Hybrid hydrogels for culturing endometrial and stromal organoid cells

Abstract

Hybrid hydrogels for organogenesis support organogenesis from mammalian cells, including human cells. The hybrid hydrogels typically include a network of crosslinked branched synthetic polymers including a branched polyalkylene oxide polymer and a thiolated extracellular matrix polymer, preferably hyaluronic acid. These polymers are crosslinked to adhesion ligand peptides. Also provided are methods of making the hybrid hydrogels and methods of using the hybrid hydrogels for organogenesis.

Description

[0001] HYBRID HYDROGELS FOR CULTURING ENDOMETRIAL AND STROMAL ORGANOID CELLS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] None.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under EB029132 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] REFERENCE TO SEQUENCE LISTING

[0007] The Sequence Listing submitted as a text file named “MIT_25192_PCT.xml” created on October 17, 2023, and having a size of 56,011 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.834(c)(1).

[0008] FIELD OF THE INVENTION

[0009] The invention is in the field of hybrid hydrogels for culture of endometrial cells, especially endometrial epithelial cells and stromal cell organoids.

[0010] BACKGROUND OF THE INVENTION

[0011] Organoids are three-dimensional structures derived from stem cells with organ- specific cell types and microarchitecture similar to the tissue of origin. These models offer a closer look into organogenesis unique to human that are not possible with animal models. In addition, human organoids allow for personalized medicine and autologous transplants using patient- derived crypts or stem cells cultured and expanded in vitro. However, stem cell-derived organoids suffer several limitations, primary of which is the reliance on natural hydrogels as a 3D scaffold, such as MATRIGEL®

[0012] MATRIGEL® is a commercial product widely used to provide the 3D scaffold for the growth of organoids of all cell types. However, the reliance on MATRIGEL®, or similar naturally derived biopolymer matrices, as the scaffold for organoid growth introduces a number of significant limitations into the study and use of the resultant organoids, and severely limits further development of the field. MATRIGEL® is derived from a basement membrane extracellular matrix (ECM)-rich mouse sarcoma and therefore introduces a significant risk of immunogen or pathogen transfer if given to a patient. Additionally, the batch-to-batch variability of MATRIGEL® may lead to inconsistent cell behaviors, introducing unknown and potentially confounding variables that complicate the interpretation of both basic and translational research. Further, the role of MATRIGEL® in organoid formation remains poorly understood. ECM is known to implicate in the regulation of tissue development and function. The specific roles of ECM factors are difficult to ascertain using MATRIGEL® given that its molecular components cannot be readily manipulated. It is unclear whether MATRIGEL® serves merely as a passive 3D scaffold providing physical support to the growing organoid, or actively influences organoid formation by providing essential biological cues. The cumbersome process to remove MATRIGEL® and recover the organoids for downstream analysis is an additional challenge.

[0013] Additionally, organoids derived from induced pluripotent stem cells (iPSC) result in complex tissue structures (for example, iPSC-derived intestinal organoids typically have smooth muscle and stroma surrounding epithelial cells (McCauley et al., Dev. 144:958-962 (2017)), but cell maturation is limited to fetal stages. There is great interest in organoids derived from postnatal tissue biopsies, as these capture features of (patho)- physiological states of mature tissues, such as epigenetic changes and somatic mutations associated with donor tissue’s natural or diseased state.

[0014] PCT / US2020 / 044067 entitled “Synthetic Hydrogels for Organogenesis’’ filed by Massachusetts Institute of Technology on luly 29, 2020, describes a synthetic gel performing as well as MATRIGEL but with more consistency and no potentially tumorigenic ingredients. The gel is formed of a branched synthetic polymer, typically an 8 kD polyethylene glycol, extracellular matrix binding (ECM) peptides that are functionalized to crosslink the PEG, and peptides comprising adhesion. The adhesion ligands are typically cell-binding peptides, for example, integrin binding peptides. The integrin binding peptides can be derived from collagen, fibronectin, and / or laminin. Preferred gels include the amino acid sequence GFOGER (SEQ ID NO:1). The ECM binders are typically synthetic peptides with affinity to extracellular matrix components, preferably the ECM polymers. For example, an ECM binder can be a peptide having affinity to fibronectin, collagen, laminin, other ECM components, or combinations thereof.

[0015] There remains a need for fully-defined hydrogels supporting organogenesis from biopsied human endometrial cells and other cells with more complex responses to their environment, such as neural cells, with control over complex cell-matrix interactions.

[0016] Therefore, it is an object of the present invention to provide hybrid hydrogels for organogenesis from endometrial and stromal cells and organoids.

[0017] It is another object of the present invention to provide methods of making and using the hybrid hydrogels.

[0018] SUMMARY OF THE INVENTION

[0019] A polymeric hydrogel material formulation has been developed for use as a three-dimensional (3D) culture substrate in cell and organoid culture. The hydrogel includes a branched polyalkylene glycol, preferably PEG, functionalized with synthetic adhesion ligand peptides which allow the binding of cell surface receptors as well as cell-secreted proteins, and crosslinked with a chemically functionalized extracellular matrix polymer such as thiolated hyaluronic acid. A critical feature of this formulation is that the peptides are bound to the multi-arm PEG, not the hyaluronic acid. There are a plethora of gels present in the literature with adhesion peptides directly bound to hyaluronic acid, which limits the mobility of the peptides compared to peptides bound to PEG

[0020] It has been found that this hydrogel containing chemically modified hyaluronic acid (HA), a glycosaminoglycan that is a significant component of the extracellular matrix (“ECM”) in mammalian tissues, instead of crosslinking peptides, alters the properties of the hydrogel and induces changes in the cells and organoids cultured therein.

[0021] Other ECM could be used together with the HA, or in place of the HA. The inclusion of HA changes the biological properties of the gel by supporting the binding of cell surface HA receptors and remodeling in response to HA-degrading hyaluronidase enzymes. The inclusion of HA also changes the physiochemical properties of the gel by modifying the ligand distribution (compared to a gel made from only PEG + crosslinking and adhesion ligand binding peptides), decreasing the swelling of the gel in aqueous media, which is important for inclusion in microfluidic platforms, and decreasing the total mass of polymer required to obtain the same ligand concentration and bulk mechanical properties. Due to the difference in physiochemical properties of PEG and HA, identifying compositions where the two polymers are interspersed and able to gel required significant testing. Inclusion of the bioactive peptide ligands on the arms of the PEG molecule increases their accessibility for binding by cell surface proteins, as opposed to alternative gel formulations which attach peptide ligands to a HA backbone. Another advantage of the inclusion of the HA is that it makes the matrix hyaluronidase-degradable but not MMP-degradable.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 is a schematic overview of making the crosslinked HA adhesive gel.

[0024] FIG. 2A is a graph of the Gel shear storage modulus (G') (Pa) and shear loss modulus (Pa), showing that it increases with increasing concentrations of hyaluronic acid (HA). FIG. 2B-2E are micrographs of the cells in culture, showing morphology as a function of HA concentration.

[0025] FIG. 3A-3E show that epithelial organoids exhibit similar morphology in soft gels, but different morphology in stiff gels. FIG. 3A is a graph of the shear storage moduli of gel formulations used for EEO culture (PEG-peptide, PEG-HA (0.25%), stiff PEG-peptide, and stiff PEG-HA (0.38% HA-SH). FIG. 3B-3E is the morphology of patient-derived human endometrial epithelial cells (“EEOs”) in 3D in soft PEG-peptide (FIG. 3B), stiff PEG-peptide (FIG. 3C), soft PEG-peptide-HA (FIG. 3D), and stiff PEG-peptide-HA hydrogels (FIG. 3E). Scale bar = 200 um. References: (1) Edelstam GAB. J Histochem Cytochem. 39(8): 1131-1135 (1991). (2) Fouladi-Nashta AA. Reproduction. 2017;153(2):R43-R58. (3) Harden SL. Front Cell Dev Biol. 9:626619 (2021). (4) Hernandez-Gordillo V. Biomater. 254: 120125 (2020).

[0026] FIG. 4A-B is a comparison of hybrid hydrogel formulations made with varied concentrations and molecular weights of hyaluronic acid (HA), including the volumetric swelling factor (FIG. 4A) and shear storage modulus (FIG. 4B).

[0027] FIG. 5A-C are fluorescence micrograph images of primary human adipocytes encapsulated in three different hydrogel formulations: commercially available HYSTEM-C (FIG. 5A), a peptide-crosslinked PEG hydrogel containing cell- and ECM-adhesive binder peptides based on the prior art (FIG. 5B), and the hybrid PEG-HA hydrogel (FIG. 5C). The cells are stained with a LIVE / DEAD viability stain (Invitrogen).

[0028] FIG. 6A-E are representative fluorescence micrograph images from a pilot study comparing the performance of three different hydrogel formulations in a nerve-on-a-chip model using spheroids of human induced pluripotent stem cell-derived neurons. The samples were stained for beta-3- tubulin (green), a marker of neural phenotype, and f- actin (red), a marker of non-neuronal cells. The images depict commercially available MATRIGEL (FIG. 6A), a peptide-crosslinked PEG hydrogel containing cell- and ECM- adhesive binder peptides based on the prior art (FIG. 6B, 6C), and the hybrid PEG-HA hydrogel (FIG. 6D,6E) used for culture of the spheroids over the course of 37 days. DETAILED DESCRIPTION OF THE INVENTION

[0029] Synthetic polymeric hydrogels are a fundamental tool in tissue engineering, offering increased user tunability and reproducibility over natural polymer hydrogels. However, the development of a hybrid hydrogel which can support the co-culture of multiple different primary human cell types, especially hormonal responsive cell types such as endometrial epithelial cells and organoids thereof, remains challenging. Previous hydrogel substrates for 3D cell and organoid culture made from synthetic polymers, artificially produced polymers from organic small molecule precursors, have included protein-mimetic peptide domains to promote cell binding and interaction. However, these gels do not include extracellular polysaccharides, naturally occurring carbohydrate polymers that are ubiquitous in animal tissues. Hyaluronic acid (HA) is a naturally occurring linear polysaccharide which can be produced industrially and is chemically modifiable and endogenously bioactive. Additionally, HA is a component of the human endometrial extracellular matrix and its metabolism by endometrial stromal cells and perivascular cells is altered in decidualized cells in vitro.

[0030] A hybrid hydrogel, consisting of both naturally derived and synthetic polymers with both comprising a substantial portion of the hydrated polymer mesh structure (at least 5% either naturally derived polymers or synthetic polymers by mass), was developed using a combination of bioactive peptide- functionalized multi-arm poly(ethylene glycol) (PEG) and chemically modified HA. This gel has demonstrated effective use as a 3D culture substrate for primary human cells, including endometrial stromal and epithelial cells and adipocytes, as well as model peripheral neurons derived from human induced pluripotent stem cells.

[0031] I. Definitions

[0032] As used herein, the term “hydrogel” refers to a substance formed when an organic polymer (natural or synthetic) is cross-linked via covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure which entraps water molecules to form a gel. Biocompatible hydrogel refers to a polymer forms a gel which is not toxic to living cells and allows sufficient diffusion of oxygen and nutrients to the entrapped cells to maintain viability.

[0033] As used herein, the term “synthetic”, in the context of polymer, refers to a polymer that is produced solely through artificial chemical synthesis from organic precursor molecules. That is, the molecule is not produced by the metabolism of any living organism.

[0034] As used herein, the term “natural”, in the context of polymer, refers to a polymer that can be produced by the metabolism of a living organism. Natural polymers can be derived from plant or animal tissue or can be produced via fermentation or recombinant synthesis by microorganisms.

[0035] As used herein, the term “hybrid”, in the context of hydrogels, refers to a hydrogel comprised of a combination of synthetic and natural polymers in a mass: mass ratio of at least 5% natural to synthetic or vice versa.

[0036] As used herein, the term “biodegradable”, in the context of polymer, refers to a polymer that will degrade or erode by enzymatic action and / or hydrolysis under physiologic conditions to smaller units or chemical species that are capable of being metabolized and / or eliminated.

[0037] As used herein, the term “binder” refers to peptides, preferably synthetic peptides, with affinity to one or more proteins. The proteins may be cell-associated proteins and / or cell-secreted proteins. Typically, binders, in the context of components of the hydrogel, include adhesion ligands and ECM polymer-binding peptides.

[0038] As used herein, the terms “inhibitor”, “apoptosis inhibitor” refers to small molecules with known inhibitory effects on cellular apoptosis. The inhibitor may inhibit any stage of cellular apoptosis. Preferably the inhibitor inhibits dissociation-induced apoptosis. The reduction in apoptosis in a cell population may be compared to a control. The control may include the same type, or similar cells, grown under the same conditions, in the absence of the inhibitor. As used herein, the term “molecular weight”, in the context of polymers, refers to generally refers to the relative average chain length of the bulk polymer, unless otherwise specified. In practice, molecular weight can be estimated or characterized using various methods including gel permeation chromatography (GPC) or capillary viscometry. GPC molecular weights are reported as the weight-average molecular weight (Mw) as opposed to the number- average molecular weight (Mn). Capillary viscometry provides estimates of molecular weight as the inherent viscosity determined from a dilute polymer solution using a particular set of concentration, temperature, and solvent conditions.

[0039] As used herein, the term “molecular weight”, in the context of biological molecules, such as peptides, proteins, glycoproteins, etc., generally refers to the molar mass of the molecule in g / mol, or Daltons, and may be measured by any suitable technique, such as electrophoresis, or mass spectrometry.

[0040] As used herein, the term “functionalize” refers to modifying in a manner that results in the attachment of a functional group or moiety. For example, a molecule may be functionalized by the introduction of a molecule which makes the molecule a strong nucleophile or strong electrophile. For example, a molecule, such as hyaluronic acid, may be functionalized to become a thiol, amine, acrylate, or quinone.

[0041] As used herein, the term “peptide” refers to a biological molecule, which may include “polypeptide,” and / or “oligopeptide,” and refers to a chain of natural and / or synthetic amino acid residues linked together by covalent bonds (<?.g., peptide bonds). The length of the peptide is limited at the lower end only by the minimum number amino acids required to form a self-assembling peptide.

[0042] As used herein, the term “adhesion ligand” refers to a synthetic peptide ligand for affinity to cellular proteins, typically to cell adhesion molecules (CAMs). Typical cell adhesion molecules include integrins, immunoglobulin superfamily (IgSF) CAMS, cadherins, and selectins. The adhesion ligands may derive from proteins of the ECM. The adhesion ligands may be linear or branched.

[0043] As used herein, the term “extracellular matrix’’, “ECM” refers to the components and / or the network of extracellular macromolecules, such as proteins, enzymes, and glycoproteins, that provide structural and biochemical support of surrounding cells. The extracellular matrix includes the interstitial matrix and the basement membrane components of the ECM include proteoglycans heparan sulfate, chondroitin sulfate, keratan sulfate; non-proteoglycan polysaccharide hyaluronic acid, and proteins collagen, elastin, fibronectin, and laminin.

[0044] As used herein, the term “extracellular matrix -binding peptide” refers to a synthetic peptide with affinity to ECM components.

