, over 100,000 doses with one round of secondary expansion, and over 900,000 doses with two rounds of secondary expansion. [00537] In some embodiments the pipeline can comprise of four distinct processes: Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT - a collection of differentiation protocols, all of which employ the xeno- and serum- free SC-RPE differentiation medium and the RPE Maturation Medium that both promotes RPE differentiation and maturation, from which a suitable method can be identified for a given stem cell line; - a process for purifying differentiated SC-RPE based on the expression of a novel set of cell surface markers and/or the presence of melanin through the SC-RPE enrichment method of the disclosure; - a process for the production of frozen stocks of SC-RPE that use a novel combination of medium additives to preserve the RPE phenotype through the preparation of frozen SC-RPE stock method of the disclosure; and - an optimized procedure, that also employs the forementioned medium additives, which allows for increased final yields by culture expansion by suppressing cell replication dependent persistent epithelial-to-mesenchymal transition and promoting the development of a mature RPE phenotype through the SC-RPE expansion method of the disclosure. [00538] Depending on the grade of reagents employed it can be used for manufacture of clinical-grade cellular drug substance or to produce high-quality research-grade cells as will be understood by a skilled person. [00539] All or parts of the method can be automated using technology known in the art. Cell culturing, manipulation, sorting, etc. can be performed by machines in order, for example, to streamline the process for generating larger tissues or multiple tissue samples. Examples include Tecan Labwrex™, Miltenyi Biotec MACSQuant^® Tyto^® cell sorter, Pall Allegro™ STR Bioreactor, and RBI LabDroid™ robotics, as will be understood by a skilled person. [00540] In some embodiments, the present disclosures comprises SC-RPE cells obtainable or obtained by the SC-RPE differentiation method, the SC-RPE maturation method, the SC-RPE enrichment method, the SC-RPE expansion method, or the SC-RPE derivation of method of the disclosure. In particular SC-RPE cell populations obtained with the methods and systems of the disclosure can have a percentage purity level of SC-RPE differentiated cells and/or SC- RPE mature cells, in the obtained cell population increased with respect to SC-RPE cells obtained in outcome corresponding differentiation methods, maturation methods, enrichment methods expansion methods, and/or derivation methods as will be understood by a skilled Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT person upon reading of the present disclosure. [00541] SC-RPE cells are described obtainable or obtained by the SC-RPE differentiation method, the SC-RPE maturation method, the SC-RPE enrichment method, the SC-RPE expansion method, or the SC-RPE derivation of method of the disclosure. As a result of the use of these methods the population of obtained SC-RPE has a number of salient features. More specifically: 1) The differentiation method allows for the identification of best conditions to maximize yield for a given stem cell line. This maximization of yield contributes to improved purity levels and phenotype homogeneity; 2) The maturation method also contributes to improved yields and subsequently improved purity levels and phenotype homogeneity; 3) In addition, as a result of its effect on pigment accumulation the maturation method enables and/or enhances the use of cell sorter instruments for selection based on pigmentation, a hallmark functional feature of RPE, which by their nature allows for improved purity and phenotype homogeneity; 4) By virtue of its ability to obtain SC-RPE by quantitative selection based on multiple cell surface markers and the RPE functional property of pigmentation using a cell sorting instrument, the enrichment method further contributes to improved purity and phenotype homogeneity, especially in contrast with traditional methods of SC-RPE enrichment employing manual microdissection, selective protease-based enrichment, bulk separation, or growth rate-based enrichment; 5) The expansion method, and in particular its use of the RPE Maintenance Medium of the disclosure, results in SC-RPE with improved phenotype homogeneity and enhances the development and maintenance of the functional RPE phenotype (gene expression, morphology, pigmentation, establishment of tight junction), especially in cases where final yields are increased by substantial expansion of the SC-RPE; 6) As a result of the combinatorial effects of the individual methods, the SC-RPE derivation of the method of the disclosure can provide SC-RPE with purity levels >99% and homogeneous phenotype as can be determined by analysis of the expression of RPE markers and non-RPE markers as can be determined using methods such as flow cytometry, microscopy, and gene expression profiling, as would be understood by a skilled person. [00542] In some embodiments of the present disclosure SC-RPE differentiation, maturation, maintenance, expansion, enrichment and/or derivation can advantageously be performed using 2-dimensional cell culture as will be understood by a skilled person. For example, in some embodiments employing 2-dimensional culture, multilayers of cells on top of the culture surface can form, such as in the case of SC-RPE differentiation. In embodiments involving Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT culture of enriched SC-RPE the cells grow as a monolayer. This contrast with 3-dimensional culture where cells are grown in penetrable gels, complex nanostructures that the cells can grow into, embryoid bodies (small clumps of stem cells), and organoids (large groups of differentiated cells that a have features that approximate an in vivo tissue or organ). [00543] In some embodiments, the SC-RPE cells obtained by the methods of the present disclosure can be comprised in a composition together with a compatible vehicle. [00544] The term “vehicle” as used herein indicates any of various media, salt solutions, or freezing medium. SC-RPE cells herein described that are comprised in the composition as an active ingredient. In particular, the composition including the SC-RPE cells can be used in one of the methods or systems herein described for regenerating a RPE of an individual. [00545] In some embodiments, the vehicle is a pharmaceutically acceptable vehicle and the composition is a pharmaceutically acceptable composition. [00546] As used herein, the term “pharmaceutically acceptable” means not biologically or otherwise undesirable, in that it can be administered to a subject without excessive toxicity, irritation, or allergic response, and does not cause unacceptable biological effects or interact in a deleterious manner with any of the other components of the composition in which it is contained. [00547] Suitable vehicles comprise buffered basic salt solutions (e.g., Dulbecco’s phosphate buffered saline, Earle’s salt solution, Intraocular Balanced Salt Solution), simple culture media (e.g., MEM, ^-MEM, DMEM), tissue storage solution (e.g., Optisol, Optisol GS, Kerasave, Eusol-C), freezing media (e.g, CyroStor CS5 or CyroStor CS2), or complete culture media such as those described in the present disclosure (e.g., RPE-MM, RPE-MMM, RPE-DM1, or RPE-DM2). In some embodiments, the SC-RPE cells can be in a suspension, attached to a substrate, or within a gel as will be understood by a skilled person. [00548] In embodiments of the disclosure, the SC-RPE cells obtained by method of the disclosure can be administered in a method of treating degenerative eye disorders involving RPE degeneration or loss of function an individual. [00549] The term “individual” or “subject” or “patient” as used herein includes vertebrates that comprise a retinal pigment epithelium such as mammals and more particularly human beings. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00550] In particular, methods of the disclosure can be used to treat individuals who have, who are suspected of having, or who can be at high risk for developing one or more health conditions or disorders for which SC-RPE cells are known or expected to have therapeutic effect. [00551] Exemplary conditions that can be treated with SC-RPE cells obtained with methods and systems of the disclosure comprise age related macular degeneration (AMD) for example by treating AMD using SC-RPE transplantation; using allogenic or autologous SC-RPE and/or SC-RPE patch transplants. [00552] Further exemplary conditions that can be treated with SC-RPE cells obtained with methods and systems of the disclosure comprise, retinal degenerative diseases (RDD), including age-related macular degeneration (AMD), Stargardt’s macular dystrophy (SMD), Best’s disease (BEST), and some forms of retinitis pigmentosa (RP) and inherited retinal degenerations (IRD). [00553] Further exemplary conditions that can be treated with SC-RPE cells obtained with methods and systems of the disclosure comprise, diabetic retinopathy and in Gardner syndrome which is characterized by FAP (familial adenomatous polyps), osseous and soft tissue tumors, retinal pigment epithelium hypertrophy and impacted teeth as will be understood by a skilled person. [00554] The systems described herein can be provided in the form of kits of parts. In kit of parts for performing any one of the methods herein described, the SC-RPE media, SC-RPE substrate, additional culture media, reagents, compositions and/or cultureware herein described, can be included in the kit alone in the presence of additional labels for the related detection as well as additional components identifiable by a skilled person. [00555] In a kit of parts, the SC-RPE media, SC-RPE substrate, additional culture media, reagents, compositions and/or cultureware herein described and additional reagents identifiable by a skilled person are comprised in the kit independently, or possibly included in a composition together with suitable vehicle carrier or auxiliary agents whenever applicable in accordance with the present disclosure. [00556] Additional components can include labels, reference standards, and additional components identifiable by a skilled person upon reading of the present disclosure. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00557] In embodiments herein described, the components of the kit can be provided with suitable instructions and other necessary reagents, in order to perform the methods here disclosed. The kit will normally contain the compositions in separate containers. Instructions, for example written or audio instructions, on paper or electronic support such as tapes, CD- ROMs, flash drives, or by indication of a Uniform Resource Locator (URL), which contains a pdf copy of the instructions for carrying out the method, will usually be included in the kit. The kit can also contain, depending on the particular method used, other packaged reagents and materials (e.g., wash buffers and the like). [00558] The serum-free SC-RPE methods and system herein described and related cells, compositions, methods and systems herein described can be used in various applications alone and/or in combination with additional agents in as will be understood by a skilled person in medical and/or research including basic biology research. [00559] Further details concerning the serum-free SC-RPE methods and system herein described and related cells, compositions, methods and systems will become more apparent hereinafter from the following detailed disclosure of examples by way of illustration only with reference to an experimental section. EXAMPLES [00560] The serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods and systems herein disclosed are further illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting. [00561] In particular, the following examples illustrate exemplary methods and systems for providing differentiated and/or mature SC-RPE cells. A person skilled in the art will appreciate the applicability and the necessary modifications to adapt the features described in detail in the present section, to additional cells, compositions, methods and systems according to embodiments of the present disclosure. [00562] The materials used in the exemplary embodiments reported in this section are reported in Table 9 below with the corresponding catalog number. Table 9 Reagents used in experimental of the disclosure Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Table 9 Reagents used in experimental of the disclosure Example 1: Preparation of Complete CTS-E8 [00563] Complete CTS-E8 medium is prepared by mixing 500 mL of the CTS-Essential 8 ^TM base medium with 10 mL of the CTS-Essential 8^TM Supplement per the directions of the manufacturer and 500 ^L of 1000X normocin is added per 510 mls of medium. Once prepared Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Complete CTS-E8 ^TM has a shelf life of 2 weeks when stored at 4^{^}C. For longer term storage aliquots of Complete CTS-E8 ^TM should be kept frozen -20^{^}C [43]. Example 2: Preparation of RPE Differentiation Medium 1 (CTS-KO-DMEM/20%CTS- KOSR Medium) [00564] A RPE Differentiation Medium 1 (CTS-KO-DMEM/20%CTS-KOSR Medium) was prepared by supplementing the base medium KnockOut™ DMEM with KnockOut™ Serum Replacement Medium, GlutaMAX™, nonessential amino acid supplement, and ^- mercaptoethanol. [00565] In particular, in the experiments of the present example, CTS-KO-DMEM/20%CTS- KOSR was prepared according to the manufacturer’s instructions by combining 389 mL of CTS-KnockOut ^TM DMEM, 100 mL of CTS-KnockOut^TM SR XenoFree Medium, 5 mL CTS- GlutaMAX™, 5 mL 100X NEAA (non-essential amino acids), 935 ^L 55 mM ^- mercaptoethanol, and 500 ^L 1000X normocin. [00566] The RPE-DM1 so prepared was stable for at least 10 days at 4^{^}C. Smaller volumes of the medium can be prepared by freezing sub-aliquots of the KnockOut^TM SR Xeno-Free Medium and adjusting the volume of the other components accordingly. [00567] Variations in the formulation the RPE-DM1 of this example comprise substitution of glutamine for GlutaMAX™ and/or elimination of normocin or substitution with another antibiotics, as will be understood by a skilled person upon reading of the present disclosure. [00568] Additional variations expected to be functional comprise changes in the amounts and relative ratios of the various components to the extent that the final formulation supports stem cell viability and growth with expected impact on RPE yield, as will also be understood by a skilled person upon reading of the present disclosure (see e.g. Table 3 and related RPDM1 discussion, such as in paragraphs ([00143], [00146], [00147], [00148], and [00149]). [00569] Further variations expected to be functional and possibly advantageous comprise addition of a buffer suitable for tissue culture such as 10-20 mM HEPES to counter acidification of the Complete KO-DMEM Medium by the cells which can lead to significant cell death if the medium is not replaced with sufficient frequency or volume. Example 3: Preparation of RPE Differentiation Medium 2 (X-VIVO™ 10/XF-B27) Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00570] A RPE Differentiation Medium 2 of the present disclosure (X-VIVO™ 10/XF-B27 Medium) was prepared by supplementing the commercially available complete medium X- VIVO™ 10 with the commercially available B27 Supplement. [00571] In the experiment of the present example X-VIVO™ 10/XF-B27 Medium was prepared by adding 10 mL of the B27 Supplement, XenoFree and 500 ^L of Normocin to 500 mL of X-VIVO™ 10 (TheraPeak cGMP with rhTransferrin). X-VIVO™ 10/XF-B27 Medium is stable for at least 10 days at 4^{^}C. Smaller volumes of the medium can be prepared by freezing smaller aliquots of the B27 Supplement, XenoFree and adjusting the volume of the other components accordingly. Example 4: Preparation of X-VIVO10/XF-B27+Activin A-SC-RPE Differentiation Medium [00572] The X-VIVO™ 10/XF-B27 RPE Differentiation Medium 2 was prepared by supplementing the complete medium X-VIVO™ 10 with B27, Xeno-Free Supplement and Activin A. [00573] The X-VIVO™ 10 Media formulations used in this example is the X-VIVO™ 10 Media commercially available at the filing date of the present disclosure with product number BEBP02-055Q by manufacturer Lonza. [00574] The B27 supplement used in this example is B27 Supplement, XenoFree commercially available by manufacturer ThermoFisher Scientific at the filing date of the present disclosure with product number A1486701. [00575] The Activin A used in the present example is PeproGMP Recombinant Human Activin A commercially available at the filing date of the present disclosure from manufacturer PeproTech with PeproTech # GMP120-14E. [00576] In particular, X-VIVO™ 10/XF-B27+ActivinA RPE Differentiation Medium 2 was prepared freshly by adding Activin A to X-VIVO™ 10/XF-B27 Medium to a final concentration of 140 ng/mL. [00577] In particular, X-VIVO™ 10/XF-B27+ActivinA Medium was prepared freshly by adding 1.4 ^L of 100 ng/^L Activin A to 1 mL of X-VIVO™ 10/CTS-B27 Medium prepared as described in Example 3 to a final concentration of 140 ng/mL. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00578] 100 ng/^L Activin A is prepared by dissolving 50 ^g of PeproGMP Recombinant Human Activin A in 500 ^L of sterile filtered water to yield final concentration of 100 ng/^l. Store aliquots at -80^{^} C and minimize freeze/thawing. [00579] Lower dosages of Activin A are expected to result in a decrease in RPE differentiation. Significantly higher dosages are expected to possibly result in poor yields as Activin A can inhibit proliferation in some cell types with an EC50 (dose of a medication that produces a desired pharmacologic effect in 50% of the studied patient population that takes the medication) of approximately 1 ng/mL. Other members of the TGFB superfamily such as but not limited to, TGFB1, GDF11, and GDF8 are expected to be able to substitute for Activin A when used at comparable concentration. Addition of a ROCK inhibitor (such as Y-27632 (1- 25 ^M, possibly 2-20 ^M)) would be expected to augment the activity of Activin A. Example 5: Stem Cell Culture [00580] Stem cells were propagated and cultured using standard methodologies that support the maintenance of a nondifferentiated state. [00581] In the experiments reported, in this example this was done under feeder-free conditions using Complete Essential 8 Medium and truncated vitronectin (rh-VTN-N) coated cultureware. Stem cells were passaged at subconfluence as cell clumps of roughly 5-20 cells per clump using EDTA based solutions (Versene), with procedures identifiable by a skilled person [43]. Example 6: SC-RPE Differentiation Method and System: Substrate coating [00582] In an exemplary, differentiation method and related system, cultureware can be coated with a substrate of the present disclosure before contacting the stem cells with the SC-RPE Differentiation medium of the disclosure such as the differentiation media of Example 2 or Example 3 or Example 4. [00583] An exemplary substrate coating step which can be performed in connection with methods and systems here described was performed with CTS-DPBS and exemplary substrate recombinant truncated vitronectin (rh-VTN-N), according to the exemplary protocol outlined below. [00584] T25 and T75 cell culture flasks for stem cell expansion are coated with 3 and 9 mL of Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT 5 ^g/mL of CTS-rhVTN-N diluted in CTS-DPBS for at least 16 hr at 4^{^}C. For SC-RPE differentiation, 6-well culture plates are coated with 2 mL/well of 5 ^g/mL CTS-rhVTN-N or Cultrex Human Vitronectin diluted in CTS-DPBS or 5 ^g/mL Biolaminin 521 CTG diluted in CTS-DPBS, calcium, magnesium for at least 16 hr at 4^{^}C. Substrate coated culture flasks and plates can be stored for at least 1 week at 4^{^}C. [00585] A skilled person will understand that the previous protocol can be performed replacing culture substrate, recombinant truncated vitronectin (rh-VTN-N), with culture substrate laminin 521 (LAM 521) or plasma vitronectin (PC-VTN). Additionally, base culture media for stem cell culture such as CTS-Essential 8 or KnockOut^TM DMEM can be substituted for CTS- DPBS or CTS-DPBS, calcium, magnesium as will be understood by a skilled person. Example 7: SC-RPE Differentiation Method and System: Thawing and plating stem cell bank before differentiation [00586] In an exemplary, differentiation method and related system, the method and system can encompass a thawing and/or plating step to perform contacting of a starting stem cell line with a cell cultureware preferably already coated with a substrate of the present disclosure (see e.g., Example 6). [00587] An exemplary thawing and plating step which can be performed in connection with methods and systems here described, was performed with the exemplary Complete CTS-E8 Medium and Complete CTS-E8 + 10 ^M Y-27632 Medium, according to the exemplary protocol outlined below. [00588] Rapidly thaw a single vial at room temperature with gentle mixing until it is approximately 90% thawed. This can be most easily accomplished using a room temperature water bath and frequent mixing by swirling or inversion of the vial. Each frozen vial contains the equivalent number of cells from 10 cm² of a 60-80% confluent master or working cell bank culture in 0.5-1 mL of freezing medium. Once the frozen cells are nearly thawed, very gently mix by pipetting and transfer the cell suspension to a 15 mL conical tube containing 10 times the volume of Complete CTS-E8 Medium. Then collect the thawed cells by centrifugation for 3 min at 300 × g and remove the supernatant by aspiration. [00589] Next, resuspend the cell pellet in 5 mL of Complete CTS-E8 + 10 ^M Y-27632 Medium by first flicking the tube to soften the pellet followed by gentle pipetting using a 5 mL Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT serological pipette. [00590] Plate the resulting cell suspension in the CTS-rhVTN-N coated T25 flask of Example 6 and culture the cells in a cell culture incubator at 37^{^}C and 5% CO₂. After approximately 24 hours post-plating the cells are fed daily with 5 mL of Complete CTS-E8 (without Y-27632) using a 90-100% medium exchange until they reach 60-90% confluence (generally 1-3 days depending on specific cell line growth rates). Example 8: SC-RPE Differentiation Method and System: One-to-six stem cell culture expansion before differentiation [00591] In an exemplary, differentiation method and related system, the method and system can encompass an optional cell expansion step of plated stem cells performed before contacting the cells with a differentiation medium of the disclosure (e.g., following a thawing and/or plating steps exemplified in Example 7). [00592] An exemplary cell expansion protocol which can be performed in connection with methods and systems here described, was performed according to the exemplary protocol outlined below. [00593] Upon reaching 60-90% confluence gently rinse the T25 flask culture twice with 5 mL of CTS-DPBS. After removal of the last CTS-DPBS rinse, then add 2 mL of CTS-Versene^TM to the flask and place the culture in the cell culture incubator at 37^{^}C and 5% CO₂. [00594] After 5 min examine the cells under the microscope to determine the extent of dissociation of the cells. If holes are apparent in the stem cell colonies, gently triturate the cells 3 to 5 times with a 5 mL serological pipette and transfer to a 15 mL conical tube. If no or few holes are apparent, continue to incubate the cells in the cell culture incubator, observe every minute or two, and harvest the cells after holes have formed as described above. [00595] After the cells have been collected, examine the flask under the microscope to determine the percentage of cells remaining attached to the plate. If fewer than one third of the cells remain attached, gently wash the flask two times with 3 mL of CTS-DPBS to recover any remaining suspended cells and combine the washes with the original cell suspension. If more than one third of the cells remain attached, add 3 mL of CTS-DPBS to the flask and harvest the remaining attached cells using a cell scraper. After scraping, add the resulting cell suspension to the previously recovered cells, wash the flask once with 3 mL of CTS-DPBS, Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT and add the wash to the previously collected pool of cells. If the cells lift as intact colonies in CTS-Versene^TM without significant hole formation, transfer the suspended colony solution to a 15 mL conical tube and gently triturate the colony suspension 3-5 times using a 5 mL serological pipette. Recover any remaining colonies in the flask by washing the flask 2 times with 3 mL of CTS-DPBS and combine the washes with the previously collected and triturated cell suspension. [00596] Once the cells have been harvested using any of the above variations of the method, collect the cells by centrifugation for 3 min at 300 × g, remove the supernatant by aspiration, soften the pellet by flicking the tube, and resuspend the cells in 1 mL of Complete CTS-E8 Medium + 10 ^M Y-27632 by gently triturating 3 times using a P1000 pipette. Finally, add 5 mL of Complete CTS-E8 Medium + 10 ^M Y-27632 to the cell suspension and gently mix the resulting cell suspension using a 5 mL serological pipette. [00597] Next, place a drop of the resulting cells on a microscope slide or hemocytometer and roughly determine the cell clump size distribution by observation on a microscope. For optimal cell suspensions most cells should be in clumps ranging in size of 5-20 cells per clump. [00598] Should most of the cell clumps be larger than desired, the cells can be triturated further as described above in 6 mL of Complete CTS-E8 Medium + 10 ^M Y-27632 until the optimal clump size distribution is achieved. [00599] After obtaining a cell suspension of the desired clump size distribution, add 3 mL of cells suspension each of two CTS-rhVTN-N coated T75 flasks containing 12 mL of Complete CTS-E8 Medium + 10 ^M Y-27632 medium, distribute the cells by manual rocking of the dish, immediately place the flask in the cell culture incubator at 37^{^}C and 5% CO2, and leave undisturbed overnight. After approximately 24 hours post-plating the cells are fed daily with 15 mL of Complete CTS-E8 Medium (without Y-27632) using a 90-100% medium exchange until they reach 60-90% confluence (generally 1-3 days depending on specific cell line growth rates). Example 9: SC-RPE Differentiation Method and System: Plating and culturing of the stem cells on SC-RPE differentiation substrates of the disclosure [00600] In differentiation methods and related systems herein described, starting stem cells are contacted with one of the SC-RPE differentiation substrates of the disclosure in stem cell Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT medium and cultured until reaching a desired degree of confluence and/or desired length of time. [00601] In particular, stem cells are plated as a mixture of single cells and 2-25 cell clumps at a density of 5000 to 70,000 cell/cm² of culture surface area and maintained in 2-3 mL of stem cell medium per 10 cm² of culture surface until they reach 50% to just confluent (typically for 3-4 days). The optimal density, clump size, extent of confluence, and length of culture time in stem cell medium to achieve the best yield of SC-RPE can vary depending on stem cell line, substrate, and RPE differentiation medium as would be understood