WO2015008275A1 - Méthodes de production à grande échelle de cellules souches - Google Patents

Méthodes de production à grande échelle de cellules souches Download PDF

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WO2015008275A1
WO2015008275A1 PCT/IL2014/050622 IL2014050622W WO2015008275A1 WO 2015008275 A1 WO2015008275 A1 WO 2015008275A1 IL 2014050622 W IL2014050622 W IL 2014050622W WO 2015008275 A1 WO2015008275 A1 WO 2015008275A1
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cells
cell
stem cells
microcarriers
pluripotent stem
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PCT/IL2014/050622
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Arye HASSON
Gallya YIRME
Neta LAVON
Chen GEFFEN-SHALEV
Kfir Molakandov
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Kadimastem Ltd
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Publication of WO2015008275A1 publication Critical patent/WO2015008275A1/fr
Priority to IL243491A priority Critical patent/IL243491A0/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/99—Serum-free medium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • the invention in some embodiments thereof, relates to methods for large scale generation of stem cells.
  • stem cells A considerable amount of interest has been generated in the fields of regenerative medicine and gene therapy by recent work relating to the isolation and propagation of stem cells.
  • Pluripotent stem cells are ideal candidates for the treatment of a variety of human diseases, including diabetes, myocardial infarction, ischemic stroke, various autoimmune diseases, etc.
  • Exemplary stem cells including but not limited to, ES or ESCs (embryonic stem cells), human embryonic stem cells (hESC) derived from either surplus pre-implantation genetic diagnosis (PGD) in vitro fertilization (ivf) embryos or surplus ivf embryos, human induced pluripotent stem cells (hiPSCs), human pluripotent stem cells (hPSC; hESCs and hiPSC are jointly called hPSC) neural stem cells, ADSCs (adipocyte stem cells), non-human primate ES cells, etc., are useful for downstream therapeutic applications such as cell therapy.
  • stem cell culture systems need to completely exclude animal serum during culture for a regulatory compliant end-product. Therefore, despite the existence of systems for growing stem cells in culture, there remain many difficulties in obtaining large scale systems for stem cells growth which maintain their sternness, and are effective for therapeutic use.
  • pancreatic islet cells and insulin producing cells are required to conduct clinical trials for diabetes, drug discovery and also to develop potential future cell therapies.
  • human pluripotent stem cells hPSC
  • hPSC human pluripotent stem cells
  • a method of expanding pluripotent stem cells to high density in suspension comprising:
  • the pluripotent cells are embryonic stem cells (ESCs) or induced pluripotent stem cell (iPSCs).
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cell
  • the feeder-free conditions utilize an ECM substrate selected from the group consisting of Matrigel, CELLstart, laminin, gelatin, vitronectin and fibronectin.
  • the ECM attachment substrate is selected from the group consisting of Matrigel, CELLstart, laminin, gelatin, vitronectin and fibronectin.
  • the feeder-free conditions utilize a serum free media (SFM) selected from the group consisting of STEMPRO ® hESC SFM, E8, E6, AF NutriStem ® hESC XF and KO-SR XF.
  • SFM serum free media
  • the ECM attachment substrate is feeder cell-free.
  • the container is a bioreactor or an Erlenmeyer.
  • the agitation is performed at a speed of between 20 and 200 rpm.
  • the culturing continues for at least four days.
  • the expanding continues for at least three passages.
  • the SFM is added every one to two days.
  • the pluripotent stem cells are attached to the microcarriers in a medium containing a Rock inhibitor.
  • the pluripotent stem cells are attached to the new volume of microcarriers in a medium containing a Rock inhibitor.
  • the pluripotent stem cells are dissociated into single cells by a reagent selected from the group consisting of EDTA, EGTA, BAPTA, TrypLE, Accutase and Versene.
  • the pluripotent stem cells are removed from the microcarriers by a reagent selected from the group consisting of EDTA, EGTA, BAPTA, TrypLE, Accutase and Versene.
  • the pluripotent stem cells are removed from the new volume of microcarriers by a reagent selected from the group consisting of EDTA, EGTA, BAPTA, TrypLE, Accutase and Versene.
  • the microcarriers consist of a positively charge cross-linked dextran matrix (Cytodex 1).
  • the microcarriers consist of a collagen covalent bound to a cross-linked dextran matrix (Cytodex 3).
  • the container is kept substantially still for two to three days.
  • the method further comprising the step of inducing differentiation of the stem cells obtained after step (f), wherein the method comprises placing the microcarrier-stem cell complexes under conditions which induce the differentiation of the stem cells.
  • the method comprises the step of separating stem cells from the microcarriers and culturing the separated stem cells in non-microcarrier culture under conditions which induce differentiation of the stem cells.
  • the method comprises inducing islet cell differentiation.
  • FIGs. 1A-C illustrate Matrigel coating layer on the microcarriers 1A: Positive to Anti Laminin antibody, IB: Control-using only the secondary antibody, 1C: Control- microcarriers without Matrigel coating layer.
  • FIG. 2 illustrates the growth of aggregates of cells on Cytodex 3 or Cytodex 1 microcarriers at the end of seeding stage (day 2) and at the end of the passage during static phase.
  • FIG. 3 illustrates cell concentration, cell pluripotency and cell expansion factor of KS-4 hESC cell line on Cytodex 1 or Cytodex 3 microcarriers seeded with single cells or cell clumps. The results are shown as mean values ( ⁇ SD) of samples obtained from two different experiments performed in duplicates.
  • FIG. 4 illustrates thawing of KS-4 frozen cells on Matrigel coated Cytodex 3 or Cytodex 1 microcarriers. Morphology of the cell/microcarrier aggregates thawed on Matrigel coated plate during four cultivation days with STEMPRO hESC SFM (Life Technologies Corporation, USA).
  • FIG. 5 illustrates comparison of different types of growth media and splitting procedures: StemPro versus NutriStem and CoUagenase versus TrypLE. Morphology of the cell/microcarrier aggregates; growth, pluripotency and specific Glucose consumption rates curves during two passages with different growth media and different splitting methodologies. The results are shown as mean values ( ⁇ SD) of samples obtained from two different experiments performed in duplicates.
  • FIGs. 6A-B illustrate comparison of different splitting methodologies: Dispase versus Mechanical splitting. The morphology of the cell/microcarrier aggregates are shown during 2-3 passages. 6A- the cells were grown in an Erlenmeyer; 6B- the cells were grown in a bioreactor.
  • FIGs. 7A-C illustrate splitting procedures of Matrigel coated Cytodex 3 microcarrier/cells aggregates using different EDTA concentrations.