[0045] As used herein, the term “hydrogel matrix” typically refers to the network of cross-linked polymers forming the hydrogel. The hydrogel matrix may or may not include the binders.

[0046] As used herein, the term “crosslinker” refers to a molecule containing two or more reactive moieties which are used to connect two or more polymer molecules via chemical bonds into a larger 3D mesh structure characteristic of a hydrogel. The bonds may be in the form of covalent bonds or ionic bonds.

[0047] As used herein, the term “reactive functional moiety” refers to a moiety containing any known suitable chemical functional group that reacts with another chemical functional group in the given context (i.e., reaction conditions) to form a bond, as exemplified herein. The bond may be in the form of covalent bonds or ionic bonds.

[0048] As used herein, the term “amino acid” refers to standard nomenclature, amino acid residue as denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (He, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Vai, V).

[0049] As used herein, the term “cleavable motif’, in the context of proteins, refers to a sequence of amino acids, such as to a sequence of two, three, four, five, six, etc., amino acids that are recognized by one or more proteolytic enzymes, such as proteases, proteinases, peptidases, and transpeptidases, such as protein sorting transpeptidases. The cleavable motif may be a synthetic or a naturally occurring recognition site for the proteolytic enzymes.

[0050] “Mammalian cell” refers to any cell derived from a mammalian subject suitable for transplantation into the same or a different subject. The cell may be xenogeneic, autologous, or allogeneic. The cell can be a primary cell obtained directly from a mammalian subject. The cell may also be a cell derived from the culture and expansion of a cell obtained from a subject. For example, the cell may be a stem cell. Immortalized cells are also included within this definition. In some embodiments, the cell has been genetically engineered to express a recombinant protein and / or nucleic acid.

[0051] “Autologous” refers to a transplanted biological substance taken from the same individual.

[0052] “Allogeneic” refers to a transplanted biological substance derived from a different individual of the same species.

[0053] “Xenogeneic” refers to a transplanted biological substance taken from a different species.

[0054] As used herein, the term “variant” refers to a peptide or a polypeptide that differs from a reference peptide or a polypeptide but retains the same mechanism of activity. A typical variant of a peptide or a polypeptide differs in amino acid sequence from another, reference peptide or a polypeptide. Generally, differences are limited so that the sequences of the reference peptide or a polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide or a polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a peptide or a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.

[0055] Modifications and changes can be made in the structure of the peptide or a polypeptide of in disclosure and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide’s biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties.

[0056] In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (- 3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0057] It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, and antigens. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within + 2 is preferred, those within + 1 are particularly preferred, and those within + 0.5 are even more particularly preferred.

[0058] Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly, where the biological functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 + 1); glutamate (+3.0 + 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5 + 1); threonine (-0.4); alanine (-0.5); histidine (- 0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within + 2 is preferred, those within + 1 are particularly preferred, and those within + 0.5 are even more particularly preferred.

[0059] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (He: Leu, Vai), (Leu: He, Vai), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Vai: He, Leu). In particular, embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide of interest.

[0060] Use of the term "about" is intended to describe values either above or below the stated value in a range of approx. + / - 10%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / - 5%.

[0061] IL Hybrid Hydrogels

[0062] As shown in FIG. 1 , the hydrogels have three main components: (1 ) a branched polyalkylene glycol such as an 8 arm polyethylene (glycol) (“PEG”), (2) peptide binders bound to the PEG to form a polyalkylene glycol-peptide adduct, and (3) thiolated ECM polymer such as hyaluronic acid, but may be a different ECM or in combination with a different ECM crosslinking the polyalkylene glycol-peptide adduct.

[0063] The hybrid hydrogels formed from synthetic polymers with binding agents (or “binders”) and ECM polymers provide chemically defined and versatile cell cultures as alternatives to natural, animal-derived matrices such as MATRIGEL®. The composition of these hybrid hydrogels can be tailored to the micro-environmental properties and components of specific tissues or particular biological states, such as varying phases of developmental, points in time-dependent physiological processes such as reproductive cycles, or disease states. These can be used to create fully defined hydrogel matrix for the expansion of cells and organoids with a hydrogel of precise biomechanical and biological properties. These hybrid hydrogels also offer a window of opportunity for large-scale production of clinical-grade cells and tissues, especially human cells and tissues. The biomechanical and biological properties of the hybrid hydrogels can be “tuned” or optimized by varying polymer structures, polymer molecular weight, and binders to support organoid growth from different types of cells, normal or tumor- derived. For example, hybrid hydrogels formed from multi-arm polyethylene glycol (PEG), integrin binding peptides, and approximately 300 kDa molecular weight hyaluronic acid (HA) support human endometrial epithelial cell growth.

[0064] The components of the hybrid polymer hydrogel can be provided either separately or combined, in dry form, such as powders, or in solution form. The hybrid polymer hydrogel forms when cross linking conditions are applied. For example, the components of the hybrid hydrogel are dissolved in a liquid, such as water or medium, and the solution temperature is increased to induce cross linking and form the hydrogel.

[0065] A. Supporting Synthetic Polymer, Preferably branched PEG

[0066] An exemplary synthetic polymer is PEG. Typically, the PEG has a molecular weight greater than 10 kDa, such as between about 10 kDa and 100 kDa. The molecular weight of the PEG may be between about 10 kDa and about 90 kDa, between about 10 kDa and about 80 kDa, between about 10 kDa and about 70 kDa, between about 10 kDa and about 60, such as about 50 kDa, about 40 kDa, about 30 kDa, or about 20 kDa. Preferably, the molecular weight of the PEG is about 20 kDa or about 40 kDa.

[0067] The plurality of synthetic polymers may include PEGs of various molecular weights, for example, a mixture of 20 kDa PEG and 40 kDa PEG. The plurality of synthetic polymers may include PEGs of at least two molecular weights, a lower molecular weight PEG and a higher molecular weight PEG, where the ratio of the lower molecular weight PEG to a higher molecular weight PEG is about 1:0.1, about 1:0.25, about 1:0.5, about 1:1, about 1: 1.5, about 1:2; about 1:2.5, about 1:3, or more. For example, in a hydrogel containing a 20 kDa 8-arm PEG and a 40 kDa 8-arm PEG, the ratio of the 20 kDa to 40 kDa PEG is about 1:0.1, about 1:0.25, about 1:0.5, about 1:1, about 1:1.5, about 1:2; about 1:2.5, about 1:3, or more, preferably about 1:1.

[0068] The synthetic polymer are branched (i.e. multi-armed) or multifunctional, preferably branched polymers, most preferably an 8 arm PAG, most preferably an 8 arm PEG. In polymer chemistry, multiple methods for synthesis can be used to yield a branched polymer. Briefly, there are several general strategies for synthesis, that is, descriptions of how the substituent molecules may be assembled to create the final product without specifying the reactants, reaction conditions, or desired product, which may be employed to create the branched synthetic polymer. Branching can occur by the replacement of a substituent, e.g., a hydrogen atom, on a monomer subunit, by another covalently bonded chain of that polymer; or, in the case of a graft copolymer, by a chain of another type. Branching can also occur by sequential covalent addition of monomers starting at a reactive site on a multifunctional initiator (core) and subsequently at the initiating site at the end of each branched polymer arm until the desired molecular weight is attained and the reaction is quenched. Branching may result from the formation of carbon-carbon or various other types of covalent bonds. Branching by ester and amide bonds is typically by a condensation reaction, producing one molecule of water (or HC1) for each bond formed.

[0069] The branching index measures the effect of long-chain branches on the size of a macromolecule in solution. It is defined as g = <sb2> / <sl2>, where sb is the mean square radius of gyration of the branched macromolecule in a given solvent, and si is the mean square radius of gyration of an otherwise identical linear macromolecule in the same solvent at the same temperature. A value greater than 1 indicates an increased radius of gyration due to branching.

[0070] There are two critical features of the PEG for this gel formulation, one being the molecular weight, and the other being the presence of more than two multiple functional groups. It has to be more than two for this gel, because the multiarm PEG molecule serves to bind to the binder peptides and connect to at least two HA molecules to crosslink the gel. The synthetic polymer may be linear and multifunctional, consisting of several (more than 2) reactive functional groups. Linear multifunctional synthetic polymers may be synthesized through a plurality of methods. Synthetic polymers can be functionalized via covalent modification of at one reactive functional group to yield the desired reactive functional group. Alternatively, multifunctional polymers can be obtained through synthesis of a co-polymer including monomers containing the desired functional group as a side chain (i.e. not participating in the polymerization reaction).

[0071] A preferred synthetic polymer is polyalkylene glycol, such as polyethylene glycol (“PEG:). Preferred PEGs are branched polymers with at least 3, or 4 arms, for example, 4 arms, 6 arms, or 8 arms. Typically, the arm has a length from the backbone of the polymer to the terminus of the arm between 1 and 100 nm, between 10 and 90 nm, or between 10 and 50 nm, for example, 20 nm.

[0072] Representative PEG molecular weights include 10 kDa, 15 kDa, 20 kDa, 30 kDa, 50 kDa, and 100 kDa. In preferred embodiments, the PEG has a molecular weight between about 10 kDa and about 100 kDa, for example, 20 kDa or 40 kDa. PEG of any given molecular weight may vary in other characteristics such as length, density, and branching. In preferred embodiments, the PEG is 8-arm with a MW of 20 kDa. In some embodiments, the PEG is 8-arm with a MW of 40 kDa. In some embodiments, PEGs of various molecular weights can be used, for example, a mixture of 20 kDa PEG and 40 kDa PEG.

[0073] In some embodiments, copolymers of PEG or other polymers described above may be used to make the hybrid hydrogel. In certain embodiments, the synthetic polymer used to make the hydrogel may be a copolymer partially composed of PEG, the other polymers described above, or a combination. The co-polymer may be a random co-polymer, with the monomers distributed randomly throughout the molecule, a block copolymer, with the monomers distributed in sections of several like monomers clustered together in the chain, or a graft co-polymer, synthesized from a homogenous polymer molecule consisting of one monomer covalently grafted to a homogenous polymer molecule consisting of a different monomer.

[0074] Assembly of the hydrogel requires mutually reactive functional moieties present on the synthetic polymer, the ECM polymer, and the binders which participate in covalent bonds that assemble the polymer network of the gel and attach binders within the gel structure. A plurality of reaction chemistries exist that could be used to assemble the hydrogel in the presence of cells, most of which are commonly classified in the literature as ‘click’ chemistry reactions and are classified by Sharpless et al. Agnew. Chemie. 2001 (https: / / doi.org / 10.1002 / 1521-

[0075] 3773(20010601)40: l l%3C2004::AID-ANIE2004%3E3.0.CO;2-5), who list several such reactions. Reactions classified as ‘click’ reactions which may be applicable for this material are those which 1) proceed under mild conditions, i.e. room or physiologic temperatures (~22-37°C), in aqueous solvent, and at physiologic pH, 2) produce inoffensive or no by-products, and 3) are stereospecific and 4) high-yielding. Some such reactions include thiol-ene reactions, inverse electron-demand Diels Alder reactions, and strain-promoted azide-alkyne click reactions. The choice of chemistry used to assemble the hydrogel precursors will motivate the selection of reactive functional moieties (“linking moieties”) included on the synthetic polymer, ECM polymer, and binders. The choice of chemistry used to assemble the hydrogel precursors will also motivate the selection of particular reaction conditions, such as temperature, pH, and inclusion or exclusion of a catalyst such as a photo initiator (i.e., a molecule which serves as a source of reaction-initiating free radicals when exposed to specific wavelengths of light). In the preferred embodiment, Michael addition thiol-ene chemistry is used. The participating reactive functional moieties used in the preferred embodiment are thiol (sulfhydryl) groups and an alkene that includes an adjacent electron- withdrawing group, in the preferred embodiment specifically vinyl sulfone. The reactive functional groups can be identified as an electrophilic group or a nucleophilic group, depending on whether within the reaction they serve as an electron acceptor or electron donor, respectively. Exemplary electrophilic groups include, but are not limited to, azide, cyano, trifluoromethyl, vinyl groups or vinyl-containing groups (e.g. vinyl sulfone), maleimide. In some embodiments, the electrophilic group is vinyl sulfone or maleimide. Exemplary nucleophilic groups include, but are not limited to, thiol, hydroxyl, alkoxy or aroxy (e.g., methoxy, benzyloxy), or primary, secondary, or tertiary amine. In some embodiments, the nucleophilic group is a thiol group.

[0076] In preferred embodiments, the synthetic polymers are functionalized with a first reactive functional moiety. The first reactive functional moiety may be present at a side chain functional group of the polymer or at a terminal functional group of the polymer. The molar degree of substitution of the first reactive functional moiety on a synthetic polymer can be varied. For example, at least 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 16, 20, 24, and 32 first linking moieties may be included per molecule in the polymer, irrespective of the molecular weight of the polymer. The first reactive functional moiety can be a reactive group that reacts with binders functionalized with a second reactive functional moiety to form polymer-binder adducts. Additionally, and alternatively, the first reactive functional moiety can be a reactive group that is involved in crosslinking either between different synthetic polymers or between the synthetic polymers / polymer-binder adducts and the ECM polymer. The first reactive functional moiety can be the same or different in the plurality of synthetic polymers.

[0077] In some embodiments, the reactive group can be an electrophilic group or a nucleophilic group. Exemplary electrophilic groups include, but are not limited to, azide, cyano, trifluoromethyl, vinyl groups or vinylcontaining groups (e.g. vinyl sulfone), maleimide. In some embodiments, the electrophilic group is vinyl sulfone or maleimide. Exemplary nucleophilic groups include, but are not limited to, thiol, hydroxyl, alkoxy or aroxy (e.g., methoxy, benzyloxy), or primary, secondary, or tertiary amine. In some embodiments, the nucleophilic group is a thiol group.

[0078] In preferred embodiments, the synthetic polymer has a weight / volume percentage in the hydrogel of between 1% and 50%, between 2% and 25%, or between 2% and 10%, for example, about 2%, about 3%, about 4%, or about 5%. B. Crosslinkable ECM Polymers

[0079] Crosslinkable chemically functionalized ECM polymers such as thiolated HA are used to crosslink the branched PAG such as the 8 arm PEG. The multifunctional chemical structure of linear and branched natural polymers found in the ECM enable robust crosslinking with a multifunctional synthetic polymer. According to Flory-Stockmayer gelation theory in polymer chemistry, the greater the number of reactive functional groups per polymer molecule of the reactants included in the gelation reaction, the lower the reaction efficiency required to obtain gelation (“critical conversion”) (Flory, P.J. (1941). "Molecular Size Distribution in Three Dimensional Polymers I. Gelation". J. Am. Chem. Soc. 63, 3083) (Stockmayer, Walter H.(1944). "Theory of Molecular Size Distribution and Gel Formation in Branched Polymers II. General Cross Linking". Journal of Chemical Physics. 12,4, 125).

