Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
  • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L31/145—Hydrogels or hydrocolloids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
  • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
  • A61L29/14—Materials characterised by their function or physical properties, e.g. lubricating compositions
  • A61L29/16—Biologically active materials, e.g. therapeutic substances
• • 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]
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/483—Physical analysis of biological material
  • G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
  • A61L2300/80—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special chemical form
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/00596—Solid-phase processes
• • 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
  • C12N2533/20—Small organic molecules
• • 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
  • C12N2533/30—Synthetic polymers
• • 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
  • C12N2537/00—Supports and/or coatings for cell culture characterised by physical or chemical treatment
  • C12N2537/10—Cross-linking
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2610/00—Assays involving self-assembled monolayers [SAMs]

Definitions

• the present disclosure relates generally to methods for preparing biomaterial compositions and methods for using the biomaterial compositions. More particularly, the present disclosure relates to hydrogel compositions and to methods for using the hydrogel compositions to promote cell expansion and cell differentiation.
• mesenchymal stem cells may be differentiated in vitro into osteoblasts, chondrocytes, myoblasts, adipocytes, neurons, and endothelial cells by exposure to a variety of growth factors. Routine cellular expansion and differentiation protocols rely on high concentrations of expensive recombinant growth factors. Substantial progress has been made in the development of defined media, but only more recently has the role of substrates and cell- substrate adhesion on cell growth been examined.
• tissue culture polystyrene is the "gold standard” for cellular expansion and differentiation during in vitro cell culture, particularly, for human mesenchymal stem cells (hMSCs); however, TCPS does not allow the user to control substrate stiffness and growth factor regulation. Stiffness has been demonstrated to be important in controlling cellular proliferation, lineage specification, commitment, and maturation. Accordingly, there exists a need for methods for preparing biomaterial compositions that will support survival and growth of cells in culture, and particularly, to provide specific molecules that promote cellular expansion, cellular differentiation and regulate cellular behavior. It would further be advantageous if the biomaterial compositions allowed for control over both biomaterial substrate stiffness and growth factor signaling.
• the present disclosure relates generally to biomaterial compositions and methods for using the biomaterial compositions. More particularly, the present disclosure relates to hydrogel compositions and methods for promoting cellular expansion and cellular differentiation using the hydrogel compositions.
• hydrogel compositions of the present disclosure can also be used for two-dimensional (2D) and three-dimensional (3D) cell culture.
• the hydrogel compositions of the present disclosure can further be used for two-dimensional and three-dimensional enrichment of biomolecules such as, for example, biomolecules to cell surfaces using soluble factor binders.
• the hydrogel compositions further offer design control over both hydrogel substrate stiffness and growth factor signaling, allow for attachment with phenotypes consistent with those offered by conventional MATRIGEL®, and growth factor regulation.
• the present disclosure is directed to a method of promoting cellular expansion.
• the method includes preparing a hydrogel composition, wherein the hydrogel composition includes a polyethylene glycol functionalized with norbornene, a crosslinking peptide, and a cell adhesion peptide; contacting a cell with the hydrogel composition; and culturing the cell.
• the present disclosure is directed to a method of promoting cellular differentiation.
• the method includes preparing a hydrogel composition, wherein the hydrogel composition includes a polyethylene glycol functionalized with norbornene, a crosslinking peptide, and a cell adhesion peptide; contacting a cell the hydrogel composition; and culturing the cell.
• FIGS. 1A-1B are schematic illustrations of the steps for preparing a hydrogel array of the present disclosure.
• FIG. 2A is a schematic illustration of the steps for patterning a metal-coated substrate used in the method for preparing a hydrogel array of the present disclosure.
• FIG. 2B are end view drawings of the metal-coated substrate during the steps for patterning a metal-coated substrate shown in FIG. 2A.
• FIG. 3 is a photograph of a hydrogel array with 64 individual hydrogel spots prepared using the methods of the present disclosure.
• FIG. 4 is a graph illustrating the surface roughness of a hydrogel array as determined by atomic force microscopy.
• FIG. 5 illustrates high magnification top-view images showing different shapes of individual hydrogel spots.
• FIG. 6 is a side-on image showing individual hydrogel spots having different heights.
• FIG. 7 is a hydrogel array showing differential patterning of individual hydrogel spots by increasing the density of a fluorescently-tagged peptide and increasing the density of encapsulated fluorescent microspheres, as discussed in Example 2.
• FIG. 8 is a graph illustrating control of the modulus of individual hydrogel spots of a hydrogel array by changing the total concentration of PEG-NB (w/w ) in the hydrogel precursor solution using the methods of the present disclosure.
• FIG. 9 is a schematic illustrating the steps for preparing a hydrogel array and further assembling the hydrogel array with a microwell add-on using the methods of the present disclosure.
• FIG. lOA-lOC are photographs of hMSCs cultured on hydrogel arrays prepared using 4 wt. % (FIG. 10A), 6 wt. % (FIG. 10B) and 8 wt. % (FIG. IOC) polyethylene glycol and presenting linear RGD peptide, as discussed in Example 2.
• Scale bar 100 ⁇ .
• FIG. 1 lA-11C are photographs of hESCs cultured on hydrogel arrays prepared using 4 wt. % (FIG. 11 A), 6 wt. % (FIG. 11B) and 8 wt. % (FIG. 11C) polyethylene glycol and presenting varying peptide identity, as discussed in Example 2.
• Scale bar 100 ⁇ .
• FIGS. 12-14 depict hMSC cell attachment, spreading and proliferation as analyzed in Example 3.
• FIGS. 15 & 16 depict hESC cell spreading and proliferation rates as analyzed in Example 4.
• hydrogel compositions can be prepared as a hydrogel array with individually controlled hydrogel spot modulus, hydrogel spot polymer density, hydrogel spot ligand identity and hydrogel spot ligand density and to methods for preparing the hydrogel arrays.
• the hydrogel compositions can be prepared as coatings such as for use on the surfaces of cell culture plates.