  • the morphology of the cell/microcarrier aggregates before (7 A) and after (7B) EDTA treatment is presented.
  • FIG. 8 illustrates the morphology of the cell/microcarrier aggregates; growth, attachment efficiency and pluripotency curves of cell cultures seeded from single cells (using EDTA splitting) versus cell clumps (using Dispase splitting) during two passages.
  • FIG. 9 illustrates growth, expansion factor, attachment efficiency and pluripotency curves of KS-4 cells grown on Matrigel coated Cytodex 3 microcarriers, comparing daily fed batches versus 80% repeated batches every two days (control) during two passages. The results are shown as mean values ( ⁇ SD) of samples obtained from two different experiments performed in duplicates.
  • FIGs. lOA-C illustrate morphology of the cell/microcarrier aggregates (1 OA- IB); growth, expansion factor and pluripotency curves (IOC) during two passages in two different experiments and two different working cell banks of KS-4 on Cytodex 1 microcarrier seeded with 10% human serum and daily fed. The results are shown as mean values ( ⁇ SD) of samples obtained from two different experiments performed in duplicates.
  • FIGs. 11A-B illustrate morphology of KS-2 and KS-4 cells grown on matrigel coated microcarriers (11 A) and their growth, pluripotency and metabolism curves (11B) during three passages on two microcarrier types (Cytodex 1/ Cytodex 3). The results are shown as mean values ( ⁇ SD) of samples obtained from two different experiments performed in duplicates.
  • FIG. 12 illustrates morphology of the cell/microcarrier aggregates as well as growth, pluripotency and metabolism curves during two passages using two agitate cultivation systems (Bioreactor versus Erlenmeyer). The results are shown as mean values ( ⁇ SD) of samples obtained from two different experiments performed in duplicates.
  • FIG. 13 is a process flow chart, which illustrates the scale up process in Erlenmeyer and Bioreactor.
  • the actual accumulative cell number and expansion factor were measured during four to five passages, in two separate experiments using KS-4 cells. The results are shown as mean values ( ⁇ SD) of samples obtained from two different experiments performed in duplicates.
  • FIG. 14 is a scheme describing the differentiation protocol of hESCs towards the pancreatic phenotype through step wise, well defined stages of pancreatic development.
  • FIG. 15 illustrates the expression of insulin and glucagon in the differentiated hESC cells as measured by quantitative Real time PCR (qRT-PCR).
  • qRT-PCR quantitative Real time PCR
  • FIGs. 16A-B illustrate insulin expression and C-peptide content in differentiated hESC grown on microcariers.
  • Figure 16A demonstrates immunofluorescence staining for insulin expression (Guinea pig DAKO, 1 : 100).
  • DAPI Nuclear staining.
  • FIG 16B demonstrates C-peptide content in hESC-derived insulin producing cells.
  • C-peptide content was measured by ELISA (Mercodia) in two time points; Days 24 and 32. The results were normalized to total protein content.
  • the present invention in some embodiments thereof, relates to a method for large scale generation of stem cells.
  • the present invention is directed to compositions and methods for cultivating stem cells in large-scale culture.
  • a method of expanding pluripotent stem cells to high density in suspension comprising:
  • human embryonic stem cells can be isolated from human blastocysts or delayed blastocyst stage (as described in WO2006/040763).
  • Human blastocysts are typically obtained from human in-vivo preimplantation embryos or from in-vitro fertilized (IVF) embryos.
  • IVF in-vitro fertilized
  • a single cell human embryo can be expanded to the blastocyst stage.
  • the zona pellucida is removed from the blastocyst and the inner cell mass (ICM) is isolated from the intact ICM by gentle pipetting. The ICM is then plated in a tissue culture plate containing the appropriate medium which enables its outgrowth.
  • the ICM derived outgrowth is dissociated into clumps either by a mechanical dissociation or by an enzymatic degradation and the cells are then re -plated on a fresh tissue culture vessel. Colonies demonstrating undifferentiated morphology are individually selected by micropipette, mechanically dissociated into clumps, and re- plated. Resulting ES cells are then routinely split every 3-7 days.
  • ES cells For further details on methods of preparation human ES cells see Thomson et al., [U.S. Pat. No. 5,843,780; Science 282: 1145, 1998; Curr. Top. Dev. Biol. 38: 133, 1998; Proc. Natl. Acad. Sci. USA 92: 7844, 1995]; Bongso et al, [Hum Reprod 4: 706, 1989]; Gardner et al, [Fertil. Steril. 69: 84, 1998].
  • ES cells can be purchased from the NIH human embryonic stem cells registry ( ⁇ http://escr.nih.gov>).
  • Non-limiting examples of commercially available embryonic stem cell lines are BG01, BG02, BG03, SA01, TE03 (13), TE04, TE06 (16), HES-1, HES-2, HES-3, UCOl, UC06, WA01, WA07 and WA09.
  • Induced pluripotent stem (iPS) cells are artificial stem cells derived from somatic cells, which can be prepared by introducing certain particular nuclear reprogramming substances to somatic cells in the form of DNA or protein, and have properties almost equivalent to those of ES cells, such as pluripotency and growth capacity by self-renewal (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al. (2007) Cell, 131 : 861-872; J. Yu et al. (2007) Science, 318: 1917- 1920; M. Nakagawa et al. (2008) Nat. BiotechnoL, 26: 101-106; WO 2007/069666).
  • the nuclear reprogramming substances may be genes specifically expressed in ES cells, or genes or gene products thereof that play important roles for maintaining the undifferentiated state of ES cells.
  • the nuclear reprogramming substances are not restricted, and examples thereof include Oct3/4, Klf4, Klfl, Klf2, Klf5, Sox2, Soxl, Sox3, Soxl5, Soxl7, Soxl8, c-Myc, L-Myc, N-Myc, TERT, SV40 Large T antigen, HPV16 E6, HPV16 E7, Bmil, Lin28, Lin28b, Nanog, Salll, Sall4, Glisl, Esrrb, Esrrg, Nr5a2 and Tbx3.
  • reprogramming substances may be used in combination when iPS cells are to be established.
  • the combination may contain at 2 0 least one, two or three of the above reprogramming substances, and the combination preferably contains four of the above reprogramming substances.
  • the information on the nucleotide sequences of mouse and human cDNAs of the above-described respective nuclear reprogramming substances, and the amino acid sequences of the proteins encoded by the cDNAs can be obtained by referring to the NCBI (National Center for Biotechnology Information) accession numbers described in WO 2007/069666.
  • the information on the mouse and human cDNA sequences and amino acid sequences of L-Myc, Lin28, Lin28b, Esrrb and Esrrg can be obtained by referring to the NCBI accession numbers described below.