[0080] ECM Polymers

[0081] In the preferred embodiment, the ECM polymer is hyaluronic acid (HA). HA is one of the major constituents of the skin and in extracellular tissues of various parts of the human body and is also found in a variety of other organisms. HA plays a role in several biological processes, including cell growth, migration and differentiation.

[0082] HA is a natural linear polysaccharide that has been used extensively in the biomedical field as it is a biocompatible, synthetic, nontoxic and non- immunogenic polymer with high water affinity. HA is formed by repeating units of D-glucuronic acid and N-acetyl-D-glucosamine disaccharide. The presence of multiple carboxylic acid and hydroxyl groups in the HA molecule makes it an ideal candidate for chemical modification. When modified with reactive functional groups such as thiols, HA can be used to crosslink a three-dimensional network of synthetic polymer molecules to form a hydrogel. Chemical crosslinking provides enhanced stability to HA- based materials. The HA is cleaved by naturally occurring hyaluronidases and by oxidation (such as in the presence of reactive oxygen species in injured or inflamed tissue). Hyaluronic acid is commonly used clinically as an excipient for pharmaceutical products and in tissue fillers. HA is readily available commercially and is industrially produced via batch fermentation.

[0083] Other ECM polymers include naturally occurring polysaccharide glycosaminoglycans (GAG) such as heparin / heparin sulfate, chondroitin sulfate / dermatan sulfate, and keratan sulfate.

[0084] Other ECM include collagen, fibronectin, (tropo)elastin and laminin.

[0085] Type I collagen is the most abundant structural protein in soft tissue and one of the earliest ECM components to be identified and isolated. It provides cells with a three-dimensional environment that supports cell growth and influences morphology and function. During tissue repair, collagen promotes cell adhesion and migration by engaging cell surface receptors such as integrin pi subunit, glycoprotein VI, non-specific receptors, and integrin-like receptors. The triple helix structure of collagen offered several favorable characteristics for engineering cellular functions, including thermal stability and mechanical strength.

[0086] Based on the properties mentioned above, type I collagen is the most popular native material for delivering cells or inducing specific cellular behavior for soft tissue regeneration. Collagen can self-assemble under physiological conditions, absorb onto surfaces through nonspecific interactions, and be electro spun into biomaterials. Type I collagen has been used in cell culture as a substrate to study and promote angiogenesis, as a wound dressing to treat bum wounds, as a vehicle to encapsulate stem cells to induce dermal regeneration, and as a scaffold to generate a skin substitute for grafting. Through integrin signaling and growth factor sensing at its interface with cells, a collagen matrix has been shown to maintain mesenchymal stem cells (MSCs) sternness or direct stem cells towards specific lineages for tissue regeneration. The compatibility between collagen matrices and MSCs enabled a collagen scaffold to serve as a cell delivery vehicle for diabetic wounds to promote repair via improved angiogenesis. Fibronectin promotes cell adhesion and controls cellular functions via peptide domains, including various binding sites for cell surface molecules and other biological molecules such as heparin, fibrin, collagen, and growth factors. The soluble form of fibronectin mainly circulates in plasma. The application of plasma fibronectin is limited to direct application of an aqueous solution, passive absorption, and covalent linkage to the surface of a biomaterial to promote bio-recognition and biocompatibility. These methods lack proper spatial control over deposition. Electro spraying was developed to deposit soluble fibronectin onto the surface of a material with controlled density, thickness, and pore size for optimal cell migration and adhesion.

[0087] Other ECM polymers such as (tropo)elastin and laminin present in basement membranes are useful. The different peptide sequences in elastin are employed to generate scaffolding for cell regeneration therapy. These elastin-like polypeptides can self-assemble into a gelatinous material above their transition temperature due to the unique thermo-responsive properties. Alternatively, they can be crosslinked by enzymatic reactions. Previously, such aggregates were applied in cartilage regeneration and were also used to encapsulate adipose-derived stem cells (ASCs) for full thickness dermal wound healing. The effect of an elastin construct on maintaining sternness was also interrogated; the author found that the elasticity and tensegrity of the construct were necessary to provide key mechanical cues for maintaining and expanding hematopoietic stem cells.

[0088] Laminin 111 , a major epithelial ECM component, has been used as a component of MATRIGEL® to provide a basement membrane-like scaffold. In addition, it has been used in electrospinning to construct fibrous meshes on a charged surface with tunable pore size and fiber diameter. The engineered meshes could maintain ASCs attachment and viability in vitro, and could induce them to develop neurite-like structure. Laminin 111 has also been shown to participate in other biological processes, including angiogenesis and neural differentiation. Laminin 511 was found to maintain the pluripotency of mouse embryonic stem cells through pi integrins, while laminin 411, 332, and 111 caused either rapid differentiation or cell death.

[0089] ECM polymers like collagen, fibronectin, elastin, and laminin are readily accessible commercially, through isolation or transgenic expression. Importantly, the presence of chemical functional groups such as amines, thiols, hydroxyls, and carboxylic acids on these polymers allows for chemical modification with a reactive functional group that can be used to assemble the hydrogel. In some cases, the reactive functional group may be endogenous to the molecule and not require further modification.

[0090] C. Covalently-Added Adhesion Ligand Binders

[0091] The hybrid polymer matrix further includes adhesion ligand binding peptides (“adhesion binders’’) specifically covalently attached to the multifunctional linear or branched synthetic polymer. These binders typically contain a second reactive functional moiety. The second reactive functional moiety can be a reactive group that reacts with the first reactive functional moiety to form a polymer-binder adduct. An exemplary second reactive functional moiety is a thiol group. Each polymer may have at least one binder incorporated, for example, 1 to 3 binders incorporated, so long as two or more first reactive functional moieties on the multifunctional synthetic polymer remain available to react after binder incorporation. In some embodiments, adhesion ligand binders are incorporated at the terminus of the arm of a multi-armed PEG such as an 8-arm PEG. The polymer- binder adduct can have an arm length from the polymer branch point to the terminus of the adhesion ligand between 10 and 100 nm. For example, an 8- arm 20 kDa PEG-GFOGER adduct has an arm length about 26 nm (~ 16 nm PEG and ~ 10 nm GFOGER) Each arm of the 8-arm 20 kDa PEG may have three GFOGER peptides incorporated. This is typically through formation of triple-helix of the three GFOGER peptides, where one of the peptides binds to one arm. An 8-arm 20 kDa PEG-PHKRGD adduct has an arm length about 21 nm (~ 16 nm PEG and ~ 5 nm PHKRGD). Each arm of the 8-arm 20 kDa PEG can have one PHKRGD incorporated. The number of binders incorporated on each polymer and the arm length depend on the type of polymer, the molecular weight of polymer, and the type of binder. Such parameters affect the cell’s sense of local nano-scale tensile forces and clustering. Typically, only a portion of the arms in the 8-arm polymer are linked to a binder.

[0092] The term GFOGER when used herein, bolded and italicized terms e.g., GFOGER as in GFOGER peptides” or 'PEG-GFOGER '. and PHKRGD as in “PHKRGD peptides” refers to a name given to an amino acid sequence or construct. See e.g., Table 1.

[0093] In some embodiments, one or more adhesion types of adhesion ligand binders are included in the hybrid hydrogel, for example, a collagen-I derived peptide and / or a fibronectin derived peptide. Typically, each adhesion ligand binder has a concentration between about 0.1 mM and about 5 mM, between about 0.25 mM and about 3 mM, or between about 0.5 mM and about 3 mM, for example, about 1.5 mM. In some embodiments, two or more extracellular protein-binding peptides (“ECM binders”) are included in the hybrid hydrogel, for example, a peptide having affinity to fibronectin (or “FN binder”) and a peptide having affinity to collagen and laminin (or “BM binder”). Typically, each extracellular protein-binding peptide has a concentration between about 0.1 mM and about 1 mM, about 0.1 mM and about 0.5 mM, or about 0.25 mM and about 0.5 mM, for example, about 0.25 mM.

[0094] The molar ratio of the adhesion ligand binder to the ECM binder(s) (collective molar concentration if more than one type of ECM binder is present), is typically greater than 1: 1, such as between about 1: 1 and about 5:1, between about 1.2: 1 and about 5:1, between about 1.5:1 and about 5:1, such as about 1.5:1, about 2:1, about 2.5: 1, about 3:1, about 3.5:1, about 4:1, about 4.5: 1, or about 5: 1.

[0095] Adhesion ligand binders are typically synthetic peptides with affinity to cellular proteins, typically to cell adhesion molecules (CAMs). Cell adhesion molecules include integrins, immunoglobulin superfamily (IgSF) CAMS, cadherins, and selectins. The adhesion ligand binders may derive from proteins of the ECM. The adhesion ligand binders may be linear or branched. Preferably, the adhesion ligand binders have affinity to one or more integrins, and / or to one or more integrin subunits. Adhesion ligand binders typically have two or more functional moieties allowing crosslinking to the PEG or ECM polymers.

[0096] The adhesion ligand binders may have a secondary structure. The adhesion ligand binders may be peptides having a single chain amino acid sequence. The adhesion ligand binders may be branched peptides. The adhesion ligand binders may be peptides having a single chain amino acid sequence that self assembles into dimers, trimers, etc. The secondary structure of the dimers and trimers may be any secondary structure for proteins and peptides, including alpha helices, beta sheets, beta turns, or omega loops.

[0097] The adhesion ligand binders typically contain a reactive functional moiety at one end of the peptide to form a covalent or non-covalent bond with the polymer. Preferably, the adhesion ligand binders form a covalent bond with the synthetic polymer at one of the peptide side chains.

[0098] The adhesion ligand binders may include a motif from any one of fibrillar or non-fibrillar collage. The fibrillar collagen include collagen types I, II, III, V, XI. The non-fibrillar collagen includes FACIT (Fibril Associated Collagens with Interrupted Triple Helices); collagen types IX, XII, XIV, XIX, XXI; short chain type VIII, X; basement membrane (Type IV); multiplexin (Multiple Triple Helix domains with Interruptions) (Type XV, XVIII); MACIT (Membrane Associated Collagens with Interrupted Triple Helices) (Type XIII, XVII); and others (Type VI, VII) (Ricard-Blum, Cold Spring Harb Perspect Biol, 2011;3:a004978).

[0099] Different tissues contain collagen of different type. For example, Type I is common in skin, tendon, vasculature, organs, bone (main component of the organic part of bone); Type II is most common in cartilage (main collagenous component of cartilage);Type III is most common in reticulate (main component of reticular fibers), commonly found alongside type I; Type IV is most common in basal lamina, the epithelium-secreted layer of the basement membrane; and Type V is most common on cell surfaces, hair, and placenta.

[0100] The collagen can be of any animal origin or human.

[0101] Fibronectin typically exists as a protein dimer, consisting of two nearly identical polypeptide chains linked by a pair of C-terminal disulfide bonds. Each fibronectin subunit typically has a molecular weight of 230-250 kDa and contains three types of modules: type I, II, and III. All three modules are composed of two anti-parallel -sheets resulting in a Betasandwich; however, type I and type II are stabilized by intra-chain disulfide bonds, while type III modules do not contain any disulfide bonds. The absence of disulfide bonds in type III modules allows them to partially unfold under applied force.

[0102] Three regions of variable splicing occur along the length of the fibronectin protomer. One or both of the "extra" type 111 modules (EI11A and EIIIB) may be present in cellular fibronectin, but they are never present in plasma fibronectin. A "variable" V-region exists between III14- 15 (the 14th and 15th type III module). The V-region structure is different from the type I, II, and III modules, and its presence and length may vary. The V-region contains the binding site for a4pi integrins. It is present in most cellular fibronectin, but only one of the two subunits in a plasma fibronectin dimer contains a V-region sequence.

[0103] The modules are arranged into several functional and protein-binding domains along the length of a fibronectin monomer. There are four fibronectin-binding domains, allowing fibronectin to associate with other fibronectin molecules. One of these fibronectin-binding domains, 11-5, is referred to as the "assembly domain", and it is required for the initiation of fibronectin matrix assembly. Modules III9-10 correspond to the "cellbinding domain" of fibronectin. The RGD sequence (Arg-Gly-Asp) is located in III10 and is the site of cell attachment via a5 i and aV 3 integrins on the cell surface. The "synergy site" is in III9 and has a role in modulating fibronectin's association with a501 integrins. Fibronectin also contains domains for fibrin-binding (11-5, 110-12), collagen-binding (16-9), fibulin- 1-binding (III13-14), heparin-binding and syndecan-binding (III 12— 14) (Mao et al., Matrix Biology, 24:389-399).

[0104] Laminin polyprotein includes fifteen laminin trimers. The laminins are combinations of different alpha-, beta-, and gamma-chains. The five forms of alpha-chains are: LAMA1 , LAMA2, LAMA3 (which has three splice forms), LAMA4, LAMA5. The beta-chains include: LAMB1, LAMB2, LAMB3, LAMB4. The gamma-chains are: LAMC1, LAMC2, LAMC3. Laminins were previously numbered as they were discovered, i.e. laminin- 1, laminin-2, laminin-3, etc., but the nomenclature was changed to describe which chains are present in each isoform (laminin- 111, laminin- 211, etc., Aumailley et al., Matrix Biology, 24:326 - 332 (2005)).

[0105] Integrins are cell adhesion molecules and are obligate heterodimers with two subunits: a (alpha) and 0 (beta). Integrins in mammals have twenty- four a and nine 0 subunits (Tables 2 and 3). Beta-1 integrins interact with many alpha integrin chains. Integrins work alongside other receptors such as cadherins, the immunoglobulin superfamily cell adhesion molecules, selectins and syndecans, to mediate cell-cell and cell-matrix interaction. Ligands for integrins include fibronectin, vitronectin, collagen and laminin.

[0106] Exemplary adhesion ligands include “RGD,” a fibronectin (FN)- derived adhesion peptide containing the canonical RGD motif from the 10th FN type III domain NH2-GCRE-RGDSP(Am) (SEQ ID NOG); PHSRN- K-RGD. ' a fibronectin-derived adhesion peptide containing both the RGD motif and the PHSRN (SEQ ID NOG) synergy site from the 9th FN type III repeat in a branched configuration to mimic features of the biophysical presentation in FN, (Ac)PHSRNGGGK-(Ac)GGGERCG-GGRGDSPY(Am) (SEQ ID NO:8);‘ PHSRN-K-RDG, ' a scrambled control sequence where the RGD motif was scrambled reducing integrin binding; (Ac)PHSRNGGGK-GGGERCG(Ac)-GGRDGSPY(Am) (SEQ ID NO:9); GFOGER. ' a collagen I-derived adhesion peptide, NH2- GGYGGGPG(GPP)5GFOGER(GPP)5GPC(Am) (SEQ ID NO:11), ‘ Lams ' ’ a laminin 5-derived adhesion peptide, NH2-GCRG- PPFLMLLKGSTR(Am) (SEQ ID NO: 13) ' LamS' Note that “(Ac)” is used as shorthand notation for N-terminal acetylation and “(Am)” is used as shorthand notation for C-terminal amidation of the peptides during synthesis.