• the hydrogel compositions can be prepared as microcarriers in suspension culture.
• the hydrogel compositions of the present disclosure can be functionalized with biomolecules, are compatible with cell culture and are biocompatible.
• the hydrogel compositions of the present disclosure can be used to alter (e.g., enhance, inhibit and change) cell function, and in particular, cellular expansion, maturation and differentiation.
• a hydrogel composition is a network of polymer chains that are hydrophilic in which a polymeric material and water are in an equilibrated form.
• the hydrogel composition is formed using unpolymerized starting components.
• the polymeric material can be, for example, a natural polymer material, a synthetic polymer material and combinations thereof.
• the methods for preparing hydrogel compositions of the present disclosure advantageously allows for the direct incorporation of peptides into the hydrogel network during polymerization by including a cysteine in the amino acid sequence during synthesis, which allows for eliminating the need for post-synthetic modifications.
• peptides can be utilized as crosslinkers by including cysteine on each end or they can be incorporated as pendant groups, which can be pre-coupled to the polymer backbone and mixed in varying combinations or incorporated during polymerization for simplicity.
• the present disclosure is generally directed to methods for preparing a hydrogel composition and use of the resulting compositions.
• the preparation methods When used to prepare a hydrogel array, the preparation methods generally include contacting a hydrogel precursor solution with a substrate, wherein the substrate includes a hydrophobic region and a hydrophilic region; placing a surface-modified substrate onto the hydrogel precursor solution such that the hydrogel precursor solution is located between the substrate and the surface- modified substrate; polymerizing the hydrogel precursor solution; and separating the surface - modified substrate from the substrate, to result in the hydrogel array. (See, FIGS. 1A-1B).
• the polymer hydrogel precursor solution polymerizes between the substrate and the surface-modified substrate and the resultant hydrogel transfers with the surface-modified substrate such that the surface-modified substrate includes the hydrogel array.
• the hydrogel array can be patterned to include an array of hydrogel spots surrounded by a hydrogel-free background as described in more detail below.
• the hydrogel array can be patterned such that an array of hydrogel-free spots (or pools) is formed within a hydrogel background as described in more detail below.
• the resultant hydrogel array can be patterned to result in differential wettability to define the geometry of each hydrogel spot and confine the contents of each hydrogel spot of the array, as well as define the spatial pattern of each hydrogel spot in the array in relation to neighboring spots.
• This is particularly useful for preparing hydrogel arrays for use with common microarray add-ons of different sizes and dimensions consistent with those of common multi-well plates (e.g., 96 well plates, 384 well plates, etc.)
• This is also useful for use with multichannel pipettes for enhanced-throughput cell culture, media exchange, and the like.
• the individual hydrogel spots of the array can have any desired shape (see e.g., FIG. 5).
• the shape can be circular, round, oval, quatrefoil, rectangular, triangular, star-shaped, diamond-shaped, combinations thereof, and the like.
• Patterns of hydrogel spots may also be created in rows, spirals, circles, squares, rectangles, combinations thereof, and the like.
• the shape of the individual hydrogel spot can be varied by changing the pattern of the stencil used for etching during patterning of the patterned substrate.
• the individual hydrogel-free spots can have any desired shape.
• the shape can be circular, round, oval, quatrefoil, rectangular, triangular, star-shaped, diamond-shaped, combinations thereof, and the like. Patterns of hydrogel-free spots may also be created in rows, spirals, circles, squares, rectangles, combinations thereof, and the like.
• the shape of the individual hydrogel-free spot can be varied by changing the pattern of the stencil used for etching during patterning of the patterned substrate.
• the upper size limit of the hydrogel array depends on the dimensions of the patterned substrate and/or the dimensions of the surface-modified substrate.
• the resultant hydrogel array can also be patterned to result in individual hydrogel spots and hydrogel-free spots having any desired sizes.
• the size and shape of the individual hydrogel spot and hydrogel-free spot can be varied by changing the pattern of the stencil used for etching during patterning of the patterned substrate.
• Suitable individual hydrogel spot size of the hydrogel array can be small enough to accommodate a single cell, but also large enough to accommodate many cells, for example.
• the individual hydrogel spot size of the hydrogel array can have any desired diameter.
• Particularly suitable individual hydrogel spot sizes of the hydrogel array can be about 10 ⁇ and larger.
• a patterned substrate can be prepared by creating hydrophobic regions and hydrophilic regions formed by self-assembled monolayers (SAMs), such as described in US Patent Application Serial No. 14/339,938, filed on July 24, 2014, herein incorporated by reference to the extent it is consistent herewith.
• SAMs self-assembled monolayers
• Suitable substrates for forming self- assembled monolayers are known to those skilled in the art and can be, for example, metal- coated substrates, silicon substrates, diamond substrates, polydimethylsiloxane (PDMS) substrates, and the like (as described in Love et al., Chem. Rev. 2005, 105:1103-1169, for example, which is hereby incorporated by reference to the extent its disclosure is consistent with the present disclosure).
• the patterned substrate can be prepared, for example, by forming regions with differential wettability on a substrate by immersing the substrate in a perfluorinated alkanethiol solution to allow perfluorinated alkanethiolate self-assembled monolayers (fluoraSAMs) to form.
• fluoraSAMs perfluorinated alkanethiolate self-assembled monolayers
• a stencil can be placed on the fluoraSAMs metal-coated substrate to selectively protect regions of the fluoraSAMs metal-coated substrate from plasma etching. Exposed regions of the fluoraSAMs substrate can then be etched by oxygen plasma treatment to form etched fluoraSAMs in the substrate.
• the substrate is then immersed in a hydroxyl-terminated alkanethiol solution to form a hydrophilic alkanethiolate SAM (EG 3 SAM) in the etched regions of the substrate.
• EG 3 SAM hydrophilic alkanethiolate SAM
• the resulting patterned substrate possesses differential wettability based on the hydrophobic SAMs and hydrophilic SAMs.
• the method can further include placing a spacer between the patterned substrate and the surface-modified substrate.