  • Those skilled in the art can prepare desired nuclear reprogramming substances by a conventional method based on the information on the cDNA sequences or amino acid sequences.
  • These nuclear reprogramming substances may be introduced into somatic cells in the form of protein by a method such as lipofection, binding to a cell membrane- permeable peptide, or microinjection, or in the form of DNA by a method such as use of a vector including a virus, plasmid and artificial chromosome; lipofection; use of liposomes; or microinjection.
  • a virus vector include retrovirus vectors, lentivirus vectors (these are described in Cell, 126, pp. 663-676, 2006; Cell, 131, pp. 861-872, 2007; and Science, 318, pp.
  • adenovirus vectors examples include human artificial chromosomes (HACs), yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs, PACs) (WO 2010/038904).
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs, PACs bacterial artificial chromosomes
  • plasmid examples include plasmids for mammalian cells (Science, 322:949-953, 2008).
  • the vectors may contain a regulatory sequence(s) such as a promoter, enhancer, ribosome binding sequence, terminator and/or polyadenylation site.
  • a regulatory sequence(s) such as a promoter, enhancer, ribosome binding sequence, terminator and/or polyadenylation site.
  • the promoter to be used include the EF1. alpha, promoter, CAG promoter, SR.alpha. promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR and HSV-TK (herpes simplex virus thymidine kinase) promoter.
  • the vectors may further contain, as required, a sequence of a selection marker such as a drug resistance gene (e.g., kanamycin-resistant gene, ampicillin-resistant gene or puromycin-resistant gene), thymidine kinase gene or diphtheria toxin gene; a gene sequence of a reporter such as the green-fluorescent protein (GFP), .beta.- glucuronidase (GUS) or FLAG; or the like.
  • a selection marker such as a drug resistance gene (e.g., kanamycin-resistant gene, ampicillin-resistant gene or puromycin-resistant gene), thymidine kinase gene or diphtheria toxin gene
  • a gene sequence of a reporter such as the green-fluorescent protein (GFP), .beta.- glucuronidase (GUS) or FLAG; or the like.
  • GFP green-fluorescent protein
  • GUS green-fluorescent protein
  • FLAG FLAG
  • the vector may have loxP sequences in the upstream and the downstream of these sequences.
  • a method may be employed wherein, after incorporation of the transgene(s) into a chromosome(s) using a transposon, transposase is allowed to act on the cells using a plasmid vector or an adenovirus vector, thereby completely removing the transgene(s) from the chromosome(s).
  • the transposon examples include piggyBac, which is a transposon derived from a lepidopteran insect (Kaji, K. et al., Nature, 458: 771-775 (2009); Woltjen et al, Nature, 458: 766-770 (2009); WO 2010/012077).
  • the vector may contain the origin of lymphotrophic herpes virus, BK virus or Bovine papillomavirus and sequences involved in their replication, such that the vector can replicate without incorporation into the chromosome and exist episomally.
  • Examples of such a vector include vectors containing EBNA-1 and oriP sequences and vectors containing Large T and SV40ori sequences (WO 2009/115295; WO 2009/157201; WO 2009/149233). Further, in order to introduce plural nuclear reprogramming substances at the same time, an expression vector which allows polycistronic expression may be used. In order to allow polycistronic expression, the sequences encoding the genes may be linked to each other via IRES or the foot-and- mouth disease virus (FMDV) 2A coding region (Science, 322:949-953, 2008; WO 2009/092042 2009/152529).
  • IRES foot-and- mouth disease virus
  • stem cells used in accordance with the present invention are at least 50 % purified, 75 % purified or at least 90 % purified.
  • the human ES cell colonies are separated from their feeder layer (x-ray irradiated fibroblast-like cells) such as by mechanical and/or enzymatic means to provide substantially pure stem cell populations.
  • the cells used for the assay of the present invention may be freshly generated or frozen cells. Typically, if the cells are frozen they are thawed in medium comprising growth factors (e.g. bFGF and EGF).
  • an “adherent culture” refers to a culture in which cells in contact with a suitable growth medium are present, and can be viable or proliferate while adhered to a substrate.
  • a “non-adherent culture” refers to a culture in which cells are typically in suspension with a suitable growth medium, and can be viable or proliferate while not being adhered to a substrate.
  • Microcarrier culture involves growing adherent cells on the surface of small, micron range diameter particles which are usually suspended in culture medium by gentle stirring. Microcarrier suspension culture systems are readily scalable and make it possible to achieve yields of several million cells per milliliter. Microcarrier culture has made it more economically feasible to use adherent cells for production of vaccines, protein production by animal cells and some other biotechnical products. Cells can be grown on microcarriers in a variety of formats such as suspended in spinner flasks and STR bioreactors, packed in column beds, in a fluidized bed bioreactor or even on microcarriers in micro titer plate wells.
  • microcarriers are often produced using cross linked polymers of dextran, cellulose, polyethylene or other polymers. Some such products feature polymeric coatings on glass or other net negative charged surfaces. Most cells exhibit significant net negative charge due to abundant surface carboxylic acid groups. This makes it easier for them to attach and grow at net positive charged surfaces. In many cases growth surfaces are modified with positively charged entities to promote carrier surface adherence of cells. Examples include commercially available Cytodex 1 microcarriers, prepared from cross linked dextran particles coated with diethylaminoethyl (DEAE) groups, as well as DEAE modified cotton (Cytopore 1) bead carriers from GE Healthcare Biosciences AB. In addition to non-specific (e.g.
  • Cytodex 3 microcarriers are prepared from cross linked dextran beads coated with a collagen protein layer designed to mimic the protein coated surfaces which cells bind to in the body (Microculture Cell Carrier Principles and Methods, GE).
  • Containers suitable for the method of the present invention include Erlenmeyers, bioreactors, spinners, and other containers suitable for cultivating cells.
  • bioreactor refers to a container suitable for the cultivation of eukaryotic cells, preferably animal cells, and more preferably for the cultivation of mammalian cells.
  • controlled bioreactor refers to bioreactor systems, wherein at least the oxygen partial pressure and the pH in the culture medium can be measured and regulated using known devices.
  • Suitable devices for measuring the oxygen partial pressure and pH are, for instance, oxygen and pH electrodes.
  • the oxygen partial pressure and pH can be regulated via the amount and the composition of the selected gas mixture (e.g., air or a mixture of air and/or oxygen and/or nitrogen and/or carbon dioxide).
  • Suitable devices for measuring and regulating the oxygen partial pressure are described by Bailey, J E. (Bailey, J E., Biochemical Engineering Fundamentals, second edition, McGraw-Hill, Inc. ISBN 0-07-003212-2 Higher Education, (1986)) or Jackson A T.