[0107] Binders having affinity to fibronectin are referred to herein as FN hinders, and include peptides (NH2)-GCRE-TLQPVYEYMVGV(C00H) (SEQ ID NO:6), and peptides, polypeptides, oligopeptides, or proteins containing the amino acid sequence GCRE (SEQ ID NO: 16), and / or TLQPVYEYMVGV (SEQ ID NO: 15).

[0108] Other exemplary FN binders include peptides with amino acid sequences VPQIHGQNKGNQSFEEDTE (SEQ ID NO: 17), VPQIQGQNKGNQSFEEDTE (SEQ ID NO: 18), VPQIHGQNNGNQSFEEDTE (SEQ ID NO: 19), VPQIQGQNNGNQSFEEDTE (SEQ ID NO:20), VPQIHGQNIGNQSFEEDTE (SEQ ID NO:21), VPQIQGQNIGNQSFEEDTE (SEQ ID NO:22), VPQIAGQNKGNQSFEEDTE (SEQ ID NO:23), and VPQIAGQNAGNQSFEEDTE (SEQ ID NO:24).

[0109] Binders having affinity to basement membrane proteins are referred to herein as BM binders. The BM binders typically include synthetic peptides with affinity to one or more of the components of the basement membrane. Exemplary components of the basement membrane include laminin; integrins; nidogens; dystroglycans; collagen types III, IV, and VII; perlacan; heparan sulfate; and fibrillin. The BM binders may have affinity to collagen type IV and laminin.

[0110] BM binders include the peptide NH2-GCRE- ISAFLGIPFAEPPMGPRRFLPPEPKKP(Am) (SEQ ID NO:5), and peptides, polypeptides, oligopeptides, or proteins containing the amino acid sequence GCRE (SEQ ID NO: 16), and / or

[0111] ISAFLGIPFAEPPMGPRRFLPPEPKKP(Am) (SEQ ID NO:25).

[0112] Table 1. Exemplary Adhesion Ligand Peptides

[0113] All amine terminals were acetylated (Ac) and all carboxyl terminals were amidated (Am).

[0114] GFOGER (SEQ ID NO: 1) appears to be critical for maximum efficacy, especially for the culture of epithelial cells. D. Additional Factors

[0115] The hydrogels may include additional factors, such as growth factors, their fragments, their variants and analogs. Typically, the additional factors are included to further support the growth and / or differentiation of the cells in the enteroids. The additional factors natural or synthetic peptides and small molecule. The additional factors may be co-factors, signaling molecules, and growth factors, including, but not limited to basic fibroblast growth factor (bFGF), FGF-1, FGF-2, FGF-4, FGF-7, FGF-10, transforming growth factor pi (TGF-pi), activin-A, activin-B, bone morphogenic protein 4 (BMP-4), hepatocyte growth factor (HGF), epidermal growth factor (EGF), P nerve growth factor ( NGF), retinoic acid, interleukin (IL)-3, IL-6, IL-11, Noggin, platelet derived growth factor (PDGF), stem cell factor (SCF), vascular endothelial growth factor (VEGF), Wnt- 1, Wnt-2, Wnt-la, Wnt-5a, Wnt-7a, their variants and analogs. Variants and analogs may include the factor(s) with amino acid modifications, such as EGF with Y13G modification (Reddy et al., Nature Biotechnology, 14:1696-1699 (1996)).

[0116] See also Cambria, et al., Biomacromolecules 2015; 16(8):2316-26.

[0117] III. Methods of Making the Gels

[0118] A preferred method of making the gel is shown in FIG. 1.

[0119] The multifunctional PAG is reacted with the peptide binders of choice to form a synthetic polymer-binder adduct. The ECM polymer is modified with a reactive functional moiety or most often obtained commercially pre-modified with the reactive functional moiety. The synthetic polymer-binder adduct is then crosslinked with the ECM polymer. Too few reactive functional moieties available on each molecule (either synthetic polymer-binder adduct or ECM polymer) would prevent gelation. In the preferred embodiment, branched PEG is used as the multifunctional PAG and HA is used as the ECM polymer. The space-filling, highly waterabsorbing physical properties of HA enables this hybrid hydrogel to use less total mass of polymer compared to hydrogels made with synthetic polymers alone while still maintaining robust mechanical properties.

[0120] Although in the preferred embodiment a thiol-vinyl sulfone reaction is used to form the synthetic polymer-binder adducts as well as crosslink the via HA, other crosslinking chemistries could be used, such as a stimulus- responsive (e.g., proceeds in the presence of stimulus such as light, pH change, or temperature change) reaction, such as radical-mediated thiol- norbomene click chemistry, or a dynamic covalent chemistry, such as oxime click chemistry. Some examples of dynamic covalent reactions that may be used are reviewed briefly by Uman et al. (Uman et al., J. Applied Polymer Sci., 2019, https: / / doi.org / 10.1002 / app.48668). The use of either a stimulus- responsive or dynamic covalent chemistry could enable the gel to be injectable or extrudable. This is particularly helpful in delivery of cell therapies and in 3D bioprinting. Additionally, the chemical reactions used in the first step, the preparation of the polymer-hinder adducts, and in the second step, the crosslinking of the polymer-hinder adducts using the ECM polymer, may be the same or different. For example, a thiol- vinyl sulfone Michael addition reaction may be used to react the branched PEG and the peptide binders to form the polymer-hinder adducts, and then a different reaction, such as a radical -mediated thiol-norbornene reaction, may be used to crosslink the polymer-hinder adducts using the ECM polymer. In that example, the binders may include a vinyl sulfone reactive functional group, the branched PEG may include thiol as a reactive functional group, and the ECM polymer may include norbornene as a reactive functional group. In the preferred embodiment, the two steps are completed using the same reaction, thiol-vinyl sulfone Michael addition chemistry. In the preferred embodiment, the reactive functional group of the branched PEG is vinyl sulfone and the reactive functional groups of both the binders and the ECM polymer are thiols.

[0121] Physiological conditions refer to a temperature range of 20-40°C, atmospheric pressure of 1, pH of 6-8. For example, the reactions can be performed at a temperature of 37 °C, atmospheric pressure of 1, and pH about 7.4. Typically, the hydrogel can be formed in about 60 min, 40 min, 30 min, 20 min, 10 min, 5 min, 2 min, or 1 min.

[0122] A. Polymer-Binder Adduct Preparation

[0123] Typically, the method of forming hybrid hydrogel includes (i) combining a plurality of synthetic polymers and one or more binders. The synthetic polymer contains a first reactive functional moiety. The first reactive functional moiety may locate in the interior position of the polymer or near or at the terminal positions of the arm of the polymer. The number of first reactive functional moiety on a polymer can be varied but must be more than 2. For example, multiple first linking moieties may be included in one polymer. In some embodiments, the first reactive functional moiety of the polymers can be an electrophilic and / or nucleophilic reactive group. The binder contains a second reactive functional moiety. The second reactive functional moiety can be a reactive group that reacts with the first reactive functional moiety to form a polymer-binder adduct. For example, when the synthetic polymers contain an electrophilic group, the second reactive functional moiety can be a nucleophilic group. When the synthetic polymers contain a nucleophilic group, the second reactive functional moiety can be an electrophilic group. Any electrophilic and nucleophilic groups disclosed above can be used. In preferred embodiments, the synthetic polymers can contain vinyl sulfone groups and the binders can contain free thiol groups at the terminus location. Each polymer may have at least one binder incorporated, for example, 1 or 3 binders incorporated. The incorporated binders may be on the same arm of the synthetic polymer or different arms of the synthetic polymer. Preferably, the binders are incorporated to the terminus of the arm of the synthetic polymer.

[0124] In some embodiments, the hybrid hydrogel contains a weight / volume percentage of polymers between 1% and 10%, between 2% and 25%, or between 2% and 10%, for example, about 2%, about 3%, about 4%, or about 5%. The binders preferably contain adhesion ligand binders and extracellular matrix protein-binders. The adhesion ligands may include one or more types of integrin-binding peptides, such as peptide derived from Collagen Type I and / or peptides derived from Fibronectin. Each type of adhesion ligand can have a concentration between 0. 1 and 5 mM, between 0.25 and 3 mM, or between 0.5 and 3 mM, for example, about 1.5 mM. In some embodiments, two or more extracellular protein-binding peptides are included in the hybrid hydrogel, for example, a peptide having affinity to fibronectin (or “FN binder”) and a peptide having affinity to collagen and laminin (or “BM binder”). Typically, each extracellular protein-binding peptide has a concentration between 0.1 and 1 mM, 0.1 and 0.5 mM, or 0.25 M. The molar ratio of the adhesion ligand binders to the ECM binder(s) (collective molar concentration if more than one type of ECM binder is present), is typically greater than 1: 1, such as between about 1: 1 and about 5:1, between about 1.2: 1 and about 5:1, between about 1.5:1 and about 5:1, such as about 1.5:1, about 2:1, about 2.5: 1, about 3:1, about 3.5:1, about 4:1, about 4.5: 1, or about 5: 1.

[0125] B. Crosslinking

[0126] The method can additionally include (ii) combining the adduct of step (i) and one or more cross-linkers, wherein the cross-linkers have a crosslinking reactive functional moiety. The crosslinker may be chemically- modified ECM polymer, functionalized with multiple (i.e., more than 2) crosslinking reactive functional moieties per molecule. The crosslinking moieties can react with the first linking moieties on the synthetic polymers to perform crosslinking and form hydrogel. For example, when the synthetic polymers contain an electrophilic group, the crosslinking reactive functional moiety can be a nucleophilic group. When the synthetic polymers contain a nucleophilic group, the crosslinking reactive functional moiety can be an electrophilic group. In preferred embodiments, the synthetic polymers contain vinyl sulfone and the cross-linkers are dithiols having one thiol group at each terminus. In some embodiments, the ratio of crosslinking reactive functional moiety to first reactive functional moiety is between 0.05 and 1, between 0.35 and 1, or between 0.5 and 1, for example, about 0.5. In preferred embodiments, the ratio of thiol of cross linkers to vinyl sulfone of polymers is between 0.1 and 1, between 0.35 and 1, or between 0.5 and 1, for example, about 0.15. The concentration of ECM polymer in % weight / volume can vary between 0.10% and 2.0%. In preferred embodiments using hyaluronic acid (HA) as a crosslinking ECM polymer, the average molecular weight of the HA molecule can vary between 10 kDa and 1500 kDa.

[0127] Crosslinking can be performed either between different synthetic polymers and crosslinkers or between the synthetic polymer / polymer-binder adducts and crosslinkers. Exemplary crosslinking reactions that are cellcompatible include, but are not limited to, reactions via thrombin-activated Factor XHIa under physiological conditions or by another enzymatic crosslinking mechanism known in the art, or via Michael addition, click chemistry, or by another mild chemical crosslinking mechanism known in the art.

[0128] In preferred embodiments, the crosslinking is performed between the synthetic polymer-binder adducts and crosslinkers. To achieve crosslinking between the synthetic polymers and cross-linkers by the preferred Michael addition, one or more synthetic polymers are functionalized with an electrophilic group and / or a nucleophilic group, whereas the crosslinkers carry a crosslinking reactive functional moiety that reacts with the electrophilic group and / or nucleophilic group on the polymers. For example, the synthetic polymers are modified with an electrophilic group, such as a vinyl sulfone, whereas the crosslinkers carry a nucleophilic group, such as a thiol group. In some embodiments, crosslinking is achieved via FXlIla- mediated crosslinking between the synthetic polymers and crosslinkers. For example, one or more synthetic polymers are functionalized with a lysine- bearing peptide sequence, whereas the crosslinker ECM polymer is functionalized with or includes a glutamine-bearing peptide sequence. In some embodiments, crosslinking is performed via click chemistry. For example, one or more synthetic polymer in the plurality of synthetic polymers is functionalized with a norbomene or thiol group, whereas crosslinkers contain a thiol or norbornene group.

[0129] A critical feature of this formulation is that the peptides are bound to the multifunctional synthetic polymer such as the multiarm PEG, not the ECM polymer such as the HA. The addition of adhesion peptides directly to HA limits the mobility of the peptides due to reduced motility of the HA molecule backbone and increased steric hindrance compared to peptides The following are exemplary selection of components, and ratios thereof:

[0130] The preferred formulation is 2.5% w / v PEG-vinyl sulfone, (8-arms = 8 vinyl sulfone groups per PEG molecule, 20 kDa molecular weight PEG) which is functionalized via reaction with the following adhesion ligand binders and ECM binders in the presence of 200 mM 4-(2 -hydroxy ethyl)- 1- piperazineethanesulfonic acid (HEPES) and 2X concentration Dulbecco’s phosphate buffered saline (PBS) to form a PEG-hinder adduct: 1.5 mM GFOGER, 1.5 mM PHSRN-K-RGD, 0.5 mM BM -binder, and 0.5 mM FN- binder. The resulting PEG-binder adducts are crosslinked with 0.25% w / v thiolated HA (~300kDa, 20-30% degree of substitution).

[0131] The concentration and choice of binders used in the formulation may be modified. The concentration and choice of binders used in the formulation may be modified. The preferred formulation has also been used with two additional adhesion ligand binders included in the PEG-binder adduct preparation step: 0.5 mM GCRE-GG1KVAV (SEQ ID NO:28), a sequence derived from the integrin-binding domain on laminin alpha 1; and 0.5 mM GCRE-YIGSR (SEQ ID NO:44), a sequence derived from the integrin- binding domain on laminin beta 1.

[0132] The preferred maximum amount of adhesion ligand peptides that can be added to the preferred formulation (without increasing the concentration of multiarm PEG beyond 2.5% w / v) is 5 mM total. It has been found that for a gel made with 2.5% w / v PEG-vinyl sulfone and 0.25% w / v thiolated HA, only about 1 mM of vinyl sulfone groups are required to react with the HA in order to form a gel. In the preferred embodiment, the concentration of vinyl sulfone reactive functional groups that remain after PEG-binder adduct preparation is in excess of 1 mM (5-6 mM) in order to ensure gelation is very consistent even with mild variability in the reagents.

[0133] The concentration of PEG and HA may be modified from the preferred formulation to control the stiffness and gelation speed of the resulting gel. A softer formulation relative to the preferred formulation has been used for adipocyte culture, which maintains the 10: 1 ratio of PEG to HA at a lower concentration of both (1.67% w / v PEG-VS + 0.167% thiolated HA), but with the same concentration of all the peptide binders.

[0134] A stiffer gel with a 5:1 ratio of PEG to HA (2.5%w / v PEG + 0.50% thiolated HA) and the same concentration of peptide binders has also been used. This yields a gel approximately 10-fold stiffer than the ‘standard’ formulation. The ‘stiff’ gel formulation (with a 5: 1 PEG to HA) gels about twice as fast as the ‘standard’ formulation. As the PEG concentration increases relative to the HA concentration, the gel becomes less stiff and slower to gel. For comparison, a gel with a 6.6: 1 ratio of PEG to HA (2.5% w / v PEG + 0.38% w / v thiolated HA) with the same concentration of the peptide binders has also been used. This formulation yields a gel approximately 5-fold stiffer than the ‘standard’ formulation, which is less stiff than the formulation made with a 5:1 ratio of PEG to HA. The 6.6:1 PEG:HA gel formulation was also observed to gel more slowly than the 5:1 PEG:HA gel formulation.