by a skilled person up reading of this disclosure. In general, stem cell suspensions where the cells are predominantly in clumps of 2-10 cells, are plated at cell densities of 20-40,000 cell/cm², are cultured for 3-4 days, and 60-90% confluence at the end of the culture period result in satisfactory results. [00602] Upon reaching 60-90% confluence the expanded stem cells T75 flask cultures of iPSC lines #032411, #120111, and #110211 and ESC lines H1, H9 and Shef1 were harvested using CTS-Versene^TM as described in Example 8 to achieve a cell suspension in 20 mL of Complete CTS- E8 Medium + 10 ^M Y-27632 with a clump size distribution where the majority of the cells were either single cells or in clumps of 2-10 cells and the concentration of cells was determined by cell counting. [00603] The stem cells were then plated in 2 mL of Complete CTS-E8 Medium + 10 ^M Y- 27632 into 6-well multiplates wells coated with 10 ^g/well of Cultrex Human Vitronectin at a density 400,000 cell/well or wells coated with 10 ^g/well CTS-rhVTN-N or Biolaminin 521 CTG at a density of 200,000 cell/well. After about 24 hours the plating medium was replaced with 2 mL of Complete CTS- E8 Medium (without Y-27632). Thereafter, the cells were fed daily with 2 mL of Complete CTS-E8 Medium for 3-4 days after plating depending on the growth rate of the specific cell line. Example 10: SC-RPE Differentiation Method and System: Contacting the stem cells with an SC-RPE differentiation medium of the disclosure [00604] In differentiation methods and related systems herein described, a starting stem cell is contacted and in particular incubated with a differentiation medium selected from RPE-DM1 or RPE DM2 media. [00605] The differentiation can occur spontaneously as the result of natural signals that arise Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT during culture, or it can be semi-directed by the addition of Activin A to the medium that promotes RPE differentiation by activation of specific signal transduction pathways. [00606] In particular the starting stem cells on a cultureware coated with any one of the substrates of the disclosure can be contacted with an effective amount (e.g., from 2 mL to 3 mL of each differentiation medium per 10 cm² of culture surface area) for a time (e.g. for about 5 to 60 days) to obtain differentiation of the stem cells into an SC-RPE cells of the disclosure. [00607] Different protocols including different contacting times, differentiation media, and amounts will be effective for different cell types as will be understood by a skilled person upon reading of the disclosure. [00608] Specific protocols can be identified with a related method of the disclosure where a stem cell line is combined with candidate combinations of differentiation media and differentiation substrate according to a candidate protocol to perform differentiation, and the candidate combination and candidate protocol providing SC-RPE cells with a desired pigmentation and characteristic phenotype can then be selected. [00609] For spontaneous differentiation with iPSC lines #032411, #120111, and #110211 and ESC lines H1, H9 and Shef1, the cells were contacted with CTS-KO-DMEM/20%CTS-KOSR Medium or X-VIVO™ 10/XF-B27 Medium when most replicate cultures for a given cell line:substrate combination reached about 60-90% confluence (Days-4 to -5). Cells cultured in CTS-KO-DMEM/20%CTS-KOSR Medium were then fed daily with 2 mL of medium until Day-31 to obtain spontaneous differentiation. Cells cultured in X-VIVO™ 10/XF-B27 Medium were fed daily with 2 mL until Day-7 or Day-8 (depending on the day of first contacting) for 3 days and thereafter every 2 days with 2 mL of medium until Day-31. (See Example 19 below.) [00610] For Activin A semi-directed differentiation with iPSC lines #032411, #120111, and #110211 and ESC lines H1, H9 and Shef1 the cells were contacted with X-VIVO™ 10/XF- B27 Medium when most cultures for a given cell line:substrate combination reached about 60- 90% confluence (Days-4 to -5) and maintained with a daily feeding of 2 mL until Days-7 or - 8 (3 days total). Cultures were then contacted with 2 mL of X-VIVO™ 10/XF-B27+ActivinA Medium for a total of 8 days and fed every other day. Thereafter the cells were fed every 2 days with 2 mL of X-VIVO™ 10/XF-B27 Medium until Day-31. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00611] In exemplary protocols the contacting can be performed by replacing an existing cell culture medium with one differentiation medium of the disclosure, preferably by performing a 90-100% medium exchange. Lesser percent medium exchange amounts can be possible provided the medium does not become excessively acidified and the cells remain viable. Example 11: Preparation of RPE Maturation Medium [00612] The RPE Maturation Medium was prepared by supplementing the base medium ^- MEM with additional non-essential amino acids (glycine, alanine, asparagine, aspartate, glutamate, proline, and serine), insulin, transferrin, selenite, putrescine, progesterone, serum albumin, galactose, catalase, superoxide dismutase, D,L-alpha-tocopherol, D,L-alpha- tocopherol acetate, glutathione, ethanolamine, linoleic acid, linolenic acid, biotin, carnitine, vitamin A, taurine, triiodothyronine, corticosterone and hydrocortisone. The components galactose, catalase, superoxide dismutase, D,L-alpha-tocopherol, D,L-alpha-tocopherol acetate, glutathione, carnitine and hydrocortisone are expected possibly not to be essential. At least one of the NEAAs is expected not to be essential. [00613] In particular, in the experiments of the present example the RPE Maturation Medium of the disclosure was prepared as follows: 10.08 g of powdered medium ^-MEM medium and 2.2 g of sodium bicarbonate were dissolved in tissue culture grade H2O to a final volume of about 900 mL. Once dissolved, 10 mL of 100X penicillin/streptomyocin solution, 1 mL of normocin solution, 10 mL of N1 Supplement, 10 mL of 100X non-essential amino acids solution (NEAA), 20 mL of 50X Xeno-Free B27 Supplement, 0.25 g taurine, and 8 ^L of 2.5 mg/ml hydrocortisone were added, the volume was brought to a final volume of 1L and the complete SC-RPE Medium was filter sterilized. [00614] The ^-MEM Powder used was purchased from Corning catalog number #50-012-PC in the nucleoside-free formulation, However, ^-MEM from other manufactures with slight variations in formulation or those containing nucleosides are expected to be suitable and can be substituted directly. Liquid ^-MEM formulations of the media can be used as well in which case the additional media components added to 1L of the base medium or the volume of the base medium can be reduced such that a final volume of complete RPE Maturation Medium after addition of the additional components is 1L. When using alternative ^-MEM formulations the addition of sodium bicarbonate may not be required and the media may need to be supplemented with glutamine, depending on the specific formulation obtained. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00615] Additional modifications known or expected (italics) to be functional comprise the substitution of N1 with N-2, CTS-N2 or N-2 Plus, N-2-Max, cGMP N-2-Max and the substitution of B27 Supplement, XenoFree with various B27 Supplement equivalents (e.g., B27, B27 Plus, CTS-B27, or NeruoCult SM1) or derivatives (e.g., NS21, N21, N21-Max) can be used in place of B27. Since the components of N1 type supplements and hydrocortisone are all contained within B27, it is expected their addition can be omitted by increasing the amount of B27-based supplement. The use of antibiotics is also expected to be optional and dependent on the specific embodiments. [00616] For example, for cGMP (current Good Manufacturing Practices) production omit the use of antibiotics (normocin, penicillin, and streptomycin) and substitute phenol-free media bases for the media bases with phenol red. For research grade production non-cGMP grade substitutes for the various media components can be used; however, differentiation method preferences and yields can vary. Example 12: SC-RPE Maturation Method: Contacting differentiated SC-RPE cells with RPE Maturation Medium [00617] As discussed in previous sections of the disclosure differentiated SC-RPE can be advantageously maturated by incubating the cells with an RPE Maturation Medium such as the one of Example 11. [00618] In particular, differentiated cells such as the exemplary SC-RPE obtained in outcome of Example 10, were contacted with RPE Maturation Medium beginning on Day-31 of the differentiation method for a total time of about 30 days to 90 days with 3 mL/well every 3 days using a 90-100% medium exchange until they are ready to be harvested in view of the desired level of pigmentation and junction formation. [00619] For the exemplary iPSC lines #032411, #120111, and #110211 and ESC lines H1, H9, and Shef1 while longer maturation times can result in a modest increase in the percentage of pigmented RPE in the culture as well as an increase in the amount of pigment, the RPE become progressively harder to harvest over time due to tight junction formation which can adversely affect final yields. Example 13: SC-RPE Differentiation and Maturation Methods and Systems: Contacting expanded stem cell with an SC-RPE differentiation medium and RPE Maturation Medium Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00620] iPSC lines #032411, #120111, and #110211 and ESC lines H1, H9, and Shef1 were differentiated with differentiation protocols comprising the steps outlined in Examples 6 to 10 with various combinations of - One of culture substrate plasma vitronectin (PC-VTN), recombinant truncated vitronectin (rh- VTN-N), or laminin 521 (LAM521) - One of culture media CTS-KO-DMEM/20%CTS-KOSR Medium, X-VIVO™ 10/XF-B27 Medium prepared as indicated in Examples 2 and 3 and X-VIVO™ 10/XF-B27+ActivinA Medium treatment prepared as indicated in Example 4, and then contacted with the RPE Maturation Medium of Example 11 with the protocol exemplified in Example 12, as shown in Figure 1. Each cell line:differentiation medium:substrate combination was done using 3-6 replicates. [00621] The results illustrated in Figure 1 show that different stem cell lines have unique differentiation media and extracellular matrix preferences. [00622] In particular, the results in Figure 1 show that the preferred culture medium for differentiation of iPSC #303411 cells is X-VIVO™ 10/XF-B27 in combination with full- length vitronectin (PC-VTN) with slightly lower yield of RPE being obtained with the combination of CTS-KO-DMEM/20%CTS-KOSR medium and full-length vitronectin (PC- VTN). [00623] In particular, the results in Figure 1 show that the preferred culture medium for differentiation of iPSC #120111 cells is CTS-KO-DMEM/20%CTS-KOSR in combination with full-length vitronectin (PC-VTN). [00624] In particular, for iPSC #110211 cells the results in Figure 1 show that the preferred culture medium for differentiation is CTS-KO-DMEM/20%CTS-KOSR in combination with full-length vitronectin (PC-VTN). [00625] In particular, the results of Figure 1 show that the preferred culture medium for differentiation and maturation of H1 cells is the X-VIVO™ 10/XF-B27 medium in combination with vitronectin (PC-VTN) or laminin 521 (LAM521). In particular, the results of Figure 1 show that for H1 cells a differentiation according to the disclosure performed with X-VIVO™ 10/XF-B27 medium and laminin 521 (LAM521) provide the preferred degree and homogeneity of the cells pigmentation following maturation performed with the RPE Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Maturation Medium of the disclosure. [00626] The results of Figure 1 also show that preferred culture medium for H9 cells is the CTS-KO-DMEM/20%CTS-KOSR medium, in combination with recombinant truncated vitronectin (rh-VTN-N) or laminin 521 (LAM521). In particular, the results of the results of Figure 1 show that for H9 cells a differentiation according to the disclosure performed with CTS-KO-DMEM/20%CTS-KOSR and recombinant truncated vitronectin (rh-VTN-N) provides a preferred degree and homogeneity of pigmentation following maturation performed with the RPE Maturation Medium of the disclosure. [00627] The results of Figure 1 further show that more than one culture medium of the disclosure can be effectively used for differentiation and/or maturation Shef1 cells. All culture media tested resulted to be effective in the differentiation of Shef1 cells as would be understood by a skilled person upon review of Figure 1. [00628] In particular, effective combinations for differentiation of Shef1 cells are - the KO-DMEM/20%CTS-KOSR medium, in combination with any one of vitronectin (PC- VTN)), recombinant truncated vitronectin (rh-VTN-N) and laminin 521 (LAM521) - the X-VIVO™ 10/XF-B27 medium in combination with laminin 521 (LAM521); and - X-VIVO™ 10/XF-B27 medium + Activin A treatment in combination with of vitronectin (PC- VTN)), or recombinant truncated vitronectin (rh-VTN-N). [00629] Beyond the combinations of medium and substrate that provide the best results for each cell lines other combinations also give substantial quantities of RPE that would be expected to give rise to significant quantities of SC-RPE after enrichment. All such combinations along with the preferred combinations are indicated by a black image border. [00630] In addition to demonstrating that varying RPE Differentiation Medium and substrate combinations of the disclosure result in optimal SC-RPE differentiation efficiencies in a cell line dependent fashion, experiments were performed to demonstrate the utility of the use of the RPE Differentiation Medium and Maturation Medium in combination. Example 14: SC-RPE Derivation without the use of RPE Maturation Medium and Methods: Contacting expanded stem cells with an RPE differentiation medium alone [00631] Shef1 cells were differentiated with differentiation protocols comprising the steps Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT outlined in Examples 6 to 10 with various combinations of - One of the culture substrates plasma vitronectin (PC-VTN), recombinant truncated vitronectin (rh-VTN-N), or laminin 521 (LAM521) and - One of culture media CTS-KO-DMEM/20%CTS-KOSR Medium, X-VIVO™ 10/XF-B27 Medium prepared as indicated in Examples 2 and 3 and X-VIVO™ 10/XF-B27+ActivinA Medium treatment prepared as indicated in Example 4 and - Without contacting RPE Maturation Medium (No RPE-MM: DM) or - With contacting RPE Maturation Medium of Example 11 with a protocol exemplified in Example 12 (No RPE-MM: DM+MM) as shown in Figure 2. [00632] The results in Figure 2 illustrate that when using Shef1 cells efficient production of SC-RPE when using CTS-KO-DMEM/20%CTS-KOSR as the differentiation medium is only achieved after contact with RPE Maturation Medium (DM+MM), irrespective of the substrate. [00633] In contrast, when using X-VIVO™ 10/XF-B27 as the differentiation medium the percentage of pigmented cells in the culture is no different with (DM+MM) or without (DM) contact with RPE Maturation Medium. However, although the yield is no different, the intensity of the pigmentation is greater when RPE Maturation Medium is used. In addition, while not apparent in the grey scale images, after contact with RPE Maturation Medium the color of the pigmentation is black and punctate as opposed golden brown and diffuse without RPE Maturation Medium, suggesting that contact with RPE Maturation Medium results in the production of more mature melanosomes. Example 15: SC-RPE Derivation without the use of RPE Differentiation Medium: Contacting expanded stem cell with an RPE Maturation Medium alone [00634] iPSC #110211 cells were differentiated with differentiation protocols comprising the steps outlined in Examples 6 to 10 with various combinations of - One of the culture substrates plasma vitronectin (PC-VTN), recombinant truncated vitronectin (rh-VTN-N), or laminin 521 (LAM521) and - RPE Maturation Medium of Example 11 with a protocol exemplified in Example 12 (No RPE- DM: MM) or - One of culture media CTS-KO-DMEM/20%CTS-KOSR Medium, X-VIVO™ 10/XF-B27 Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Medium prepared as indicated in Examples 2 and 3 and X-VIVO™ 10/XF-B27+Activin A Medium treatment prepared as indicated in Example 4 and subsequent contact with the RPE Maturation Medium of Example 11 with the protocol exemplified in Example 12 (No RPE- DM: DM+MM). as shown in Figure 2. For cultures using the RPE Differentiation Media of the disclosure the cultures were subsequently contacted with RPE Maturation Medium of Example 11 on Day- 31 according to the protocol exemplified in Example 12. Cultures differentiated in RPE Maturation Medium were maintained in said media for the entire period of RPE differentiation and maturation. [00635] The results in Figure 2 illustrate that when iPSC #110211 cells are differentiated in RPE Maturation Medium alone numerous pigmented foci of pigmented cells form on both full- length vitronectin (PC-VTN) and laminin 521 (LAM521). Similar pigmented foci also appear on recombinant truncated vitronectin (rh-VTN-N); although, to a lesser extent. However, in no case did the use of RPE-Maturation Medium by itself result in substantial percentage of the cells becoming pigmented. Rather the pigmented foci appear with in the first few weeks after contacting at a time when no pigmentation was observed in the cultures contacted with the different RPE Differentiation Media and no further spread of pigmentation was seen thereafter (data not shown). Example 16: SC-RPE Maturation Method: Contacting with RPE Maturation Medium at different times after contact with RPE Differentiation Medium [00636] Shef1 cells were plated on one of the culture substrates plasma vitronectin (PC-VTN), recombinant truncated vitronectin (rh-VTN-N), or laminin 521 (LAM521) in Complete CTS- E8 Medium of Example 1 according to the methods of Examples 6 to 9. At Day-3 after plating the cells on each of the substrates were the contacted with - RPE Maturation Medium of Example 11 (RPE-MM at Day-3) or - the RPE Differentiation Medium, CTS-KO-DMEM/20%CTS-KOSR, prepared as indicated in Example 2, and the cells were maintained in accordance with the protocols outlined in Example 10. At Days 31 and 59 cells in differentiation medium on the three different substrates were contacted with Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT RPE Maturation Medium of Example 11 (RPE-MM at Day-31 and RPE-MM at Day-59 respectively) and the cells were maintained according to the protocol described in Example 12. A single set cultures on each of the three substrates was maintained in CTS-KO- DMEM/20%CTS-KOSR for the duration of the experiment using daily feedings (No RPE- MM). [00637] As shown in Figure 3, with exception of cells on laminin 521 (LAM521) cells differentiated using CTS-KO-DMEM/20%CTS-KOSR medium and subsequently contacted with RPE Maturation Medium on Day-31 or Day-59 had comparable levels SC-RPE differentiation. Cells grown on laminin 521 and switched to RPE Maturation Medium at Day- 59 had a greater degree of pigmentation than those switched at Day-31; however, the degree of difference is within the range of differences often seen with experimental replicates. Therefore, it is uncertain whether this apparent increase in yield is due to the difference in the timing of the contacting with RPE Maturation Medium or simple experimental variability. [00638] As shown previously differentiation in RPE Maturation Medium alone (RPE-MM at Day-3) or the omission of contacting with RPE Maturation Medium (no RPE-MM) resulted in poor yields of SC-RPE. Example 17: SC-RPE Maturation Methods: Passaging of differentiated SC-RPE prior to SC-RPE enrichment Shef1 cells were plated on recombinant truncated vitronectin (rhVTN-N) or laminin 521 (LAM521) in Complete CTS-E8 Medium of Example 1 according to the methods of Examples 6 to 9 and cells were differentiated using CTS-KO-DMEM/20%CTS-KOSR as prepared in Example 2 with the optional Activin A treatment of Example 4 using the protocols described in Example 10. On Day-31 the resulting differentiated cells were contacted with RPE Maturation Medium of Example 11 according to the protocol exemplified in Example 12 for 56 days. [00639] On Day-87 after the initial plating, the resulting cultures which had low to moderately low numbers of pigmented SC-RPE were harvested by dissociation using proteases using the methods of Example 27 and the resulting cell suspensions were plated at 100,000 cell/cm² on laminin 521 coated culture wells and maintained for 47 days using RPE Maturation Medium of Example 11. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00640] From the whole well images and the 4-100X brightfield microscopy images of the cultures before and after passaging it is clearly evident that intermediate passage after contact with RPE Maturation Medium results in a very substantial increase in the number of pigmented cells. Comparable results can be obtained with shorter contact times (about 2 weeks) with RPE Maturation Medium prior to passage (see Example 46 and Figure 27) and can be an effective means to rescue cultures that begin to lose their attachment to the substrate, which otherwise results in substantial reduction in number of SC-RPE that can be recovered after differentiation and maturation. Interestingly, passaging after only 3 days of contact with RPE Maturation Medium has been associated with a decrease yield of SC-RPE compared to not passaging. Thus, suggesting that in addition to supporting RPE phenotype maturation RPE Maturation Medium possibly promotes RPE differentiation as well when applied directly to cells at the end of the period of contacting with RPE differentiation medium. Example 18: Effects of RPE-MM supplements and base medium on SC-RPE maturation. [00641] Additional experiments were performed on the cell line Shef1 to determine whether maturation of differentiated cells can be obtained in the absence of key components of the RPE Maturation Medium of the disclosure. [00642] In particular, in a first set of experiments Shef1-RPE that were differentiated using the CTS-KO-DMEM/20%CTS-KOSR:PC-VTN and X-VIVO™ 10/XF-B27:rhVTN-N protocols were plated at moderate density (40,000 cell/cm²) in laminin 521 coated wells of a 96-well microplate and maintained for 58 days in RPE Maturation Medium (RPE-MM) and RPE-MM missing one of its additives. Each condition was done in triplicate. 40,000 cell/cm² is a suboptimal density for RPE maturation and was chosen to maximize the possible effects of the individual medium components. [00643] The results of this first set of experiments illustrated in Figure 5 show that each of the RPE Maturation Medium components contribute to RPE phenotype maturation and pigmentation. The omission of supplementary NEAA and taurine have a modest effect and while their addition results in more homogenous RPE phenotype they are clearly not essential. Given this result it is expected that lower amounts of these two supplements can be effective. B27, whose omission has the most profound effect of RPE maturation and in some cases is essential, has all of the components of N1 at similar concentrations as well as corticosterone, a slightly less potent equivalent of hydrocortisone. The findings that removal of N1 and Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT hydrocortisone reduce the effectiveness or RPE Maturation Medium suggest that the concentration of at least one of the N1 components and hydrocortisone is insufficient in B27. A simplified formulation of RPE Maturation Medium is expected to be possible by doubling the amount of B27 and eliminating the addition of N1 and hydrocortisone, providing that higher concentration of the other components of B27 do not inhibit RPE maturation. [00644] In a second set of experiments Shef1-RPE that were differentiated using the CTS-KO- DMEM/20%CTS-KOSR:PC-VTN (RPE-DM1 SC-RPE) and X-VIVO™ 10/XF-B27:rhVTN- N (RPE-DM2 SC-RPE) protocols were plated at 40,000 cell/cm² in laminin 521 coated wells of a 96-well microplate and maintained for 58 days in RPE Maturation Medium (RPE-MM), CTS-KO-DMEM/20%CTS-KOSR (RPE-DM1) differentiation medium, and XVIVO X- VIVO™ 10/XF-B27 (RPE-DM2) differentiation medium, and CTS-KO-DMEM/20%CTS- KOSR, and X-VIVO™ 10/XF-B27, KO-DMEM, and KO-DMEM/F12 with all of the RPE- MM supplements and additives. Each condition was done in triplicate. [00645] The results of the second set of experiments reported in Figure 6 show that ^-MEM is a preferred base medium for RPE maturation and pigmentation. As shown in the illustration of Figure 6 both ^-MEM (RPE-MM) and X-VIVO™ 10 (RPE-DM2) based media formulations support RPE pigmentation. Media prepared using KO-DMEM or KO-DMEM/F12 as the base do not support pigmentation, although the addition of the additional RPE-MM components does result in a slight increase in pigmentation for CTS-KO-DMEM/20%CTS-KOSR. B) 20X brightfield images of cells maintained in RPE-MM and X-VIVO™ 10/XF-B27 media. While X-VIVO™ 10/XF-B27 supports pigmentation, the pigment is diffuse, the cell:cell junctions are less defined, and the apical-basal cell height (not shown) is greater than in cells cultured in RPE Maturation Medium. Further supplementation of the X-VIVO™ 10/XF-B27 with RPE- MM additives results in slight improvement in morphology as would be understood by a skilled person upon reading of the present disclosure. Example 19: SC-RPE Differentiation and Maturation Protocol [00646] Exemplary steps of an SC-RPE differentiation and maturation method are schematically illustrated in Figure 7, different steps comprising - Thaw and Plate Stem Cell Bank, - Primary stem cell expansion, Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT - Cell differentiation, and - Cell maturation. [00647] Exemplary protocols for each of the above steps are illustrated in Example 6, Example 7, Example 8, Example 9, Example 10, and Example 12 respectively as will be understood by a skilled person upon reading of the present disclosure. Example 20: SC-RPE Differentiation and Maturation Protocol [00648] Exemplary steps of an SC-RPE differentiation and maturation method are schematically illustrated in Figure 8, different steps comprising - Performing cell harvesting and plating and control of length of the period of growth to achieve a desired cell confluency, followed by - Performing SC-RPE cell differentiation and by - Performing SC-RPE cell maturation. [00649] Exemplary protocols for each of the above steps are illustrated in Examples 21 to Example 24. Example 21: SC-RPE Differentiation and Maturation Protocol: Stem Cell Harvesting and Plating. [00650] The exemplary SC-RPE differentiation and maturation method schematically illustrated in Figure 7, and related system, encompass a stem cell harvesting and plating step, which can be performed as outlined below. [00651] As shown in the schematic illustration of Figure 8, on Day-0 a Stem Cell Harvesting and Plating step can be performed. Using a 60 to 80% confluent T-75 flask of stem cells, rinse the cells twice with 15 mL of CTS-DPBS and discard the rinses. Then add 4 mL of CTS- Versene^TM to the flask and incubate the cells in the cell culture incubator at 37^{^}C and 5% CO₂. [00652] After 5 min examine the cells under the microscope to determine the extent of dissociation of the cells. If holes are apparent in the stem cell colonies, gently triturate the cells 3 to 5 times with a 5 mL serological pipette and transfer the cell suspension to a 15 mL conical tube. If no or few holes in the colonies are apparent, continue to incubate the cells at 37^{^}C, observing every minute or two, and harvest the cells after holes have formed as described above. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00653] After the cells have been collected, examine the culture flask under the microscope to determine the percentage of cells remaining attached to the plate. If fewer than one third of the cells remain attached, gently wash the flask two times with 4 mL of DPBS to recover any remaining suspended cells and combine the washes with the original cell suspension. [00654] If more than one third of the cells remain attached, add 4 mL of CTS-DPBS to the flask and harvest the remaining attached cells using a cell scraper. After scraping, add the resulting cell suspension to the previously recovered cells, wash the flask once with 4 mL of CTS-DPBS, and add the wash to the previously collect pool of cells. If the cells lift as intact colonies in CTS-Versene^TM without significant hole formation, transfer the suspended colony solution to a 15 mL conical tube. Then gently triturate the colony suspension 3-5 times using a 5 mL serological pipette. Finally, recover any remaining colonies in the flask by washing the flask 2 times with 4 mL of CTS-DPBS and combine the washes with the triturated cell suspension. [00655] Once the cells have been harvested using any of the above variations of the method, collect the cells by centrifugation for 3 min at 300 × g. After removal of the supernatant, resuspend the cells by flicking the pellet and gentle trituration 3 times in 1 mL of Complete CTS-E8 Medium + 10 ^M Y-27632 using a P1000 pipette. Then dilute the resuspended and triturated cells using 9 mL of Complete CTS-E8 Medium + 10 ^M Y-27632, determine the cell density using automated or manual cell counting, and estimate cell clump size distribution by visual inspection. For optimal cell suspensions it is recommended that most cells are in clumps ranging in size of 2-10 cells per clump. Should most of the cell clumps be larger than desired, the cells can be triturated further until the optimal clump size distribution is achieved. Finally, add 2 mL of Complete CTS-E8 Medium + 10 ^M Y-27632 to each well of a 6-well multiwell plate that has been coated with CTS-rhVTN-N, Cultrex Human Vitronectin, or Biolaminin 521 CTG and seed each well with 200,000 cells for CTS-rhVTN-N and Biolaminin 521 CTG coated wells and 400,000 cells for the Cultrex Human Vitronectin coated wells using the appropriate volume of the cell suspension, distribute the cells by manual rocking of the dish, immediately place the dish in the cell culture incubator at 37^{^}C and 5% CO2, and leave undisturbed for 24 hours. Example 22: SC-RPE Differentiation and Maturation Protocol: Stem Cell Expansion in Stem Cell Medium [00656] The exemplary SC-RPE differentiation and maturation method schematically Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT illustrated in Figure 8, and related system, encompass a step of control of the length of the period of stem cell growth in stem cell medium without Y-27632 to achieve a desired cell confluency prior to the initiation of differentiation as outlined below. [00657] As shown in the schematic illustration of Figure 8, at Day-1 & 2: Removal of Y-27632 and feeding can be performed. Feed each well daily with Complete CTS-E8 Medium. (2 mL/well) using a 90-100% medium exchange until most of the replicates cultures for a given cell:media:substate combination reach approximately 50-90% confluence. Substrate choice will dramatically affect confluency at this stage. Cells grown on laminin 521 form loosely packed large colonies with little space between colonies. Cells grown on full-length vitronectin form tightly packed smaller colonies with more space between colonies. [00658] For some stem cell line:substrate combinations this stage will take 2 days. However, slow growing cell line:substrate cultures can take an additional 1-2 days to achieve the desired cell density. In cases where the growth period is longer than 3-days the timing of the following differentiation and maturation steps can be shifted accordingly. Due to the variability in growth rates and in combination with cell plating technical error it is expected that some replicates or even some replicate sets can be 90% to just confluent on the intended day of proceeding to the next step. These do not need to be discarded and can be carried forward, especially when carrying out a method screening. Example 23: SC-RPE Differentiation and Maturation Protocol: Contacting stem cells with differentiation media. [00659] The exemplary SC-RPE differentiation and maturation method schematically illustrated in Figure 8, and related system, encompass a step of contacting stem cells with differentiation media, which can be performed as outlined below. The values reported (such as timing of media changes, target clump size distribution, and stem cell plating densities) are the actual values used or obtained. These parameters can be adjusted, as described in the detailed description of the methods of this disclosure, to obtain the best possible SC-RPE differentiation efficiency for a given stem cell line:differentiation medium:substrate combination. [00660] As shown in the schematic illustration of Figure 8, at Days 4-6: Switch to CTS-KO- DMEM/20%CTS-KOSR Medium (RPE-DM1) or X-VIVO™ 10/XF-B27 Medium (RPE- DM2) can be performed. Feed each well daily with CTS-KO-DMEM/20%CTS-KOSR or X- Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT VIVO™ 10/XF-B27 media (2 mL/well) using a 90-100% medium exchange. [00661] At Day-7 to Day-14 – Spontaneous Differentiation: For cultures being differentiated by spontaneous differentiation and maintained in CTS-KO-DMEM/20%CTS-KOSR Medium (RPE-DM1) continue to feed daily using 2 mL of medium per well. For spontaneously differentiated cells maintained in X-VIVO™ 10/XF-B27 Medium (RPE-DM2) feed every other day using 2 mL of medium per well. [00662] At Day-7 to Day-14 – Semi-Directed Differentiation: For semi-directed differentiation cells remove 90-100% of the medium from a culture being maintained in CTS- KO-DMEM/20%CTS-KOSR or X-VIVO™ 10/XF-B27 media and replace with 2 mL of the corresponding medium plus 140 ng/mL Activin A (RPE-DM1+Activin A or RPE- DM2+Activin A). Feed every day using 2 mL of CTS-KO-DMEM/20%CTS- KOSR+ActivinA or every other day with X-VIVO™ 10/XF-B27+ActivinA media per well. [00663] At Day-15 to Day-31: For cells maintained in CTS-KO-DMEM/20%CTS-KOSR or CTS-KO-DMEM/20%CTS-KOSR+ActivinA media continue to feed the cells daily with CTS- KO-DMEM/20%CTS-KOSR Medium (2 mL/well). For cells maintained in X-VIVO™ 10/XF- B27 or X-VIVO™ 10/XF-B27+ActivinA media, beginning on Day-15, feed each well every 2 or 3 days with 2 or 3 mL XVIVO X-VIVO™ 10/XF-B27 Medium using a 90-100% medium exchange. Example 24: SC-RPE Differentiation and Maturation Protocol: Contacting differentiated SC-RPE with the RPE Maturation Medium [00664] The exemplary SC-RPE differentiation and maturation method schematically illustrated in Figure 8, and related system, encompass a step of contacting stem cells with RPE Maturation Medium of the disclosure, which can be performed as outlined below. [00665] As shown in the schematic illustration of Figure 8, From Day 31 on: Feed with RPE Maturation Medium (3 mL/well) every 3 days using a 90-100% medium exchange until they are ready to be harvested for SC-RPE enrichment. In general, SC-RPE can be enriched between Days-60 and -90, depending on the progression of RPE maturation which can vary between stem cell lines. While longer maturation times can result in a greater percentage of pigmented RPE in the culture, the RPE become progressively harder to harvest over time due to tight junction formation which can adversely affect final yields. The ideal length of SC-RPE Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT enrichment should be determined empirically, and generally corresponds to the time when the increase in the percentage of clearly pigmented cells is near plateau or just reaches plateau. The percentage of pigmented culture area can be determined based on whole culture images collected over time and quantification of using automated image analysis, as would be understood by a skilled person, but estimates of the time of plateau based on simple observation of the time course images are generally sufficient. [00666] Due to well-to-well fluctuations in the yield of SC-RPE, it is recommended that at least 2 replicates are used for purpose of determining the optimal differentiation medium-substrate combination for a given stem cell line. For large-scale SC-RPE production using a single stem cell line and method as many as forty 6-well differentiation plates can be managed by a single technician in a non-cGMP research setting. [00667] From a single vial of frozen stem cells this protocol typically generates sufficient cells to produce at least twenty 6-well culture plates when using CTS-rhVTN-N or Biolaminin 521 CTG as the substrate or ten 6-well culture plates when using Cultrex Human Vitronectin as the substrate. [00668] The use of alternative cultureware formats than described in the methods is possible; however, the cell numbers and volumes of medium should be scaled accordingly based on the culture surface area. Furthermore, due to potential edge effects and possible differences in gas exchange rates changes culture format can impact final differentiation yields. Additionally, the concentration of the media components, the amount of substrate used for coating, the clump size and plating density, and the timing of the media switches should not be considered as absolutes. Rather they are recommended values that generally yield satisfactory results. Adjustment to the recommended values can result in improved SC-RPE yields and quality or protocol preference for a specific stem cell line. For some stem cell line:differentiation protocol combinations there can be substantial yields of SC-RPE differentiation, but the cells detach from the substrate over time. In these cases, it can be possible to rescue the culture by dissociating the detached cell sheets with proteases such as trypsin, TrypLE™, or Accumax™ and replating the cells on matrix coated wells in RPE Maturation Medium as described in Example 17. Maintaining floating sheets of cells for the entire differentiation and maturation protocol is difficult since they can be easily loss when feeding and even when it has been done without loss of the sheet it has always been associated Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT with poor yields of enriched SC-RPE, even when there is a substantial degree of SC-RPE differentiation. If there are other differentiation medium combinations that give better SC-RPE differentiation efficiencies, it is advisable that they be used to obtain SC-RPE. If this is not the case, or if there are other user specific reasons for favoring one differentiation medium:substrate combination over another, then using an intermediate passage during the maturation phase is expected to result in favorable outcomes. This can be done on a replicate culture basis when detachment is observed or for maintaining a common protocol it can be done on all replicates when one replicate starts to detach. Example 25: Exemplary complete SC-RPE Differentiation and Maturation Protocol [00669] An exemplary protocol to determine optimal medium:substrate combinations using the three different exemplary media and three different exemplary substrate coatings of the present disclosure is outlined below. Stem Harvest, Plating, and Growth [00670] Day 0 – Stem Cell Harvest and Plating: Using subconfluent stem cell cultures (50- 90% confluent) harvest the cells while still maintaining cell clumps. Optimally, most of the clumps are within the range of 5-20 cells per clump. Typically, this is done using an EDTA- based cell dissociation reagents, but physical- or protease-based methods are expected to work as well. After generation of the cell clump suspension, transfer the cells to the stem cell medium of choice (typically, Essential 8 Medium is used but others that support feeder free stem cell culture, such as mTESR can be used as well). The cell/clumps are then plated onto separate cultureware coated with one of the three different substrates (Cultrex Human Vitronectin, rhVTN-N, or Biolaminin 521). For method screening and using Essential 8 Medium, it is recommended that the cells be plated at a density of 40,000 cell/cm² on Cultrex Human Vitronectin which yields smaller more tightly packed colonies, and that they should be seeded at 20,000 cell/cm² on both rhVTN-N and Biolaminin 521, which promote cell flattening and larger colony size. Generally, Y-27632 is added to the medium as well to enhance cell survival, but it is not essential. [00671] Days 1-3(4): Culture maintenance in stem cell medium. Feed the cells with the stem cell medium of choice in keeping with the requirements of the medium. Generally, this will be every day but for some media, such as media with stabilized bFGF, feeding every other day Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT can be acceptable. If Y-27632 was used in the plating medium it is recommended that the cells be fed with medium without Y-27632 the following day. Continue to feed the cultures with stem cell medium until most cultures for a given substrate reach 50-90% confluence. This will generally occur on Day-4 or -5, depending on the cell line and stem cell medium. If it falls outside of this range, seeding at a different cell density can be considered. ] Contacting with Differentiation Medium [00672] Days 4 or 5: – Switch to RPE-DM1 or RPE-DM2. On Day-4, for cultures where most replicates have reached 50-90% confluence switch one-third of the cultures to being fed with CTS-KO-DMEM/20%CTS-KOSR and two-thirds of the cultures to being fed with X-VIVO™ 10/XF-B27. For any cultures with growth rates about half that of the fastest growing cultures carry out the same media switches on Day-5. For the first 3 days after the switch, it is recommended that the cells be fed daily. Longer times can result in culture loss due to medium acidification or depletion. [00673] Day-5(or 6) to Day-14(or 15) – Spontaneous Differentiation: For cultures being differentiated solely by spontaneous differentiation and maintained in CTS-KO- DMEM/20%CTS-KOSR Medium it is recommended to continue to feed the cultures daily. For spontaneously differentiated cells maintained in X-VIVO™ 10/XF-B27 the cells can be fed every other day. [00674] Day-7(or 8) to Day-14(or 15) – Activin A Semi-Directed Differentiation: For semi- directed differentiation, 3 days after switching to X-VIVO™ 10/XF-B27 Medium switch half of the cultures to the corresponding media plus Activin A. For the next 6 days it is recommended that cells be fed every other day for cells in X-VIVO™ 10/XF-B27+ActivinA for a total of 4 feedings. Thereafter, switch the cells back to being fed with their original media (X-VIVO™ 10/XF-B27). The effects of starting the Activin A treatment earlier or a longer treatment period are not known. Optionally, one can also add the Activin A treatment step to cells in CTS-KO-DMEM/20%CTS-KOSR. This is generally not necessary to find a protocol that works for a given cell line, but has been seen to improve yield in certain CTS-KO- DMEM/20%CTS-KOSR Medium:substrate combinations for some cell lines. [00675] Day-15(or 16) to Day-31 – Continuation of SC-RPE Differentiation: Continue to feed the cultures using their corresponding medium. For cells maintained in CTS-KO- Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT DMEM/20%CTS-KOSR it is recommended that they are fed daily. For cells maintained in X- VIVO™ 10/XF-B27 the cultures can be fed every other day. Contacting with Maturation Medium [00676] From Day 32(or 33) on – RPE Maturation: Feed all cultures with RPE Maturation Medium. The frequency of feeding can be based on the extent of medium acidification. With normal volumes of culture medium this is typically at least every 3 days for the first week or two after the medium switch. Thereafter feeding twice a week (every 3-4 days) is possible. The optimal differentiation protocol is best identified by the determining the percentage of pigmented cells in each culture or alternatively by quantifying the amount of PEDF in the media as would be understood by a skilled person upon reading of this disclosure. In general, SC-RPE can be enriched between Days-60 and 90 with optimal results for further evaluation or use. [00677] Early enrichment is associated with lower yields and possible difficulties in isolating the RPE depending on the mode of enrichment. Longer maturation times can result in a greater percentage of pigmented RPE in the culture, but the RPE become progressively harder to harvest over time which can adversely affect final yields. The ideal length of SC-RPE enrichment for a given cell line:medium:substrate combination should be determined empirically by monitoring the extent of pigmented cell accumulation. [00678] Optional passages are expected to be applicable depending on the specific embodiments of the methods herein described as will be understood by a skilled person. For some cell line:medium:substrate combinations the cells will spontaneously detach as sheets. While it is possible to continue to maintain the cultures as floating sheets the yield of RPE is often poor, even when there is a significant amount of pigmented RPE. In this case it is possible to rescue the culture at the time of detachment, preferably before full detachment, by dissociating the sheets using proteases (e.g., trypsin, TrypLE™, Accutase™, or Accumax™) and culturing the resulting cell suspensions at a preferable passage ratio of 1:2 to 1:4 using a substrate of choice (e.g., laminin or vitronectin, Matrigel, etc.) and RPE Maturation Medium according to the harvesting and recovery protocols of the SC-RPE enrichment methods of the disclosure. After about 30-60 days the rescued SC-RPE can be enriched. Alternatively, when it is known based previous experience that detachment is a problem with particular cell:medium:substrate it is possible to proactively passage the cells at the time of the switch to Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT RPE Maturation Medium, preferably in the days following the switch. [00679] Additional media, cell lines and substrates can be used performing modifications of the above protocol as would be understood by a skilled person upon reading of this disclosure. For example for production of SC-RPE using different numbers of media or coatings or for production of SC-RPE using a specific media:substrate combination adjust the protocol accordingly. [00680] Also note that the feeding frequencies listed are not absolutes. The should be frequent enough to keep up with the acidification and depletion of the medium, but too frequent of changes can remove cell-derived factors that play a role in the differentiation process. For this reason it is recommended that base media formulations that include a pH indicator (phenol red) are used during the method screening process. When necessary, it is possible to extend the feeding frequency by a day by increasing the volume of medium, but this is not recommended as a routine practice. Example 26: Maturated SC-RPE Enrichment Method and System [00681] An exemplary method to perform an SC-RPE Enrichment method of the disclosure is schematically illustrated in Figure 9. [00682] Exemplary steps of an SC-RPE Enrichment method of the disclosure are schematically illustrated in Figure 9, in which the steps comprise - Harvesting maturated SC-RPE, followed by - Cell staining of maturated SC-RPE, by - Cell sorting of stained maturated SC-RPE, and by - Post sorting enriched maturated SC-RPE recovery [00683] Exemplary protocols for each of the above steps are illustrated in Examples 27 to Example 30. Example 27: SC-RPE Enrichment Method and System: Cell Harvesting [00684] The exemplary SC-RPE Enrichment method schematically illustrated in Figure 9, and related system, encompass a step of cell harvesting of the disclosure, which can be performed as outlined below. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00685] General Guidelines: At 60-90 days post plating for differentiation, the cells should be ready for harvest. At this time there should be substantial accumulation of pigment and sufficient expression of the cell surface markers used for sorting. The goal of the cell harvesting is to generate maximal yields of single cell suspensions of cells for sorting. The trick is to strike a balance between efficiently dissociating the adherent cell sheets and not killing the cells in the process by overly rough handling or excessive protease digestion. Up to a point, it is best to let the proteases do the work rather than using manual force. The times and the extent of manual force will need to adjusted depending on the cell line, the specific differentiation protocol, and run-to-run variability in the differentiation process. If you see a lot of gooeyness due to cell lysis you are being too rough, or the digestion times are too long. For 120 cm² of differentiated cell surface area expect to allow 8-12 hours for harvesting and sorting using sorting rates of 3000 cells/sec. [00686] Aspirate the media and wash the cells twice with a volume of CTS-DPBS equivalent to the volume of medium that they were being fed. Add 1 mL of CTS-TrypLE™ per 10 cm² culture surface area and incubate at 37^{^}C for 30-60 min with intermittent mixing by swirling. Check on the progress of lifting after 30 minutes and continue until the cells round up or lift off the plate as sheets on their own or with very gentle trituration and/or teasing of the edges of the cell sheet using a pipette tip or pipette of appropriate size. Do not suck up an intact cell sheet into a pipette, as it can be very difficult to expel it. The greater the number and the more pigmented and mature the RPE are, the harder it will be to lift. [00687] Once the cells have been detached from the dish, transfer the cell suspension and/or cell sheet to a 50 mL tube conical bottom centrifuge tube. The cells from an entire 6-well plate or three T25 flasks can be combined in one tube. Add an equal volume of CTS-DPBS to each culture well or flask and gently triturate to recover remaining cells. Transfer the resulting cell suspension to the tube containing the first harvest of cells and gently mix by inversion of the tube. (In the event that many pigmented cells remain attached, a fresh aliquot of CTS- TrypLE™ can be added and incubated for an additional 30 min. Then repeat the cell collection process. If a large number of RPE still remain attached it is expected tobe necessary to increase the strength of trituration, or to use a cell scrapper, but expect that there will be a decrease in cell viability and yield.) Add CTS-DPBS to the pooled cell suspension to bring the volume to 50 mL and recover the harvested cells by centrifugation at 300 × g in a swinging bucket rotor for 5 minutes at room temperature. Remove the supernatant carefully using a serological pipette Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT taking care not to disrupt the pellet. If you begin to suck up cells, stop and expel the cells back into the tube. Add enough CTS-DPBS to nearly fill the tube and gently suspend the pellet. (The goal here is to dilute out the TrypLE; it is not necessary to achieve a uniform cell suspension.) Recover the cells by centrifugation at 300 × g in a swinging bucket rotor for 5 minutes at room temperature and discard the supernatant. [00688] After the supernatant is removed, tap/flick the pellet to loosen it and add 1 mL of room temperature Accumax™ per 10 cm² of original culture surface area. Incubate at room temperature for 15-60 min with frequent mixing by manual rocking or continuous mixing on a rocking platform. The goal is to provide enough agitation to the cell suspension to disrupt cell clumps and dissociate cell sheet associated cells, but not so much agitation that you kill cells. (Accumax™ is inactivated at elevated temperatures. Do not incubate at 37^{^}C.). [00689] When a relatively homogenous cell suspension is achieved, or few pigmented cells remain associated with intact sheets filter the cell suspension through a 70 ^m cell strainer into a 50 mL conical bottom centrifuge tube. If there is a lot of pigmented gooey cell mass left, gently pick it up out of the top of the filter with a 1 mL pipetman tip and put it back in the 50 mL tube for a second round of Accumax™ digestion as just described. Also, if the cell strainer becomes clogged it is expected to be necessary to use multiple strainers. Rinse the cell strainer(s) with an equal volume of the RPE Maturation Medium. If a second round of Accumax™ digestion has been done, filter the cell suspension and rinse the filters with RPE Maturation Medium as described above. Add sufficient RPE Maturation Medium to the pooled cell suspension such that the Accumax™ is diluted by at least 6-fold. Finally, collect all cells by centrifugation at 300 × g in a swinging bucket rotor for 5 minutes at 4^{^}C. [00690] After discarding the supernatants, flick the tubes to soften the pellets and resuspend in 10 mL of RPE Maturation Medium. Count the cells and then repellet in a 15 mL conical centrifuge tube by centrifugation at 300 × g in a swinging bucket rotor for 5 minutes at 4^{^}C. Typical expected yields are in the range of 2-5 × 10⁵ cells per cm² differentiation culture. Remove and discard the supernatant. The cell pellets can be stored for a short period of time (preferably less than 1 hour) at 4^{^}C prior to beginning the staining process. Example 28: SC-RPE Enrichment Method and System: Cell Staining [00691] The exemplary SC-RPE Enrichment method schematically illustrated in Figure 9, and Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT related system, encompass a step of cell staining of the disclosure, which can be performed as outlined below. [00692] For 1.0 × 10⁷ cells prepare the following labeling mixture (For more than 1.0 × 10⁷ cells adjust volumes proportionally. For fewer cells it is recommended that no adjustments to the staining mixture volumes be made. Anti-CD49b-APC (REA188, Miltenyi #130-100-348): 10 µL Anti-CD104-PE (REA236, Miltenyi #130-123-756): 5 µL Anti-CD57-PE/Vio770 (REA769 Miltenyi #130-111-812): 10 µL RPE Maturation Medium: 175 µL [00693] Gently resuspend the cells in the appropriate volume of labeling mixture to yield a concentration of 1.0 × 10⁷ cell/200 ^L in the 15 mL tube. [00694] Incubate for 30 min at 4°C with gentle intermittent mixing by slightly flicking the tube (every 5-10 min). Avoid widespread distribution of the cells onto the walls of the tube. [00695] Add 10 volumes of RPE Maturation Medium and pellet cells for 5 min at 200 × g in a swinging bucket rotor. [00696] Discard the supernatant and resuspend the cells in 2 mL of RPE Maturation Medium per 10⁷ cells. [00697] Pellet cells for 5 min at 200 × g in a swinging bucket rotor. [00698] Discard supernatant and resuspend the cells in 2 mL of RPE Maturation Medium per 10⁷ cells. [00699] Pellet cells for 5 min at 200 × g in a swinging bucket rotor. [00700] Discard the supernatant and resuspend the cells in 300 µL RPE Maturation Medium per 10⁷ cells (For fewer than 10⁷ cells use a minimum volume of 300 µL). Alternatively, for cGMP sorting using a MACSQuant Tyto cell sorter the cells can be resuspended in an appropriate volume of MACS GMP Tyto Running Buffer that is freshly prepared by mixing