  • a typical cultivation volume of a controlled bioreactor is between 500 ml and 30,000 L.
  • Other suitable containers include spinners.
  • Spinners are regulated or unregulated bioreactors, which can be agitated using various agitator mechanisms, such as glass ball agitators, impeller agitators, and other suitable agitators.
  • the cultivation volume of a spinner is typically between 20 ml and 500 ml.
  • “Large scale”, as used herein with regard to cell cultivation and expansion, refers to the cultivation of SC under conditions which permit at least the doubling of cells after four weeks.
  • the term may be used to denote cultures of both undifferentiated pluripotent stem cells and cultures of differentiated cells derived from stem cells (either by directed differentiation or by spontaneous differentiation).
  • Long term refers to the cultivation of SC for at least three weeks, preferably, for at least ten weeks and more preferably, for at least 20 weeks.
  • single cell suspension refers to a hES cell single cell suspension or a hES -derived single cell suspension by any mechanical or chemical means.
  • methods and culture media conditions capable of supporting single-cell dissociation of hES cells is useful for expansion, cell sorting, and defined seeding for multi-well plate assays and enable automatization of culture procedures and clonal expansion.
  • one embodiment of the invention provides methods for generating a stable single-cell dissociation hES cell or hES-derived cell culture system capable of supporting long-term maintenance and efficient expansion of undifferentiated, pluripotent hES cell or differentiated hES cells.
  • survival is meant any process by which a cell avoids death.
  • survival also refers to the prevention of cell loss as evidenced by necrosis, apoptosis, or the prevention of other mechanisms of cell loss.
  • Increasing survival as used herein indicates a decrease in the rate of cell death by at least 10%, 25%, 50%>, 75%, 100%, or more relative to an untreated control.
  • the rate of survival may be measured by counting cells capable of being stained with a dye specific for dead cells (e.g., propidium iodide) in culture.
  • the term “to a greater extent” is meant between 2 to 100 fold.
  • flow cytometry refers to an assay in which the proportion of a material (e.g. hES comprising a particular maker) in a sample is determined by labeling the material (e.g., by binding a labeled antibody to the material), causing a fluid stream containing the material to pass through a beam of light, separating the light emitted from the sample into constituent wave lengths by a series of filters and mirrors, and detecting the light.
  • dispersing agents include, but are not limited to EDTA, dispase, collagenase, accutase, TrypLE and trypsin. Alternatively, or additionally trituration may also be performed to increase the dispersal of the cells.
  • differentiate refers to the production of a cell type that is more differentiated than the cell type from which it is derived. The term therefore encompasses cell types that are partially and terminally differentiated. Differentiated cells derived from hES cells are generally referred to as hES -derived cells or hES-derived cell aggregate cultures, or hES-derived single cell suspensions, or hES-derived cell adherent cultures and the like.
  • differentiation refers to a change that occurs in cells to cause those cells to assume certain specialized functions and to lose the ability to change into certain other specialized functional units.
  • Cells capable of differentiation may be any of totipotent, pluripotent or multipotent cells. Differentiation may be partial or complete with respect to mature adult cells.
  • pluripotent cell refers to a cell derived from an embryo produced by activation of a cell containing DNA of all female or male origin that can be maintained in vitro for prolonged, theoretically indefinite period of time in an undifferentiated state that can give rise to different differentiated tissue types, i.e., ectoderm, mesoderm, and endoderm.
  • the pluripotent state of the cells may be maintained by culturing inner cell mass or cells derived from the inner cell mass of an embryo produced by androgenetic or gynogenetic methods under appropriate conditions, for example, by culturing on a fibroblast feeder layer or another feeder layer.
  • the pluripotent state of such cultured cells can be confirmed by various methods, e.g., (i) confirming the expression of markers characteristic of pluripotent cells; (ii) production of chimeric animals that contain cells that express the genotype of the pluripotent cells; (iii) injection of cells into animals, e.g., SCID mice, with the production of different differentiated cell types in vivo; and (iv) observation of the differentiation of the cells (e.g., when cultured in the absence of feeder layer) into embryoid bodies and other differentiated cell types in vitro.
  • the pluripotent state of the cells produced by the present invention can be confirmed by various methods.
  • the cells can be tested for the presence or absence of characteristic ES cell markers.
  • characteristic ES cell markers In the case of human ES cells, examples of such markers are identified surface markers, and include TRA-1-60, SSEA-4, SSEA-3, and TRA-1-81 and are known in the art.
  • endoderm cells refers to a population of cells wherein at least 50 % thereof, more preferably at least 70 % thereof express at least one of the two markers Sox 17 or FoxA2. According to a preferred embodiment, less than 20 % of the cells, more preferably less than 10 % of the cells express markers for pluripotency, e.g. Oct4. Methods of determining expression levels of Soxl7, FoxA2 or Oct4 are known in the art and include for example RT-PCR, Immunohistochemistry and the like.
  • endoderm cells may be generated via embryoid bodies.
  • hES cells may be cultured in suspension without FGF to generate embryoid bodies.
  • the endodermal cells may be selected out of the EBs, see for example (Segev, Fischman, Ziskind et al, Stem cells, 2004;22(3):265-74.
  • the differentiation into endodermal cells is carried out in the presence of activin A.
  • concentration ranges of activin A include 1-500 ng/ml, more preferably 1-250 ng/ml, more preferably 50-200 ng/ml, such as for example 100 ng/ml.
  • the pluripotent stem cells are differentiated into endodermal cells by initial culture (e.g. for about 2 days) in a medium comprising activin A and a Wnt-3 ligand and subsequent culture (e.g. 1 day) in a medium comprising activin A, but devoid of Wnt- 3.
  • the first culture medium there may be a lower concentration of serum, relative to the second culture medium.
  • Increasing the serum concentration in the second culture medium increases the survival of the cells, or, alternatively, may enhance the proliferation of the cells.
  • the serum concentration of the first medium may be in the range of about 0 % to about 10 %.
  • the serum concentration of the first medium may be in the range of about 0 % to about 2 %.
  • the serum concentration of the first medium may be in the range of about 0 % to about 1 %.
  • the serum concentration of the first medium may be about 0.5 %.
  • both the first culture medium and the second culture medium are devoid of serum.
  • a replacement is added in place of serum.
  • Such replacements may be provided at various concentrations, such as a concentration of at least 0.1 %, e.g., a concentration of at least 0.2 %, at least 1 %, at least 1.5 % or at least 2 %.
  • Serum replacements are widely available - for example from Invitrogen (knock-out serum replacementTM and Sigma-Aldrich).