• the spacer placed onto the patterned substrate while performing the method functions to define the height (or thickness) of the hydrogel forming the hydrogel array.
• a spacer may be particularly desirable when preparing higher (i.e., thicker) hydrogel arrays.
• the hydrogel array can have any desirable height (see e.g., FIG. 6). Suitable heights of the hydrogel array can be from about 20 micrometers ( ⁇ ) to about 1 millimeter, however, hydrogel arrays can be made much higher than 1 millimeter if desired.
• the spacer also functions to prevent direct contact between the surface of the patterned substrate and the surface-modified substrate during formation of the hydrogel.
• the spacer used in the method can be any suitable material known to those skilled in the art.
• a particularly suitable spacer can be, for example, polydimethylsiloxane (PDMS).
• PDMS polydimethylsiloxane
• the height the hydrogel array can be determined, for example, using a microscope to focus from the top of the hydrogel down to the substrate, using a microscope to focus from the substrate up to the top of the hydrogel, and by measuring the surface roughness of a hydrogel array as determined by atomic force microscopy (see e.g., FIG. 4).
• the preparation method further includes contacting a hydrogel precursor solution with the patterned substrate.
• the hydrogel precursor solution is contacted with the hydrophilic regions of the patterned substrate.
• the hydrophobic regions of the patterned substrate serve as a barrier between neighboring hydrophilic regions and also allow for the isolation of each hydrophilic region.
• the hydrogel precursor solution can be, for example, a combination of a polymer and a multifunctional polymer crosslinker.
• preparation methods When used as a hydrogel coating composition, preparation methods generally include contacting a hydrogel precursor solution with a substrate to be coated (e.g., surface of a cell culture plate).
• a substrate to be coated e.g., surface of a cell culture plate.
• Suitable polymers for use in the hydrogel precursor solution are known by those skilled in the art and can include, for example, poly(ethylene glycol), hyaluronic acid, gelatin, collagen, MATRIGEL®, dithiol polymers (e.g., acrylamide), click-based composite hydrogels (as discussed in Polizzotti et al. Biomacromolecules 2008, 9: 1084-1087, which is hereby incorporated by reference to the extent its disclosure is consistent with the present disclosure), poly(ethylene glycol)-diacrylate, poly(ethylene glycol)-vinyl sulfone, and the like.
• Particularly suitable polymers can be, for example, poly(ethylene glycol).
• Particularly suitable polymers can be, for example, functionalized polymers.
• a particularly suitable functionalized polymer can be, for example, eight-arm poly(ethylene glycol) with terminal hydroxyl (-OH) groups (commercially available from JenKem Technology USA, Allen, TX) that is functionalized with norbornene. Eight-arm poly(ethylene glycol) can be functionalized with norbornene as described in Fairbanks et al. (Adv. Mater. 2009, 21 :5005-5010).
• polystyrene resin poly(ethylene glycols) that may be functionalized using click chemistry.
• Click chemistry is an extremely versatile method for chemically attaching biomolecules, which is used to describe the [3+2] cycloaddition between alkyne and azide functional groups. Azides and alkynes are largely inert towards biological molecules and aqueous environments, which allows the use of the Huisgen 1,3- dipolar cycloaddition to yield stable triazoles that are very difficult to oxidize or reduce. Both the copper(I)-catalyzed and copper-free strained- alkyne variant reactions are mild and very efficient.
• the hydrogel precursor solutions include concentrations of polymer of up to, and including, 200 mg/mL, including from about 36 mg/mL to about 160 mg/mL, and including from about 36 mg/mL to about 70 mg/mL.
• Suitable multifunctional polymer crosslinkers for use in the hydrogel precursor solution are known by those skilled in the art.
• Particularly suitable bi-functional polymer crosslinkers and multifunctional polymer crosslinkers can be, for example, polyethylene glycol dithiol (PEG-DT), protease-degradable crosslinkers and multi-arm poly(ethylene glycol) terminated with thiol (e.g., 4-arm PEG terminated with thiol).
• Suitable protease-degradable crosslinkers can be, for example, matrix metalloproteinase (MMP)- degradable crosslinkers as described in Nagase and Fields (Biopolymers 1996, 40:399-416, which is hereby incorporated by reference to the extent it is consistent with the present disclosure). More particularly, suitable MMP-degradable crosslinking peptides for use in the hydrogel precursor solution include KCGGPQGIWGQGCK (SEQ ID NO:27) and KCGGPQGIAGQGCK (SEQ ID NO:28).
• MMP matrix metalloproteinase
• the hydrogel precursor solution can further include an initiator.
• an initiator As known by those skilled in the art hydrogel polymerization can occur in the absence of an initiator. An initiator can, however, induce polymerization and/or decrease the polymerization rate.
• Suitable initiators are known to those skilled in the art and can be, for example, chemical initiators and photoinitiators. Particularly suitable photoinitiators can be, for example, IRGACURE 2959 photoinitiator (commercially available from Ciba/BASF, Ludwigshafen, Germany) and Eosin Y. Polymerization to form the hydrogel can also be performed by temperature change.
• the hydrogel precursor solution can include a cell adhesion peptide.
• a "cell adhesion peptide” refers to an amino acid sequence obtained from an adhesion protein to which cells bind via a receptor-ligand interaction. Varying the cell adhesion peptide and concentrations thereof in the solution allow for the ability to control the stability of the cellular attachment to the resulting hydrogel composition.