MACS GMP PBS/MgCl2 Buffer and MACS GMP Tytonase according to the manufacturer’s Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT instructions. [00701] Remove cell clumps using a 5 mL round bottom polystyrene FACS tube with a 35 ^m strainer cap. Example 29: SC-RPE Enrichment Method and System: Cell Sorting [00702] The exemplary SC-RPE Enrichment method schematically illustrated in Figure 9, and related system, encompass a step of cell sorting of the disclosure, which can be performed as outlined below. [00703] Sort the cells with relative high side scatter (SSC) to forward scatter (FSC) ratio or high light absorbance depending on the capability of the sorter, negative CD49b expression, and positive CD104 and CD57 expression into 15 mL conical centrifuge tubes containing 1.5 mL of -RPE Maturation Medium using an appropriate nozzle size and event rate depending upon the capability of the sorter and preferred sort strategy (e.g., high purity single pass sort or high yield double pass sort). [00704] For sorting on a Becton Dickinson FACSAria II SORP sorter using 2-tube high purity sorting, a nozzle size of 100 ^m and event rate of 3000 should yield satisfactory results. Faster sorting rates can be achieved using smaller nozzle sizes; however, there can be a reduction in final yield due to loss of viability. The exact setting for the gates will need to be determined empirically and can vary depending on stem cell line and differentiation method. Some guidance can be gotten by comparing the labeling pattern of the pigmented (high SSC/light absorbance) and non-pigmented populations. Additional guidance can be obtained by staining with non-specific negative control antibodies and by staining negative control cells such as undifferentiated stem cells or fibroblast. See Figure 10 for an example set of cytometry scatter plots and sorting gates. [00705] Figure 10 shows results of a possible SC-RPE gating strategy to sort SC-RPE. In the experiments resulting in the illustration of Figure 10 pigmented SC-RPE are identified based on their relatively high side scatter (SSC) to forward scatter (FSC) ratio, positive expression of CD104 and CD57, and no expression of CD49b. Depending on the nature and extent of contaminating cell types, which can vary with stem cell line and differentiation protocol, other gating strategies such as Pigmentation ^ CD104/CD49b ^ CD57 can better resolve the SC- RPE from non-RPE cells. Comparing the labeling pattern of the pigmented and non-pigmented Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT populations can also aid in the decision as to the optimal sorting strategy and positioning of the gates. [00706] Sorting based on all four parameters will give the best results with respect of final purity. However, using a subset of markers can yield satisfactory results in cases of cell lines that have high differentiation efficiencies, where yield is a priority over purity, or where it is desirable to reduce cost and complexity. In general, the ranking of importance of the markers is pigmentation > CD104 > CD49b > CD57. [00707] Cell sorting based on pigmentation and cell surface markers can result in the isolation of highly pure populations of SC-RPE as shown by the illustration of Figure 11. [00708] In the experiments providing the results reported in Figure 11, at the end of the differentiation protocol the culture was dissociated with TrypLE™ and Accumax™ and the resulting cell suspensions were stained with fluorescent label antibodies directed against CD104, CD57, and CD49b. [00709] In Figure 11, Panel A the Top Row shows a Sorting based on cell surface markers alone. Single cells were identified based on forward and side light scattering (FSC & SSC) profiles and SC-RPE were sorted based on expression of both CD104 and CD57 and lack of expression of CD49b. The far-right panel shows the light scatter profiles of the CD104 Positive/CD57 Positive/CD49b Negative cells. Cells that fell within the “Pigmented” gate were small and highly pigmented. Cells that fell within the Non-Pigmented gate were larger and had little or no pigment (data not shown). In Figure 11, Panel A, the Bottom Row: Cells sorting based on pigmentation and cell surface marker expression. When all four parameters (pigmentation, CD104, CD57, and CD49b) are used a highly homogenous population of pigmented RPE are obtained with very few non-pigmented cells. While many of the non- pigmented cells with a proper RPE marker staining profile are immature RPE and will give rise to mature RPE upon subsequent culture, some are RPE in the early stages of persistent EMT and will not give rise to mature RPE and if not removed will result in an enriched SC-RPE population of lesser quality. [00710] In Figure 11, Panel B results are shown of an SC-RPE Purity analysis of cells sorted based on pigmentation and surface marker expression. After sorting the enriched SC-RPE were plated at high density and maintained in RPE Maturation Medium for 1 month. The cells were Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT then harvested by protease digestion, fixed with formaldehyde, and stained with an FITC labeled antibody directed against the RPE marker, premelanosome protein (PMEL). Example 30: SC-RPE Enrichment Method: Post Sort Recovery [00711] The exemplary SC-RPE Enrichment method schematically illustrated in Figure 9, and related system, encompass a step of cell Post-Sort Recovery of the disclosure, which can be performed as outlined below. [00712] Collect the sorted cells by centrifugation for 5 min at 200 × g in a swinging bucket rotor. Discard the supernatant and based on the number of sort events resuspend the cells in an appropriate volume of RPE Maintenance Medium (RPE Maturation Medium containing 5 ^M Y-27632, 0.1% DMSO, and 2 ng/mL bFGF) to achieve an approximate cell concentration of 10⁶ cells/ml. Determine the cell concentration by automated cell counting or using a hemocytometer and pellet the cells by centrifugation for 5 min at 200 × g in a swinging bucket rotor. Discard the supernatant and resuspend the cells in an appropriate volume of culture medium in accordance with the Experimental design. [00713] At this point the cells can be used directly for their intended purpose, but best results are achieved by allowing the cells to recover in culture for a short period of time to allow for the cells to reenter the cell cycle, to allow for the removal of cells damaged during the cell enrichment process, and to increase the yield of SC-RPE. For post-sort recovery preferably plate the cells at 75,000-150,000 cell/cm² on RPE substrate coated cultureware in 3 mL of RPE Maintenance Medium per 10 cm² of cell culture surface area. At this stage laminin 521 is preferred due to its reported inhibition of EMT, but other substrates such as truncated recombinant vitronectin, full-length vitronectin, or Matrigel^TM work as well. Thereafter, feed the cells every 2-3 days with RPE Maintenance Medium. When the cells reach near or just confluence (about 4-7 days) the cells can be harvested by protease digestion (e.g., TrypLE™, Accumax™, or trypsin) for use in their intended downstream application. Shorter times, 2-3 days, are possible but will result in the generation of fewer cells. Longer times up to about 2 weeks will result in slight increases in yield, but the cells will become increasingly more difficult to harvest due to tight junction formation and with significantly longer times the cells can be resistant to cell cycle reentry. [00714] The combination of Y-27632, DMSO, and bFGF acts to both facilitate proliferation Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT and minimize proliferation-dependent epithelial-to-mesenchymal transition. It is possible to omit bFGF from the medium or to only use Y-27632 for the first 24 hours; although one should expect to have reduced growth rates and a possible increase in mesenchymal RPE. If Y-27632 is only used for the first 24 hours, it is typically used at a concentration of 10 ^M. Using bFGF alone will increase growth rates, but there is little effect on passage associated epithelial-to- mesenchymal transition. Use of RPE Maturation Medium alone is also possible, but reduced yields can occur due to decreased survival and cell cycle reentry. Example 31: RPE Maturation Medium Supplemented for SC-RPE Cell Expansion [00715] RPE Maturation Medium of the disclosure can be supplemented with DMSO, ROCK inhibitor, and bFGF to obtain a medium optimized for cell expansion. [00716] In the first set of experiments an RPE Maintenance Medium (RPE Maturation Medium of Example 11 containing 0.1% DMSO and 2 ng/ml bFGF has been provided and tested on Shef1 cells. [00717] In particular, frozen Shef1-RPE manufactured using the CTS-KO-DMEM/20%CTS- KOSR:rhVTN-N differentiation protocol was thawed and plated in RPE Maturation Medium + 5 ^M Y-27632 according to the standard protocol. One day after the plating the medium was switched to RPE Maturation Medium, and the cells were fed again on Day-3. Four days after plating the cells were harvested by protease digestion and plated on laminin 521 coated macroporous membranes or culture plastic at 100,000 cell/cm² in RPE Maturation Medium with and without 2 ng/mL bFGF + 0.1% DMSO. The cultures were fed every 2-4 days and the bFGF/DMSO was removed from a subset of the cultures after 7, 14, and 66 days. At the indicated timepoints the amount of pigmentation in the cells plated on plastic was determined by measuring the absorbance of 510 nm light using 3×3 multipoint scan (n=1) and RPE barrier function was determined by measuring the transepithelial electrical resistance of the macroporous insert cultures (mean +/- SEM, n=3). [00718] The results illustrated in the diagrams of Figure 12A and Figure 12B show that basic FGF plus DMSO promotes pigmentation and the establishment and maintenance of RPE barrier function. In particular, the data of Figure 12A and Figure 12B indicate that treatment with bFGF/DMSO results in increased levels of pigmentation, however long-term treatment is less effective than transient treatment for 7 or 14 days. Basic FGF plus DMSO treatment also Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT improved barrier function, but in contrast to pigmentation barrier function is highest with constant treatment and withdrawal of treatment leads to a steady decrease to levels similar to cells treated for 7 or 14 days. [00719] In a second set of experiments, frozen purified iPSC #110211-RPE were thawed and plated in RPE Maturation Medium according to the standard protocol. Prior to reaching confluence they were harvested by TrypLE™ digestion and plated in matrix coated 96-well microplates at low density (4000 cell/cm²) in RPE Maturation Medium plus 0.1% DMSO, 2 ng/ml bFGF, or 0.1% DMSO + 2 ng/ml bFGF and the resulting cultures were maintained in their respective media for one month. In the absence of any treatment RPE will undergo EMT and fail to mature when plated at low density. [00720] The results reported in Figure 13 show that addition of the combination of DMSO and bFGF to RPE Maturation Medium results in more homogenous SC-RPE pigmentation. In particular, in accordance with the indications of Figure 13 the addition of DMSO by itself results in minimal pigmentation. Basic FGF by itself promotes pigmentation; however, there is significant heterogeneity within a replicate culture well as well as significant variability between replicate wells. In contrast, addition of both DMSO and bFGF results in more uniform pigmentation within each replicate culture and a concomitant increase in average replicate pigmentation among replicate cultures. Example 32: SC-RPE Cell Expansion: Contacting of maturated SC-RPE cell with RPE Maturation Medium or RPE Maintenance Medium [00721] SC-RPE cell expansion can be performed in the embodiment of the disclosure by contacting maturated SC-RPE with RPE Maturation Medium or RPE Maintenance Medium of the disclosure. [00722] In a set of experiments, performed in accordance with the schematic of Figure 14A, a frozen stock vial of iPSC #1102111-RPE was thawed and plated in RPE Maturation Medium (RPE-MM) on matrix coated culture flasks at 6333 cell/cm². After a recovery and growth period of 4 days the cells were harvested by trypsin digestion and aliquots of the cells were plated in a single well of a matrix Matrigel^TM coated 6-well multiplate at 4000 cell/cm² for serial passaging and in triplicate wells of a Matrigel^TM coated 96-well multiplate at 80,000 cell/cm² for observation of SC-RPE maturation. Cells were cultured in RPE Maturation Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Medium with 0.1% DMSO, DMSO+2 ng/ml bFGF, DMSO+5 ^M Y-27632, or DMSO+Y- 27632+bFGF (i.e. RPE Maintenance Medium). When the 6-well multiplate culture reached approximately 80% confluence they were harvested by trypsin digestion and the passaging at low density and plating for maturation at high density was repeated. Images of the 96-well cultures were collected regularly throughout the experimental period. This process was repeated for a total of six passages. [00723] The results are illustrated in the representative 4X brightfield images of Figure 14B in which an image taken on the day indicated of a replicate well of SC-RPE cultured in each of the media are shown as a function of passage. For each passage, the relative level of pigmentation at Day-34 of maturation was calculated by quantifying the gray scale intensity of each image and the value was plotted versus the cumulative number of total population doublings at each passage as determined by cell counting of the harvested cells (mean +/- SEM, n=3) as shown in illustration of Figure 14C. A table of calculated yields at a level of pigmentation equal to 90% of the level observed at Passage 1 is shown in Figure 14D. [00724] The results shown in Figure 14B to Figure 14D indicate that the continual treatment with the combination of DMSO, bFGF, and Y-27632 significantly delays the onset of proliferation associated persistent EMT and senescence and hence allows for increased yield of differentiation competent RPE. In RPE Maturation Medium with DMSO cells can undergo about 4 additional rounds of cell division after enrichment, primary expansion and freezing before they lose the capacity to mature and proliferate. As DMSO which has not been observed to have an effect on RPE proliferation associated EMT and senescence in other experiments (not shown) sets the level of possible expansion with RPE Maturation Medium. The addition of both bFGF and DMSO to RPE Maturation Medium has minimal effect on the onset of EMT but increases the number of population doublings before the onset of senescence. In contrast, Y-27632 + DMSO allows for about two additional population doublings before there is any loss in the ability of SC-RPE to mature and increases the number of rounds of cell division that can be obtained before the cells senesce to similar degree as bFGF. Despite its lack of ability to delay the onset of EMT by itself, the addition of bFGF to the combination of Y-27632 and DMSO unexpectedly results in further delay in the onset of EMT by about 4 population doublings and a delay in senescence of over 7 population doublings. Based on these observations, when limited expansion SC-RPE is needed or in cases where the use of bFGF and Y-27632 is not desired RPE Maturation Medium can be suitable medium for RPE Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT expansion. In cases where the addition of bFGF is possibly not desired, RPE Maturation Medium + Y-27632 can allow for moderate increases in the degree to which SC-RPE can be expanded and still mature. For maximal yields and minimal risk of EMT, RPE-Maintenance Medium, in particular, RPE Maturation Medium supplemented with Y-27632, bFGF, and DMSO is preferred for all culture of SC-RPE after enrichment. Furthermore, due to the unexpected effect of DMSO on RPE maturation in combination with bFGF (Example 31, Figure 13) it is preferred, but not essential, to include DMSO in the medium whenever bFGF is used. Example 33: SC-RPE Primary Expansion and Preparation of Frozen Stocks Method and System [00725] An exemplary method to perform an SC-RPE Primary Cell expansion and preparation of frozen stocks method of the disclosure is schematically illustrated in Figure 15. [00726] Exemplary steps of an SC-RPE Primary Cell expansion method of the disclosure are schematically illustrated in Figure 15, in which the steps comprise - Harvesting recovered SC-RPE, followed by - Performing primary expansion of recovered SC-RPE, by - Pre-freeze harvesting of SC-RPE, and by - Freezing of harvested SC-RPE. [00727] Exemplary protocols for each of the above steps are illustrated in Examples 34 to Example 37. Example 34: SC-RPE Primary Expansion Method and Preparation of Frozen Stocks Method and System: Harvesting recovered SC-RPE [00728] The exemplary SC-RPE primary cell expansion and preparation of frozen stocks method schematically illustrated in Figure 15, and related system, encompass a step of harvesting recovered SC-RPE, which can be performed as outlined below. [00729] After 3-14 days of post-sorting recovery culture, wash the SC-RPE twice with a volume of CTS-DPBS equal to the volume of culture medium. Shorter times in culture will reduce the total number of cells, whereas longer times in culture will increase the difficulty of harvesting cells due to cell:cell tight junction formation. In general, 7 days of post-sort recovery Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT will yield satisfactory results. For a single well of a 6-well multiplate add 1 mL of CTS- TrypLE™ (1 mL/10 cm²) and incubate at 37^{^} C. After approximately 10 min examine the cells using a microscope. If the cells are rounded up and detaching from the culture surface proceed to the next step. Otherwise return the cultures to the incubator and monitor periodically (every 5-10 min) until most of the cells have detached. [00730] Using a 1 mL pipette gently resuspend the cells without trituration in the TrypLE™ solution and transfer to a 15 mL conical centrifuge tube. Using gentle trituration recover any remaining cells in the culture device using 1 mL of RPE Maturation Medium, combine with the cells in TrypLE, and add an additional 12 mL of RPE Maturation Medium to the combined cell suspension to further dilute the TrypLE. As an alternative to RPE Maturation Medium, CTS-DPBS can be used to triturate the cells and as a TrypLE^TM diluent. When harvesting the cells, it is important to minimize mechanical damage and it is not essential to achieve a single cell suspension. If substantial numbers of cells remain attached to the culture plate the TrypLE™ digestion can be repeated. Depending on intended SC-RPE usage, other proteases such as trypsin or Accumax™ can be used as alternatives to TrypLE™ with appropriate modifications to the harvesting protocol. [00731] Recover the cells by centrifugation at 300 × g for 5 min and discard the supernatant. Gently disrupt the cell pellet by flicking the bottom of the centrifuge tube and then resuspend the cells in 10 mL of RPE Maintenance Medium (RPE Maturation Medium + 5 ^M Y-27632, 0.1% DMSO, and 2 ng/ml bFGF). Pellet the resuspended cells for 5 min at 300 × g and discard the supernatant. Example 35: SC-RPE Primary Expansion and Preparation of Frozen Stocks Method and System: Primary Expansion of recovered enriched SC-RPE [00732] The exemplary SC-RPE primary cell expansion and preparation of frozen stocks method schematically illustrated in Figure 15, and related system, encompass a step of primary cell expansion of SC-RPE, which can be performed as outlined below. [00733] Gently disrupt the cell pellet by flicking the bottom of the centrifuge tube and then resuspend the cells in 10 mL of RPE Maturation Medium and transfer the cell suspension to a matrix coated (e.g., laminin 521 or vitronectin) T75 flask and add an additional 5 mL of RPE Maintenance Medium. Evenly distribute the cells by rocking the culture flask and then incubate Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT the cultures at 37^{^} C and 5% CO_2. Feed the cells every two days with 15 mL of RPE Maintenance Medium using a 90-100% medium exchange. [00734] RPE Maturation Medium, RPE Maturation Medium + Y27632, or RPE Maturation Medium + bFGF and DMSO can be used as an alternative to RPE Maintenance Medium with expected effects on the degree that the SC-RPE can be expanded before loss of the ability to obtain a characteristic RPE phenotype as would be understood by a skilled person up reading of this disclosure. [00735] When fewer cells are required for downstream use or when it is desired to minimize the number of frozen stocks the Primary Expansion can be omitted and frozen stocks can be prepared directly without expansion after Post-Sort Recovery. Example 36: SC-RPE Primary Expansion and Preparation of Frozen Stocks Method and System : Pre-freeze Harvest [00736] The exemplary SC-RPE primary cell expansion and preparation of frozen stocks method schematically illustrated in Figure 15, and related system, encompass a step of pre- freeze harvesting of SC-RPE, which can be performed as outlined below. [00737] When the cells are approximately 80% confluent (2-4 days after plating) or less preferably near or just confluent (2-5 days after plating) harvest the cells for freezing. Wash the T75 flask twice with 15 mL of CTS-DPBS, add 5 mL of CTS-TrypLE™ to the flask, and incubate at 37^{^} C. After approximately 5-10 min examine the cells using a microscope. If the cells are rounded up and detaching from the culture surface proceed to the next step. Otherwise return the cultures to the incubator and monitor periodically (every 5-10 min) until most of the cells have detached. Depending on intended SC-RPE usage, other proteases such as trypsin or Accumax™ can be used as alternatives to TrypLE™ with appropriate modifications to the harvesting protocol. [00738] Using a serological pipette gently resuspend the cells without trituration in the TrypLE™ solution and transfer to a 50 mL conical centrifuge tube. Using gentle trituration recover any remaining cells in the culture device using 10 mL of RPE Maturation Medium and combine with the cells in TrypLE. If substantial cells remain in the flask, they can be recovered with an additional wash using RPE Maturation Medium. Bring the volume of the pooled cell suspension to 50 mL using RPE Maturation Medium. As an alternative to RPE Maturation Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Medium, CTS-DPBS can be used to triturate the cells and as a TrypLE™ diluent. When harvesting the cells, it is important to minimize mechanical damage and it is not essential to achieve a single cell suspension. [00739] Collect the cells by centrifugation at 300 × g for 5 min and discard the supernatant. Loosen the cell pellet by gently flicking the bottom of the tube and the gently resuspend the cells in 10 mL of CTS-DPBS or RPE Maturation Medium. Count the cells using a hemocytometer or automated cell counter and pellet the cells by centrifugation at 300 × g for 5 min and discard the supernatant. [00740] When greater numbers of frozen cells are desired the SC-RPE can be further expanded by repeating the Primary Expansion Methods, keeping in mind that this will decrease extent that the cells can be expanded after freezing before they lose the ability to acquire a mature RPE phenotype. Example 37: SC-RPE Primary Expansion and Preparation of Frozen Stocks Method and System : Cell Freezing [00741] The exemplary SC-RPE Primary Cell expansion and preparation of frozen stocks method schematically illustrated in Figure 15, and related system, encompass a step of freezing SC-RPE cells, which can be performed as outlined below. [00742] SC-RPE are typically frozen at a concentration of 0.5-5 × 10⁶ or 0.5-20 × 10⁶ cell/mL in volumes of 0.5 mL or greater. The choice of the cell concentration and aliquot size should be determined based on the number of cells required in the downstream applications. [00743] Disrupt the cell pellet by gentle flicking of the bottom of the centrifuge tube and gently resuspend the cells in an appropriate volume of chilled (^4^{^} C) CryoStor CS10 freezing medium and transfer desired size aliquots to an appropriate size cryovial or bag. During the aliquoting process it is best to maintain the cells at 4^{^} C until they are frozen. [00744] After the aliquoting process is complete, freeze the cells using a controlled rate of approximately -1^{^} C/min to a final set point of roughly -80^{^} C. At the completion of the freezing process transfer the cells to -125 to -200^{^} C for long term storage. [00745] Other xeno- or serum-free freezing media, such as CryoStor CS5, CryoStor CS2, CTS Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Synth-a-Freeze or mFreSR can be used with similar results with respect to post-thaw viability (generally >90%) and SC-RPE quality. Example 38: SC-RPE Patch Production Method and System [00746] An exemplary method to perform an SC-RPE Thaw of Frozen Stocks, Secondary Expansion & Patch production method of the disclosure is schematically illustrated in Figure 16. [00747] Exemplary steps of an SC-RPE Thawing of Frozen Stocks, Secondary Expansion & Patch Production method of the disclosure are schematically illustrated in Figure 16, in which the steps comprise - Thawing and recovering of SC-RPE cells by - Harvesting recovered cells and - Performing patch production. [00748] Exemplary protocols for each of the above steps are illustrated in Examples 39 to Example 41. Example 39: SC-RPE Patch Production method and systems: Thawing and Recovering SC-RPE cells [00749] The exemplary SC-RPE Thawing of Frozen Stocks, Secondary Expansion & Patch production method schematically illustrated in Figure 16, and related system, encompass a step of Harvesting recovered SC-RPE, which can be performed as outlined below. [00750] Quickly thaw a stock vial or container at room temperature with intermittent mixing by gentle inversion until only a small portion remains frozen. This can be most easily accomplished for example using a room temperature water bath and frequent mixing by swirling or inversion of the vial. [00751] For a 1 mL volume of frozen cells, transfer the partially thawed cell suspension to a 15 mL conical centrifuge tube by pipetting or decanting. Rinse the cryotube with 1 mL of RPE Maturation Medium and add the rinse to the transferred frozen cell suspension. Then add an additional 10 mL of RPE Maturation Medium to the cell suspension and mix by gentle inversion. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00752] Recover the thawed cells by centrifugation at 300 × g for 5 min and aspirate the supernatant. Soften the remaining cell pellet by gentle flicking of the bottom of the tube and gently resuspend the cells in 2 mL of RPE Maintenance Medium (RPE Maturation Medium + 5 ^M Y-27632, 0.1% DMSO, and 2 ng/ml bFGF). Determine the number of recovered viable cells using a staining method of choice (e.g., trypan blue or propidium iodide) and counting on a hemocytometer or using an automated cell counter. [00753] At this stage the SC-RPE can be used directly in the intended downstream application, but for optimal results and for increased total yield it is best to allow the cells to recover and expand after thawing. For 1 million cells dilute the cell suspension with 13 mL of RPE Maintenance Medium and transfer 7.5 mL to each of two SC-RPE substrate (e.g., laminin 521 or vitronectin) coated T75 flasks. Add an additional 7.5 mL of RPE Maintenance Medium to each flask, mix the cell suspension by gentle rocking to uniformly distribute the cells, and place the flask in a cell culture incubator at 37^{^} C and 5% CO₂. (If fewer cells are needed the cells can be plated at higher density by adjusting the protocol accordingly.) Feed the cells every 2-3 days with RPE Maintenance Medium. Example 40: SC-RPE Patch Production Method and System: Harvesting recovered SC- RPE cells [00754] The exemplary SC-RPE thawing of Frozen Stocks, Secondary Expansion & Patch Production method schematically illustrated in Figure 16, and related system, encompass a step of harvesting of SC-RPE cells, which can be performed as outlined below. [00755] When the cells reach near or just confluence, generally 3-4 days, wash the SC-RPE twice with a volume of CTS-DPBS equal to the volume of culture medium. (Shorter or longer times in culture are possible. Shorter times will result in reduced yield, whereas longer times in culture will increase the difficulty of harvesting cells due to cell:cell tight junction formation. In general, up to 7 days of post-thaw recovery will yield satisfactory results.) After removing the second CTS-DPBS wash, add 4 or 5 mL of CTS-TrypLE™ and incubate at 37^{^} C. After approximately 10 min examine the cells using a microscope. If the cells are rounded up and detaching from the culture surface proceed to the next step. Otherwise return the cultures to the incubator and monitor periodically (every 5-10 min) until most of the cells have detached. [00756] For each flask, using a serological pipette, gently resuspend the cells in the TrypLE™ Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT solution without trituration and transfer to a 50 mL conical centrifuge tube. Using gentle trituration recover any remaining cells in the culture device using 10 mL of RPE Maturation Medium or CTS-DPBS, combine with the cells in TrypLE, and add an additional 35 mL of RPE Maturation Medium or CTS-DPBS to the combined cell suspension to further dilute the TrypLE. Depending on intended SC-RPE usage, other proteases such as Trypsin or Accumax™ can be used as alternatives to TrypLE™ with appropriate modifications to the harvesting protocol. [00757] Recover the cells by centrifugation at 300 × g for 5 min. Carefully remove the supernatant, soften the cell pellet by gentle flicking of the bottom of the tube, resuspend the cells in 1 mL of RPE Maturation Medium, combine the two cell suspensions, and count the cells using an automated cell counter or a hemocytometer. From a single vial of 1 million cells expect have approximately 6 million cells when the cells are harvested at near confluence. If additional numbers of cells are desired, the culture can be further expanded by repeating the above plating and harvesting steps and using RPE Maintenance Medium as the culture medium, providing the cells are not expanded to the point where they will no longer obtain a normal mature RPE phenotype. Otherwise proceed to the patch production stage. For further expansion, cells should be plated at a minimum of 6500 cell/cm². The extent that a specific lot of SC-RPE can be expanded without significant loss in quality will vary; however, upward of 10 total population doublings from a frozen stock vial prepared according to the SC-RPE Primary Expansion and Preparation of Frozen Stocks Method and System are commonly possible when using RPE Maintenance Medium (RPE Maturation Medium + 5 ^M Y-27632, 0.1% DMSO, and 2 ng/ml bFGF) as the culture medium. Example 41: SC-RPE Patch Production Method and System: Performing production of SC-RPE patches [00758] The exemplary SC-RPE Thawing of Frozen Stocks, Secondary Expansion & Patch Production method schematically illustrated in Figure 16, and related system, encompass a step of performing the production of SC-RPE Patches cells, which can be performed as outlined below. [00759] Seed the cells onto matrix coated (laminin 521 or vitronectin) porous membranes or films in RPE Maintenance Medium (RPE Maturation Medium + 5 ^M Y-27632, 0.1% DMSO, and 2 ng/ml bFGF) such that the density of cells on the membrane will be at a density of about Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT 100,000 cell/cm². The total number of cells, the plating volume, and feeding volume will be dependent on the culture device and the relative surface area of the membrane in the culture device. [00760] Feed the cultures every 2-3 days with RPE Maintenance Medium. If desired, after 2-3 weeks the cultures can be switched to a feeding schedule of every 3-4 days. Additionally, after 2-3 weeks the addition of Y-27632, DMSO and/or bFGF to the medium can be omitted. Removal of bFGF/DMSO will result in a slight increase in the level of pigmentation. However, the establishment and maintenance of RPE:RPE tight junctions is bFGF-dependent and removal of bFGF/DMSO will result in reduced RPE barrier function as will be understood by a skilled person. In addition, the entire process can be done using RPE Maturation Medium or variations of RPE Maintenance Medium (e.g., without bFGF and DMSO, without Y-27632) with expected results on RPE phenotype and function as will be understood by skilled person. [00761] For the production of SC-RPE monolayers for implantation the cells are plated on a membrane support of choice and they are typically maintained for a sufficient period of time such that they become largely quiescent and at least partially differentiated prior to final release. [00762] When the intended use of the cells is for something other than production of SC-RPE patches such as production of cell suspensions for therapeutic use or research studies one can use the SC-RPE Patch Production Method and System to produce those cells, save for the step of plating the cells on the membranes or films. Example 42: RPE Maturation Media Testing and Selection: Comparison to serum supplemented RPE medium and comparison of RPE Maturation Medium with and without vitamin A and supplementation with N1 or N2. [00763] To evaluate the utility of the RPE Maturation Medium as a serum-free alternative for the differentiation, maturation, and post-enrichment culturing methods, or recovery methods of the disclosure several alternative formulations of RPE Maturation Media were prepared and tested. [00764] Two iPSC lines derived from primary fetal RPE cells were expanded in accordance with the methods of the disclosure, seeded onto Matrigel coated culture wells, and differentiated using the semi-directed method where X-VIVO 10/Neurocult SM1 was the Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT differentiation medium, with the one exception that the activin A differentiation agent was provided in combination with nicotinamide. At about one month, at which time there was minimal observed pigmentation, perhaps for the occasional golden brown tinted cell foci, the cells were switched from the differentiation medium to the preferred embodiment of RPE Maturation Medium (see Example 11) and where the B27-based supplement was SM1- Neruocult Neuronal Culture Supplement (with vitamin A) or SM1-Neruocult Without Vitamin A Supplement, or it was replaced with 5% FBS and where the N1-type supplement was N1 or N2-Supplement. Cells were maintained in their respective RPE Maturation Media until the increase in the percentage of pigmented surface area and the intensity of pigmentation reached plateau (Figure 17). As can be seen, the efficiency of differentiation for the #110211 cells was very robust in all six formulations, with perhaps some slight increase in differentiation in RPE- MM minus vitamin A, that was also seen with the replicate wells. Similarly, the #082809 cells showed no to minimal RPE-MM preference, although the efficiency of differentiation was significantly lower. The slight variation in yields between conditions was in the range of variability seen between replicates. [00765] The pigmented SC-RPE were then isolated in accordance to the enrichment method of the disclosure using a post-sort recovery period of about one month in their corresponding RPE-MM. At the end of the recovery period the cells were harvested for either gene expression profiling and for culture on microporous transwells to assess their ability to establish tight junctions by measuring their transepithelial electrical resistance at about 30 day post-seeding. [00766] Figure 18 shows the results obtained for the #110211 cell line N1/SM1 Neurocult cells compared to primary fetal RPE in the in SM1 minus and plus vitamin A media. Both cells had typical TEERs of a 1 month old culture. Interestingly the fetal RPE cultures had significantly higher TEERS in the SM1 without vitamin A. The media dependent difference was not observed with the SC-RPE but their resistance was equivalent to that seen with fetal RPE. [00767] When observed in the light microscope the cells had the expected RPE morphology with perhaps some subtle differences that would be overlooked and that could not be captured in images. To obtain a more rigorous assessment of the phenotype of cells grown in media with and without vitamin A in media supplemented with N1, these two lines along with 4 other lines were compared by gene expression analysis. [00768] Using a pairwise analysis, it was found that 3.8% of the expressed genes were Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT differentially expressed (>2-fold, FDR< 0.001) in cells differentiated and cultured with and without vitamin A of which 40% were upregulated by vitamin A (see Figure 19) for a graphical representation of the effect of vitamin A on the expression of all expressed genes). Based on gene ontology analysis and manual examination of the differentially expressed genes (DEG) it was determined that the vitamin A upregulated genes were enriched for known RPE genes and the downregulated genes were enriched for genes associated with wound responses, reversible EMT associated with wound repair, and genes associated with persistent EMT associated RPE dedifferentiation. [00769] In Figure 20, the effects of vitamin A on the gene expression of select RPE genes associated with various RPE functions is shown for the SC-RPE and fetal RPE. As is readily apparent, most well-known RPE genes are minimally affected by vitamin A such as key genes involved in pigmentation and cell:cell adhesion (RPE barrier function; nor are the best recognized RPE transcriptional regulators MITF, LHX2, OTX2, and RAX. The one exception being EYA2, supporting the conclusion that EYA2 may be involved the vitamin A mediated regulation of RPE gene expression. Not surprisingly two genes (LRAT and ABCA4) of the visual cycle, which is responsible for retinoid metabolism are highly affected by Vitamin A. [00770] Figure 21 shows the results of transferring cells grown in the presence of vitamin A and then switching to media without vitamin A (Plus-to-Minus) or vice versa (Minus-to-Plus). As can be seen the effects of vitamin A are at least in part reversible. Two of the genes shown are the previously mentioned visual cycle genes LRAT and ABCA4; CFI, CFH, and C1S are components of the complement cascade; and the gene product of SSP1 colocalizes with an AMD risk-associated complement reactivity in drusen, the hallmark pathology of age-related macular degeneration. All these genes have been genetically linked to degenerative disease in the eye and/or elsewhere. Regulation of these classes of genes by vitamin A, a method enabled by RPE-MM, can provide cell culture disease models for study or drug discovery. [00771] Vitamin A regulates gene expression in a dose dependent manner (Figure 22). A 2- fold serial dilution series of vitamin A by dilution of RPE-MM Plus Vitamin A with RPE-MM Minus Vitamin A and fetal RPE were seeded and maintained in the media for 1 month, after which their gene expression profiles were determined by sequencing. The gene GNGT1 codes for the gamma subunit of transducin, which is a central player in the phototransduction cascade. TGFBI is transforming growth factor inducible, a key marker of wound response. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00772] Based on these unexpected findings enabled by RPE-MM, vitamin A can be used as a result effective variable in RPE-Maturation to produce an SC-RPE or RPE cell of a desired phenotype, whether that is for research, drug discovery or production of clinical cell therapy. Example 43: Evaluation of SC-RPE Gene Expression: Comparison of the enriched SC-RPE and fetal RPE transcriptome profiles and known RPE gene expression. [00773] To assess the quality of the SC-RPE obtained from the differentiation, maturation, and enrichment methods of the disclosure eight of the ESC differentiation cultures shown in Figure 1 were enriched by cell sorting based on pigment light scatter and negative CD49b, positive CD104, and positive CD57 staining. After enrichment they were seeded onto laminin 521 coated culture wells in RPE-MM:CTS-N2/CTS-B27 with 10 ^M Y-27632 for 1-2 days and RPE-MM without additional supplements thereafter. After a one-month recovery period the matured RPE cultures were harvested and analyzed by whole transcriptome RNA AmpliSeq. The resulting transcriptome profiles were contrasted with an existing data set comprised of four fetal RPE (fRPE) lines seeded on Matrigel coated plates and maintained with RPE- MM:SM1/N1 for one-month and four pluripotent RPE-derived iPSC lines grown in Essential 8 stem cell medium. The four iPSC and four fetal RPE cell lines were sequence using a single replicate and each of the SC-RPE lines were sequenced in triplicate. After obtaining the average expression level for the >14,000 expressed genes the expression data for the SC-RPE lines was compared to the undifferentiated stem cell and fRPE by plotting the data on scatter plots (Figure 23A). As is evident from the scatter plots, the SC-RPE have a transcriptome profile very similar to fRPE in comparison to the iPSC lines. [00774] To drill down further into the gene expression data the expression of the Top-500 up- regulated genes associated with RPE maturation [21]. for each of the enriched SC-RPE lines were examined by comparison with the average fRPE data (Figure 23B). In the top leftmost scatter plot the data for fRPE and iPSC cells are compared to each other. As can be seen most, but not all genes, in the Top-500 RPE gene set are preferential expressed in fRPE compared to stem cells. While some of these markers are expressed at higher level in stem cells, these genes are not necessarily RPE markers. In contrast to the comparison with stem cells, when the SC- RPE lines are compared to fRPE the data falls on the diagonal with very few outliers. indicating again that the SC-RPE produced by the methods of this disclosure have a gene expression profile very similar to fRPE. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00775] In Figure 24A the average expression of a set of 90 curated RPE markers in SC-RPE and fRPE are compared to each other. As can be readily seen the pattern of expression of all 90 RPE-Signature genes in SC-RPE are qualitatively indistinguishable from that of fRPE with all genes being expressed. In Figure 24B both the SC-RPE and fRPE expression levels relative to the expression in stem cells is shown for 33 select RPE genes that play a role in key RPE functions are shown. Except for the four genes, EYA2, ABCA4, LRAT, and CXCL14 there is no significant difference between SC-RPE and fetal RPE gene expression for these well-known RPE genes, Interestingly, EYA2, ABCA4, and LRAT are part of the vitamin A responsive gene class. Given that the fetal RPE and SC-RPE were cultured using RPE-MM supplemented with different B27-based supplements (SM1 Neurocult vs. CTS-B27 XF) a difference in expression in the vitamin responsive genes is not surprising. CXCL14 in one the most up-regulated genes in the macula, suggesting that the SC-RPE of the disclosure can have a gene expression pattern more similar to macular RPE than extramacular RPE. All of the RPE marker genes are expressed at substantially higher levels in RPE cells compared stem cells and are suitable markers to follow the monitor the differentiation and maturation methods of the disclosure. Example 44: Cell Sorting [00776] An example setup for flow cytometry based cell sorting in the SC-RPE enrichment method is shown in Figure 25. A mixture of pigmented and unpigmented cells is piped (9905) into a reservoir of sheath fluid (9910), which is separated into drops (9915a, 9915b) statistically containing one cell per drop into an analysis tube (9911). A laser (9920) tuned to the absorption of the pigmentation passes a beam (9925) through the analysis tube (9911) and the scattering by the drops (9915a, 9915b) are measured by a forward scatter detector (9930) and a side scatter detector (9935). [00777] The detectors’ readings are fed into an analyzer (9940) that determines if the drop being analyzed is contains a pigmented cell (9915b) or not (9915a) based on absorption and other characteristics. A charge induction device (9945) imparts an electric charge on a drop leaving the analysis tube (9911) based on the prior determination made by the analyzer (9940), in this example giving a charge to droplets containing pigmented cells. Electrically charged plates (9950a, 9950b) deflect the charged drops into collection bins (9960a, 9960b) while the uncharged drops go into waste (9965). Other configurations would be understood by one skilled in the art, as well as other sorting methods (fluorescent-activated, magnetic-activated, Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT buoyancy-activated, selective media, filtering, etc.). Example 45: Transplantation devices [00778] Transplantation of mature SC-RPE cells in suspension or as a monolayer can be performed with various devices that are configured to place cells in target ocular areas of an individual, such as a subretinal space of the individual. [00779] A schematic representation of exemplary devices that can be used to perform SC-RPE transplantation methods are shown by example in Figure 26 and can comprise: a trephine surgical kit (2605); a microfluidic and actuator-based transplantation device (2610); an extracellular matrix-scaffold-supported transplantation device (2615); an ophthalmic cannula for insertion of RPE strips (2620); and additional devices identifiable by a skilled person. Example 46: SC-RPE Maturation Methods: Effect of timing and expansion ratio on SC- RPE yield after intermediate passage [00780] It has been found that passaging maturing SC-RPE during the period of SC-RPE maturation can result in significant increases in SC-RPE yields, especially in cases where the yields are less than desired due to a poor cell line:medium:substrate combination (see Example 17 and Figure 4). In this experiment the effect of the timing of the intermediate passage after contacting the differentiated stem cells with RPE-Maturation Medium and the passage split ratio on SC-RPE yield was examined. [00781] Shef1 stem cells were differentiated according to the semi-directed differentiation method of the disclosure using laminin 521 as the substrate and X-VIVO™ 10/CTS-B27 as the differentiation medium with the exception that Activin A was used in combination with nicotinamide. At day-35 the cells were contacted with RPE-MM:N1/CTS-B7 and at 3, 16, and 36 days after the medium switch the duplicate culture wells of maturing cells were harvested by protease digestion and the cells were replated on laminin 521 coated plates using a 1:2 or 1:4 split ratio and RPE-MM:N1/CTS-B7 or RPE-MM:N1/CTS-B7 + 2 ng/mL bFGF and 0.1% DMSO as the RPE maturation medium. At about 115 days after the initial stem cell plating the cells were sorted according to the SC-RPE enrichment methods of the disclosure using light scatter and Cd49b, CD104, and CD57b staining. The yields were then determined by cell counting and the corrected yields were calculated by division with the split ratio to account for expected difference in yield between a split ratio of 1:2 and 1:4 (Figure 27). Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00782] As shown in the figure, passage at later times after contacting with RPE-MM (16 and 36 days) after contacting with RPE-MM results in substantially higher yields (about 3-fold) compared to passaging after only 3 days and the yields using 1:2 and 1:4 were comparable. In addition, no improvement in corrected yields were seen when using RPE-MM supplemented with bFGF and DMSO, irrespective of the timing of the passage or the split ratio. Addition of bFGF to the medium did have one effect and that was that the cells were notably more difficult to harvest, likely due to its promotion of tight junction formation (see Example 31 and Figure 12B). Of course, more total numbers of cells are obtained with a split ratio of 1:4 versus 1:2, but this is at an increase cost of materials and time and doubles the number of cells to enriched. If greater numbers of cells are needed it is preferable that cells be further expanded after sorting and period of about 2-weeks in RPE-MM give best yields, with shorter times being possible. In addition to providing guidance about the preferred timing and conditions for intermediate passage to increase yields, these findings also support the conclusion that in the differentiation and maturation method that the environment, that is the cells in direct association with the presumptive RPE, at the time of contacting with RPE-MM, plays a role in the transition of a partially differentiated or immature SC-RPE to a determined SC-RPE cell in concert with RPE-MM. [00783] Example 47: Derivation method of human pluripotent stem cells [00784] In some embodiments of the SC-RPE derivation methods and systems of the disclosure frozen vials of SC-RPE suitable for the use in clinical therapeutics are prepared as follows. [00785] Using the differentiation and maturation methods and systems of the disclosure human pluripotent stem cells (Shef1) are differentiated according to the spontaneous method to obtain differentiated SC-RPE cultures where the stem cells are subjected to the derivation method according to the following exemplary workflow. [00786] The stem cells are seeded on recombinant human truncated vitronectin (preferably CTS-Vitronectin (VTN-N) recombinant human protein, truncated) coated cultureware in complete CTS-Essential 8 medium (preferably supplemented with 5-10 ^M Y-27632 for the first 24-48 hours). [00787] After a culture period of generally 2-5 days the stems cells are then contacted with a RPE-DM1 in the sense of the disclosure (preferably CTS-KO-DMEM/20%CTS-KOSR or KO- Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT DMEM/20%KOSR). [00788] After about 28 days after the initial seeding the cultures are contacted with an RPE- MM in the sense of the disclosure (preferably liquid alpha MEM, without phenol red and with nucleosides, 1X CTS-B27 XenoFree, 1X CTS-N2, 2 mM taurine, 100 ^M NEAA, and 55 nM hydrocortisone). [00789] One to two weeks after contacting with RPE-MM the cells are passaged using protease digestion and replated at split ration of about 1:2 on recombinant human truncated vitronectin (preferably CTS-Vitronectin (VTN-N) recombinant human protein, truncated) coated cultureware and cultured using RPE-MM for about 1 month or until the percentage of pigmented cells in the culture begins reach plateau to obtain a culture of matured SC-RPE. [00790] Example 48: Enrichment of human stem cells [00791] According to the disclosure, maturated SC-RPE such as the cells obtained in outcome of the procedure of Example 47 can be enriched with the enrichment method and system of the disclosure. [00792] For example using the SC-RPE enrichment and recovery methods and systems of the disclosure the SC-RPE are enriched by cell sorting by selection of SC-RPE based on the presence of pigment using a pigment detection method of the disclosure (preferably light absorption), the absence of CD49b, and/or the presence of CD104 and/or CD59b. [00793] The enriched SC-RPE can then undergo a post-sort recovery method wherein the enriched SC-RPE are seeded on an SC-RPE substrate accordingly to the disclosure, preferably recombinant human truncated vitronectin (CTS-Vitronectin (VTN-N) recombinant human protein, truncated) coated cultureware and wherein the recovery medium is an RPE-MMM or RPE-MM accordingly to the disclosure (preferably liquid alpha-MEM, without phenol red and with nucleosides, 1X CTS-B27 XenoFree, 1X CTS-N2, 2 mM taurine, 100 ^M NEAA, and 55 nM hydrocortisone, 5 ^M Y-27632, 2-4 ng/mL bFGF, and 0.1% DMSO) and the cells are maintained in the recovery medium for about 1 week. Example 49: preparation of intermediate frozen stock of human stem cells [00794] Using the SC-RPE Freezing and Expansion methods of the disclosure an intermediate Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT frozen stock of SC-RPE can be obtained by harvesting post-sort recovered cells such as the post recovered cells obtained by the procedure of Example 48, expanding the cells, and cryopreserving the cells according to the following exemplary workflow. [00795] The recovered enriched SC-RPE can be harvested using protease and frozen vials of cells at a desired density and amount are prepared by freezing in cryopreservation medium (preferably CryoStor® CS10, CryoStor® CS5, or CryoStor® CS2). [00796] The recovered enriched SC-RPE can also be harvested using protease and seeded onto an SC-RPE substrate in the sense of the disclosure, preferably recombinant human truncated vitronectin (CTS-Vitronectin (VTN-N) recombinant human protein, truncated) coated cultureware using an RPE-MMM or RPE-MM in the sense of the disclosure (preferably liquid alpha-MEM, without phenol red and with nucleosides, 1X CTS-B27 XenoFree, 1X CTS-N2, 2 mM taurine, 100 ^M NEAA, and 55 nM hydrocortisone, 5 ^M Y-27632, 2-4 ng/mL bFGF, and 0.1% DMSO). [00797] After a culture expansion period of about 3-4 days and/or at a time when the cells are near confluence the recovered enriched SC-RPE are harvested using protease and frozen vials of cells at a desired density and amount are prepared by freezing in cryopreservation medium (preferably CryoStor® CS10, CryoStor® CS5, or CryoStor® CS2). Example 50: Preparation of a Frozen SC-RPE Drug Product [00798] Using the SC-RPE Expansion and Freezing methods and system of the disclosure a Frozen SC-RPE Drug Product can be obtained from RPE mature cells such as the post-sort recovered cells obtained by the procedure of Example 48. [00799] An exemplary workflow comprises thawing a vial of the intermediate frozen SC-RPE stock and further expanding SC-RPE by seeding the cells with on cultureware coated with any one of the SC-RPE substrate in the sense of the disclosure, using an RPE-MMM or RPE-MM in the sense of the disclosure . [00800] Preferably the SC-RPE substrate is recombinant human truncated vitronectin (CTS- Vitronectin (VTN-N) recombinant human protein, truncated). [00801] Preferably the seeding is performed with liquid alpha MEM, without phenol red and Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT with nucleosides, 1X CTS-B27 XenoFree, 1X CTS-N2, 2 mM taurine, 100 ^M NEAA, and 55 nM hydrocortisone, 5 ^M Y-27632, 2-4 ng/mL bFGF, and 0.1% DMSO). [00802] When the cultures reach near confluence, about 3-10 days (e.g., about 3-4 days), the SC-RPE are harvested using protease and frozen vials of cells at a desired density and amount are prepared by freezing in cryopreservation medium (preferably CryoStor® CS2, CryoStor® CS5, or CryoStor® CS10). [00803] In summary, provided herein are serum-free methods for the derivation of stem