  • An additional agent that may be used to replace serum is albumin, for example human recombinant albumin.
  • pancreatic progenitor cells refers to a population of cells which are not fully differentiated into pancreatic cells, yet are committed to differentiating towards at least one type of pancreatic cell - e.g. beta cells that produce insulin; alpha cells that produce glucagon; delta cells (or D cells) that produce somatostatin; and/or F cells that produce pancreatic polypeptide.
  • pancreatic progenitor cells express some of the phenotypic markers that are characteristic of pancreatic lineages (e.g. GLUT2, PDX-1 ⁇ , PCl/3, Beta2, Nkx2.2 and PC2). Typically, they do not produce progeny of other embryonic germ layers when cultured by themselves in vitro, unless dedifferentiated or reprogrammed. It will be appreciated that it is not implied that each of the cells within the population have the capacity of forming more than one type of progeny, although individual cells that are multipotent pancreatic progenitor cells may be present.
  • islet cells refers to a cell that synthesizes at least one of the following islet-specific polypeptide hormones - insulin, glucagon, somatostatin and pancreatic polypeptide.
  • the islet cells generated according to the methods of the present invention may be construed as beta cells that produce insulin; 2) alpha cells that produce glucagon; 3) delta cells (or D cells) that produce somatostatin; and/or F cells that produce pancreatic polypeptide.
  • the islet cells of this aspect of the present invention store the hormones in secretary vesicles in the form of secretory granules.
  • populations of islet cells may be generated, the relative amounts of each cell type reflecting those in naturally occurring islets (i.e. two thirds insulin producing cells and one third glucagon producing cells).
  • diabetes refers to a disease resulting either from an absolute deficiency of insulin (type 1 diabetes) due to a defect in the biosynthesis or production of insulin, or a relative deficiency of insulin in the presence of insulin resistance (type 1 diabetes)
  • diabetes i.e., impaired insulin action
  • the diabetic patient thus has absolute or relative insulin deficiency, and displays, among other symptoms and signs, elevated blood glucose concentration, presence of glucose in the urine and excessive discharge of urine.
  • extracellular matrix (ECM) substrates refer to a surface beneath cells which supports growth.
  • ECM substrates include, but are not limited to, Matrigel, laminin, gelatin, vitronectin and fibronectin substrates.
  • such substrates may comprise collagen IV, entactin, heparin sulfate proteoglycan, to include various growth factors (e.g., bFGF, epidermal growth factor, insulin-like growth factor- 1, platelet derived growth factor, nerve growth factor, and TGF-.beta.-l).
  • the term “substantially” refers to a great extent or degree, e.g. “substantially similar” in context is used to describe one method which is to a great extent or degree similar to or different than another method.
  • the term “substantially free”, e.g., “substantially free” or “substantially free from contaminants,” or “substantially free of serum” or “substantially free of insulin or insulin like growth factor” or equivalents thereof means that the solution, media, supplement, excipient and the like, is at least 98%, or at least 98.5%, or at least 99%, or at least 99.5%, or at least 100% free of serum, contaminants or equivalent thereof.
  • a defined culture media with no serum, or 100% serum-free, or substantially free of serum in one embodiment, there is provided a defined culture media with no serum, or 100% serum-free, or substantially free of serum.
  • substantially similar or equivalents thereof means that the composition, process, method, solution, media, supplement, excipient and the like is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similar to that previously described in the specification herein, or in a previously described process or method incorporated herein in its entirety.
  • the term "enriched" refers to a cell culture that contains more than approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the desired cell lineage.
  • the term "express” refers to the transcription of a polynucleotide or translation of a polypeptide in a cell, such that levels of the molecule are measurably higher in a cell that expresses the molecule than they are in a cell that does not express the molecule.
  • Methods to measure the expression of a molecule are well known to those of ordinary skill in the art, and include without limitation, Northern blotting, RT-PCR, in situ hybridization, Western blotting, and immunostaining.
  • the term “isolated” refers to being substantially separated from the natural source of the cells such that the cell, cell line, cell culture, or population of cells are capable of being cultured in vitro.
  • the term “isolating” is used to refer to the physical selection of one or more cells out of a group of two or more cells, wherein the cells are selected based on cell morphology and/or the expression of various markers.
  • the cell culture environments and methods of the present invention comprise plating the cells in an adherent culture.
  • the terms “plated” and “plating” refer to any process that allows a cell to be grown in adherent culture.
  • adherent culture refers to a cell culture system whereby cells are cultured on a solid surface, which may in turn be coated with an insoluble substrate that may in turn be coated with another surface coat of a substrate, such as those listed below, or any other chemical or biological material that allows the cells to proliferate or be stabilized in culture.
  • the cells may or may not tightly adhere to the solid surface or to the substrate.
  • the substrate for the adherent culture may comprise any one or combination of polyornithine, laminin, poly-lysine, purified collagen, gelatin, fibronectin, tenascin, vitronectin, entactin, heparin sulfate proteoglycans, poly glycolytic acid (PGA), poly lactic acid (PLA), hyaluronan hydrogel and poly lactic-glycolic acid (PLGA).
  • the substrate for the adherent culture may comprise the matrix laid down by a feeder layer, or laid down by the pluripotent human cell or cell culture.
  • extracellular matrix encompasses solid substrates such as but not limited to those described above, as well as the matrix laid down by a feeder cell layer or by the pluripotent human cell or cell culture.
  • the cells are plated on matrigel-coated plates or microcarriers.
  • the cells are plated on vitronectin-coated plates.
  • serum can be placed in the medium for up to 24 hours to allow cells to plate to the plastic. If using serum to promote the attachment of the cells, the media is then removed and the compositions, which are essentially serum-free, are added to the plated cells.
  • compositions and methods of the present invention contemplate that the differentiable cells are cultured in conditions that are essentially free of a feeder cell or feeder layer.
  • a "feeder cell” is a cell that grows in vitro, that is co- cultured with a target cell and stabilizes the target cell in its current state of differentiation.
  • a "feeder cell layer” can be used interchangeably with the term “feeder cell.”
  • the term "essentially free of a feeder cell” refers to tissue culture conditions that do not contain feeder cells, or that contain a de minimus number of feeder cells. By “de minimus”, it is meant that number of feeder cells that are carried over to the instant culture conditions from previous culture conditions where the differentiable cells may have been cultured on feeder cells.
  • conditioned medium is obtained from a feeder cell that stabilizes the target cell in its current state of differentiation.
  • the defined medium is a non-conditioned medium, which is a medium that is not obtained from a feeder cell.