• Suitable cell adhesion peptides include, for example, RGD, RODS (SEQ ID NO: l), CRGDS (SEQ ID NO:2), CRGDSP (SEQ ID NO:3), PHSRN (SEQ ID NO:4), GWGGRGDSP (SEQ ID NO:5), SIDQVEPYSSTAQ (SEQ ID NO:6), GRNIAEIIKDI (SEQ ID NO:7), DITYVRLKF (SEQ ID NO: 8), DITVTLNRL (SEQ ID NO: 9), GRYVVLPR (SEQ ID NO: 10), GNRWHSIYITRFG (SEQ ID NO: 11), GASIKVAVSADR (SEQ ID NO: 12), GTTVKYIFR (SEQ ID NO: 13), GSIKIRGTYS (SEQ ID NO: 14), GSINNNR (SEQ ID NO: 15), SDPGYIGSR (SEQ ID NO:16), YIGSR (SEQ ID NO:17), GTPGPQ
• the concentration of cell adhesion peptide in the hydrogel precursor solution will depend on the specific cell adhesion peptide being used as well as the other components in the hydrogel precursor solution. Typically, however, the hydrogel precursor solution includes from about 0.125 mM to about 4 mM cell adhesion peptide, including from about 0.25 mM to about 2 mM cell adhesion peptide. In one suitable embodiment, the cell adhesion peptide is CRGDS (SEQ ID NO:2), and the hydrogel precursor solution includes from about 0.25 mM to about 4 mM CRGDS (SEQ ID NO:2).
• the cell adhesion peptide is a cyclic RGD
• the hydrogel precursor solution includes from about 0.125 mM to about 2 mM cyclic RGD, particularly cyclic RGD ⁇ Fd ⁇ C (SEQ ID NO:33).
• the hydrogel precursor solution can include a soluble factor binder.
• a peptide for binding a soluble factor contained in a cell culture medium is included in the hydrogel precursor solution.
• the density (concentration) of the soluble factor binder in a hydrogel composition can be controlled by altering the concentration of the soluble factor binder in the hydrogel precursor solution. Examples of particularly suitable soluble factor binders are provided in Table 1 , below.
• the concentration of soluble factor binder in the hydrogel precursor solution will depend on the specific soluble factor binder being used as well as the other components in the hydrogel precursor solution.
• the hydrogel precursor solution can further include a cell.
• Suitable cells are known to those skilled in the art and can include, for example, an embryonic stem cell, an embryonic stem cell-derived neuron, an embryonic stem cell-derived neural progenitor cell, an embryonic stem cell-derived astrocyte, an embryonic stem cell-derived microglial cell, an embryonic stem cell-derived endothelial cell, an embryonic stem cell-derived retinal pigment epithelial cell, an induced pluripotent stem cell, an induced pluripotent stem cell-derived neural progenitor cell, an induced pluripotent stem cell-derived astrocyte, an induced pluripotent stem cell-derived microglial cell, an induced pluripotent stem cell-derived endothelial cell, a mesenchymal stem cell, an umbilical vein endothelial cell, an NIH 3T3 fibroblast, a dermal fibroblast, a fibrosarcoma cell, a valvular interstitial
• the hydrogel precursor solution can further include a microsphere carrier (i.e., microcarrier).
• Microsphere carriers can contain molecules such as, for example, cells, biomolecules, dyes and other molecules known to those skilled in the art. Microspheres can be degradable microspheres that dissolve or degrade to release the contents of the microsphere.
• the hydrogel precursor solution is contacted with a substrate
• a patterned surface-modified substrate e.g., a patterned surface-modified substrate, surface of a cell culture plate, etc.
• the surface-modified substrate can be, for example, mica, glass, silicon, diamond and metal oxide surfaces.
• the surface-modified substrate can be prepared, for example, by functionalizing a surface such as a glass coverslip having a silane monolayer.
• a particularly suitable surface-modified substrate can be, for example, a glass slide.
• a particularly suitable method for functionalizing the substrate can be, for example, silanization.
• the substrate can be surface- modified by activating both sides of the surface in oxygen plasma treatment. Oxygen plasma treatment can increase the number of activated hydroxyl groups on the surface of the substrate.
• a silane monolayer can be prepared with an alkoxysilane that is dissolved in an anhydrous organic solvent such as, for example, toluene.
• suitable alkoxysilanes can be for example, aminosilanes, glycidoxysilanes and mercaptosilanes.
• Particularly suitable aminosilanes can be, for example, (3-aminopropyl)- trriethoxysilane, (3-aminopropyl)-diethoxy-methylsilane, (3-aminopropyl)-dimethyl- ethoxysilane and (3-aminopropyl)-trimethoxysilane.
• Particularly suitable glycidoxysilanes can be, for example, (3-glycidoxypropyl)-dimethyl-ethoxysilane.
• Particularly suitable mercaptosilanes can be, for example, (3-mercaptopropyl)-trimethoxysilane and (3- mercaptopropyl)-methyl-dimethoxysilane.
• Other suitable silanes are commercially available (Sigma Aldrich, St. Louis, MO).
• Preparation of a surface-modified silane substrate can be performed using any silane having a terminal functional group that can participate in click chemistry as described herein.
• mercaptosilane contains a terminal thiol that can react with the norbornene of the PEG-norbornene.
• suitable functional surface-modified silane substrates can be, for example, acrylates and methacrylates. Following surface- modification of the substrate, non-adhesive self-assembled monolayers are formed on the surface-modified substrate.
• the method After contacting the substrate with the hydrogel precursor solution, the method includes polymerizing the hydrogel precursor solution such that polymerized hydrogel attaches (i.e., is coupled) to the substrate.
• the method can be used to form an array having "spots" or “islands” of hydrogel (referred to herein as "hydrogel spots") that are surrounded by a background that is substantially free, and even completely free, of hydrogel ("hydrogel- free").
• the hydrogel-free background corresponds to the hydrophobic regions of the patterned substrate and the hydrogel spots correspond to the hydrophilic regions of the patterned substrate. Referring to FIG. 1, the circles would represent the hydrogel spots that would be surrounded by a hydrogel-free region in this embodiment.
• the method can be used to form an array having hydrogel-free pools surrounded by a background of hydrogel (referred to herein as "a hydrogel background").
• a hydrogel background a background of hydrogel
• the circles would represent the hydrogel-free pools that would be surrounded by the hydrogel-free background in this embodiment.
• the present disclosure is directed to a hydrogel compositions including hydrogel spots having variable modulus, variable shear modulus, variable ligand identity, variable ligand density and combinations thereof.