cell- derived retinal pigment epithelial cells (SC-RPE) and related cells, compositions, methods and systems that in several embodiments allow efficient, differentiation, isolation, and/or maturation of SC-RPE from a variety of different mammalian stem cells. In particular, provided herein serum free media related method and systems for maturation, enrichment and/or expansion of SC-RPE cells, as well as additional or alternative method and system for preparation of freezing stock , preparation of SC-RPE patch or cells suspension and related uses to transplant the SC-RPE in the sub-retinal space of an individual. [00804] More particularly, according to a first aspect (A1) a method is described for differentiating a stem cell-derived (SC) retinal pigment epithelium (RPE) cells, the method comprising contacting stem cells of a starting stem cell line with a cell culture support comprising any one of the SC-RPE differentiation medium of the present disclosure and any one of the SC-RPE cell culture substrate of the present disclosure wherein the SC-RPE differentiation medium and the SC-RPE cell culture substrate are selected depending on the starting stem cell line, and wherein the contacting is performed for a time and under conditions to obtain a differentiated SC-RPE cell, such as partially differentiated SC-RPE, presumptive SC-RPE progenitors, and/or immature SC-RPE. [00805] In some embodiments (A1.1) of the method of the first aspect (A1) the SC-RPE differentiation medium is an RPE-DM1 culture medium in the sense of the disclosure. [00806] In some embodiments (A1.1.1) of any one of the embodiments (A1.1) the concentrations of the RPE-DM1 medium components are concentrations within the ranges of Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Set 1 as described in Table 3 of the present disclosure. [00807] In some embodiments (A1.1.2) of any one of the embodiments (A1.1) the concentration of the RPE-DM1 medium components are the concentrations within the ranges of Set 2 as described in Table 3 of the present disclosure. [00808] In some embodiments (A1.1.3) of any one of the embodiments (A1.1) the concentration of the RPE-DM1 medium components are the concentrations within the ranges of Set 3 as described in Table 3 of the present disclosure. [00809] In some embodiments (A1.1.4) of the of any one of the embodiments (A1.1) the concentration of the RPE-DM1 medium components are the concentrations within the ranges of Set 4 as described in Table 3 of the present disclosure. [00810] In some embodiments (A1.1.5) of the method of the embodiments (A1.1) the concentration of the RPE-DM1 medium components are the concentrations within the ranges of Set 5 as described in Table 3 of the present disclosure. [00811] In some embodiments (A1.1.6) of any one of the embodiments (A1.1) the RPE-DM1 medium is comprised of a base medium, KOSR in particular Embryonic Stem Cell Serum Replacement Serum (ESCSR)-based medium supplement in a concentration from 5 to 30%, from 0.25 to 4X NEAA supplement, ^-mercaptoethanol in a concentration from 25 to 200 mM, and glutamine and/or L-alanyl-L-glutamine dipeptide in a concentration from 1.35 to 3.71 mM. [00812] In some embodiments (A1.1.6.1) of any one of the embodiments (A1.1.6) the KOSR medium in particular Embryonic Stem Cell Serum Replacement Serum (ESCSR)-based supplement is certified for manufacture of cells for clinical use. [00813] In some embodiments (A1.1.6.2) of any one of the embodiments (A1.1.6) or (A1.1.6.1) the concentration of glutamine is 2 mM. [00814] In some embodiments (A1.1.6.3) of any one of the embodiments (A1.1.6), (A1.1.6.1), or (A1.1.6.2) the composition comprises glutamine in a concentration from 1.35 to 3.71 mM, or L-alanyl-L-glutamine dipeptide in a concentration from 1.35 to 3.71 mM, or a combination of glutamine and L-alanyl-L-glutamine dipeptide in a concentration from 1.35 to 3.71 mM. [00815] In some embodiments (A1.1.6.4) of any one of the embodiments (A1.1.6), (A1.1.6.1), Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT (A1.1.6.2), or (A1.1.6.3) the concentration of NEAA supplement is preferably 0.5-2X, more preferably from 0.75-1.25X, or even more preferably 1X. [00816] In some embodiments (A1.1.6.5) of any one of the embodiments (A1.1.6), (A1.1.6.1), (A1.1.6.2), (A1.1.6.3), or (A1.6.4), the concentration of ^-mercaptoethanol is preferably from 50 to 150 mM, more preferably from 75 to 125 mM and even more preferably 100 mM. [00817] In some embodiments (A1.1.6.6) of any one of the embodiments (A1.1.6), (A1.1.6.1), (A1.1.6.2), (A1.1.6.3), (A1.1.6.4), or (A1.1.6.5), the concentration of the KOSR in particular ESCSR based medium supplement is preferably 10-25%, more preferably between 15-20%, and even more preferably 20%. [00818] In some embodiments (A1.1.6.7) of any one of the embodiments (A1.1.6), (A1.1.6.1), (A1.1.6.2), (A1.1.6.3), (A1.1.6.4), (A1.1.6.5), or (A1.1.6.6), the base medium is KO-DMEM or CTS-KO DMEM. [00819] In some embodiments (A1.1.6.8) of any one of the embodiments (A1.1.6), (A1.1.6.1), (A1.1.6.2), (A1.1.6.3), (A1.1.6.4), (A1.1.6.5), or (A1.1.6.6), the base medium is KO- DMEM:F12 or CTS-KO-DMEM:F12. [00820] In some embodiments (A1.1.6.9) of any one of the embodiments (A1.1.6), (A1.1.6.1), (A1.1.6.2), (A1.1.6.3), (A1.1.6.4), (A1.1.6.5), or (A1.1.6.6), the base medium is ^-MEM. [00821] In some embodiments (A1.1.6.10) of any one of the embodiments (A1.1.6), (A1.1.6.1), (A1.1.6.2), (A1.1.6.3), (A1.1.6.4), or (A1.1.6.5) the base medium is RPMI1640. [00822] In some embodiments (A1.1.6.11) of any one of the embodiments (A1.1.6), (A1.1.6.1), (A1.1.6.2), (A1.1.6.3), or (A1.1.6.4) the base medium is IMDM. [00823] In some embodiments (A1.1.6.12) of any one of the embodiments (A1.1.6.10) or (A1.1.6.11), the concentration of the KOSR medium supplement is preferably from 15 to 20%, more preferably from 10 to 20%, even more preferably 20%. [00824] In some embodiments (A1.2) of any one of the first aspect (A1) the SC-RPE differentiation medium is an RPE-DM2 culture medium in the sense of the disclosure. [00825] In some embodiments (A1.2.1) of any one of the embodiments (A1.2), the RPE-DM2 Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT culture medium is comprised or consists of the cell culture medium, X-VIVO 10, preferably supplemented with a B27-based supplement at a 0.25 to 3X strength, more preferably 0.5 to 2X strength, and even more preferably 0.75 to 1.25X strength and most preferably at 1X strength. [00826] In some embodiments (A1.2.2) of any one of the embodiments (A1.2.1), the B27-based supplement is selected from any one of CTS-B27, B27, B27+, XF-B27NeuroCult SM1, NS21, N21, N21-MAX, GMP N21-MAX, Custom B27, or Custom N21. [00827] In some embodiments (A1.2.3) of any one of the embodiments (A1.2), the RPE-DM2 culture medium is solely comprised of the cell culture medium, X-VIVO 10 and thus consists of cell culture medium, X-VIVO 10. [00828] In some embodiments (A1.2.4) of any one of the embodiments (A1.2.1) to (A1.2.3), the culture medium X-VIVO 10 is replaced by any one of StemSpan SFEM, StemSpan H3000, or StemSpan ACF culture medium. [00829] In some embodiments (A1.2.5) of any one of the embodiments (A1.2.1) to (A1.2.3), the culture medium X-VIVO 10 is replaced with AIM V or CTS-AIM V culture medium. [00830] In some embodiments (A1.2.6) of any one of the embodiments (A1.2.1) to (A1.2.3), the culture medium X-VIVO 10 is replaced with IMDM supplemented with insulin, transferrin, selenate, and albumin or wherein X-VIVO 10 is replaced by IMDM and RPM140. [00831] In some embodiments (A1.2.7) of any one of the embodiments (A1.2.1) to (A1.2.3), the culture medium X-VIVO 10 is replaced with RPMI1640 supplemented with insulin, transferrin, selenate, and albumin wherein X-VIVO 10 is replaced by IMDM and RPM140. [00832] In some embodiments (A1.2.8) of any one of the embodiments (A1.2.1) to (A1.2.7), the contacting is performed to obtain a spontaneous differentiation of SC-RPE cells, and in particular partially differentiated SC-RPE, SC-RPE progenitors, and/or immature SC-RPE as will be understood by a skilled person. [00833] In some embodiments (A1.2.9) of any one of the embodiments (A1.2.8), the contacting is performed about 1-7 days after plating, preferably 2-5 days after plating, more preferably, 3- 4 days after plating. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00834] In some embodiments (A1.2.10) of any one of the embodiments (A1.2.8) or (A1.2.9), the contacting is performed for a time from about 11 to 56 days, more preferably 21-42 days, and most preferably 28-35 days. [00835] In some embodiments (A1.3) of any one of the embodiments (A1.1) to (A1.1.6.12), the SC-RPE differentiation medium is a culture medium selected from the RPE-DM1 group of media supplemented with Activin A. [00836] In some embodiments (A1.3.1) of any one of the embodiments (A1.1.3) the RPE-DM1 medium is supplemented with 20-280 ng/mL Activin A, more preferably 70-210 ng/mL, even more preferably 140 ng/mL Activin A. [00837] In some embodiments (A1.4) of any one of the embodiments (A1.2) to (A1.2.10) the SC-RPE differentiation medium is a culture medium selected from the RPE-DM2 group of media supplemented with Activin A. [00838] In some embodiments (A1.4.1) of any one of the embodiments (A1.4), the RPE-DM2 medium is supplemented with 20-280 ng/mL Activin A, more preferably 70-210 ng/mL Activin A, even more preferably 140 ng/mL Activin A. [00839] In some embodiments (A1.5) of any one of the embodiments (A1.3) or (A1.4), the contacting is performed to obtain a semi-directed differentiation of SC-RPE cells and in particular, differentiated SC-RPE, SC-RPE progenitors and/or immature SC-RPE as will be understood by a skilled person. [00840] In some embodiments (A1.5.1) of any one of the embodiments (A1.5), the contacting is initiated from 1 to 14 days after reaching 60-90% confluence, preferably 2-6 days after reaching 60-90% confluence, more preferably 2-4 days after reaching 60-90% confluence, and even more preferably 3 days after reaching 60-90% confluence. [00841] In some embodiments (A1.5.2) of any one of the embodiments (A1.5.1), the contacting is performed for a time from for 4-28 days, more preferably 6-21 days, and most preferably 8 days. [00842] In some embodiments (A1.6) of any one of the embodiments (A1.1.3) to (A1.5), the method further comprises following the contacting, the step of treating the cells with the Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT differentiation medium without Activin A. [00843] In some embodiments (A1.6.1) of any one of the embodiments (A.1.6), the treating is performed for up to 60 days, more preferably 7-35 days, most preferably 14-21 days, after which time the medium is replaced with differentiation medium without Activin A or RPE Maturation Medium. [00844] In some embodiments (A1.7) of any one of the first aspect (A1) or embodiments (A.1.1) to (A1.6), the SC-RPE cell culture substrate is selected from laminin, vitronectin, Matrigel, Geltrex, Cultrex BME, collagen IV, and combinations thereof. [00845] In some embodiments (A1.8) of any one of the first aspect (A1) or embodiments (A.1.1) to (A1.7), the SC-RPE cell culture substrate is selected from laminin 511, laminin 521, laminin 111, laminin 211, laminin 221, laminin 332, and laminin 411. [00846] In some embodiments (A1.9) of any one of the first aspect (A1) or embodiments (A.1.1) to (A1.8), the SC-RPE cell culture substrate is selected from laminin 521, truncated vitronectin, full-length vitronectin, and combinations thereof. [00847] In some embodiments (A1.10) of any one of the first aspect (A1) or embodiments (A.1.1) to (A1.9), wherein the contacting is preceded by propagating and/or culturing the stem cells. [00848] In some embodiments (A1.10.1) of any one of the embodiments (A.10), the propagating and/or culturing is performed for 1 to 4 days. [00849] In some embodiments (A1.11) of any one of the first aspect (A1) or embodiments (A.1.1) to (A1.10), the stem cells are embryonic stem cells, adult stem cells, or induced stem cells. [00850] In some embodiments (A1.12) of any one of the first aspect (A1) or embodiments (A.1.1) to (A1.11), the starting stem cell line is a human stem cell line. [00851] In some embodiments (A1.13) of any one of the first aspect (A1) or embodiments (A.1.1) to (A1.12), the starting stem cell line is H1, H9 or Shef1. [00852] In some embodiments (A1.14) of any one of the first aspect (A1) or embodiments Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT (A.1.1) to (A1.13), the starting stem cell line is Shef1. [00853] In a second aspect (A2) an SC-RPE differentiation system is described, the system comprising - at least one serum-free SC-RPE differentiation medium and - at least one SC-RPE cell culture substrate for combined use to provide a cell culture support configured to allow introduction and/or maintenance of stem cells within the cell culture support to obtain differentiated SC-RPE cells, and in particular, differentiated SC-RPE, SC-RPE progenitors and/or immature SC-RPE as will be understood by a skilled person, the combined use performed according to the method of any one of the first aspect (A1) or embodiments (A.1.1) to (A1.14). [00854] In some embodiments (A2.1) of any one of the second aspect (A2), the at least one serum-free SC-RPE differentiation medium comprises at least one of the RPE-DM1 group of media, the RPE-DM2 group of media, the RPE-DM1 group of media supplemented with Activin A, and the RPE-DM2 group of media supplemented with Activin A in the sense of the present disclosure. [00855] In some embodiments (A2.2) of any one of the first aspect (A2) or embodiments (A2.1), the at least one serum-free SC-RPE differentiation medium comprises at least one of the RPE-DM1 group of media in the sense of the present disclosure in particular any one of the RPE-DM1 media of any one of embodiments (A1.1.1) to (A1.1.6.12), and wherein the system is to perform a method for a spontaneous differentiation of SC-RPE cells. [00856] In some embodiments (A2.3) of any one of the first aspect (A2) or embodiments (A2.1) to (A2.2), the at least one serum-free SC-RPE differentiation medium comprises at least one of the RPE-DM2 group of media in the sense of the present disclosure, preferably any one of the RPE-DM2 of any one of the embodiments (A1.2.1) to (A1.2.7) and wherein the system is to perform a method for a spontaneous differentiation of SC-RPE cells. [00857] In some embodiments (A2.4) of any one of the first aspect (A2) or embodiments (A2.1) to (A2.3), the at least one serum-free SC-RPE differentiation medium comprises at least one of the RPE-DM1 group of media supplemented with Activin A, preferably any one of the RPE- DM1 media supplemented with Activin A of embodiments (A1.3) and wherein the system is Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT to perform a method for a semi-directed differentiation of SC-RPE cells. [00858] In some embodiments (A2.5) of any one of the first aspect (A2) or embodiments (A2.1) to (A2.4) , the at least one serum-free SC-RPE differentiation medium comprises at least one of the RPE-DM2 group of media supplemented with Activin A, preferably any one of the RPE- DM2 media supplemented with Activin A of embodiments (A1.4.1) and wherein the system is to perform a method for a semi-directed differentiation of SC-RPE cells. [00859] In some embodiments (A2.6) of any one of the embodiments (A2.1) to (A2.5), the RPE-DM1 is selected from any one of the RPE-DM1 media identified in any one of embodiments (A1.1.6) to (A1.6.7). [00860] In some embodiments (A2.7) of any one of the embodiments (A2.1) to (A2.6), the RPE-DM2 medium is selected from any one of the RPE-DM2 media identified in any one of embodiments (A2.1) to (A1.2.3). [00861] In some embodiments (A2.8) of any one of the embodiments (A2.1) to (A2.7), the at least one SC-RPE differentiation medium comprises at least one of the RPE-DM1 media of the methods of any one of embodiments (A1.1.6) to (A1.6.7) supplemented with 140 ng/mL Activin A, preferably 70- 210 ng/mL, less preferably 20-280 ng/mL. [00862] . In some embodiments (A2.9) of any one of the embodiments (A2.1) to (A2.8), the at least one SC-RPE cell differentiation medium comprises at least one of the RPE-DM2 media of the methods of embodiments (A2.1) to (A1.2.3) supplemented with 140 ng/mL Activin A, preferably 70- 210 ng/mL, less preferably 20-280 ng/mL. [00863] In some embodiments (A2.10) of any one of the embodiments (A2.1) to (A2.9), the at least one SC-RPE cell culture substrate comprises at least one of laminin, vitronectin, Matrigel, Geltrex, Cultrex BME, collagen IV and combinations thereof. [00864] In some embodiments (A2.11) of any one of the embodiments (A2.1) to (A2.10), the at least one SC-RPE cell culture substrate comprises at least one of full length or fragments of laminin 111, laminin 211, laminin 221, laminin 332, and laminin 411, laminin 511, and laminin 521. [00865] In some embodiments (A2.12) of any one of the embodiments (A2.1) to (A2.11), the Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT at least one SC-RPE cell culture substrate comprises at least one of laminin 521, truncated vitronectin, full-length vitronectin and combinations thereof. [00866] In a third aspect (A3) an SC-RPE differentiated cell obtained by the method of any one of the first aspect (A1) and in particular any one of embodiments (A1.1) to (A1.14) and/or with the use of the system of any one of the second aspect (A2) in particular any one of embodiments (A2.1) to (A2.12). [00867] In a fourth aspect (A4) an RPE Maturation Medium comprising the base medium ^- MEM supplemented with additional non-essential amino acids (glycine, alanine, asparagine, aspartate, glutamate, proline, and serine), insulin, transferrin, selenite, putrescine, progesterone, serum albumin, galactose, catalase, superoxide dismutase, D,L-alpha- tocopherol, D,L-alpha-tocopherol acetate, glutathione, ethanolamine, linoleic acid, linolenic acid, biotin, carnitine, vitamin A, taurine, triiodothyronine, corticosterone, hydrocortisone and optional antibiotics and antimycotics in an effective amount to promote differentiation and maturation of partially differentiated SC-RPE cells, presumptive SC-RPE progenitors, and immature SC-RPE. [00868] In some embodiments (A4.1) of the fourth aspect (A4), the maturation medium is RPE- MM in the sense of the disclosure. [00869] In some embodiments (A4.2) of the fourth aspect (A4), the maturation medium is comprised of the base medium ^-MEM, additional nonessential amino acids, taurine, hydrocortisone, one of the N1-based supplements (N1, N-2, CTS-N-2, N-2-Plus, N-2-MAX, GMP-N-2-MAX, Custom N1) and one of the B27-based supplements (B27, B27+, XF-B27, CTS-B27, NeuroCult SM1, NS21, N21-MAX, GMP N21-MAX and Custom B27) and optional antibiotics and antimycotics at amounts and concentration ranges as defined in the disclosure. [00870] In some embodiments (A4.3) of the fourth aspect (A4), the maturation medium is comprised of the base medium ^-MEM, additional nonessential amino acids, taurine, hydrocortisone, one of the B27-based supplements (B27, B27+, XF-B27, CTS-B27, NeuroCult SM1, NS21, N21-MAX, GMP N21-MAX and Custom B27) and optional antibiotics and antimycotics at amounts and concentration ranges as defined in the disclosure. [00871] In some embodiments (A4.4) of the fourth aspect (A4), the maturation medium has the composition indicated in Table 7A of the present disclosure. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00872] In some embodiments (A4.5) of the fourth aspect (A4), the maturation medium has the composition indicated in Table 7B of the present disclosure. [00873] In some embodiments (A4.6) of the fourth aspect (A4), the maturation medium has the composition indicated in Table 7C of the present disclosure. [00874] In some embodiments (A4.7) of the fourth aspect (A4), the maturation medium has the composition indicated in Table 7D of the present disclosure. [00875] In some embodiments (A4.8) of the fourth aspect (A4), the maturation medium has the composition indicated in Table 7E of the present disclosure. [00876] In a fifth aspect (A5) a method is described to maturate a differentiated stem cell- derived retinal pigment epithelial (SC-RPE) cell, the method comprising contacting differentiated SC-RPE, partially differentiated SC-RPE, SC-RPE progenitors, or immature SC-RPE cells with a cell culture maturation medium of the present disclosure and in particular a maturation medium of the fourth aspect (A4), such as any one of embodiments (A4.1) to (A4.8), for a time and under conditions to obtain matured stem cell- derived retinal pigment epithelial (SC-RPE) cell. [00877] In some embodiments (A5.1) of the fifth aspect (A5), the cell culture maturation medium is the RPE Maturation Medium of the fourth aspect (A4) in particular embodiments (A4.1) to (A4.8). [00878] In some embodiments (A5.2) of the fifth aspect (A5) or embodiments (A5.1), the contacting is performed for a time selected to obtain a target percentage of mature SC-RPE cells. [00879] In some embodiments (A5.3) of the fifth aspect (A5) or embodiments (A5.1), the contacting is performed for a time selected to obtain a target quality of mature SC-RPE cells. [00880] In some embodiments (A5.4) of the fifth aspect (A5) or embodiments (A5.1), the contacting is performed for a time selected to obtain a target percentage of mature SC-RPE cells in combination with a target quality of mature SC-RPE cells. [00881] In some embodiments (A5.5) of the fifth aspect (A5) or embodiments (A5.1) to (A5.4), the contacting is performed for a time ranging from 15 to 90 days. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00882] In some embodiments (A5.6) of the fifth aspect (A5) or embodiments (A5.1) to (A5.4), the contacting is performed for a time of about 35 days. [00883] In some embodiments (A5.7) of the fifth aspect (A5) or embodiments (A5.1) to (A5.6), the differentiated stem cells are the SC-RPE differentiated cells of the third aspect (A3). [00884] In some embodiments (A5.8) of any one of embodiments (A5.7) SC-RPE differentiated cells of any one of the third aspect (A3) are obtained by spontaneous differentiation methods and/or with a system configured for spontaneous differentiation of the present disclosure, and wherein contacting differentiated SC-RPE cells with a cell culture maturation medium is performed beginning between and about 7 to 60 days after the initial plating, preferably from 14 to 50 days, more preferably from 21 to 37 days and even most preferably on about day 30. [00885] In some embodiments (A5.9) of any one of embodiments (A5.8), SC-RPE differentiated cells of any one of the embodiments (A3) are obtained by semi-directed differentiation methods and/or with a system configured for semi-directed differentiation of the present disclosure, and wherein contacting differentiated SC-RPE cells with a cell culture maturation medium begin from 7 to 60 days after the initial plating, preferably from about 14 to 60 days after the initial plating, more preferably from 14 to 50 days after the initial plating, even more preferably between 21 and 37 days after the initial plating, most preferably about 30 days after the initial plating. [00886] In a sixth aspect (A6) a matured SC-RPE cell, obtained by the method of the fifth aspect (A5) in particular embodiments (A5.1) to (A5.9) and/or by using the RPE Maturation Medium of the fourth aspect (A4) in particular embodiments (A4.1) to (A4.8) . [00887] In a seventh aspect (A7) a method to enrich mature stem cell-derived retinal pigment epithelial (SC-RPE) cells, the method comprising providing stem cell-derived retinal pigment epithelial (SC-RPE) cells; contacting the SC-RPE cells with a probe specific for a marker selected from CD57, CD104, and/or CD49b to obtain stained mature SC-RPE cells stained for CD57, CD104, and/or CD49b markers; and sorting the stained SC-RPE cells to select the mature SC-RPE cells stained for CD57 Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT and/or CD104 markers, and discard cells stained for CD49b marker, to obtain a population of SC-RPE cells enriched in mature SC-RPE cells. [00888] In some embodiments (A7.1) of the seventh aspect (A7), the providing comprises harvesting SC-RPE cells by dissociating the cultures by protease digestion. [00889] In some embodiments (A7.2) of the seventh aspect (A7) in particular embodiments (A7.1), the probes specific for CD57, CD104, and/or CD49b comprise one or more of anti- CD104-PE (Clone: REA236, Miltenyi Biotec), anti-CD57-PE/Vio770 (Clone: REA739, Miltenyi Biotec) and anti-CD49b-APC (Clone: REA188, Miltenyi Biotec) antibodies. [00890] In some embodiments (A7.3) of the seventh aspect (A7) in particular embodiments (A7.1) to (A7.2), the sorting can be performed by Fluorescence Activated Cell Sorting or FACS and/or Immunomagnetic cell sorting. [00891] In some embodiments (A7.4) of the seventh aspect (A7) in particular embodiments (A7.1) to (A7.3), the contacting is performed with one or more probes specific for CD57 and/or CD104 and the sorting is performed to select SC-RPE cells positive for at least one of CD57 and CD104, and/or SC-RPE cells each positive for both of CD57 and CD104. [00892] In some embodiments (A7.5) of the seventh aspect (A7) in particular embodiments (A7.1) to (A7.3), the contacting is performed with one or more probes specific for CD57 and/or CD49b and the sorting is performed to select SC-RPE cells positive for CD57, SC-RPE cells negative for CD49b and/or SC-RPE cells each positive for CD57 and negative for CD49b. [00893] In some embodiments (A7.6) of the seventh aspect (A7) in particular embodiments (A7.1) to (A7.3), the contacting is performed with one or more probes specific for CD104 and/or CD49b and the sorting is performed to select SC-RPE cells positive for CD104, SC- RPE cells negative for CD49b, and/or SC-RPE cells each positive for CD104 and negative for CD49b. [00894] In some embodiments (A7.7) of the seventh aspect (A7) in particular embodiments (A7.1) to (A7.3), the contacting is performed with one or more probes specific for CD57, CD104 and/or CD49b and the sorting is performed to select SC-RPE cells positive for CD57, SC-RPE cells positive for CD104, and/or SC-RPE cells negative for CD49b. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00895] In some embodiments (A7.8) of the seventh aspect (A7) in particular embodiments (A7.1) to (A7.3), the contacting is performed with one or more probes specific for CD57, CD104 and/or CD49b and the sorting is performed to select SC-RPE cells positive for at least one of CD57 and CD104, SC-RPE cells positive for both of CD57 and CD104, SC-RPE cells each positive for CD104 and negative for CD49b, SC-RPE cells each positive for CD57 and negative for CD49b, and/or SC-RPE cells each positive for both of CD57 and CD104 and negative for CD49b. [00896] In some embodiments (A7.9) of the seventh aspect (A7) in particular embodiments (A7.1)to (A7.8), the method further comprises post-sorting cell recovery of the population of SC-RPE cells enriched in mature SC-RPE cells. [00897] In some embodiments (A7.10) of the seventh aspect (A7) in particular embodiments (A7.1) to (A7.9), the SC-RPE cells are the SC-RPE differentiated cells of claim 62 and/or the SC-RPE mature cells of claim 82. [00898] In an eighth aspect (A8), a system is described to enrich mature stem cell-derived retinal pigment epithelial (SC-RPE) cell, the system comprising at least two probes each specific for at least one marker selected from CD57, CD104, and/or CD49b for combined use to stain differentiated SC-RPE cells in a method to obtain a population of SC-RPE cells enriched in mature SC-RPE cells of the seventh aspect (A7) and in particular any one of the embodiments (A7.1). to (A7.10). [00899] In some embodiments (A8.1) of the eighth aspect (A8), the probe specific for at least one of CD57, CD104, and CD49b comprise one or more of anti-CD104-PE (Clone: REA236, Miltenyi Biotec), anti-CD57-PE/Vio770 (Clone: REA739, Miltenyi Biotec) and anti-CD49b- APC (Clone: REA188, Miltenyi Biotec) antibodies. [00900] In a ninth aspect (A9), an enriched SC-RPE mature cell population is described that is obtained with the method of any one of aspects (A5) to (A7) and in particular any one of the embodiments (A5.1) to (A5.9) and any one of the embodiments (A7.1) to (A7.10), and/or with the system of any one of aspect (A8) and in particular any one of embodiments (A8.1). [00901] In a tenth aspect (A10) An RPE Maintenance Medium comprising the RPE Maturation Medium of the present disclosure and in particular a maturation medium of aspect (A4), such as any one of embodiments (A4.1) to (A4.8), supplemented with basic fibroblast growth factor Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT (bFGF), a protein kinase inhibitor, and dimethyl sulfoxide (DMSO) in an effective amount to promote expansion of mature SC-RPE cells. [00902] In some embodiments (A10.1) of the tenth aspect (A10), the protein kinase inhibitor is Rho-Associated Coiled-Coil Containing Protein Kinase (ROCK) inhibitor. [00903] In some embodiments (A10.2) of any one of embodiments (A10.1), the ROCK inhibitor is Y-27632, thiazovivin, or RevitaCell. [00904] In some embodiments (A10.3) of the tenth aspect (A10), the protein kinase inhibitor is any one of the transforming growth factor beta receptor (TGF^R) kinase inhibitors RepSox, A-83-01, or SB-431542. [00905] In some embodiments (A10.4) of the tenth aspect (A10), the protein kinase inhibitor is any one of the group of ROCK inhibitors Y-27632, thiazovivin, or RevitaCell in combination with any one of the TGF^R kinase inhibitors RepSox, A-83-01, or SB-431542. [00906] In an eleventh aspect (A11), a method is described for expansion of stem cell-derived retinal pigment epithelial (SC-RPE) cells, the method comprising providing increased numbers of stem cell-derived retinal pigment epithelial (SC-RPE) cells; and contacting the SC-RPE cells with the RPE Maintenance Medium of the disclosure in the tenth aspect (A10) in particular embodiments (A10.1) to (A10.4) on an SC-RPE substrate of the disclosure, the contacting performed for a time and under condition to obtain a target cell density of the SC-RPE cells. [00907] In some embodiments (A11.1) of the eleventh aspect (A11), the providing comprises providing SC-RPE cells thawed and plated from frozen SC-RPE stocks. [00908] In some embodiments (A11.2) of the eleventh aspect (A11) in particular embodiments (A11.1), the method further comprises harvesting the SC-RPE cells and replating the harvested SC-RPE cells until the cultures reach the desired density. [00909] In some embodiments (A11.3) of the eleventh aspect (A11) in particular embodiments (A11.1) to (A11.2), the method further expands the SC-RPE cells. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00910] In some embodiments (A11.4) of the eleventh aspect (A11) in particular embodiments (A11.1) to (A11.3), the SC-RPE cell culture substrate is selected from laminin, vitronectin, Matrigel, Geltrex, Cultrex BME, collagen IV and combinations thereof. [00911] In some embodiments (A11.5) of the eleventh aspect (A11) in particular embodiments (A11.1) to (A11.4), the SC-RPE cell culture substrate is selected from laminin 511, laminin 521, laminin 111, laminin 211, and laminin 411. [00912] In some embodiments (A11.6) of the eleventh aspect (A11) in particular embodiments (A11.1) to (A11.5), the SC-RPE cell culture substrate is selected from laminin 521, truncated vitronectin, full-length vitronectin and combinations thereof. [00913] In some embodiments (A11.7) of the eleventh aspect (A11) in particular embodiments (A11.1) to (A11.6), the culture medium is a variant of RPE Maintenance Medium omitting any one or combination of DMSO, bFGF, a ROCK inhibitor, and/or TGF^R kinase inhibitor. [00914] In some embodiments (A11.8) of the eleventh aspect (A11) in particular embodiments (A11.1) to (A11.7), the SC-RPE cells are the SC-RPE differentiated cells of the third aspec (A3), the matured SC-RPE cells of the sixth aspect (A6) and/or the enriched SC-RPE cell population of the ninth aspect (A9). [00915] In a twelfth aspect (A12) an SC-RPE expansion system is described for expansion of stem cell-derived retinal pigment epithelial (SC-RPE) cells, the system comprising the RPE Maintenance Medium, and an SC-RPE substrate of the disclosure for combined use in a method to perform expansion of the SC-RPE cells of the disclosure. [00916] In some embodiments (A12.1) of the twelfth aspect (A12), the RPE Maintenance Medium comprises the RPE Maturation Medium of the fourth aspect (A4) in particular embodiments (A4.1) to (A4.8). [00917] In some embodiments (A12.2) of the twelfth aspect (A12), the culture medium is a variant of RPE Maintenance Medium omitting any one or combination of DMSO, bFGF, a ROCK inhibitor and/or TGF^R kinase inhibitor. [00918] In some embodiments (A12.3) of the twelfth aspect (A12) in particular embodiments (A12.1) to (A12.2), the SC-RPE cell culture substrate is selected from laminin, vitronectin, Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Matrigel, Geltrex, Cultrex BME, collagen IV and combinations thereof. [00919] In some embodiments (A12.4) of the twelfth aspect (A12) in particular embodiments (A12.1) to (A12.3), the SC-RPE cell culture substrate is selected from laminin 511, laminin 521, laminin 111, laminin 211, and laminin 411. [00920] In some embodiments (A12.5) of the twelfth aspect (A12) in particular embodiments (A12.1) to (A12.4), the SC-RPE cell culture substrate is selected from laminin 521, truncated vitronectin, full-length vitronectin and combinations thereof. [00921] In a thirteenth aspect (A13) a method is described to prepare frozen stock of stem cell- derived retinal pigment epithelial (SC-RPE) cells, the method comprising - performing expansion of SC-RPE cells with the method of the eleventh aspect (A11) in particular embodiments (A11.1) to (A11.8), to obtain SC-RPE cells having a target cell density - harvesting the SC-RPE cells having the target cell density to obtain harvested SC-RPE cells; and - freezing the harvested SC-RPE cells. [00922] In a fourteenth aspect (A14), a system is described for frozen stock preparation of stem cell-derived retinal pigment epithelial (SC-RPE) cell, the system comprising the SC-RPE maturation medium of the fourth aspect (A4) in particular embodiments (A4.1) to (A4.8) and/or preferably the RPE Maintenance Medium of the tenth aspect (A10) in particular embodiments (A10.1) to (A10.4) and/or the SC-RPE substrate of the present disclosure and freezing media, for combined use in a method to provide frozen stock of the eleventh aspect (A11) in particular embodiments (A11.1) to (A11.8). [00923] In some embodiments (A14.1) of the fourteenth aspect (A14), the RPE Maintenance Medium is a variant of RPE Maintenance Medium omitting any one or combination of DMSO, bFGF, a ROCK inhibitor and/or TGF^R kinase inhibitor in accordance with the present disclosure. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00924] In a fifteenth aspect (A15), a method is described to perform the production of stem cell-derived retinal pigment epithelial (SC-RPE) patches (SC-RPE Patch), the method comprising - providing enriched SC-RPE or frozen SC-RPE stock - performing optional expansion of SC-RPE cells to obtain cells having a target cell density; and - contacting the SC-RPE cells having the target cell density with a physical support (for example a membrane support and/or a microcarrier support) and RPE Maturation Medium and/or preferably the RPE Maintenance Medium to obtain a population of quiescent partially mature and/or mature SC-RPE cell monolayers. [00925] In a sixteenth aspect (A16), a system is described to perform the production of stem cell-derived retinal pigment epithelial (SC-RPE) patches (SC-RPE Patch), the method comprising enriched SC-RPE of the ninth aspect (A9) or frozen SC-RPE stock of the methods and systems of the fourteenth aspect (A14) and in particular embodiments (A14.1), or the fifteenth aspect (A15); performing optional expansion of SC-RPE cells with the method of the eleventh aspect (A11) in particular of any one of embodiments (A11.1) to (A11.8) to obtain SC-RPE cells having the potential to mature and having a target cell density; contacting the SC-RPE cells having the target cell density with a physical support, for example a membrane support and/or a microcarrier support) and at least one of the RPE Maturation Media of the fourth aspect (A4) in particular embodiments (A4.1) to (A4.8), and/or preferably the RPE Maintenance Media of the tenth aspect (A10) in particular embodiments (A10.1) to (A10.4), to obtain a population of quiescent partially mature and/or mature SC-RPE cells monolayers. [00926] In some embodiments (A16.1) of the sixteenth aspect (A16), the RPE Maintenance Medium is a variant of RPE Maintenance Medium omitting any one or combination of DMSO, bFGF, a ROCK inhibitor and/or TGF^R kinase inhibitor. [00927] In a seventeenth aspect (A17), a method is described to derive retinal pigment Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT epithelial (RPE) cell, from a starting stem cell (SC), the method comprising differentiating the stem cell (SC) to obtain partially differentiated stem cell-derived retinal pigment epithelial (SC-RPE) cells presumptive SC-RPE progenitors and/or immature SC-RPE; and maturating the differentiated SC-RPE cells to obtain matured SC-RPE cells, the method optionally further comprising at least one of performing enrichment of matured SC-RPE stem cells, preparing a frozen stock of the SC-RPE, performing expansion of the SC-RPE and/or providing the matured SC-RPE on a physical support to obtain a population of quiescent partially matured or matured SC-RPE cells monolayers; wherein the differentiating is performed by the method of the first aspect (A1) in particular embodiments (A1.1.) to (A1.13), the maturating, is performed by the method of the fifth aspect (A5) in particular embodiments (A5.1) to (A5.9), the performing enrichment is performed by the method of the seventh aspect (A7) in particular embodiments (A7.1) to (A7.10), the performing expansion is performed by the method of the eleventh aspect (A11) in particular embodiments (A11.1) to (A11.8), the preparing a frozen stock is performed by the method of the thirteenth aspect (A13) and/or the providing the quiescent partially mature and/or mature SC-RPE on a physical support is performed according to method of the fifteenth aspect (A15). [00928] In an eighteenth aspect (A18), A derivation system comprising at least two of at least one serum-free SC-RPE differentiation medium of the disclosure at least one SC-RPE cell culture substrate of the disclosure at least one serum-free RPE Maturation Medium of the disclosure Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT at least one serum-free RPE Maintenance Medium of the disclosure at least one probes each specific for a marker selected from CD57, CD104, and/or CD49b for combined use in a derivation method of the seventeenth aspect (A17). [00929] In some embodiments (A18.1) of the eighteenth aspect (A18), the at least one serum- free SC-RPE differentiation medium comprises at least one of RPE-DM1 family of media in the sense of the present disclosure. [00930] In some embodiments (A18.2) of the eighteenth aspect (A18), in particular embodiments (A18.1), the at least one serum-free SC-RPE differentiation medium comprises at least one of the RPE-DM2 family of media in the sense of the present disclosure. [00931] In some embodiments (A18.3) of the eighteenth aspect (A18) in particular embodiments (A18.1) to (A18.2), the at least one serum-free SC-RPE differentiation medium comprises at least one of the RPE-DM1 group of media supplemented with Activin A. [00932] In some embodiments (A18.4) of the eighteenth aspect (A18) in particular embodiments (A18.1) to (A18.3), the at least one serum-free SC-RPE differentiation medium comprises at least one of the RPE-DM2 group of media supplemented with Activin A. [00933] In some embodiments (A18.5) of the eighteenth aspect (A18) in particular embodiments (A18.1) to (A18.4), the at least one of RPE-DM1 family of media, comprises one or more of the RPE-DM1 media identified in any one or any one of embodiments (A1.1.6) to (A1.6.7). [00934] In some embodiments (A18.6) of the eighteenth aspect (A18) in particular embodiments (A18.1) to (A18.5), the at least one of RPE-DM2 family of media, comprises one or more of the RPE-DM2 type of medium identified in any one of embodiments (A2.1) to (A1.2.3). [00935] In some embodiments (A18.7) of any one of embodiments (A18.1) to (A18.6), the RPE-DM1 medium supplemented with Activin is any one of the RPE-DM1 of any one of embodiments (A1.1.6) to (A1.6.7) supplemented with Activin A; preferably at a concentration of 20-280 ng/mL, more preferably 70-210 ng/mL, and most preferably 140 ng/ml. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT [00936] In some embodiments (A18.8) of any one of embodiments (A18.4) to (A18.6), the RPE-DM2 medium supplemented with Activin is any one of the RPE-DM2 of embodiments (A2.1) to (A1.2.7) supplemented with Activin A; preferably at 20-280 ng/mL, more preferably 70-210 ng/mL, and most preferably 140 ng/ml. [00937] In some embodiments (A18.9) of the eighteenth aspect (A18) in particular embodiments (A18.1) to (A18.8), the at least one SC-RPE cell culture substrate comprises at least one of laminin, vitronectin, Matrigel, Geltrex, Cultrex BME, collagen IV and combinations thereof. [00938] In some embodiments (A18.10) of the eighteenth aspect (A18) in particular embodiments (A18.1) to (A18.9), the at least one SC-RPE cell culture substrate comprises at least one of laminin 511, laminin 521, laminin 111, laminin 211, and laminin 411. [00939] The SC-RPE derivation system of the eighteenth aspect (A18) in particular embodiments (A18.1) to (A18.10), wherein the at least one SC-RPE cell culture substrate comprises at least one of laminin 521, truncated vitronectin, full-length vitronectin and combinations thereof. [00940] In some embodiments (A18.11) of the eighteenth aspect (A18) in particular embodiments (A18.1) to (A18.10), the at least one maturation medium comprises the maturation medium RPE-MM as defined by the present disclosure. [00941] In some embodiments (A18.12) of the eighteenth aspect (A18) in particular embodiments (A18.1) to (A18.10), the at least one maturation medium comprises the maturation medium of the embodiments (A4.2). [00942] In some embodiments (A18.13) of the eighteenth aspect (A18) in particular embodiments (A18.1) to (A18.112), the at least one maturation medium comprises a maturation medium of the embodiments (A4.3). [00943] In some embodiments (A18.14) of the eighteenth aspect (A18) in particular embodiments (A18.1) to (A18.10), the at least one maturation medium comprises a maturation medium of any one of the embodiments (A4.4) to (A4.8). [00944] In some embodiments (A18.15) of the eighteenth aspect (A18) in particular Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT embodiments (A18.1) to (A18.14), the at least one maintenance medium comprises an expansion medium of the tenth aspect (A10) in particular of any one of embodiments (A10.1) to (A10.4). [00945] In some embodiments (A18.16) of the eighteenth aspect (A18) in particular embodiments (A18.1) to (A18.15), the at least one expansion medium is a variant of RPE Maintenance Medium omitting any one or combination of DMSO, bFGF, a ROCK inhibitor and/or TGF^R kinase inhibitor. [00946] In some embodiments (A18.17) of the eighteenth aspect (A18) in particular embodiments (A18.1) to (A18.16), the at least one probe specific for CD57 CD104, and/or CD49b comprise anti-CD104-PE (Clone: REA236, Miltenyi Biotec), anti-CD57-PE/Vio770 (Clone: REA739, Miltenyi Biotec) and anti-CD49b-APC (Clone: REA188, Miltenyi Biotec) antibodies. [00947] The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the of the serum- free methods for the derivation of SC-RPE cells and related cells, compositions, methods, and systems of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure. Those skilled in the art will recognize how to adapt the features of the exemplified serum-free methods for the derivation of SC-RPE cells and related cells, compositions, methods, and systems according to various embodiments and scope of the claims. [00948] All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the disclosure pertains. [00949] The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background, Summary, Detailed Description, and Examples is hereby incorporated herein by reference. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually. However, if any inconsistency arises between a cited reference and the present disclosure, the present disclosure takes precedence. [00950] The terms and expressions which have been employed herein are used as terms of Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed. Thus, it should be understood that although the disclosure has been specifically disclosed by embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure as defined by the appended claims. [00951] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. The term “plurality” includes two or more referents unless the content clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. [00952] When a Markush group or other grouping is used herein, all individual members of the group and all combinations and possible sub-combinations of the group are intended to be individually included in the disclosure. Every combination of components or materials described or exemplified herein can be used to practice the disclosure, unless otherwise stated. One of ordinary skill in the art will appreciate that methods, system elements, and materials other than those specifically exemplified may be employed in the practice of the disclosure without resort to undue experimentation. All art-known functional equivalents, of any such methods, device elements, and materials are intended to be included in this disclosure. [00953] Whenever a range is given in the specification, for example, a concentration range, a temperature range, a frequency range, a time range, or a composition range, all intermediate ranges and all subranges, as well as, all individual values included in the ranges given are intended to be included in the disclosure. Any one or more individual members of a range or group disclosed herein may be excluded from a claim of this disclosure. The disclosure illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. [00954] A number of embodiments of the disclosure have been described. The specific Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT embodiments provided herein are examples of useful embodiments of the disclosure and it will be apparent to one skilled in the art that the disclosure can be carried out using a large number of variations of the materials, cells, reagents, system components, and methods steps set forth in the present description. As will be obvious to one of skill in the art, methods and systems useful for the present serum- free methods for the derivation of SC-RPE cells and related cells, compositions methods and systems may include a large number of optional composition and processing elements and steps. [00955] In particular, it will be understood that various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are within the scope of the following claims. REFERENCES 1. Google_English_Dictionary. 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Claims

Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT CLAIMS 1. An RPE Maturation Medium comprising the base medium ^-MEM supplemented with additional non-essential amino acids (glycine, alanine, asparagine, aspartate, glutamate, proline, and serine), insulin, transferrin, selenite, putrescine, progesterone, serum albumin, galactose, catalase, superoxide dismutase, D,L-alpha-tocopherol, D,L-alpha-tocopherol acetate, glutathione, ethanolamine, linoleic acid, linolenic acid, biotin, carnitine, vitamin A, taurine, triiodothyronine, corticosterone, hydrocortisone and optional antibiotics and antimycotics in an effective amount to promote differentiation and maturation of partially differentiated SC-RPE cells, presumptive SC-RPE progenitors, and immature SC-RPE. 2. The RPE Maturation Medium of claim 1, wherein the maturation medium has composition Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT 3. The RPE Maturation Medium of claim 1, wherein the maturation medium is comprised of the base medium ^-MEM, additional nonessential amino acids, taurine, hydrocortisone, one of the N1-based supplements (N1, N-2, CTS-N-2, N-2-Plus, N-2-MAX, GMP-N-2-MAX, Custom N1) and one of the B27-based supplements, optionally comprising B27, B27+, XF- B27, CTS-B27, NeuroCult SM1, NS21, N21-MAX, GMP N21-MAX and Custom B27 and optional antibiotics and antimycotics at amounts and concentration ranges as defined in the disclosure. 4. The RPE Maturation Medium of claim 1, wherein the maturation medium is comprised of the base medium ^-MEM, additional nonessential amino acids, taurine, hydrocortisone, one of the B27-based supplements (B27, B27+, XF-B27, CTS-B27, NeuroCult SM1, NS21, N21- MAX, GMP N21-MAXand Custom B27) and optional antibiotics and antimycotics at amounts and concentration ranges as defined in the disclosure. 5. The RPE Maturation Medium of claim 1, wherein the maturation medium has the composition Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT . 6. The RPE Maturation Medium of claim 1, wherein the maturation medium has the composition Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT 7. The RPE Maturation Medium of claim 1, wherein the maturation medium has the composition Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT . 8. The RPE Maturation Medium of claim 1, wherein the maturation medium has the composition Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT . 9. The RPE Maturation Medium of claim 1, wherein the maturation medium has the composition Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT . 10. The RPE Maturation Medium of claim 1, wherein the maturation medium has the composition Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT . 11. The RPE Maturation Medium of claim 1, wherein the maturation medium has the composition Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT . 12. A method to maturate a differentiated stem cell-derived retinal pigment epithelial (SC- RPE) cell, the method comprising contacting differentiated SC-RPE, with a cell culture maturation medium of the present disclosure for a time and under conditions to obtain matured stem cell-derived retinal pigment epithelial (SC-RPE) cell. 13. The method of claim 12, wherein the cell culture maturation medium is the RPE Maturation Medium of any one of claims 1 to 11. 14. The method of claim 12 or 13, wherein the contacting is performed for a time selected to obtain a target percentage of mature SC-RPE cells. 15. The method of claim 12 or 13, wherein the contacting is performed for a time selected to obtain a target quality of mature SC-RPE cells. 16. The method of claim 12 or 13, wherein the contacting is performed for a time selected to obtain a target percentage of mature SC-RPE cells in combination with a target quality of mature SC-RPE cells. 17. The method of any one of claims 12 to 16, wherein the contacting is performed for a time ranging from 15 to 90 days. 18. The method of any one of claims 12 to 16, wherein the contacting is performed for a time of about 35 days. 19. The method of any one of claims 12 to 18, wherein the differentiated stem cells are SC- RPE differentiated cells obtained by Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT contacting stem cells of a starting stem cell line with a cell culture support comprising an SC-RPE differentiation medium an SC-RPE cell culture substrate wherein the SC-RPE differentiation medium and the SC-RPE cell culture substrate are selected depending on the starting stem cell line, and wherein the contacting is performed for a time and under conditions to obtain the differentiated SC-RPE cell. 20. The method of claim 19, wherein SC-RPE differentiated cells are obtained by spontaneous differentiation methods and/or with a system configured for spontaneous differentiation, and wherein contacting differentiated SC-RPE cells with a cell culture maturation medium is performed beginning between and about 7 to 60 days after the initial plating; preferably from 14 to 50 days, more preferably from 21 to 37 days and even most preferably on about day 30. 21. The method of claim 19 wherein SC-RPE differentiated cells of claim are obtained by semi- directed differentiation methods and/or with a system configured for semi-directed differentiation, and wherein contacting differentiated SC-RPE cells with a cell culture maturation medium begin from 7 to 60 days after the initial plating, preferably from about 14 to 60 days after the initial plating, more preferably from 14 to 50 days after the initial plating, even more preferably between 21 and 37 days after the initial plating, most preferably about 30 days after the initial plating. 22. A matured SC-RPE cell population, obtained by the method of any one of claims 12 to 19 and/or by using the RPE Maturation Medium of any one of claims 1 to 11. 23. A method to enrich mature stem cell-derived retinal pigment epithelial (SC-RPE) cells, the method comprising providing the matured stem cell-derived retinal pigment epithelial (SC-RPE) cells population of claim 22; contacting the matured SC-RPE cells population with a probe specific for a marker selected from CD57, CD104, and/or CD49b to obtain stained mature SC-RPE cells stained for CD57, CD104, and/or CD49b markers; and sorting the stained SC-RPE cells to select the mature SC-RPE cells stained for CD57 and/or CD104 markers, and discard cells stained for CD49b marker, to obtain a population of Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT SC-RPE cells enriched in mature SC-RPE cells. 24. The method of claim 23, wherein the providing comprises harvesting SC-RPE cells by dissociating the cultures by protease digestion. 25. The method of claim 23 or 24 wherein the probes specific for CD57, CD104, and/or CD49b comprise one or more of anti-CD104-PE (Clone: REA236, Miltenyi Biotec), anti-CD57- PE/Vio770 (Clone: REA739, Miltenyi Biotec) and anti-CD49b-APC (Clone: REA188, Miltenyi Biotec) antibodies. 26. The method of any one of claims 23 to 24 wherein the sorting can be performed by Fluorescence Activated Cell Sorting or FACS and/or Immunomagnetic cell sorting. 27. The method of any one of claims 23 to 26, wherein the contacting is performed with one or more probes specific for CD57 and/or CD104 and the sorting is performed to select SC-RPE cells positive for at least one of CD57 and CD104, and/or SC-RPE cells each positive for both of CD57 and CD104. 