[0135] In some embodiments, the method additionally includes combining cells, one or more inhibitors of dissociation-induced apoptosis, and the adduct of the first step. The incorporation of inhibitors is important for preventing death of encapsulated cells, especially for human organoid culture. In some embodiments, the inhibitor is at a concentration between 1 nM and 1 mM, between 10 nM and 100 pM, or between 100 nM and 50 pM, for example, about 10 pM. Cross-linking of the hydrogel is preferably performed in the presence of cells to be cultured within the hydrogel. This way the cells or cell aggregates are encapsulated by the formed hydrogel matrix, i.e. are residing in a distinct cell culture microenvironment. Alternatively, the cells and inhibitors of dissociation-induced apoptosis can be combined with the product of the second step.

[0136] The cells are typically incorporated at a cell density between about 1 x KF cells / ml and about 1 x 109cells / ml as the final density in the polymer- binder-crosslinker solution (“pre-gel mixture”). Exemplary suitable ranges for the cell density in the final solution include between about 1 x 105cells / ml and about 1 x 108cells / ml, between about 5 x 105cells / ml and about 5 x 10scells / ml, between about 5 x 105cells / ml and about 1 x 10scells / ml, preferably between about 1 x 106cells / ml and about 1 x 108cells / ml, more preferably between about 1 x 106cells / ml and about 1 x 107cells / ml.

[0137] IV. Methods of Using the Hybrid Hydrogels

[0138] The hybrid hydrogels are suitable for 3D cell culture, organoid formation, drug discovery, inter-organ interaction characterization, and multi-organ drug responses. The hybrid hydrogels are particularly suitable for supporting culture of primary human cells and interrogating the biological response of cells in culture to hyaluronic acid. These are especially useful for culturing endometrial epithelial cells and organoids thereof, as well as some other types such as primary human adipocytes and neural cells.

[0139] A. Exemplary Method for Cell Encapsulation

[0140] The cells are typically incorporated with the synthetic polymer-binder adducts at the time of addition of the crosslinker ECM polymer. Certain cell types, such as endometrial epithelial cells, are incorporated in the presence of an apoptosis inhibitor. The cells may be at any suitable density (that is, concentration of cells per unit volume of gel) and are not harmed by the subsequent crosslinking reaction. The crosslinking step occurs at physiological conditions, e.g., at about neutral pH, and 37 °C.

[0141] An exemplary method includes the following steps. Hydrogels may be fabricated as gel droplets in the base of a non-tissue culture treated polystyrene 96-well plate using Michael-type reaction chemistry. First, the reaction mixture for a concentrated PEG-binder adduct solution was made. 5% w / v 20kDa, 8-arm poly(ethylene glycol)-vinyl sulfone (PEG-VS), 200 mM HEPES / 2X PBS, and the peptide binders 3.0 mM PHSRN-K-RGD, 3.0 mM GFOGER, 1.0 mM BM-binder, and 1.0 mM FN -binder were combined in a microtube and vortexed to mix. The mixture was allowed to react at room temperature for 30 min to form the PEG-binder adduct. After 30 min, the concentrated PEG-binder adduct solution was moved to ice until further use.

[0142] The cells were detached from their culture surface, such as a T75 culture flask, using a dissociation reagent such as trypsin / EDTA. The cells were collected and pelleted via centrifugation, then the supernatant containing trypsin / EDTA was removed. The cells were then resuspended in growth media, such as DMEM / F12 / FBS, and counted. The cell solution is then split into microtube aliquots, with the number of cells per tube determined by the desired final cell density and batch size (i.e., final volume of gel) for the gel samples. An exemplary cell suspension aliquot would contain 3xl05cells to produce a final volume of 30 pl of gel with a final cell density of I lO7cells / ml of gel. The aliquots of cells were then centrifuged to obtain cell pellets.

[0143] To encapsulate the cells in the gel, the supernatant was carefully and completely removed from one cell pellet. The cells were resuspended in sufficient sterile PBS to achieve the desired final volume of the mixture after adding the PEG-binder adduct solution and the thiolated HA (HA-SH) stock solution. The concentrated PEG-binder adduct solution was then added to the cell suspension so that the PEG-binder adduct solution will be diluted 2-fold in the final pre-gel mixture. It is critical that the PEG-binder adduct solution is diluted in PBS before the addition of the HA-SH stock solution, because if the two are mixed together without dilution the mixture will gel instantaneously, presumably via physical interactions. Pipette the mixture up and down repeatedly to mix. The HA-SH stock solution was added to the mixture with the cells and pipetted up and down repeatedly to form the pregel mixture. The final concentrations of each component in the pre-gel mixture were: 2.5% w / v PEG-VS, 100 mM HEPES / 1X PBS, 1.5 mM PHSRN-K-RGD, 1.5 mM GFOGER 0.5 mM BM-binder, 0.5 mM FN- binder, and 0.25% w / v HA-SH. Immediately after combining the HA-SH, 3 pl droplets of pre-gel mixture were deposited per well in a 96- well, non- tissue culture-treated polystyrene plate. The well plate was moved to a humidified incubator at 37°C and 5% CO2 for 30 min to polymerize.

[0144] After gelation is complete, growth media, such as DMEM / F12 / FBS, may be added to the apical (top) (100 pL) and basolateral (bottom) (600 pL) sides of the TRANSWELL® to achieve hydrostatic equilibrium. Cultures are typically maintained in a humidified incubator at 37 °C, 95% air, 5% CO2.

[0145] B. Hydrogel Dissolution

[0146] The hybrid hydrogels with cells or organoids are formed of materials permitting direct assaying of the cells or organoids without the need to remove the hydrogel components. The hybrid hydrogels with cells or organoids may be subject to dissolution to remove a portion, or substantially all of the components of the hydrogel.

[0147] The removal of hydrogel components occurs through hydrogel dissolution. Hydrogel dissolution includes incubating the hybrid hydrogels with cells or organoids in the presence of dissolution agents such as hyaluronidase. Typically, the incubation is for at least about 5 min, about 10 min, about 15 min, about 20 min, about 25 min, about 30 min, about 35 min, about 40 min, about 45 min, about 50 min, about 55 min, or about 60 min. In preferred embodiments, the dissolution is complete within about 10 min, about 15 min, about 20 min, about 25 min, about 30 min, about 35 min, about 40 min, about 45 min. Typically, the incubation occurs at physiological conditions, such as 37 °C, 5% CO2, humidified atmosphere, and includes growth media used to support survival of the cells or organoids.

[0148] In summary, hybrid hydrogels for organogenesis from mammalian cells, preferably biopsied human cells, and especially epithelial cells, have been developed. The hybrid hydrogels are tunable matrices with components select to support growth of cells of different origins (e.g., cells of mouse and human origin) and from different organs and tissues (e.g., endometrial cells, or intestinal cells). The hybrid hydrogels provide a matrix where the complex cell-matrix interactions can be controlled, which are not possible with complex natural hydrogels. Preferred ECM binders typically have affinity to fibronectin, collagen type IV, and / or laminin. The ECM binders are typically functional at one end of the peptide to form a covalent or non-covalent bond with the polymer. Preferably, the ECM binders form a covalent bond with the polymer at one of the peptides. a. Peptides Having Affinity to Fibronectin

[0149] Binders having affinity to fibronectin are referred to herein as FN binders, and include peptides (NH2)-GCRE-TLQPVYEYMVGV(C00H) (SEQ ID NO:6), and peptides, polypeptides, oligopeptides, or proteins containing the amino acid sequence GCRE (SEQ ID NO: 16), and / or TLQPVYEYMVGV (SEQ ID NO: 15).

[0150] Other exemplary FN binders include peptides with amino acid sequences VPQIHGQNKGNQSFEEDTE (SEQ ID NO: 17), VPQIQGQNKGNQSFEEDTE (SEQ ID NO: 18), VPQIHGQNNGNQSFEEDTE (SEQ ID NO: 19), VPQIQGQNNGNQSFEEDTE (SEQ ID NO:20), VPQIHGQNIGNQSFEEDTE (SEQ ID NO:21), VPQIQGQNIGNQSFEEDTE (SEQ ID NO:22), VPQIAGQNKGNQSFEEDTE (SEQ ID NO:23), and VPQIAGQNAGNQSFEEDTE (SEQ ID NO:24). b. Peptides Having Affinity to Basement Membrane Proteins

[0151] Binders having affinity to basement membrane proteins are referred to herein as BM binders. The BM binders typically include synthetic peptides with affinity to one or more of the components of the basement membrane. Exemplary components of the basement membrane include laminin; integrins; nidogens; dystroglycans; collagen types III, IV, and VII; perlacan; heparan sulfate; and fibrillin. The BM binders may have affinity to collagen type IV and laminin. polypeptides, oligopeptides, or proteins containing the amino acid sequence

[0152] GCRE (SEQ ID NO: 16), and / or

[0153] ISAFLGIPFAEPPMGPRRFLPPEPKKP(Am) (SEQ ID NO:25).

[0154] Table 2. Exemplary binders for the synthetic hydrogels.

[0155] All amine terminals were acetylated (Ac) and all carboxyl terminals were amidated (Am).

[0156] 3. Second Reactive functional moiety

[0157] The binder typically contains a second reactive functional moiety. The second reactive functional moiety can be a reactive group that reacts with the first reactive functional moiety of the synthetic polymer to form a polymer-binder adduct. The second reactive functional moiety can contain any suitable reactive groups described above. An exemplary second reactive functional moiety is a thiol group. The thiol group is preferably a free thiol group at the terminus of the binders. For example, the binders are adhesion ligands and extracellular protein-binding peptides, where each peptide was synthesized to have a free thiol group on a cysteine residue near the N- terminus.

[0158] C. Additional Factors

[0159] The hydrogels may include additional factors, such as growth factors, their fragments, their variants and analogs. Typically, the additional factors are included to further support the growth and / or differentiating the cells in the enteroids. The additional factors natural or synthetic peptides and small molecule. The additional factors may be co-factors, signaling molecules, and growth factors, including, but not limited to basic fibroblast growth factor (bFGF), FGF-1, FGF-2, FGF-4, FGF-7, FGF-10, transforming growth factor pi (TGF- i), activin-A, activin-B, bone morphogenic protein 4 (BMP-4), hepatocyte growth factor (HGF), epidermal growth factor (EGF), nerve growth factor (PNGF), retinoic acid, interleukin (IL)-3, IL-6, IL-11, Noggin, platelet derived growth factor (PDGF), stem cell factor (SCF), vascular endothelial growth factor (VEGF), Wnt-1 , Wnt-2, Wnt-l a, Wnt-5a, Wnt-7a, their variants and analogs. Variants and analogs may include the factor(s) with amino acid modifications, such as EGF with Y13G modification (Reddy et al., Nature Biotechnology, 14: 1696-1699 (1996)). See also Cambria, et al., Biomacromolecules 2015; 16(8):2316-26.

[0160] F. Cells, Tissues, and Organs

[0161] Typically, the hybrid hydrogels support attachment, growth, and differentiation of primary cells, that is, cells isolated directly from living organs and tissues. The hybrid hydrogels may also be used to expand organoids, that is, grow from single cells to multicellular structures with a well-defined composition, structure, and morphology, or culture organoids derived from primary tissue, that is, multicellular structures with heterogenous cell composition. The hybrid hydrogels are suitable for encapsulating cells derived from stem cells, such as differentiated induced pluripotent stem cells, as well as established cell lines.

[0162] Generally, the cells in hybrid hydrogels form tissues and organs with differentiation, architecture, cellular composition and cellular organization characteristic of epithelial tissues, gastric tissues, intestinal tissues, endometrial tissues, cardiac tissues, vascular tissues, renal tissues, pulmonary tissues, immune cell interactions, neuronal tissues, osteochondral tissues, and muscle tissues.

[0163] The cells or organoids formed in the hybrid hydrogels may be suitable for forming or studying the organs, tissues, and their interactions in the reproductive system, cardio-vascular system, renal system, pulmonary system, digestive system, immune system, nervous system, and / or musculoskeletal system.

[0164] G. Hydrogel Properties

[0165] As shown in the examples, the hybrid hydrogel supports human endometrial epithelial cell growth in the form of organoids, spherical layers of single cells that expand in a 3D culture matrix. The organoids formed in the synthetic hydrogel show correct polarity, exhibit appropriate architecture, and proliferate. Additionally, other cell types, such as primary human adipocytes and induced pluripotent stem cell-derived neurons, have been cultured in the hybrid hydrogel.

[0166] The biomechanical and biological properties of the synthetic hydrogels can be tuned by varying the mass ratio of synthetic polymer to ECM polymer, concentration of total polymer, synthetic or ECM polymer molecular weight, and type and concentration of binder, to support growth, proliferation, and / or differentiation of multiple cell types, normal or tumor- derived. For example, hybrid hydrogels formed from 2.5% w / v 8-arm polyethylene glycol (PEG) with 20 kDa molecular weight, integrin binding peptides, FN binders, BM binders, and 0.25% w / v -300 kDa hyaluronic acid (HA) functionalized with thiol groups support human endometrial epithelial cell growth.

[0167] The biomechanical properties of the synthetic hydrogels can be measured using relevant features, such as storage modulus, swelling, and pore size. Storage modulus is an indication of hydrogel’s ability to store deformation energy in an elastic manner, and is a measurement of what is colloquially referred to as “stiffness”. Storage modulus is directly related to the degree of crosslinking, i.e., the higher the number of crosslinking bonds formed between the ECM polymer and the synthetic polymer-binder adducts, the greater the storage modulus. In some embodiments, the hybrid hydrogel has a storage modulus between about 50 Pa and about 2000 Pa, such as between about 50 Pa and about 1800 Pa, between about 50 Pa and about 1500 Pa, between about 50 Pa and 1000 Pa, between about 500 and about 2000 Pa, between about 600 and about 1200 Pa, between about 700 and about 1000 Pa, or between about 600 and about 1050 Pa. The storage modulus can be tuned by the mass ratio of synthetic polymer to ECM polymer, concentration of total polymer, synthetic or ECM polymer molecular weight, and / or type and concentration of binders.

[0168] Swelling is inversely related to the degree of crosslinking, i.e. the more crosslinking bonds present in the hydrogel, the more swelling is restricted and the less the hydrogel swells. Hydrogel swelling is typically measured via changes in hydrogel mass or volume over time in aqueous conditions. Final swollen volume is measured at equilibrium, i.e., the point at which the mass and / or volume of the gel is no longer changing. In some embodiments, the synthetic hydrogel exhibits a volumetric swelling ratio (the ratio of gel volume after swelling at equilibrium to the volume of the hydrogel right after crosslinking) of less than or about 3.00, less than or about 2.00, less than or about 1.20, or less than or about 1.00 when exposed to an aqueous fluid.

[0169] For example, different biomechanical properties (i.e. storage modulus and volumetric swelling factor) was determined with varying concentrations and molecular weights of hyaluronic acid, as described in the examples.