  • the term "stabilize,” when used in reference to the differentiation state of a cell or culture of cells, indicates that the cells will continue to proliferate over multiple passages in culture, and preferably indefinitely in culture, where most, if not all, of the cells in the culture are of the same differentiation state.
  • the stabilized cells divide, the division typically yield cells of the same cell type or yield cells of the same differentiation state.
  • a stabilized cell or cell population in general does not further differentiate or de-differentiate if the cell culture conditions are not altered, and the cells continue to be passaged and are not overgrown.
  • the cell that is stabilized is capable of proliferation in the stable state indefinitely, or for at least more than 2 passages.
  • the cells are stable for more than 3 passages, 4 passages, 5 passages, 6 passages, 7 passages, 8 passages, 9 passages, more than 10 passages, more than 15 passages, more than 20 passages, more than 25 passages, or more than 30 passages.
  • the cell is stable for greater than approximately 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, or 11 months of continuous passaging.
  • the cell is stable for greater than approximately 1 year of continuous passaging.
  • stem cells are maintained in culture in a pluripotent state by routine passage in the defined medium until it is desired that they be differentiated.
  • the term "proliferate” refers to an increase in the number cells in a cell culture.
  • the cell medium compositions of the present invention are refreshed at least once every day, but the medium can be refreshed more often or less often, depending of the specific needs and circumstances of the suspension culture.
  • cells are usually grown in culture media in a batch mode and exposed to various media conditions.
  • the cells exist in a dish-culture as either adherent cultures or as microcarrier/cell aggregates in suspension, and maintained in contact with a surrounding culture medium.
  • the culture medium may be refreshed about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, or any fraction thereof.
  • the medium may be refreshed less often such as, but not limited to, every 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or every 2 or more days, or any time frame in between.
  • the cells that are cultured in suspension in the medium compositions of the present invention are "split" or “passaged” every week or so, but the cells can be split more often or less often, depending on the specific needs and circumstances of the suspension culture.
  • the cells may be split every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days, or any time frame in between.
  • the term "split" or "passaged” in the context of cell culture is used as it is in the art. Namely, cell culture splitting, or passaging, is the collection of cells from a previous culture and subsequent transfer the collected (harvested) cells into a new cell culture vessel. In general, passaging cells allows the cells to continue to grow in a healthy cell culture environment.
  • One of skill in the art will be familiar with the process and methods of cell culture passaging, which may, but not necessarily, involve the use of enzymatic or non-enzymatic methods that may be used to disaggregate cells that have clumped together during their growth expansion.
  • the cells can be passaged using enzymatic, non- enzymatic, or manual dissociation methods prior to and/or after contact with the defined medium of the invention.
  • enzymatic dissociation methods include the use of proteases such as trypsin, TrypLE, collagenase, dispase, and accutase.
  • the resultant culture can comprise a mixture of singlets, doublets, triplets, and clumps of cells that vary in size depending on the enzymatic method used.
  • a non-limiting example of a non-enzymatic dissociation method is a cell dispersal buffer or EDTA.
  • the disaggregation solution used in the methods of the present invention can be any disaggregation solution capable of breaking apart or disaggregating the cells into single cells, without causing extensive toxicity to the cells.
  • disaggregation solutions include, but are not limited to, accutase, 0.25% Trypsin/EDTA, TrypLE, 0.05% EDTA or VERSENETM (EDTA) and trypsin.
  • the methods of the present invention need not result in every cell of the confluent layer or suspension being disaggregated into single cells, provided that at least a few single cells are disaggregated and capable of being re-cultured.
  • a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • KS-4 & KS-2 cell lines, passages 20-42) and IPS cells were cultured on a feeder layer of inactivated ( ⁇ -irradiated),, human foreskin fibroblast (HFF).
  • Cells were maintained in growth medium consisting of 84% KnockOut® DMEM, 14% (v/v) KnockOut® SR, 1 mM Glutamax, 0.1 mM - ⁇ - mercaptoethanol, 1% nonessential amino acids (all from Gibco, Life Technologies, USA), and 8 ng/ml bFGF (R&D Systems, USA); the medium was replaced every other day. Under these conditions the cells were kept undifferentiated.
  • the cells were harvested every 4-7 days in a ratio of 1 : 2-4 by a treatment with lmg/ml CoUagenase type IV (Gibco, Life Technologies, USA) followed by mechanical dissociation to achieve small cell clumps, and reseeded on freshly prepared HFF feeders.
  • Undifferentiated hESCs and IPS grown to confluency were adapted to grow at feeder free conditions.
  • the cells were removed from their feeder layer by a treatment with CoUagenase type IV (O.lml/cm 2 ) and mechanically dissociated into small clumps.
  • Cells were re-seeded as colonies in a concentration of -50 colonies /cm 2 onto plates coated with matrix (BD MatrigelTM (BD Biosciences, USA), Vitronectin (Peprotech) or Cellstart (Life technologies, USA) and cultured with serum free medium (STEMPRO ® hESC SFM (Life Technologies Corporation, USA) with 0.1 mM - ⁇ -mercaptoethanol and 8 ng/ml bFGF, AF NutriStem® hESC XF (Biological Industries, Israel), E8 (Life technologies, USA) or KO-SR XF (Life technologies, US A)). The medium was replaced every other day.
  • matrix BD MatrigelTM (BD Biosciences, USA)
  • Vitronectin Peprotech
  • Cellstart Cellstart
  • serum free medium STEMPRO ® hESC SFM (Life Technologies Corporation, USA) with 0.1 mM - ⁇ -mercaptoethanol and 8 ng/ml bFGF, AF NutriStem®
  • the cells were harvested every 4-6 days in a ratios of 1 to 2-4 by a treatment with Dispase (Gibco, Life Technologies, USA) or 0.05% EDTA (Biological Industries, Israel) or TrypLE Select (Gibco Life Technologies, USA), followed by mechanical dissociation to achieve small cell clumps, and reseeded on freshly prepared plates coated with matrix (up to 15 passages). Under these conditions cells were kept undifferentiated.
  • Adapted undifferentiated hESCs and IPS grown to confluency were used to grow the cells on microcarriers at feeder free conditions.
  • the cells fed with fresh medium including Rock inhibitor (Y-27632, Sigma) 4-6 hr prior the split.
  • the cells were removed from their feeder free matrix by treatment with 0.05% EDTA or TrypLE Select followed by mechanical dissociation to achieve single cells.
  • Cells were re-seeded on Cytodex 1 and Cytodex 3 microcarriers coated with feeder free matrix, at cell concentration of 0.08 ⁇ 0.04X10 6 viable cell/cm 2 , and cultured with the relevant serum free medium.
  • Rock inhibitor (10 ⁇ ) was added at seeding.