• Hydrogel compositions having variable modulus, variable shear modulus, variable ligand identity, variable ligand density and combinations thereof can be prepared according to the methods described herein above.
• Suitable ligands are known to those skilled in the art and can be, for example, any biomolecule containing a cysteine and/or functionalized with a thiol. Thiol- functionalizing of ligands can be performed using commercially available kits (e.g., Traut's Reagent (2-iminothiolane»HCl), Thermo Fischer Scientific, Rockford, IL).
• Suitable ligands can be, for example, proteins, peptides, nucleic acids, polysaccharides, lipids, biomimetic materials and other molecules, and combinations thereof.
• Particularly suitable proteins can be, for example, adhesion proteins.
• Particularly suitable adhesion proteins can be, for example, fibronectin, cadherin and combinations thereof.
• Particularly suitable peptides can be, for example, cell adhesion peptides and/or soluble factor binders, as described herein above.
• the hydrogel compositions of the present disclosure include combinations of cell adhesion peptides and soluble factor binders that are suspected of binding or interacting with a cell to affect cell attachment, spreading, migration, maturation, proliferation, differentiation, and formation of cellular structures (e.g., tubules).
• Hydrogel compositions may further include variable moduli.
• Hydrogel compositions can have a range of stiffness (expressed herein as substrate elastic moduli).
• hydrogels with different moduli can be prepared by ch25anging the concentration of the polymer and/or changing the stoichiometric ratio of the multifunctional polymer (e.g., the bifunctional polymer thiol-polyethylene glycol-thiol (SH-PEG-SH)) to polymer ratio in the hydrogel precursor solution (see e.g., FIG. 8).
• Suitable ratios can be from about 1:1 to about 4:1 (molar ratio).
• the patterned hydrogel array can be further assembled with a microarray add-on whereby the patterned hydrogel array is prepared with dimensions to accommodate add-ons of any size.
• Suitable microarray add-ons are commercially available (Grace Bio Labs, Bend, OR).
• a microarray add-on can allow for the isolation of each individual hydrogel spot and hydrogel-free pool of the hydrogel array such that soluble factor presentation can be controlled.
• the microarray add-on can include the same number of openings as the number of individual hydrogel spots and hydrogel-free pools of the hydrogel array such that each hydrogel spot and hydrogel-free pool can be independently interrogated with soluble factor presentation.
• the microarray add-on can have larger openings that can accommodate more than one individual hydrogel spot and more than one individual hydrogel-free pool.
• a microarray add-on can have openings large enough to accommodate a single hydrogel spot or a single hydrogel-free pool.
• the present disclosure is directed to methods of using the hydrogel compositions to promote cellular expansion, maturation and cellular differentiation.
• the methods include preparing the hydrogel compositions; contacting a cell with the hydrogel compositions; and culturing the cells.
• the hydrogel compositions are prepared as described above and typically include a polymer (e.g., a polyethylene glycol functionalized with norbornene), a multifunctional polymer crosslinker (e.g., MMP-degradable crosslinking peptide, non-degradable PEG-dithiol crosslinker), and a cell adhesion peptide as described more fully above.
• the method further includes contacting a cell with the hydrogel composition.
• contacting a cell refers to seeding the cells with the purpose of culturing the cells.
• a cell suspension is typically transferred to a substrate and cells are given sufficient time to adhere to the substrate.
• cells can be incorporated into the hydrogel of the hydrogel compositions using a hydrogel precursor solution that includes the polymer, the crosslinker, the cell adhesion peptide, and the cell.
• the cells are then cultured for a desired time such as, for example, about one hour to about 30 days.
• cells can be analyzed by microscopy such as, for example, immunofluorescence microscopy, phase contrast microscopy, light microscopy, electron microscopy and combinations thereof.
• microscopy such as, for example, immunofluorescence microscopy, phase contrast microscopy, light microscopy, electron microscopy and combinations thereof.
• Cells can be analyzed for cell attachment, cell spreading, cell morphology, cell proliferation, cell migration, cell expansion, cell differentiation, protein expression, cell-to-cell contact formation, sprouting, tubulogenesis, formation of structures, and combinations thereof.
• Suitable cells can be any cell known by those skilled in the art. Particularly suitable cells can include, for example, an embryonic stem cell, an embryonic stem cell- derived neuron, an embryonic stem cell-derived neural progenitor cell, an embryonic stem cell-derived astrocyte, an embryonic stem cell-derived microglial cell, an embryonic stem cell-derived endothelial cell, an embryonic stem cell-derived retinal pigment epithelial cell, an induced pluripotent stem cell, an induced pluripotent stem cell-derived neural progenitor cell, an induced pluripotent stem cell-derived astrocyte, an induced pluripotent stem cell-derived microglial cell, an induced pluripotent stem cell-derived endothelial cell, an induced pluripotent stem cell-derived retinal pigment epithelial cell, a mesenchymal stem cell, an umbilical vein endothelial cell, an NIH 3T3 fibroblast, a dermal fibroblast, a fibrosarcom
• the cell is a circulating angiogenic cell CAC.
• CACs are pro- angiogenic cell population that fulfills many roles in vascular biology including the formation of new blood vessels during healing. While CACs are a promising tool for treatment of multiple cardiovascular disorders including peripheral ischemia and restoration of damaged or dysfunctional endothelium; harvesting of CACs is challenging due to their scarcity in the blood stream, so only small numbers of CACs can be isolated at any one time.
• hydrogel compositions of the present disclosure prepared using hydrogel precursor solutions that encourage proliferation of recruited CACs, the above problem of low initial numbers of recruited CACs and limited CAC expansion is addressed.
• the hydrogel compositions when used with CACs, include 8-arm, 20 kDa poly(ethylene glycol) (PEG) functionalized with norbornene, a MMP degradable crosslinking peptide, and a cell adhesion peptide.