[0170] The biomechanical properties of the hybrid hydrogels can be measured using relevant features, such as storage modulus, swelling, and pore size. Storage modulus is an indication of hydrogel’s ability to store deformation energy in an elastic manner. Storage modulus is directly related to the extent of cross-linking, i.e., the higher the degree of cross-linking, the greater the storage modulus. In some embodiments, the hybrid hydrogel has a storage modulus between about 50 Pa and about 2000 Pa, such as between about 50 Pa and about 1800 Pa, between about 50 Pa and about 1500 Pa, between about 50 Pa and 1000 Pa, between about 500 and about 2000 Pa, between about 600 and about 1200 Pa, between about 700 and about 1000 Pa, or between about 600 and about 1050 Pa. The storage modulus can be tuned by varying the polymer structure, polymer molecular weight, and / or type of binders.

[0171] Swelling is directly related to the degree of cross-linking, i.e. the more cross-linking, the more swelling is restricted. In some embodiments, the hybrid hydrogel exhibits a swelling ratio (the ratio of gel volume after swelling to the volume of the hydrogel right after cross-linking) of less than or about 500%, less than or about 400%, less than or about 300%, less than or about 250%, less than or about 200%, less than or about 190%, or less than or about 180% when exposed to a fluid.

[0172] In some embodiments, the mean pore size of the hybrid hydrogels can be between about 10 nm and about 1 pm, between about 15 nm and about 500 nm, between about 15 nm and about 100 nm, between about 15 nm and about 50 nm, or between about 15 nm and about 35 nm.

[0173] For example, different biomechanical properties (i.e. storage modulus and volumetric swelling factor) was determined with varying concentrations and molecular weights of hyaluronic acid, as described in the examples.

[0174] Advantages of the hybrid hydrogels include high cell viability comparable to other gel types; usefulness with human and mouse stem cells; usefulness with intestinal stem cells from various regions of the same organ; allows incorporating other cell types (intestinal myofibroblast, immune cells, etc.), non-proteolytic synthesis and breakdown; mechanically robust and easy to tailor to a desired tissue organogenesis; highly reproducible composition and inexpensive and broadly accessible.

[0175] III. Methods of Making the Hybrid hydrogels

[0176] Methods for forming the hybrid hydrogels are also provided. The formulation methods are highly tailorable and thus can be applied to various synthetic polymers, binders, and ECM polymers. The reactions are performed under cell-compatible conditions. Cell-compatible reactions refer to reactions which are not significantly toxic to living tissue and / or cells. Exemplary cell-compatible reactions include, but are not limited to reactions via thrombin-activated Factor Xllla under physiological conditions or by another enzymatic addition mechanism known in the art, via Michael addition or other click chemistry reactions, or by another mild chemical addition mechanism known in the art.

[0177] Physiological conditions refer to a temperature range of 20-40°C, atmospheric pressure of 1, pH of 6-8. For example, the reactions can be performed at a temperature of 37 °C, atmospheric pressure of 1, and pH about 7.4. Typically, the hydrogel can be formed in about 60 min, 40 min, 30 min, 20 min, 10 min, 5 min, 2 min, or 1 min.

[0178] A. Polymer-Binder Adduct Preparation

[0179] Typically, the method of forming synthetic hydrogel includes (i) combining a plurality of synthetic polymers and one or more binders. The synthetic polymer contains a first reactive functional moiety. The first reactive functional moiety may locate in the interior position of the polymer or near or at the terminal positions of the arm of the polymer. The number of first reactive functional moiety on a polymer can be varied but must be more than 2. For example, multiple first linking moieties may be included in one polymer. In some embodiments, the first reactive functional moiety of the polymers can be an electrophilic and / or nucleophilic reactive group. The binder contains a second reactive functional moiety. The second reactive functional moiety can be a reactive group that reacts with the first reactive functional moiety to form a polymer-binder adduct. For example, when the synthetic polymers contain an electrophilic group, the second reactive functional moiety can be a nucleophilic group. When the synthetic polymers contain a nucleophilic group, the second reactive functional moiety can be an electrophilic group. Any electrophilic and nucleophilic groups disclosed above can be used. In preferred embodiments, the synthetic polymers can contain vinyl sulfone groups and the binders can contain free thiol groups at the terminus location. Each polymer may have at least one binder incorporated, for example, 1 or 3 binders incorporated. The incorporated binders may be on the same arm of the synthetic polymer or different arms of the synthetic polymer. Preferably, the binders are incorporated to the terminus of the arm of the synthetic polymer.

[0180] In some embodiments, the synthetic hydrogel contains a weight / volume percentage of synthetic polymer(s) between 1% and 10%, between 2% and 25%, or between 2% and 10%, for example, about 2%, about 3%, about 4%, or about 5%. The binders preferably contain adhesion ligand binders and ECM protein binders. The adhesion ligands may include one or more types of integrin-binding peptides, such as peptide derived from Collagen Type I and / or peptides derived from Fibronectin. Each type of adhesion ligand can have a concentration between 0. 1 and 5 mM, between 0.25 and 3 mM, or between 0.5 and 3 mM, for example, about 1.5 mM. In some embodiments, two or more extracellular protein-binding peptides are included in the hybrid hydrogel, for example, a peptide having affinity to fibronectin (or “FN binder”) and a peptide having affinity to collagen and laminin (or “BM binder”). Typically, each extracellular protein-binding peptide has a concentration between 0.1 and 1 mM, 0.1 and 0.5 mM, or 0.25 and 0.5 mM, for example, about 0.25 mM.

[0181] The molar ratio of the adhesion ligand binders to the ECM binder(s) (collective molar concentration if more than one type of ECM binder is present), is typically greater than 1:1, such as between about 1:1 and about 5:1, between about 1.2: 1 and about 5: 1, between about 1.5:1 and about 5:1, such as about 1.5: 1, about 2:1, about 2.5:1, about 3:1, about 3.5: 1, about 4:1, about 4.5: 1, or about 5:1.

[0182] B. Crosslinking

[0183] The method can additionally include (ii) combining the adduct of step (i) and one or more crosslinkers, wherein the crosslinkers have a crosslinking reactive functional moiety. The crosslinker may be chemically-modified ECM polymer, functionalized with multiple (i.e., more than 2) crosslinking reactive functional moieties per molecule. The crosslinking moieties can react with the first linking moieties on the synthetic polymers to perform crosslinking and form hydrogel. For example, when the synthetic polymers contain an electrophilic group, the crosslinking reactive functional moiety can be a nucleophilic group. When the synthetic polymers contain a nucleophilic group, the crosslinking reactive functional moiety can be an electrophilic group. In preferred embodiments, the synthetic polymers contain vinyl sulfone and the crosslinkers are thiol-functionalized HA. In some embodiments, the ratio of crosslinking reactive functional moiety to first reactive functional moiety is between 0.05 and 1, between 0.25 and 1, or between 0.5 and 1 , for example, about 0.15. In preferred embodiments, the ratio of thiol of cross linkers to vinyl sulfone of synthetic polymers is between 0.1 and 1, between 0.25 and 1, or between 0.5 and 1, for example, about 0. 15. The concentration of ECM polymer in % weight / volume can vary between 0.10% and 2.0%. In preferred embodiments using hyaluronic acid (HA) as a crosslinking ECM polymer, the average molecular weight of the HA molecule can vary between 10 kDa and 1500 kDa.

[0184] Crosslinking can be performed either between different synthetic polymers and crosslinkers or between the synthetic polymer / polymer-binder adducts and crosslinkers. Exemplary crosslinking reactions that are cellcompatible include, but are not limited to, reactions via thrombin-activated Factor Xllla under physiological conditions or by another enzymatic crosslinking mechanism known in the art, or via Michael addition, click chemistry, or by another mild chemical crosslinking mechanism known in the art.

[0185] In preferred embodiments, the crosslinking is performed between the synthetic polymer-binder adducts and crosslinkers. To achieve crosslinking between the synthetic polymers and cross-linkers by the preferred Michael addition, one or more synthetic polymers are functionalized with an electrophilic group and / or a nucleophilic group, whereas the crosslinkers carry a crosslinking reactive functional moiety that reacts with the electrophilic group and / or nucleophilic group on the polymers. For example, the synthetic polymers are modified with an electrophilic group, such as a vinyl sulfone, whereas the crosslinkers carry a nucleophilic group, such as a thiol group. In some embodiments, crosslinking is achieved via FXIIIa- mediated crosslinking between the synthetic polymers and crosslinkers. For example, one or more synthetic polymers are functionalized with a lysine- bearing peptide sequence, whereas the crosslinker ECM polymer is functionalized with or includes a glutamine-bearing peptide sequence. In some embodiments, crosslinking is performed via click chemistry. For example, one or more synthetic polymer in the plurality of synthetic polymers is functionalized with a norbomene or thiol group, whereas crosslinkers contain a thiol or norbornene group.

[0186] Crosslinking can be performed either between different synthetic polymers or between the synthetic polymer / polymer-binder adducts and cross-linkers. Exemplary crosslinking reactions that are cell-compatible include, but are not limited to, reactions via thrombin-activated Factor Xllla under physiological conditions or by another enzymatic crosslinking mechanism known in the art, or via Michael addition, click chemistry, or by another mild chemical crosslinking mechanism known in the art.

[0187] In preferred embodiments, the crosslinking is performed between the synthetic polymer-binder adducts and crosslinkers. To achieve crosslinking between the synthetic polymers and cross-linkers by the preferred Michael addition, one or more synthetic polymers are functionalized with an electrophilic group and / or a nucleophilic group, whereas the crosslinkers carry a crosslinking reactive functional moiety that reacts with the electrophilic group and / or nucleophilic group on the polymers. For example, the synthetic polymers are modified with an electrophilic group, such as a vinyl sulfone, whereas the crosslinkers carry a nucleophilic group, such as a thiol group. In some embodiments, crosslinking is achieved via FXIIIa- mediated crosslinking between the synthetic polymers and crosslinkers. For example, one or more synthetic polymers are functionalized with a lysine- bearing peptide sequence, whereas the crosslinkers include a glutamine- bearing peptide sequence. In some embodiments, crosslinking is performed via click chemistry. For example, one or more synthetic polymer in the plurality of synthetic polymers is functionalized with a norbornene or thiolene group, whereas cross-linkers contain a thiolene or norbornene group.

[0188] Alternatively, or additionally, the crosslinking may be performed between synthetic polymers. For example, to achieve crosslinking between different synthetic polymers by the preferred Michael addition, at least one synthetic polymer in the plurality of synthetic polymers is functionalized with an electrophilic group, such as a vinyl sulfone or maleimide group, whereas at least another synthetic polymer in the plurality of synthetic polymers is functionalized with a nucleophilic group, such as a thiol group. In some embodiments, crosslinking is achieved via FXIIIa-mediated crosslinking between different synthetic polymers, at least one synthetic polymer in the plurality of synthetic polymers is functionalized with a lysine- bearing peptide sequence, whereas at least another synthetic polymer in the plurality of synthetic polymers is functionalized with a glutamine-bearing peptide sequence. In some embodiments, crosslinking is performed via click chemistry, e.g., norbomene / thiolene click chemistry. At least one synthetic polymer in the plurality of synthetic polymers is functionalized with a norbomene group, whereas at least another synthetic polymer in the plurality of synthetic polymers is functionalized with a thiolene.

[0189] C. Combining Cells and Inhibitors

[0190] In some embodiments, the method additionally include combining cells, one or more inhibitors of dissociation-induced apoptosis, and the adduct of the first step immediately before the crosslinking reaction with chemically modified ECM polymer in the second step. The incorporation of inhibitors can be important for preventing death of encapsulated cells, especially for human organoid culture. In some embodiments, the inhibitor is at a concentration between 1 nM and 1 mM, between 10 nM and 100 p M, or between 100 nM and 50 pM, for example, about 10 pM. Cross-linking of the hydrogel is preferably performed in the presence of cells to be cultured within the hydrogel. This way the cells or cell aggregates are encapsulated by the formed hydrogel matrix, i.e. residing in a distinct cell culture microenvironment. Alternatively, the cells and inhibitors of dissociation- induced apoptosis can be combined with the product of the second step.

[0191] The cells are typically incorporated at a cell density between about 1 x I O cells / ml and about 1 x 109cells / ml as the final density in the polymer- binder-crosslinker solution (“pre-gel mixture”). Exemplary suitable ranges for the cell density in the final solution include between about 1 x 105cells / ml and about 1 x 108cells / ml, between about 5 x 105cells / ml and about 5 x 108cells / ml, between about 5 x 105cells / ml and about 1 x 108cells / ml, preferably between about 1 x 106cells / ml and about 1 x 108cells / ml, more preferably between about 1 x 106cells / ml and about 1 x 107cells / ml.

[0192] IV. Methods of Using the Hybrid hydrogels

[0193] The hybrid hydrogels are suitable for 3D cell culture, organoid formation, drug discovery, inter-organ interaction characterization, and multi-organ drug responses. The hybrid hydrogels are particularly suitable for supporting culture of primary human cells and interrogating the biological response of cells in culture to hyaluronic acid. These are especially useful for culturing endometrial epithelial cells and organoids thereof, as well as some other types such as primary human adipocytes and neural cells.

[0194] Commercial Applications:

[0195] The most immediate application of this platform is for use in cell and organoid culture in research. The most commonly used hydrogel for 3D culture on the market today is MATRIGEL® (Coming), which is derived from the basement membrane proteins secreted by Engelbreth-Holm-Swarm murine sarcoma cells. Several companies now offer their own version of basement-membrane based protein hydrogel (such as GELTREX® from Thermo Fisher Scientific and ULTIMATRIX from Bio-Techne). However, these materials obtained from different manufacturers but are not interchangeable, and even users of MATRIGEL® alone report significant lotto-lot variability. Since the gel is poorly characterized and a natural product, it is difficult to identify the source of variability. The PEG-peptide-HA gel is well characterized and has exhibited highly consistent performance in the culture of endometrial epithelial organoids across lots of PEG, peptides, and HA and with cells sourced from multiple donors.

[0196] Natural polymer hydrogels, such as collagen gels or polysaccharide (alginate, cellulose, dextran) based gels have been developed as an alternative, but have been used significantly less commonly compared to MATRTGEL®. These gels have a more controlled composition, hut have limited physiological relevance and tend to perform poorly for primary cells in comparison to MATRIGEL®. The hybrid hydrogel described herein is well characterized and has exhibited highly consistent performance in the culture of endometrial epithelial organoids across lots of PEG, peptides, and HA and with cells sourced from multiple donors. Additionally, the hybrid hydrogel uses a combination of synthetic peptides and HA to imitate both protein and glycan molecular features of the extracellular matrix microenvironment.