  • the medium was added every day at ⁇ 10%> of the working volume, up to total volume of ⁇ 30%> of the working volume. At day 2-3 full amount of bFGF and ⁇ -mercaptoethanol was added. The cells were harvested every 4-7 days with 0.01-0.05% EDTA or TrypLE Select followed by mechanical dissociation to achieve small cell/microcarrier aggregates and reseeded on freshly prepared feeder free microcarriers (up to 10 Passages). Under these conditions cells were kept undifferentiated.
  • the dynamic process was confirmed in three cultivation systems - non adherent 6-well plates, Erlenmeyers (125ml, 250ml and 500ml, Corning Incorporated, Corning NY, USA) and Bioreactor (500ml, CellSpin of Integra Biosciences, Fernwald, Germany).
  • the shaking and agitation values were adjusted with the cultivation system and the working volume to the range of 65-95RPM.
  • the expansion process included static seeding period of two days followed by 2-5 days of growth period.
  • hESC/microcarriers aggregates (KS#4, P35PFF4E8) were collected, washed, and re-suspended in fresh StemPro medium.
  • the hESC/microcarriers were treated similar to cryopreserved hESC colonies, with the exception that the cells were not removed from the microcarriers prior to freezing.
  • hESC/microcarriers were suspended in DMSO (10%), KO-SR (30%), Rock inhibitor ( ⁇ ) and 60% HESM at 3X10 6 cells/mL (0.11 and 0.27X10 6 cells/cm 2 , Cytodex 1 or Cytodex 3, respectively). The cells were divided into cryovials, frozen inside the Mr. Frosty Freezing container in a freezer (-80°C), and transferred into liquid nitrogen within 24 hrs.
  • StemPro medium and Rock inhibitor were added into the vials to dilute the cryoprotectants. The cells were then pelleted and washed in the culture medium. Finally, the cells were re-suspended in StemPro and Rock inhibitor and plated onto a Matrigel coated culture plates, without detached from the microcarriers prior to re- plating.
  • the microcarriers were prepared and sterilized, following the manufacturer's instructions. Briefly, the dry Cytodex 1 or Cytodex 3 microcarriers (GE healthcare, USA) were swollen in PBS for at least three hours at room temperature. The supernatant was decanted and the microcarriers were washed twice for a few minutes in fresh PBS, and sterilized by autoclaving. At this stage the sterile microcarriers may be stored at 4°C for about two months (based on the manufacturer's recommendation). Prior to use, the sterilized microcarriers were allowed to settle and the PBS was decanted.
  • the dry Cytodex 1 or Cytodex 3 microcarriers GE healthcare, USA
  • BD MatrigelTM 1050 ⁇ 1 cold BD MatrigelTM was dissolved in cold DMEM and added to 70-210 mg (dry weight) cold, sterile microcarriers. DMEM was added to bring the final volume to 70 ml. Then the mixture was transferred into the 125ml erlenmeyer and shacked continuously overnight at 70 RPM. The Matrigel coated microcarriers may be stored at 4°C up to seven days. The Matrigel coated microcarriers were washed with DMEM/F12 before seeding of hESCs or IPS.
  • Homogenously suspended cell/microcarrier aggregates were harvested in ⁇ lml growth medium (at-least two separate samples for each cultivation vessel). The samples were analyzed for total cell concentration with NucleoCounter ® NC-100 (ChemoMetec, Denmark) while attached to the microcarriers. For viability measurement the medium was removed, cell/microcarrier aggregates were washed with PBS and incubated for five minutes with two ml TrypLE Select (Gibco, Life technologies, USA) at 37°C and re-suspended to achieve a single cell suspension. Cells' viability was determined by NucleoCounter ® NC-100.
  • the cultivation systems were removed from the shaker to allow the cell/microcarrier aggregates to settle.
  • Samples from the utilized medium were immediately analyzed for pH. Determination of glucose and lactic acid concentrations was performed by using RQfiexlO analyzer (MERCK, Germany). Glucose consumption rates (GCR) and lactate formation rates (LFR) were calculated.
  • hESCs grown on cytodex microcarriers as described in example 1 1 were washed once with Day 1 differentiation media without differentiation factors. Following gravity sedimentation (1-2 minutes) the supernatant was discarded and the cells were dissolved in Day 1 differentiation media including Activin A (100 ng/ml) and Wnt3a (25 ng/ml) (R&D), in a concentration of lxl 0 6 cells/ml. The cells were then transferred to low adherent 6 well plates (Corning) in static conditions (2xl0 6 cells/well). Media change was done according to Table 1 by tilting the plate, sedimentation of cells and replacing about 90% of the media.
  • the content of human insulin C-peptide was measured with an ELISA kit (Mercodia,Upsalah, Sweden, ultrasensitive c-peptide kit (minimal detection 5pM) or Mercodia c-peptide ELISA (minimal detection 90pM).
  • Samples in M-PER were diluted 3 fold, or more in the kit buffer, and 20 ⁇ of the 1/3 dilution were assayed. Results were expressed per mg total protein (as measured by Bradford assay) and per million cells (counted in a NucleoCounter). Other cell pellets were suspended in 0.7 ml buffer RLT (RNAeasy, Qiagen) per million cells, for RNA extraction.
  • Human insulin mRNA was quantitated by RT-qPCR (Taq-Man, Applied Biosystems, Step One) using the Hypoxanthine-guanine phosophoribosyl transferase (HPRT) gene as reference.
  • RT-qPCR Taq-Man, Applied Biosystems, Step One
  • HPRT Hypoxanthine-guanine phosophoribosyl transferase
  • Static ultra-low attachment 6-well plates were inoculated with 0.5xl0 6 cells/4mg microcarriers (Cytodex 1 or Cytodex 3). 0.03-0.05xl0 6 cells/cm 2 were seeded into each well. As control, the cells were seeded at the same concentrations into 2D- 6-well plate covered with Matrigel. As shown in Figure 2 large aggregates were established due to static conditions. Most of the cells are a live (>65%).
  • Single cells were harvested from colonies grown at least three passages on Matrigel/Stempro 2D feeder free growth system, using TrypLE in the presence of ROCK inhibitor, while small clumps were harvested using TrypLE (dilutedl :5) in the presence of ROCK inhibitor.
  • Figure 3 shows that there is no advantage of seeding cell clumps as compared to single cells. In contrary, the diluted TrypLE was not able to dissociate efficiently the cell colonies to cell clumps.
  • Cytodex 3 and Cytodex 1 growth systems which were seeded with single cells, there were no distinct differences in terms of cell expansion and pluripotency.