• Particularly suitable cell adhesion peptides include immobilized RGD-containing peptides, including CRGDS (SEQ ID NO:2), Acetylated-GCYGRGDSPG (SEQ ID NO:31); cyclic ⁇ RGD(Fd)C ⁇ (SEQ ID NO:33); CRGD-(G)13-PHSRN (SEQ ID NO:29); and CPHSRN-(SG)5-RGD (SEQ ID NO:30).
• the hydrogel compositions include at least about 1 mM cell adhesion peptide, including from about 1 mM to about 4 mM cell adhesion peptide. Further, the hydrogel compositions may include from about 20 mg/mL to about 100 mg/mL PEG concentration.
• the hydrogel compositions are prepared to include crosslinking to an extent of at least 35%, including at least 45%, and including from about 35% to about 75%, and including from about 45% to about 50%.
• the hydrogel compositions for use with CACs including a shear modulus in the range of from about 1.8 kPa to about 12 kPa, including from about 2 kPa to about 12 kPa.
• the cell is a human mesenchymal stem cell (hMSC). It has been found that PEG-hydrogel compositions support hMSC adhesion and expansion. Further, these hydrogel compositions offer combinatorial control over substrate stiffness, cell adhesion, and growth factor regulation.
• hMSC human mesenchymal stem cell
• the hydrogel compositions when used with hMSC, include 8-arm, 20 kDa poly(ethylene glycol) (PEG) functionalized with norbornene, a MMP degradable crosslinking peptide, and a cell adhesion peptide.
• Particularly suitable cell adhesion peptides include immobilized RGD-containing peptides, including CRGDS (SEQ ID NO:2), Acetylated-GCYGRGDSPG (SEQ ID NO:31); cyclic ⁇ RGD(Fd)C ⁇ (SEQ ID NO:33); CRGD-(G)13-PHSRN (SEQ ID NO:29); and CPHSRN-(SG)5-RGD (SEQ ID NO:30).
• the hydrogel compositions include at least about 0.25 mM cell adhesion peptide, including from about 0.25 mM to about 4 mM cell adhesion peptide, and including from about 1 mM to about 4 mM cell adhesion peptide.
• the hydrogel compositions may include from about 40 mg/mL to about 160 mg/mL PEG concentration.
• the hydrogel compositions for use with hMSCs possess a shear modulus of at least 1.8 kPa, including a shear modulus of from about 1.8 kPa to about 33 kPa, and including from about 1.8 kPa to about 10.9 kPa.
• the cell includes a human pluripotent stem cell (hPSC) such as, for example, human embryonic stem cells (hESC) and human induced pluripotent stem cells.
• hPSC human pluripotent stem cell
• hMSC human embryonic stem cells
• hydrogel compositions including (PEG) functionalized with norbornene, a MMP degradable crosslinking peptide, and a cell adhesion peptide (e.g., cyclic ⁇ RGD(Fd)C ⁇ (SEQ ID NO:33), offer substrate stiffness control, cell adhesion control, and growth factor regulation, thereby supporting hPSC adhesion and expansion.
• the hydrogel compositions include at least about 0.25 mM cell adhesion peptide, including from about 0.25 mM to about 4 mM cell adhesion peptide, and including from about 2 mM to about 4 mM cell adhesion peptide.
• hydrogel compositions for use with hPSCs possess a shear modulus of at least 3 kPa, including a shear modulus of from about 3 kPa to about 16 kPa, and including from about 3 kPa to about 10 kPa.
• the hydrogel compositions further include immobilized low molecular weight heparin.
• the hydrogel composition when present, includes low molecular weight heparin in amounts ranging from about 0.1 mM to about 2 mM.
• the method may further include contacting the cell with a soluble molecule by including the soluble molecule in the culture medium in which the cells are cultured.
• soluble molecules can be growth factors and proteoglycans.
• Suitable growth factors can be, for example, proteins from the transforming growth factor beta superfamily, fibroblast growth factor family of growth factors, platelet derived growth factor family of growth factors and combinations thereof.
• Particularly suitable growth factors can be, for example, vascular endothelial growth factor, bone morphogenetic proteins, fibroblast growth factor, insulin-like growth factor and combinations thereof.
• Suitable proteoglycans and be, for example, proteoglycans with heparin, heparin sulfate, and/or chondroitin glycosaminoglycan side chains.
• DCC ⁇ , ⁇ '- Dicyclohexylcarbodiimide
• DCM anhydrous dichloromethane
• the filtrate was then precipitated in 900 mL cold diethyl ether and 100 mL hexane.
• the solids were collected on qualitative grade filter paper and air dried overnight.
• the PEG-NB product was purified by dialysis against 4L of dH 2 0 at 4°C for 72 hours (with water change every 8 hours) using rehydrated SNAKESKIN dialysis tubing to remove residual norbornene acid and subsequently freeze dried.
• Hydrogel arrays used for these experiments were composed of hydrogel spots immobilized on silanized glass substrates. Hydrogel spots were formed using gold surfaces patterned to possess regions with differential wettability, whereby the pattern was defined by an elastomeric stencil. The method of preparing the hydrogel arrays is further described below. Glass Silanization
• HC1 solution Glass coverslips and hydrochloric acid (HC1) solution were purchased from Thermo Fisher Scientific (Waltham, MA). Toluene, methanol, ethanol, 3-mercaptopropyl trimethoxysilane (3-MPTS), and dithiothreitol (DTT) were purchased from Sigma Aldrich (St. Louis, MO). A low pressure plasma system was purchased from Diener Electronic (Ebhausen, Germany).
• Glass coverslips were silanized with 3-MPTS to create substrates presenting thiol groups capable of participating in thiol-ene reaction with PEG-NB and subsequently enable covalent immobilization of PEG-NB hydrogels (Seo et al. Colloids Surf B Biointerfaces 2012, 98:1-6). Liquid-phase silanization was performed as previously described (Seo et al. Colloids Surf B Biointerfaces 2012, 98:1-6; Halliwell et al. Anal Chem 2001, 73:2476-2483; and Cras et al. Biosens Bioelectron 1999, 14:683-688).