[0197] Synthetic polymer hydrogels have been developed, but there are few commercially available options at present. A synthetic, PEG-based gel platform described in PCT / US2020 / 044067 “Synthetic Hydrogels for Organogenesis” Massachusetts Institute of Technology, has demonstrated similar success for endometrial epithelial organoid expansion and culture. However, the hybrid hydrogel has demonstrated advantages over this platform. The first, and most important, is that it can be used to interrogate the biological effects of HA in the tissue microenvironment. Second, by crosslinking the gel with a multifunctional thiolated HA as opposed to a bifunctional peptide crosslinker, the molecular architecture allows for less sensitivity to gel stoichiometry. In terms of performance, this translates to less lot-to-lot and user-to-user variability. Third, preliminary data suggests that certain cell types, such as primary adipocytes, exhibit better viability in an environment containing HA. Fourth, hybrid hydrogels exhibit minimal swelling, which makes them especially well-suited for use in microfluidic platforms.

[0198] The crosslinking chemistry of this gel in its preferred embodiment (vinyl sulfone Michael addition) also allows for some degree of control of gelation time through control of the pH of the pre-gel solution. The crosslinking chemistry used to assemble the hydrogel could be adapted for extrusion or injection. Additionally, the protein-free, xeno-free nature of the gel is beneficial for human use. Xenogenic protein products present complications when translating to use in human patients because of potential antigenicity as well as the potential transmission of infectious agents from animals to humans. Hyaluronic acid is a natural product, but is commonly produced via fermentation and is already commonly used as a drug delivery excipient and as tissue fillers for human patients. Furthermore, hyaluronic acid has a simple molecular structure that is conserved throughout the animal kingdom, i.e., pure hyaluronic acid from an animal such as a mouse is chemically indistinguishable from pure hyaluronic acid in a human. For all these reasons, while the primary application is for use in 3D culture systems, this hydrogel has potential applications in drug and cell therapy delivery, tissue engineering, and regenerative medicine.

[0199] While the monofunctional bioactive (cell- adhesive and proteinadhesive) peptide binders are the same as the material described in PCT / US2020 / 044067, the fundamental structure of the gel is quite different and required development from scratch. First, the hybrid hydrogel in its preferred embodiment uses HA, with several thiols per molecule to crosslink the 8-arm PEG molecules together, in place of the enzymatically degradable peptide with one thiol at each end in the material described in PCT / US2020 / 044067. Using two molecules with several reacting groups on each as reactants when forming the hydrogel makes the hydrogel less sensitive to differences in the ratio between the reacting groups. Also, this strategy essentially the reverse of other published HA-based hydrogels. The standard practice is to use a difunctional PEG to crosslink the HA molecules (which are most of the polymer included in the hydrogel by mass) together. Instead, the hybrid hydrogels use a smaller amount of multi-functional HA to crosslink PEG together. This is because the mass ratio between the PEG and the HA is extremely important and requires thorough optimization as the physical interactions of two different polymers in combination in a hydrogel are difficult to predict.

[0200] Through a semi-empirical approach, the biomechanical properties of the hydrogels can be tailored to support the culture of cells and organoids from various organs, or regions of organs. The examples below show the growth of primary human endometrial epithelial cells, primary human adipocytes, and human neurons derived from induced pluripotent stem cells (iPSC) in the hybrid hydrogels. They also demonstrate the tuning of the biomechanical properties of the hydrogels by tuning the mass ratio of synthetic polymer to ECM polymer, concentration of total polymer, and ECM polymer molecular weight.

[0201] The hybrid hydrogels are also useful in drug discovery. The examples show that organoids emerging in the hybrid hydrogels respond to drug stimulation to a similar extent as organoids emerging in MATRIGEL®

[0202] A. Exemplary Method for Cell Encapsulation

[0203] The cells are typically incorporated with the synthetic polymer-binder adducts at the time of addition of the crosslinker ECM polymer. Certain cell types, such as endometrial epithelial cells, are incorporated in the presence of an apoptosis inhibitor. The cells may be at any suitable density (that is, concentration of cells per unit volume of gel) and are not harmed by the subsequent crosslinking reaction. The crosslinking step occurs at physiological conditions, e.g., at about neutral pH, and 37 °C.

[0204] An exemplary method includes the following steps. Hydrogels may be fabricated as gel droplets in the base of a non-tissue culture treated polystyrene 96-well plate using Michael-type reaction chemistry. First, the reaction mixture for a concentrated PEG-binder adduct solution was made. 5% w / v 20kDa, 8-arm poly(ethylene glycol)-vinyl sulfone (PEG-VS), 200 mM HEPES / 2X PBS, and the peptide binders 3.0 mM PHSRN-K-RGD, 3.0 mM GFOGER, 1.0 mM BM-binder, and 1.0 mM FN-binder were combined in a micro tube and vortexed to mix. The mixture was allowed to react at room temperature for 30 min to form the PEG-binder adduct. After 30 min, the concentrated PEG-binder adduct solution was moved to ice until further use.

[0205] Meanwhile, the cells were detached from their culture surface, such as a T75 culture flask, using a dissociation reagent such as trypsin / EDTA. The cells were collected and pelleted via centrifugation, then the supernatant containing trypsin / EDTA was removed. The cells were then resuspended in growth media, such as DMEM / F12 / FBS, and counted. The cell solution is then split into microtube aliquots, with the number of cells per tube determined by the desired final cell density and batch size (i.e., final volume of gel) for the gel samples. An exemplary cell suspension aliquot would contain 3xl05cells to produce a final volume of 30 pl of gel with a final cell density of IxlO7cells / ml of gel. The aliquots of cells were then centrifuged to obtain cell pellets.

[0206] To encapsulate the cells in the gel, the supernatant was carefully and completely removed from one cell pellet. The cells were resuspended in sufficient sterile PBS to achieve the desired final volume of the mixture after adding the PEG-binder adduct solution and the thiolated HA (HA-SH) stock solution. The concentrated PEG-binder adduct solution was then added to the cell suspension so that the PEG-binder adduct solution will be diluted 2-fold in the final pre-gel mixture. It is critical that the PEG-binder adduct solution is diluted in PBS before the addition of the HA-SH stock solution, because if the two are mixed together without dilution the mixture will gel instantaneously, presumably via physical interactions. Pipette the mixture up and down repeatedly to mix. The HA-SH stock solution was added to the mixture with the cells and pipetted up and down repeatedly to form the pregel mixture. The final concentrations of each component in the pre-gel mixture were: 2.5% w / v PEG- VS, 100 mM HEPES / 1X PBS, 1.5 mM PHSRN-K-RGD, 1.5 mM GFOGER, 0.5 mM RM -binder. 0.5 mM FN- binder, and 0.25% w / v HA-SH. Immediately after combining the HA-SH, 3 pl droplets of pre-gel mixture were deposited per well in a 96- well, nontissue culture-treated polystyrene plate. The well plate was moved to a humidified incubator at 37°C and 5% CO2 for 30 min to polymerize.

[0207] After gelation is complete, growth media, such as DMEM / F12 / FBS, may be added to the well along with the gel droplet (100 pL). Cultures are typically maintained in a humidified incubator at 37 °C, 95% air, 5% CO2.

[0208] B. Hydrogel Dissolution

[0209] The hybrid hydrogels with cells or organoids are formed of materials permitting direct assaying of the cells or organoids without the need to remove the hydrogel components. The hybrid hydrogels with cells or organoids may be subject to dissolution to remove a portion, or substantially all of the components of the hydrogel.

[0210] The removal of hydrogel components occurs through hydrogel dissolution. Hydrogel dissolution includes incubating the hybrid hydrogels with cells or organoids in the presence of dissolution agents such as hyaluronidase. Typically, the incubation is for at least about 5 min, about 10 min, about 15 min, about 20 min, about 25 min, about 30 min, about 35 min, about 40 min, about 45 min, about 50 min, about 55 min, or about 60 min. In preferred embodiments, the dissolution is complete within about 10 min, about 15 min, about 20 min, about 25 min, about 30 min, about 35 min, about 40 min, about 45 min. Typically, the incubation occurs at physiological conditions, such as 37 °C, 5% CO2, humidified atmosphere, and includes growth media used to support survival of the cells or organoids.

[0211] 1. Dissolution agents

[0212] Dissolution agents typically include enzymes, such as hydrolases such as hyaluronidase, as well as competitive substrates and acceptor substrates.

[0213] The dissolution agents may be in a solution at a concentration between about 0.01 pM and about 100 pM, such as between about 0.1 pM and about 100 pM, between about 1 pM and about 100 pM, between about 1 pM and about 75 pM, preferably between about 1 pM and about 50 pM, most preferably about 10 pM, about 30 pM, about 50 pM. Competitive substrates and / or acceptor substrates may be used at similar concentrations, and may be added together with the enzyme(s), or after the addition of the enzyme(s).

[0214] V. Kits

[0215] Kits including the components of the hydrogel are also provided. The kits may include every component of the hydrogel in a separate container, or a combination of components in the same container. The kits may also provide the cells to be encapsulated. The kits are typically provided with instructions for preparing the hydrogels and encapsulating cells.

[0216] The components of the hydrogels provided in the kit may be any one, or a combination of, the polymer(s), the adhesion ligand binders, crosslinkers), dissolution agent(s), and cells.

[0217] The present invention will be further understood by reference to the following non-limiting examples.

[0218] Examples

[0219] Example 1: Synthesis of Synthetic PEG-crosslinked pep tide-adhesion peptide ECM supports human-derived endometrial organoid culture (Prior Art PCT / US2020 / 044067)

[0220] The endometrium is a highly dynamic mucosal barrier tissue which sheds about 1 cm of glandular and luminal epithelia along with supporting stroma each month and then regenerates the tissue from remaining pools of stem and progenitor cells. Regeneration and growth is primarily fueled by estradiol, and tissue differentiation processes (i.e. decidualization) commences upon the onset of progesterone after ovulation. A study to include human endometrial organoids was conducted, as they exhibit many similarities to intestinal organoids when cultured in MATRIGEL®: a single layer of epithelium surrounding a lumen; strongly proliferative in a culture medium favoring stem cells; responsive to differentiation cues; robust to serial passage for 5+ months without changes in DNA; and robust recovery after a freeze / thaw cycle.

[0221] Materials and Methods

[0222] Multi-arm polyethylene glycol (PEG) polymers of various molecular weights, small peptides, and inhibitors were identified as the components formulating the hybrid hydrogels. Specifically, 20 kDa 8-arm PEG, 40 kDa 8-arm PEG, or a combination of both were identified for forming PEG hydrogels. The PEG arms were functionalized with vinyl sulfone (VS) groups.

[0223] Integrin binding peptides derived from collagen type I (containing the sequence GFOGER (SEQ ID NO:1)) and fibronectin (containing the sequence PHSRN (SEQ ID NO:2)) were selected for cell adhesion. The peptide derived from collagen type I has high affinity for integrin a | ] , ai Pi , aio0i, and an Pi. The peptide derived from fibronectin has high affinity for integrin avp3 and moderate affinity for integrin asPi. The integrin binding peptides were synthesized to contain a free thiol group at the N-terminus. The collagen type I- and fibronectin- derived adhesion peptides can be used individually or in combination in the hydrogel formulation.

[0224] ECM binding peptides having affinity for secreted collagen and laminin BM binders ") (NH2-GCRE- ISAFLGIPFAEPPMGPRRFLPPEPKKP(Am)) (SEQ ID NO:5), and fibronectin (“FN binders”) (NH2-GCRE-TLQPVYEYMVGV-COOH) (SEQ ID NO:6)), were selected. These extracellular protein binding peptides were designed to capture fibronectin produced by endometrial stromal fibroblasts throughout the cycle and both laminin and type IV collagen basement membrane proteins that are normally produced by the epithelium during all menstrual cycle stages and locally produced and accumulated in the pericellular region of hormonally responsive fibroblasts in the stroma (Cook, et al. , Integrative Biology, 9:271-289 (2017)). The extracellular protein binding peptides were synthesized to contain a free thiol group at the N- terminus.

[0225] 57

[0226] SUBSTITUTE SHEET (RULE 26) Peptides that are cleavable by a matrix-metalloproteinase (MMP) (Ac)GCRD-GPQGIAGQ-DRCG(Am) (SEQ ID NO: 10) or Sortase A transpeptidase (Sortase) are identified as crosslinkers to form the final crosslinked hydrogels.

[0227] The PEG polymer arms were functionalized with vinyl sulfone groups (purchased from JenKem Technology, Beijing). The integrin-binding peptides and extracellular protein binding peptides were synthesized to contain a free thiol group at the N-terminus. The crosslinkers are dithiols with one thiol group at each terminus. The PEG hydrogels (3.5-10%, weight / volume) were assembled via sequential Michael-type addition reactions between the vinyl sulfone groups on the PEG polymer arms and the terminal thiol groups of the integrin binding peptides (0.25-3 mM) and extracellular protein binding peptides (0.25-0.5 mM) (FIG. 1). Cells and ROCK inhibitors were then mixed with the peptide-PEG polymer adducts. The peptide-PEG polymer adducts were crosslinked with crosslinkers (thiol of cross linkerVS, 0.35-1), such as dithiol-MMP cleavable peptides. The PEG hydrogels are fully assembled after 30 minutes at pH 7.4 at 37 C. The Michael type addition reaction occurs at physiological conditions thus cells are not under stress during cell-encapsulation). Endometrial organoids were established using published protocols and cultured in endometrial organoid medium (EnOM) (Boretto et al., Nat. Cell Biol. 21:1041-1051 (2019), Turco et al., Nat. Cell Biol. 19:568-577 (2017)).

[0228] PEG macromers and peptides. 8-arm PEG macromers with vinyl sulfone (VS) terminal groups (40,000 MwPEG-VS, “PEG-40” and 20,000 MwPEG-VS, “PEG-20”) were obtained from lenKem Technology (Beijing). All peptides were custom synthesized and purified by Boston Open Labs (Cambridge, MA), GenScript (Piscataway, NI), or CPC Scientific (Sunnyvale, CA). Peptides sequences and their relevant features are included in the Table 3. XL-MMP \ a crosslinker containing a matrix metalloproteinase (MMP)-sensitive substrate, “XL-MMP-SrtA” a crosslinker containing a MMP-sensitive substrate and a Sortase-sensitive recognition site (LPRTG), “XL-SrtA,'’ a crosslinker containing only the Sortase-sensitive site, RGDF a fibronectin (FN)-derived peptide containing the canonical RGD motif from the 10th FN type III domain, PHSRN-K-RGD ” a FN- derived peptide containing the RGD motif and the PHSRN (SEQ ID NO:2) synergy site from the 9th FN type III repeat in a branched configuration;

[0229] GFOGERN a collagen I-derived peptide presented as a triple helix (Wojtowicz et al., Biomaterials, 31:2574-2582 (2010), Carafoli et al., PLoS ONE, 7(7):e42473 (2012)), “GFOGDR,” a collagen I-derived peptide with E- D point mutation that reduces integrin binding (Knight et al., J. Biol. Chem. 275:35-40 (2000)) , “G11RGD ” an extended ligand of RGD with eleven Gly spacers, “CMPRGD. ' an extended and clustered RGD-containing ligand using a triple helical collagen mimetic peptide (Hernandez- Gordillo et al., Biomaterials, 35:7363-7373 (2014)), “FN-binder,” a peptide with affinity for FN, and BM-binderN a peptide with affinity for collagen IV (C-IV) and laminin (LMN) (Johnson et al., Biochem. Biophys. Res. Commim., 319:448-455 (2004)). All peptides were reconstituted in acidic (pH 5.2) Milli-Q water (Millipore). The concentration of free thiols in all peptides was determined using Ellman’s reagent (Sigma). Other peptides were as described in Zustiak and Leach, Biomacromolecules . 11:1348-1357 (2010); Kassis et al., Sei. Rep. 9: 12479 (2019); and Schindelin et al, Nat. Methods. 9:676-682 (2012).