28. The method of any one of claims 23 to 27, wherein the contacting is performed with one or more probes specific for CD57 and/or CD49b and the sorting is performed to select SC-RPE cells positive for CD57, SC-RPE cells negative for CD49b and/or SC-RPE cells each positive for CD57 and negative for CD49b. 29. The method of any one of claims 23 to 27, wherein the contacting is performed with one or more probes specific for CD104 and/or CD49b and the sorting is performed to select SC- RPE cells positive for CD104, SC-RPE cells negative for CD49b, and/or SC-RPE cells each positive for CD104 and negative for CD49b. 30. The method of any one of claims 23 to 29, wherein the contacting is performed with one or more probes specific for CD57, CD104 and/or CD49b and the sorting is performed to select SC-RPE cells positive for CD57, SC-RPE cells positive for CD104, and/or SC-RPE cells negative for CD49b. 31. The method of any one of claims 23 to 29, wherein the contacting is performed with one or more probes specific for CD57, CD104 and/or CD49b and the sorting is performed to select SC-RPE cells positive for at least one of CD57 and CD104, SC-RPE cells positive for both of CD57 and CD104, SC-RPE cells each positive for CD104 and negative for CD49b, SC-RPE Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT cells each positive for CD57 and negative for CD49b, and/or SC-RPE cells each positive for both of CD57 and CD104 and negative for CD49b. 32. The method of any one of claims 23 to 31, further comprising detecting pigmentation in stained mature SC-RPE cells to identify pigmented stained mature SC-RPE cells and sorting the stained SC-RPE cells to select the stained mature SC-RPE cells with detected pigmentation and discard the stained SC-RPE cells with no detected pigmentation. 33 The method of claim 32, wherein the detection is performed by qualitative macroscopic detection of pigmented cells on a suitable background. 34 The method of claim 32 or 33, wherein the detection is performed by performing by image analysis, such analysis of images by performing macroscopic and/or light microscopy. 35. The method of any one of claims 32 to 34, wherein the detection is performed by measuring light absorbance readings at wavelength of at least 400 nm, preferably between 480-600 nm to quantitively detect pigmented culture area. ^ 36 The method of any one of claims 32 to 35, wherein the detection is performed by light scattering, preferably by detecting relative light side scatter to forward scatter ratio following directing a light beam towards the cell culture area. 37. A method to enrich mature stem cell-derived retinal pigment epithelial (SC-RPE) cells, the method comprising providing the matured stem cell-derived retinal pigment epithelial (SC-RPE) cells population of claim 22; detecting pigmentation in SC-RPE cells of the mature SC-RPE cell and sorting the mature SC-RPE cell populations to select pigmented mature SC-RPE cells and discard mature SC-RPE cells with no detected pigmentation. 38. The method of claim 37, wherein the providing comprises harvesting SC-RPE cells by dissociating the cultures by protease digestion. 39. The method of claim 37 or 38, further comprising contacting the pigmented mature SC-RPE cells. with a probe specific for a marker Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT selected from CD57, CD104, and/or CD49b to obtain stained pigmented mature SC-RPE cells stained for CD57, CD104, and/or CD49b markers; and sorting the stained SC-RPE cells to select the pigmented mature SC-RPE cells stained for CD57 and/or CD104 markers, and discard pigmented cells stained for CD49b marker, to obtain a population of SC-RPE cells enriched in mature SC-RPE cells. 40. The method of any one of claims 23 to 39, wherein the method further comprises post- sorting cell recovery of the population of SC-RPE cells enriched in mature SC-RPE cells. 41. A system to enrich mature stem cell-derived retinal pigment epithelial (SC-RPE) cell of claim 22, the system comprising at least two probes each specific for at least one marker selected from CD57, CD104, and/or CD49b for combined use to stain differentiated SC-RPE cells in a method to obtain a population of SC-RPE cells enriched in mature SC-RPE cells of any one of claims 23 to 31. 42. The system of claim 41, further comprising an imaging device and/or a cell sorter for combined use to detect pigmented mature SC-RPE cells in a method to obtain a population of SC-RPE cells enriched in mature SC-RPE cells of any one of claims 32 to 40. 43. The system of claim 41 or 42, wherein the probe specific for at least one of CD57, CD104, and CD49b comprise one or more of anti-CD104-PE (Clone: REA236, Miltenyi Biotec), anti- CD57-PE/Vio770 (Clone: REA739, Miltenyi Biotec) and anti-CD49b-APC (Clone: REA188, Miltenyi Biotec) antibodies. 44. A system to enrich the mature stem cell-derived retinal pigment epithelial (SC-RPE) cell of claim 20, the system comprising mature stem cell-derived retinal pigment epithelial (SC- RPE) cell of claim 22, in combination with an imaging device and/or a cell sorter for combined use in a method to obtain a population of SC-RPE cells enriched in mature SC-RPE cells of any one of claims 32 to 40. 45. An enriched SC-RPE mature cell population obtained with the method of any one of claims 23 to 40 and/or with the system of claims 41 to 44. 46. An RPE Maintenance Medium comprising the RPE Maturation Medium of any one of claims 12 to 21 supplemented with basic fibroblast growth factor (bFGF), a protein kinase inhibitor, and dimethyl sulfoxide (DMSO) in an effective amount to promote expansion of mature SC-RPE cells. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT 47. The RPE Maintenance Medium of claim 46, wherein the protein kinase inhibitor is Rho- Associated Coiled-Coil Containing Protein Kinase (ROCK) inhibitor. 48. The RPE Maintenance Medium of claim 47, wherein the ROCK inhibitor is Y-27632, Thiazovivin, or RevitaCell. 49. The RPE Maintenance Medium of claim 46, wherein the protein kinase inhibitor is any one of the transforming growth factor beta receptor (TGF^R) kinase inhibitors RepSox, A-83-01, or SB-431542. 50. The RPE Maintenance Medium of claim 46, wherein the protein kinase inhibitor is any one of the group of ROCK inhibitors Y-27632, Thiazovivin, or RevitaCell in combination with any one of the TGF^R kinase inhibitors RepSox, A-83-01, or SB-431542. 51. A method for expansion of stem cell-derived retinal pigment epithelial (SC-RPE) cells, the method comprising providing increased numbers of stem cell-derived retinal pigment epithelial (SC-RPE) cells; and contacting the SC-RPE cells with the RPE Maintenance Medium of any one of claims 46 to 50 on an SC-RPE substrate, the contacting performed for a time and under condition to obtain a target cell density of the SC-RPE cells. 52. The method of claim 51, wherein the providing comprises providing SC-RPE cells thawed and plated from frozen SC-RPE stocks. 53. The method of claim 51 or 52, wherein the method further comprises harvesting the SC- RPE cells and replating the harvested SC-RPE cells until the cultures reach the desired density. 54. The method of any one of claims 51 to 53, wherein the method is further performed to expand the SC-RPE cells. 55. The method of any one of claims 51 to 54, wherein the SC-RPE cell culture substrate is selected from laminin, Vitronectin, Matrigel, Geltrex, Cultrex BME, Collagen IV and combinations thereof. 56. The method of any one of claims 51 to 55, wherein the SC-RPE cell culture substrate is Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT selected from laminin 511, laminin 521, laminin 111, laminin 211, laminin, laminin 332, and laminin 411. 57. The method of any one of claims 51 to 56, wherein the SC-RPE cell culture substrate is selected from laminin 521, truncated vitronectin, full-length vitronectin and combinations thereof. 58. The method of any one of the claims 51 to 57, wherein the culture medium is a variant of RPE Maintenance Medium omitting any one or combination of DMSO, bFGF, a ROCK inhibitor and/or TGF^R kinase inhibitor. 59. The method of any one of claims 51 to 58, wherein the SC-RPE cells are SC-RPE differentiated cells, the mature SC-RPE cells of claim 22 and/or the enriched SC-RPE cell population of claim 45. 60. An SC-RPE expansion system for expansion of stem cell-derived retinal pigment epithelial (SC-RPE) cells, the system comprising the RPE Maintenance Medium any one of claims 46 to 50, and an SC-RPE substrate of for combined use in a method to perform expansion of the SC- RPE cells of any one of claims 51 to 59. 61. The system of claim 60, wherein the RPE Maintenance Medium comprises the RPE Maturation Medium of any one of claims 1 to 9. 62. The system of claim 60 or 61, wherein the culture medium is a variant of RPE Maintenance Medium omitting any one or combination of DMSO, bFGF, a ROCK inhibitor and/or TGF^R kinase inhibitor. 63. The system of any one of claims 60 to 62, wherein the SC-RPE cell culture substrate is selected from laminin, Vitronectin, Matrigel, Geltrex, Cultrex BME, Collagen IV and combinations thereof. 64. The system of any one of claims 60 to 63, wherein the SC-RPE cell culture substrate is selected from laminin 511, laminin 521, laminin 111, laminin 211, laminin 332, and laminin 411. 65. The system of any one of claims 60 to 64, wherein the SC-RPE cell culture substrate is selected from laminin 521, truncated vitronectin, full-length vitronectin and combinations thereof. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT 66. A method to prepare frozen stock of stem cell-derived retinal pigment epithelial (SC-RPE) cells, the method comprising - performing expansion of SC-RPE cells with the method of any one of claims 51 to 59 to obtain SC-RPE cells having a target cell density - harvesting the SC-RPE cells having the target cell density to obtain harvested SC-RPE cells; and - freezing the harvested SC-RPE cells. 67. A system for frozen stock preparation of stem cell-derived retinal pigment epithelial (SC- RPE) cell, the system comprising the SC-RPE maturation medium of any one of claims 1 to 11 and/or preferably the RPE Maintenance Medium of any one of claim 46 to 50 and/or an SC-RPE substrate of the present disclosure and freezing media, for combined use in a method to provide frozen stock of claim 66. 68. The system for frozen stock preparation of claim 67, wherein the RPE Maintenance Medium is a variant of RPE Maintenance Medium omitting any one or combination of DMSO, bFGF, a ROCK inhibitor and/or TGF^R kinase inhibitor. 69. A frozen stock preparation of (SC-RPE) cell obtainable or obtained with the method of claim 66, or with the system of claims 67 or 68. 70. A method to perform the production of stem cell-derived retinal pigment epithelial (SC- RPE) patches (SC-RPE Patch), the method comprising - providing enriched SC-RPE cell population preferably the enriched cell population of claim 45, or a frozen SC-RPE stock, preferably the SC-RPE frozen stock of claim 69; - performing optional expansion of SC-RPE cells to obtain cells having a target cell density; and - contacting the SC-RPE cells having the target cell density with a physical support, RPE Maturation Medium of any one of claims 1 to 11, and/or preferably the RPE Maintenance Medium of any one of claims 46 to 50 to obtain a population of quiescent partially mature and/or mature SC-RPE cell monolayers. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT 71. A system to perform the production of stem cell-derived retinal pigment epithelial (SC- RPE) patches (SC-RPE Patch), the method comprising enriched SC-RPE of claim 45 or frozen SC-RPE stock of claim 69, performing optional expansion of SC-RPE cells with the method of any one of claims 51 to 59 to obtain SC-RPE cells having the potential to mature and having a target cell density; contacting the SC-RPE cells having the target cell density with a physical support, for example a membrane support and/or a microcarrier support) and at least one of the RPE Maturation Media of any one of claims 1 to119, and/or preferably the RPE Maintenance Medium of any one of claims 46 to 49 to obtain a population of quiescent partially mature and/or mature SC-RPE cells monolayers. 72. The system for SC-RPE Patch production of claim 71, wherein the RPE Maintenance Medium is a variant of RPE Maintenance Medium omitting any one or combination of DMSO, bFGF, a ROCK inhibitor and/or TGF^R kinase inhibitor. 73. A method to derive retinal pigment epithelial (RPE) cell, from a starting stem cell (SC), the method comprising differentiating the stem cell (SC) to obtain differentiated stem cell-derived retinal pigment epithelial (SC-RPE); and maturating the differentiated SC-RPE cells to obtain matured SC-RPE cells, the method optionally further comprising at least one of performing enrichment of matured SC-RPE stem cells, preparing a frozen stock of the SC-RPE, performing expansion of the SC-RPE and/or providing the matured SC-RPE on a physical support to obtain a population of quiescent partially matured or matured SC-RPE cells monolayers wherein the maturating, is performed by the method of any one of claims 12 to 21, the performing enrichment is performed by the method of any one of claims 23 to 40, the performing expansion is performed by the method of any one of claims 51 to 59, Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT the preparing a frozen stock is performed by the method of claim 66, and/or the providing the quiescent partially mature and/or mature SC-RPE on a physical support is performed according to method of claim 70. 74. The method of claim 73, wherein the differentiating is performed by contacting stem cells of a starting stem cell line with a cell culture support comprising a SC-RPE differentiation medium an SC-RPE cell culture substrate wherein the SC-RPE differentiation medium and the SC-RPE cell culture substrate are selected depending on the starting stem cell line, and wherein the contacting is performed for a time and under conditions to obtain a differentiated SC-RPE cell. 75. The method of claim 74, wherein the SC-RPE differentiation medium is a RPE-DM1 culture medium KnockOut™ DMEM supplemented with KnockOut™ Serum Replacement Medium, GlutaMAX™, nonessential amino acid supplement, ^-mercaptoethanol, and normocin. 76. The method of claim 75, wherein the concentrations of the RPE-DM1 medium has composition Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT . 77. The method of claim 75, wherein the RPE-DM1 has composition Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT . 78. The method of claim 75, wherein the concentration of the RPE-DM1 medium has composition Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT . 79. The method of claim 75, wherein the concentration of the RPE-DM1 medium has composition Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT 80. The method of claim 75, wherein the concentration of the RPE-DM1 medium has composition Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT . 81. The method of claim 75, wherein the RPE-DM1 medium is comprised of a base medium, KOSR medium supplement in a concentration from 5 to 30%, from 0.25 to 4X NEAA supplement, ^-mercaptoethanol in a concentration from 25 to 200 mM, and glutamine and/or by L-alanyl-L-glutamine dipeptide in a concentration from 1.35 to 3.71 mM. 82. The method of claim 81, wherein the KOSR medium supplement is clinical grade (CTS- certified for manufacture of cells for clinical use). 83. The methods of claims 81 or 82, wherein the concentration of glutamine is 2mM. 84. The methods of any one of claims 81 to 83, wherein the RP-DM1 medium comprises glutamine in a concentration from 1.35 to 3.71 mM, or L-alanyl-L-glutamine dipeptide in a concentration from 1.35 to 3.71 mM, or a combination of glutamine and L-alanyl-L-glutamine dipeptide in a concentration from 1.35 to 3.71 mM. 85. The methods of any one of claims 81 to 84, wherein the concentration of NEAA supplement is preferably 0.5-2X, more preferably from 0.75-1.25X, or even more preferably 1X. 86. The methods of any one of claims 81 to 84, wherein the concentration of ^-mercaptoethanol is preferably from 50 to 150 mM, more preferably from 75 to 125 mM and even more Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT preferably 100 mM. 87. The methods of any one of claims 81 to 86, wherein the concentration of the KOSR medium supplement is preferably 10-25%, more preferably between 15-20%, and even more preferably 20%. 88. The methods of any one of the claims 81 to 87, wherein the base medium is KO-DMEM or CTS-KO DMEM. 89. The methods of any one of the claims 81 to 88, wherein the base medium is KO- DMEM:F12 or CTS-KO-DMEM:F12. 90. The methods of any one of the claims 81 to 87, wherein the base medium is ^-MEM. 91. The methods of any one of the claims 81 to 87, wherein the base medium is RPMI1640. 92. The methods of any one of claims 81 to 87, wherein the base medium is IMDM. 93. The methods of claims 91 and 92, wherein the concentration of the KOSR medium supplement is preferably from 15 to 20%, more preferably from 10 to 20%, even more preferably 20%. 94. The method of claim 74, wherein the SC-RPE differentiation medium is an RPE-DM2 culture medium having composition Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT possibly comprising or consisting of the cell culture medium, X-VIVO 10, preferably supplemented with a B27-based supplement at a 0.25 to 3X strength, more preferably 0.5 to 2X strength, and even more preferably 0.75 to 1.25X strength and most preferably at 1X strength. 95. The method of claim 94, wherein the B27-based supplement is selected from any one of CTS-B27, B27, B27+, XF-B27, NeuroCult SM1, NS21, N21, N21-MAX, GMP N21-MAX, Custom B27, or Custom N21. 96. The method of claim 94, wherein the RPE-DM2 culture medium is solely comprised of the cell culture medium, X-VIVO 10 and thus consists of cell culture medium, X-VIVO 10. 97. The methods of claims 94 to 96, wherein the culture medium X-VIVO 10 is replaced by any one of StemSpan SFEM, StemSpan H3000, or StemSpan ACF culture medium. 98. The methods of claims 94 to 96, wherein the culture medium X-VIVO 10 is replaced with AIM V or CTS-AIM V culture medium. 99. The methods of claims 94 to 96, wherein the culture medium X-VIVO 10 is replaced with IDMM supplemented with insulin, transferrin, selenate, and albumin. 100. The methods of claims 94 to 96, wherein the culture medium X-VIVO 10 is replaced with RPMI1640 supplemented with insulin, transferrin, selenate, and albumin. 101. The method of any one of claims 75 to 100, wherein the contacting is performed to obtain a spontaneous differentiation of SC-RPE cells. 102. The method of claim 100 wherein the contacting is performed for a time from about 11 to 56 days, more preferably 21-42 days, and most preferably 28-35 days. 103. The method of any one of claims 77 to102, wherein the SC-RPE differentiation medium is a culture medium selected from the RPE-DM1 group of media supplemented with Activin A. 104. The methods of claim 103, wherein the RPE-DM1 medium is supplemented with 20-280 ng/mL Activin A, more preferably 70- 210 ng/mL, even more preferably 140 ng/mL Activin A. 105. The method of any one of claims 77 to 102, wherein the SC-RPE differentiation medium is a culture medium selected from the RPE-DM2 group of media supplemented with Activin A. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT 106. The methods of claim 105, wherein the RPE-DM2 medium is supplemented with 20-280 ng/mL Activin A, more preferably 70-210 ng/mL Activin A, even more preferably 140 ng/mL Activin A. 107. The method of any one of claims 102 to 106, wherein the contacting is performed to obtain a semi-directed differentiation of SC-RPE cells. 108. The method of claim 107, wherein the contacting is initiated from 1 to 14 days after reaching 60-90% confluence, preferably 2-6 days after reaching 60-90% confluence, more preferably 2-4 days after reaching 60-90% confluence, and even more preferably 3 days after reaching 60-90% confluence. 109. The method of claim 108, wherein the contacting is performed for a time from for 4-28 days, more preferably 6-21 days, and most preferably 8 days. 110. The method of any one of claims 102 to 109, wherein the method further comprises following the contacting, the step of treating the cells with the differentiation medium without Activin A. 111. The method of claim 110, wherein the treating is performed for up to 60 days, more preferably 7-35 days, most preferably 14-21 days, after which time the medium is replaced with differentiation medium without Activin A or RPE Maturation Medium. 112. The method of any one of claims 74 to 111, wherein the SC-RPE cell culture substrate is selected from laminin, vitronectin, Matrigel, Geltrex, Cultrex BME, collagen IV, and combinations thereof. 113. The method of any one of claims 74 to 111, wherein the SC-RPE cell culture substrate is selected from laminin 511, laminin 521, laminin 111, laminin 211, laminin 221, laminin 332, and laminin 411. 114. The method of any one of claims 74 to 113, wherein the SC-RPE cell culture substrate is selected from laminin 521, truncated vitronectin, full-length vitronectin, and combinations thereof. 115. The method of any one of claims 74 to 114, wherein the contacting is preceded by propagating and/or culturing the stem cells. 116. The method of claim 115, wherein the propagating and/or culturing is performed for 1 to 7 days, possibly 2 to 5 days and preferably 3 to 4 or 5 days. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT 117. The method of claim 116, wherein the propagating and/or culturing is performed for 1 to 4 days, or 5 to 7 days. 118. The method of any one of claims 74 to 117, wherein the stem cells are embryonic stem cells, adult stem cells, or induced stem cells. 119. The method of any one of claims 74 to 118, wherein the starting stem cell line is a human stem cell line. 120. The method of any one of claims 74 to 119, wherein the starting stem cell line is H1, H9 or Shef1. 121. The method of any one of claims 74 to 120, wherein the starting stem cell line is Shef1. 122. A derivation system comprising at least two of at least one serum-free RPE Maturation Medium of any one of claims 1 to 11 and/or at least one serum-free RPE Maintenance Medium of any of claims 46 to 50 optionally in combination with at least one serum-free SC-RPE differentiation medium at least one SC-RPE cell culture substrate at least one probes each specific for a marker selected from CD57, CD104, and/or CD49b for combined use in a method to derive retinal pigment epithelial (RPE) cell, from a starting stem cell (SC), of claim of any one of 73 to 121. 123. The SC-RPE derivation system of claim 122, wherein the at least one serum-free SC-RPE differentiation medium comprises at least one of RPE-DM1 comprised of a base medium, KOSR medium supplement in a concentration from 5 to 30%, from 0.25 to 4X NEAA supplement, ^-mercaptoethanol in a concentration from 25 to 200 mM, and glutamine and/or by L-alanyl-L-glutamine dipeptide in a concentration from 1.35 to 3.71 mM. 124. The SC-RPE derivation system of claim 122 or 123, wherein the at least one serum-free SC-RPE differentiation medium comprises at least one of the RPE-DM2 is comprised or consists of the cell culture medium, X-VIVO 10, preferably supplemented with a B27-based supplement at a 0.25 to 3X strength, more preferably 0.5 to 2X strength , and even more preferably 0.75 to 1.25X strength and most preferably at 1X strength. Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT 125. The SC-RPE derivation system of any one of claims 122 to 124, wherein the at least one serum-free SC-RPE differentiation medium comprises at least one of the RPE-DM1 group of media supplemented with Activin A. 126.The SC-RPE derivation system of any one of claims 122 to 125, wherein the at least one serum-free SC-RPE differentiation medium comprises at least one of the RPE-DM2 group of media supplemented with Activin A. 127. The SC-RPE derivation system of any one of claims 122 to 126, wherein the at least one of RPE-DM1 family of media, comprises one or more RPE-DM1 media identified in any one of claims 75 to 93. 128. The SC-RPE derivation system of claim 123 to 127 wherein the at least one of RPE-DM2 family of media, comprises one or more of the RPE-DM2 type of medium identified in any one of claims 94 to 100. 129. The SC-RPE derivation system of the claim 125 to 128, wherein the RPE-DM1 medium supplemented with Activin is any one of the RPE-DM1 of claims 75 to 93 supplemented with Activin A; preferably at a concentration of 20-280 ng/mL, more preferably 70-210 ng/mL, and most preferably 140 ng/ml. 130. The SC-RPE derivation system of the claim 127 to 121, wherein the RPE-DM2 medium supplemented with Activin is any one of the RPE-DM2 of claims 94 to 100 supplemented with Activin A; preferably at 20-280 ng/mL, more preferably 70-210 ng/mL, and most preferably 140 ng/ml. 131. The SC-RPE derivation system of any one of claims 122 to 130, wherein the at least one SC-RPE cell culture substrate comprises at least one of laminin, vitronectin, Matrigel, Geltrex, Cultrex BME, collagen IV and combinations thereof. 132. The SC-RPE derivation system of any one of claims 122 to 130, wherein the at least one SC-RPE cell culture substrate comprises at least one of laminin 511, laminin 521, laminin 111, laminin 211, laminin 332 and laminin 411. 133. The SC-RPE derivation system of any one of claims 122 to 132, wherein the at least one SC-RPE cell culture substrate comprises at least one of laminin 521, truncated vitronectin, full- length vitronectin and combinations thereof. 134. The SC-RPE derivation system of any one of claims 122 to 133 wherein the at least one Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT maturation medium comprises the maturation medium RPE-MM of any one of claims 1 to 11. 135. The SC-RPE derivation system of any one of claims 122 to 134, wherein the at least one maturation medium comprises the maturation medium of claim 11. 136. The SC-RPE derivation system of any one of claims 122 to 135, wherein the at least one maturation medium comprises a maturation medium of claim 4. 137. The SC-RPE derivation system of any one of claims 122 to 136, wherein the at least one maturation medium comprises a maturation medium of any one of claims 2 to 11. 138. The SC-RPE derivation system of any one of claims 122 to 137, wherein the at least one maintenance medium comprises a maintenance medium of any one of claims 46 to 50. 139. The SC-RPE derivation system of any one of claims 112 to 138, wherein the at least one maintenance medium is a variant of RPE Maintenance Medium omitting any one or combination of DMSO, bFGF, a ROCK inhibitor and/or TGF^R kinase inhibitor. 140. The SC-RPE derivation system of any one of claims 12 to 139, wherein the at least one probe specific for CD57 CD104, and/or CD49b comprise anti-CD104-PE (Clone: REA236, Miltenyi Biotec), anti-CD57-PE/Vio770 (Clone: REA739, Miltenyi Biotec) and anti-CD49b- APC (Clone: REA188, Miltenyi Biotec) antibodies. 141. A matured SC-RPE cell population obtained by the derivation method of any one of claims 74 to 121 and/or by using the RPE Derivation System of any one of claims 122 to 140. 142. A matured SC-RPE cell population of claim 22, for use in transplanting an individual with the SC-RPE mature cell to treat and/or prevent retinal conditions in the individual. 143. The enriched SC-RPE mature cell population of claim 45, for use in transplanting an individual with the SC-RPE mature cell to treat and/or prevent retinal conditions in the individual. 144. The matured SC-RPE cell population of claim 141, for use in transplanting an individual with the SC-RPE mature cell to treat and/or prevent retinal conditions in the individual.