• Coverslips were sonicated for 45 minutes in 1 :1 methanol to HC1 to remove bulk contaminants. Immediately prior to silanization, coverslips were activated by oxygen plasma treatment at 40 seem and 50 W for 5 minutes on each side to increase the number of activated hydroxyl groups on the surface. Activated coverslips were placed in a coplin jar containing 2.5% v/v 3-MPTS in toluene for 4 hours. Excess silanes were removed from the surface of the coverslips by rinsing with toluene, 1:1 ethanol/ toluene, and ethanol and dried with N 2 gas.
• Silanized coverslips were placed in an airtight chamber, purged with N 2 gas, and cured at 100°C for 1 hour to crosslink the silanes coupled to the surface and reduce their susceptibility to hydrolysis. Silanized coverslips were stored in the N 2 gas purged chamber and protected from light until use. Prior to use, silanized glass coverslips were treated with 10 mM DTT in PBS for 30 minutes at 37°C to reduce disulfides formed on the surface and to increase free thiols available at the surface (Vistas et al. Appl Surf Sci 2013, 286:314-318).
• Silicon wafers were purchased from WRS Materials (San Jose, CA).
• SU-8 100 photoresist was purchased from MicroChem (Newton, MA).
• Sylgard 184 silicone elastomer kit was purchased from Dow Corning Corporation (Midland, MI).
• Polydimethylsiloxane (PDMS) elastomeric stencils were created using soft lithography as previously described (Jo et al. J Microelectromechanical Syst 2000, 9:76-81).
• the layout and geometries for the stencil were drawn using Adobe Illustrated, printed onto transparency films using a high resolution commercial laser printing service provided by ImageSetter (Madison, WI).
• ImageSetter Modison, WI
• the transparency film was used as a photo mask in combination with conventional photolithography techniques to create master molds with SU-8 negative- tone UV photoresist spin-coated on silicon wafers.
• the curing agent and PDMS pre-polymer solution from the Sylgard elastomer kit were thoroughly mixed in a 1:10 weight ratio, spread onto the master mold, and cured at 80°C for 6 hours. After curing, the PDMS stencils were peeled off from the master mold, briefly cleaned with ethanol, and dried with N 2 gas.
• Gold-coated test slides 1,000 A gold on 50 A titanium metal thin films on 25 mm x 75 mm x 1 mm glass
• Perfluorinated alkanethiol HS-(CH 2 )n-0-(CH 2 ) 2-(CF 2 ) 5 -CF 3
• Hydroxyl-terminated alkanethiol HS-Cn-(0-CH 2 - CH 2 ) 3 -OH
• Gold-coated slides were patterned with hydrophobic and hydrophilic self- assembled monolayers (SAMs) of alkanethiolates to form regions with differential wettability. Differential wettability patterning served two purposes simultaneously: 1) defined the geometries of the hydrogel spots and 2) confined the contents of each hydrogel spot in the array.
• SAMs self- assembled monolayers
• Gold-coated slides were immersed in ethanol and sonicated for ⁇ 2 minutes, rinsed with ethanol, and dried with N 2 gas to remove contaminants and gold oxide layers.
• Gold-coated slides were immersed in a 1 mM perfluorinated alkanethiol in ethanol solution for >2 hours to allow for perfluorinated alkanethiolate SAMs (fluoraSAMs) formation.
• fluoraSAMs gold-coated slides were cleaned with ethanol and dried with N 2 gas.
• PDMS stencils were placed on the fluoraSAMs gold-coated slides to selectively protect areas of the slides from plasma etching.
• the spatial and geometric patterning of the exposed regions on the fluoraSAMs gold-coated slides were defined by the pattern of the PDMS stencil, which, in turn, defined the geometry and spatial patterning of the hydrogel spots that the arrays could comprise.
• Exposed regions of the fluoraSAMs gold-coated slides were etched by oxygen plasma treatment at 40 seem and 50 W for 1 minute.
• the etched gold-coated slides were cleaned with ethanol and dried with N 2 gas and immersed in a 0.1 mM hydroxyl-terminated alkanethiol in ethanol solution for >2 hours so that hydrophilic alkanethiolate SAMs (EG 3 SAMS) were formed in the selectively-etched regions of the gold-coated slides.
• the resulting gold-coated slides with differential wettability were cleaned with ethanol and dried with N 2 gas before hydrogel formation.
• PEG-DT crosslinker (3.4 kDa) was purchased from Laysan Bio (Arab, AL). IRGACURE 2959 photoinitiator was purchased from Ciba/BASF (Ludwigshafen, Germany). Cysteine - terminated peptides were purchased from GenScript USA (Piscataway, NJ). Omnicure Series 1000 UV spot cure lamp (365 nm wavelength), light guide, and collimating adapter were purchased from Lumen Dynamics Group (Ontario, Canada). PDMS spacers with thickness dimensions corresponding to the desired hydrogel spot heights were fabricated using the same procedure as stated above.
• Hydrogel precursor solutions were prepared by combining PEG-NB, PEG-DT, peptides, and photoinitiator and diluted to desired concentrations with phosphate buffered saline (PBS) immediately prior to hydrogel spots formation.
• PBS phosphate buffered saline
• To form each hydrogel array a patterned gold-coated slide was rinsed with ethanol and dried with N 2 gas, PDMS spacers were placed onto hydrophobic regions of the slide, and hydrogel precursor solutions were spotted onto the hydrophilic regions.
• a DTT-treated silanized glass coverslip was used to sandwich the hydrogel precursor solutions between the coverslip and the slide.
• Hydrogel precursor solutions were polymerized by UV-initiated photo-crosslinking for 2 seconds at 90 mW/cm 2 , with the light penetrating through the glass coverslip.
• the resulting polymerized hydrogel spots were covalently attached and immobilized onto the coverslip.
• the silanization procedure produced glass coverslips that were functionalized with thiol- terminated silanes that were capable of participating in the thiol-ene reaction used for hydrogel precursor solution polymerization, which effectively crosslinked the hydrogel network to the surface-bound silanes.