  • the 2D system seeded with single cells which had the same growth area of -lOcm 2 as the Cytodex 3 system, was inferior in terms of cell expansion, although the pluripotency remained similar to the pluripotency of cells grown on the microcarriers systems.
  • Cells were harvested using TrypLE in the presence of ROCK inhibitor for receiving single cells, and using collagenase for receiving small clumps from colonies grown on 2D feeder free (Matrigel) plates.
  • Erlenmeyers 125 ml were inoculated with 0.02xl0 6 cell/cm 2 seeded on Matrigel coated Cytodex 3 (3 mg/ml) and the dynamic growth phase was started following two days of the static seeding stage.
  • Figure 5 presents the morphology of the cell/microcarrier aggregates at day six. The cells were grown in two different media (StemPro® hESC SFM versus NutriStem® hESC XF) and two different splitting methodologies (single cells versus cell clumps) were used.
  • the curves of cell growth, Glucose consumption rate and pluripotency during two passages on the same two media and using the same splitting methodologies are also presented.
  • the single cell seeding gave rise to smaller aggregates.
  • the smaller aggregates presented higher cell concentration, higher expansion, lower doubling time (data not show), higher specific Glucose consumption rate and higher pluripotency.
  • Anther splitting methodology which was tested and finally elected was the use of EDTA in different conditions and concentrations in order to obtain single cells or very small aggregates.
  • EDTA concentrations as well as several splitting procedures were tested.
  • using very mild concentrations of 0.01- 0.05% EDTA for 3-4 minutes and medium neutralization was found to be ideal to re- growing of the cells on new microcarriers.
  • KS-4 were seeded on Matrigel coated Cytodex 3 and cultivated during three passages. The splitting efficiency was high when using all EDTA concentrations and reached up to 80%.
  • Cell growth on Matrigel coated Cytodex 1 or Cytodex 3 microcarriers was tested using two different cell lines (KS-2 and KS-4).
  • the optimal conditions used were as described below: the cell seeding concentration was 0.08 ⁇ 0.04X10 6 viable cells/cm 2 , StemPro medium was used, the microcarrier concentration was 1-20 mg/ml, the seeding duration was two days at static conditions followed by constant agitation (65-95 RPM), a working volume of 10-30%, daily fed-batches was used, the passage duration was 4-6 days and EDTA (0.01-0.05%) was used for splitting.
  • Figure 11A presents the morphology of the cell/microcarrier aggregates and Figure 11B demonstrates the growth and pluripotency curves during three passages using the two microcarrier types. It was found that the cell/microcarrier aggregate size was depended on two parameters: the propagation duration and the splitting efficiency. When the splitting efficiency was lower, the new seeded aggregates were larger. When the passage duration was longer, the cell/microcarrier aggregates were larger and denser, and it was harder to break them prior to the next passage. The cell concentration was 0.8xl0 6 cell/cm 2 following eight days in culture, and 0.4-0.6 cell/cm 2 following five days of culture (at seeding concentration of 0.04X10 6 viable cells/cm 2 ) .
  • the cells displayed high growth rate (doubling time of -40 hours) and high expansion factor of -10 with potential of -15.
  • the pluripotency remained over 80% during the four passages.
  • the metabolism conditions were kept in the following levels: Glucose level over 1.0 gr/L in a passage of five days; lactate level lower than 12mM and pH over 6.5, in order to avoid oxygen limitation in the cell/microcarrier aggregates and high acidification of the medium.
  • Figure 12 presents the morphology of the cell/microcarrier aggregates and the growth; metabolism and pluripotency at day 7, during two passages, in the two cultivation systems.
  • the bioreactor presented larger aggregates the expansion factor at PI was higher and the doubling time was lower (data not shown), due to higher surface area/volume ratio and a better medium replacement methodology, as was detected by the higher levels of glucose and lower levels of lactate in the spent media.
  • the decrease in the pluripotency was dramatic at both systems and can be referred to the non-efficient mechanical splitting rather than the cultivation systems.
  • the scale up process flow chart may comprise the following steps:
  • Adaptation and cultivation of pluripotent hESCs in feeder- free growth conditions Matrigel coated 2D vessels; Stempro media including 8ng bFGF; passaged every 2-8 days using Dispase; high pluripotency (over 85% EpCAM and Tra-1-60)
  • pancreatic differentiation potential of hESCs grown on microcarriers was analyzed by applying a step wise differentiation protocol developed to manipulate differentiation of hESCs towards mature pancreatic endocrine phenotype.
  • cells go through several well characterized developmental stages, yielding mature endocrine hormone producing cells, similar to human islets.
  • a flow chart of the differentiation protocol is described in figure 14.
  • the differentiated islet like cells exhibit mature ⁇ -cell characteristics manifested by the expression of insulin mRNA ( Figure 15) and the ability to produce and store insulin/C-peptide ( Figures 16 A and B).
  • Glucagon mRNA is also detected, implying that the pancreatic endocrine differentiation protocol leads also to the generation of glucagon producing a-cells.

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Abstract

L'invention concerne des méthodes de production à grande échelle de cellules souches. L'invention concerne en particulier des compositions et des méthodes permettant de cultiver des cellules souches dans le cadre d'une culture à grande échelle.
PCT/IL2014/050622 2013-07-17 2014-07-10 Méthodes de production à grande échelle de cellules souches WO2015008275A1 (fr)

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DE102019007815B3 (de) * 2019-11-12 2021-01-14 Zellwerk Gmbh Zellkulturträger für Bioreaktoren
CN113637635A (zh) * 2021-10-13 2021-11-12 北京华龛生物科技有限公司 微载体细胞培养体系中微载体聚团状态的解散方法
WO2023055247A1 (fr) 2021-09-30 2023-04-06 Warszawski Uniwersytet Medyczny Pansement pour le traitement de plaies difficiles à cicatriser et son procédé de fabrication

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DE102019001604A1 (de) * 2019-03-06 2020-09-10 Zellwerk Gmbh Zellkulturträger für Bioreaktoren
DE102019001604B4 (de) * 2019-03-06 2020-12-24 Zellwerk Gmbh Zellkulturträger für Bioreaktoren
DE102019007815B3 (de) * 2019-11-12 2021-01-14 Zellwerk Gmbh Zellkulturträger für Bioreaktoren
WO2023055247A1 (fr) 2021-09-30 2023-04-06 Warszawski Uniwersytet Medyczny Pansement pour le traitement de plaies difficiles à cicatriser et son procédé de fabrication
CN113637635A (zh) * 2021-10-13 2021-11-12 北京华龛生物科技有限公司 微载体细胞培养体系中微载体聚团状态的解散方法

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