• the gold-coated slide was separated from the coverslip, which enabled the glass-immobilized hydrogel spots to cleanly detach from the gold-coated slide.
• the resulting glass-immobilized hydrogel spots collectively referred to as the "hydrogel array" was sterilized for 1 hour in 70% ethanol and washed with PBS to remove any remaining unreacted components.
• each hydrogel spot in the array was defined by both the identity and concentration of the peptides incorporated therein.
• Peptides used in this study were CRGDS (SEQ ID NO:2), CRGD-(G)i 3 -PHSRN (SEQ ID NO:29), CRGD-(SG) 5 - PHSRN (SEQ ID NO:30), acetylated-CRGDSP (SEQ ID NO:31), cyclic (RGD ⁇ Fd ⁇ C) (SEQ ID NO:33), and a non-bioactive scrambled peptide CRDGS (SEQ ID NO:32).
• the modulus of each hydrogel spot in the hydrogel array was defined by the total concentration of PEG in the crosslinked hydrogel network. Increasingly, the concentration of PEG-NB in the hydrogel precursor solution resulted in a larger amount of PEG crosslinked into the polymerized network, which resulted in an increase in the compressive modulus (see, FIG 8).
• FIGS. 1A-1B A gold substrate was modified with a patterned alkanethiolate self-assembled monolayer (SAMs) to provide isolated hydrophilic regions separated by a surrounding hydrophobic region (as illustrated in FIGS. 1A-1B).
• SAMs alkanethiolate self-assembled monolayer
• FIG. 2A also shown in FIG. 1A
• FIG. 2B provides end views during patterning of a gold substrate at the step before hydrophobic patterning 100; of the substrate having fluoraSAMs 110; of the substrate after etching 120; and of the substrate after hydrophilic patterning 130.
• Hydrogel precursor solutions containing all components required for polymerization reactions were deposited onto the hydrophilic SAMs regions of the patterned substrate (see, FIG. IB).
• the hydrophilic regions served to both confine the contents of the solutions deposited onto each region and to define the geometries of the resulting polymerized hydrogel.
• Elastomeric spacers (with thickness dimensions equivalent to the desired hydrogel array height) were placed onto the hydrophobic areas of the patterned slide to define the height of the hydrogel array.
• the components of the hydrogel precursor solution formed a crosslinked network as well as formed covalent bonds with the end-function groups on the glass substrate.
• the polymerized hydrogels removed cleanly from the patterned gold substrate to produce a hydrogel array immobilized on the glass substrate (see, FIG. 3).
• Poly (ethylene glycol) (PEG) hydrogel arrays were formed using patterned hydrophobic/hydrophilic self-assembled monolayers on gold substrates to both define the geometry and confine the contents of each hydrogel spot in the array as described above (see, FIGS. 1A-1B).
• UV-initiated thiol-ene crosslinking simultaneously polymerized the hydrogel and immobilized the hydrogel spots on the glass to result in the hydrogel array.
• hydrogel arrays could be prepared with dimensions compatible with a 64-well microarray add-on (commercially available from Grace Bio-Labs, Bend, OR).
• Hydrogel solutions with fibronectin-derived peptides, fluorescent microspheres and a dithiol crosslinker were deposited onto the SAMs and sandwiched with a silanized glass slide. As shown in FIG. 7, individual hydrogel spots of the hydrogel array could be prepared to include varying amounts of fluorescently-tagged peptides as well as varying amounts of fluorescent microspheres. Hydrogel solutions with varying PEG or crosslinker concentration were also prepared prior to crosslinking to change the stiffness, peptide identity or peptide concentration (FIG. 8). The resultant arrays (see, FIG. 3) included 2.4 mm diameter, 150 um height posts.
• Human mesenchymal stem cells were cultured on posts with varying PEG concentrations (4 wt %, 6 wt % and 8 wt ) to change stiffness and monitored for changes in initial cell adhesion and spreading.
• Human embryonic stem cells were cultured on posts with varying peptide identity (blank, RDGS, RGDS (SEQ ID NO:l), RGD-PHSRN (SEQ ID NO:34), RGDSP (SEQ ID NO:47), and cyclic RGD) and monitored for changes in initial cell adhesion and spreading.
• 2D culture of hMSCs demonstrated cell spreading dependence in response to changes in modulus consistent with published observations (see, Engler et al. Cell 126:677 (2006)).
• 2D culture of hESCs in chemically- defined, albumin-free media demonstrated that cell adhesion was highly specific to peptide- presenting spots. Both hESC cell adhesion and spreading were dependent on the binding affinity of integrin receptors to immobilized peptides (see, FIG. 11).
• Arrays allowed for changes in hydrogel spot shape, hydrogel spot height (by changing patterned hydrogel spot shapes or adding spacers), hydrogel spot stiffness and hydrogel spot peptide concentrations, and was adaptable for both 2D and 3D cell culture.
• hydrogel arrays of the present disclosure can support cell adhesion and survival and allow for screening complex cell-environment interactions.
• hydrogel compositions of the present disclosure were prepared, human mesenchymal stem cells (hMSCs) were cultured thereon, and cell properties were analyzed.
• hMSC initial cell attachment, spreading, and proliferation were linearly correlated with immobilized CRGDS (SEQ ID NO:2) concentration.
• immobilized CRGDS SEQ ID NO:2 concentration.
• minimal hMSCs were attached to hydrogel spots that did not contain CRGDS (SEQ ID NO:2) (4 mM "scrambled” CRDGS (SEQ ID NO: 32)), indicating that initial cell adhesion was mediated by the bioactivity of the immobilized peptide.
• Increased CRGDS (SEQ ID NO:2) concentration resulted in increased hMSC cell attachment, spreading, and proliferation with maximal values on hydrogel spots presenting 4 mM CRGDS (SEQ ID NO:2).
• hydrogel compositions of the present disclosure were prepared, human embryonic stem cells (hESCs) were cultured thereof, and cell expansion was analyzed.

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