[0230] Table 3. Peptide sequences and relevant features.

[0231] Synthesis of hydrogel precursors. All synthetic matrices were assembled at 3 or 5 % PEG (w / v) using 8-arm PEG-40 or 8-arm PEG-20. The mixed PEG-40 / 20 synthetic matrix was made mixing 1:1 (v / v, 2.5% 8- arm PEG-40 and 2.5% 8-arm PEG-20). All matrices were assembled stepwise via Michael-type addition reaction. First, the PEG- VS macromer (either PEG-40, PEG-20, or the PEG-40 / 20 mix) was diluted with 10X PBS- HEPES solution (1 mM, pH 7.8); second, the matrix-binder peptides were added to the PEG reaction mixture and incubated for 30 minutes at RT; third, the integrin binder peptides (RGD, PHSRN-K-RGD, GFOGER, G11RGD, CMPRGD or GFOGDR) were added to the reaction mixture and allowed to react for an additional 30 min at RT. This sequential reaction created a PEG- functionalized mixture “fPEG-VS” that was used to resuspend the cells prior to matrix gelation (see cell encapsulation below). The nominal concentration of the matrix-binders in all matrices was 0.25 mM each, whereas the integrin binder peptides were at 1.5 mM (unless otherwise noted in the figure legends). For most experiments the fPEG-VS solution was crosslinked at 50% crosslinking density (unless otherwise noted).

[0232] Cell culture. Organoids were passaged every four days for human intestinal organoids, eight days for human endometrial organoids and three days for mouse organoids. For passaging, organoids were incubated in Cell Recovery Solution (CRS, Gibco) for Ih at 4°C, followed by trypsin treatment to generate single cells. The cell suspension was inspected under the microscope to ensure the presence of dispersed single cells and if needed, filtered through a 30 pm cell strainer to remove cell clumps. Single cells were counted using a hemocytometer and seeded in GFR MATRIGEL® (25 pL droplet) at a density of 1,000 cells / pL for human and 500 cells / pL for mouse organoids. After MATRIGEL® polymerization, 600 pL of EM or EnOM was layered on top. Media was changed every two days.

[0233] Organoid differentiation. Organoids in both MATRIGEL® and the synthetic matrix were cultured in EM for six days (human) or four days (mouse) before switching to differentiation medium. Human and mouse organoids in MATRIGEL® were differentiated as reported by Kasendra et al., Sci. Rep. 8:2871 (2018), Miyoshi et al., Nat. Protoc. 8:2471-82 (2013). Intestinal organoids in the synthetic matrix were differentiated using L-WRN conditioned medium (25% L-WRN) diluted in Adv DMEM / F12, HEPES (1 pM, Gibco), Pen / Strep (100 U / mL, 100 mg / mL, Invitrogen), N2 supplement (IX, Gibco), B27 supplement (IX, Gibco), human [Leul5]-gastrin I (10 nM, BACHEM), N-acetyl cysteine (1 mM, SIGMA) and Y-27632 (5 pM, TOCRIS). Mouse organoids in the synthetic ECM were differentiated as in MATRIGEL®.

[0234] Organoids were established derived from endometrial biopsies obtained from the stratum functionalis of human donors. In comparison to intestine, the endometrial stem cell compartment is relatively poorly defined, and the tissue exhibits dramatic steroid hormone responses. One study using in situ hybridization for LGR-5 RNA showed that the endometrium potentially has stem / progenitor pools in the functionalis region of the endometrium, from which the donor tissue was harvested, throughout the menstrual cycle. The geographical location of putative stem / progenitor cell compartments aids selection of synthetic ECM components in light of reported integrin expression profiles in the endometrium. Integrin alpha 2 beta- 1 is expressed throughout all phases of the menstrual cycle and is strongly expressed in luminal and glandular epithelia, thus motivating the inclusion of the GFOGER peptide. The RGD-binding vitronectin receptor, integrin alpha-beta3, has an unusual cyclical expression profile. In healthy women, it is expressed in mid-secretory phase throughout implantation and may play a role in embryo implantation. Its expression in the endometrial organoids would not be expected due to control over sex hormone concentrations in the in vitro model. While it is well-established that the endometrial stromal cells express the canonical fibronectin-binding receptor, integrin alpha-5-betal, its expression in the epithelia is controversial.

[0235] Integrin P6, which functions as a LMN receptor when paired with al or a4, is expressed in basolateral fashion throughout the endometrium in proliferative and secretory phases. A screen of ECMs containing PEG-20- GFOGER, PEG-20-PHSRN-K-RGD, PEG-20-RGD, PEG-20-G11RGD, and PEG-20-CMPRGD yielded a formulation suitable for culture of endometrial organoids.

[0236] Single cells obtained from dissociation of endometrial organoids were embedded in the synthetic ECMs, where they proliferated and formed lumenized cystic organoids with thick columnar cells. Among the formulations tested, the best synthetic ECM formulation for supporting robust growth of polarized endometrial organoids was identical to the best synthetic ECM for enteroids). Similar to intestinal enteroids, endometrial organoids in MATRIGEL® appeared larger and more abundant than organoids in the synthetic matrices eight days after encapsulation. In the PEG-20-GFOGER. the organoid median diameter was 85 [im, compared to 58 pm for the PEG-20-PHSRN-K-RGD. In MATRIGEL® the median diameter was 91 pm. Approximately 38% of all endometrial organoids counted in the PEG-20-GFOGER matrix reached sizes above 90 microns, compared to 21% in the PEG-20-PHSRN-K-RGD. Both synthetic ECMs, however, were lower than MATRIGEL® in which 46% of the organoid population reached diameters above 90 microns. Finally, endometrial organoids, in the PEG-20-GFOGER matrix, showed proliferative cells (EdU), epithelial EpCAM, apical actin, and basal LMN deposition similar to those grown in MATRIGEL®. Immunostaining analysis of organoids in the PEG-2Q-GFOGER matrix made at 1.5 mM (0.5 mM GFOGERth), 0.25 mM BM -binder. 0.25 FN-binder and 50% XL-MMP-SrtA crosslinker, showed proliferative cells (EdU), apical markers (actin), epithelial marker (EpCAM), and basolateral marker (LMN). Collectively, these data showed that a defined intestinal formulation of a hybrid hydrogel can be applicable to additional epithelial-rich tissues.

[0237] Hydrogel samples were prepared for cell culture and characterization using the following procedure: first, 20kDa, 8-arm end-functionalized PEG- vinyl sulfone (PEG-VS) was combined with a mixture of integrin-adhesive and secreted protein-sequestering pendant (monofunctional) peptides in a buffered solution at a pH of -8-8.4 for 30 min at room temperature. This mixture was prepared with approximately 4-fold excess of vinyl sulfone groups relative to pendant peptide thiol groups, providing free alkenes for later hydrogel crosslinking. The PEG-peptide was then diluted in neutral buffer, and that mixture was used to resuspend human endometrial epithelial cells (EECs). Human endometrial epithelial cells were cultured over the course of 7-14 days and the emergence of EEOs as well as their morphology and polarization were assessed via brightfield and confocal microscopy, A fully synthetic matrix is described that supports primary, human tissue-derived, enteroids from the upper and lower intestinal track and primary, human tissue-derived, endometrial epithelial organoids. The local biophysical presentation of an a2pi integrin-binding peptide (GFOGER) in the presence of peptides that sequester cell-produce ECM appear as the dominant variables governing epithelial cell proliferation and organoid formation in PEG-based synthetic ECMs. While the synthetic matrix does not match MATRIGEL® benchmarks by some metrics (rate of human organoid emergence and overall proliferation over 6-8 days), it is superior to MATRIGEL® by other metrics, in applications that require (i) a rigorously defined microenvironment to interrogate basic human stem cell biology; (ii) long-term growth of human organoids beyond 6 days; and (iii) in vivo human applications where MATRIGEL® would violate GMP protocols.

[0238] The synthetic ECM offers additional benefits such as on-demand dissolution to recover intact organoids and other co-cultured cells for in depth omics analysis and downstream cell-secreted metabolites quantification. This feature is of great utility to uncover complex and dynamic cell-cell communications in emerging stromal-epithelial co-culture systems.

[0239] Primary human endometrial epithelial organoids were maintained in culture in this material up to 2 weeks. The cells exhibit strong and clear polarization, which suggests that their epithelial phenotype is preserved. Example 2: Characterization of Gel Mechanical Properties and Comparison for Use in Endometrial Epithelial Organoid Culture

[0240] Synthetic polymer hydrogels are a fundamental tool in tissue engineering, offering increased user tunability and reproducibility over natural polymer hydrogels. However, the development of a synthetic hydrogel which can support the co-culture of multiple different primary human cell types remains challenging. Hyaluronic acid (HA) gels are attractive as chemically modifiable and endogenously bioactive platforms. Additionally, HA is a component of the human endometrial extracellular matrix, and its metabolism by endometrial cells is altered in the differentiation process known as decidualization in vitro. Here, a hybrid hydrogel was developed using a combination of bioactive peptide- functionalized multi-arm poly(ethylene glycol) (PEG) and chemically modified HA. The fitness of this gel as a 3D culture substrate for primary human endometrial epithelial organoids (EEOs) was evaluated.

[0241] Materials and Methods:

[0242] Materials. 8-arm, 20kDa PEG macromers (“PEG- VS”) synthesized with a tripentaerythritol core and functionalized with vinyl sulfone terminal groups (molar degree of substitution < 90%) were obtained from JenKem Technology (Beijing) and used as described without further modification. Thiol-functionalized (“thiolated”) hyaluronic acid (HA-SH) with a molecular weight of approximately 300 kDa and molar degree of substitution of approximately 20-30% under the product name “GLYCOSIL” was obtained from Advanced Biomatrix and was used as described without further modification. HA-SH was thiolated at the carboxylic acid residues of the D- glucuronic acid portion of the repeating units (“monomers”) of the polymer, consisting of one D-glucuronic acid group and one N-acetyl-D-glucosamine group. The reported molar degree of substitution is the average fraction of monomers which have been functionalized with a thiol group at that carboxylic acid residue out of the total number of monomers in the batch of polymer.

[0243] HA-SH was reconstituted at 1% w / v in sterile phosphate buffered saline (PBS), pH 7.4 and stored in aliquots at -20°C until use, without repeated freeze-thaw cycles. When stored at -20°C, the maximum shelf life of the frozen stock solutions was found to be approximately 2 months. A concentrated buffer solution of 10X Dulbecco’s phosphate buffered saline and 1 M 4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid (HEPES) was prepared by dissolving one packet of powdered Dulbecco’s PBS (without Ca2+ / Mg2+, sufficient for IL of buffer at IX) (Gibco, REF# 21600-051) in 100 mL IM 4-(2 -hydroxyethyl)-! -piperazineethanesulfonic acid (HEPES) (Gibco, REF# 15630-080), pH 7.8-8.2. The pH of the concentrated HEPES / PBS solution (“HEPES / PBS”) was adjusted to 8.0-8.2 using 2 N sodium hydroxide in MilliQ distilled water, then sterile-filtered and stored in 1 mL aliquots at 4C, covered to protect from light. SIGMACOTE® (a chlorinated organopolysiloxane, Sigma- Aldrich) was purchased and used according to the manufacturer’s specifications to treat glass surfaces to make them more hydrophobic.

[0244] Peptides: All peptides were custom synthesized and purified by GenScript (Piscataway, NJ) or CPC Scientific (Sunnyvale, CA). Peptides sequences, their function in the gel, and the abbreviations by which they are referenced are included in Table 4. Importantly, the inclusion of cysteine residues in the peptide sequences enabled addition of the molecule to the gel via the reactivity of the thiol group on the cysteine side chain with the alkene vinyl sulfone on the PEG macromers.

[0245] For the purposes of gel assembly, the thiol group on the peptide is the reactive functional group. Peptides with one thiol group (i.e., one cysteine residue) are referred to as “monofunctional” or “pendant” peptides, and peptides with two thiol groups (i.e., two cysteine residues) are referred to as “bifunctional” or “crosslinking” peptides. Note that all peptides were modified post-synthesis by the manufacturer with N-terminal acetylation (“Ac”) and C-terminal amidation (“Am”). In the peptide sequences non- canonical amino acid hydroxyproline is represented by the letter O. Peptides used include:

[0246] “LW,” a peptide with one cysteine residue at each end to facilitate crosslinking of PEG-VS macromers, containing a MMP-labile domain GPQGIWGQ (SEQ ID NO:43) and a Sortase-labile domain LPRTG (SEQ ID NO:27) / “PHSRN-K-RGD,” a fibronectin-derived peptide containing the RGD motif and the PHSRN (SEQ ID NO:2) synergy site from the 9th fibronectin type III repeat in a branched configuration; ' GFOGER P a collagen I-derived peptide presented as a triple helix (Wojtowicz et al., Biomaterials, 31:2574-2582 (2010), Carafoli et al., PLoS

[0247] “FN-binder,” a peptide with non-covalent binding affinity for fibronectin; and

[0248] “BM-binder,” a peptide with non-covalent binding affinity for collagen IV and laminin (Johnson et al., Biochem. Biophys. Res. Commun., 319:448-455 (2004)).

[0249] All peptides were reconstituted in ultrapure Water (Invitrogen), with pH adjusted using 2 N sodium hydroxide solution in MilliQ distilled water to facilitate solubility when needed.

[0250] The concentration of peptide in the reconstituted stock solution were evaluated using 205 nm absorbance, with the exception of the GFOGER peptide. Due to light scattering of the triple-helical trimer in solution, the 205 nm absorbance of the GFOGER peptide was artificially elevated, so the concentration of the stock solution was measured by proxy using the concentration of free thiols, determined using Ellman’s reagent (Sigma).

[0251] Other peptides are as described in Zustiak and Leach, Biomacromolecules. 11: 1348-1357 (2010); Kassis et al., Sci. Rep. 9:12479 (2019); and Schindelin et al, Nat. Methods. 9:676-682 (2012).

[0252]

[0253] Preparation of Gel Samples for Characterization. Acellular gel samples were prepared to identify HA-crosslinked (“PEG-HA”) and MMP- degradable peptide-crosslinked (“PEG-peptide”) formulations with comparable mechanical properties. Notably, both PEG-peptide and PEG-HA gel formulations used identical concentrations of peptides PHSRN-K-RGD, GFOGER, BM-binder, and FN-binder. Two gel formulations, one “soft” and one “stiff’ were selected and used for characterization and subsequent cell culture experiments. The detailed composition of these two PEG-peptide formulations are included in Table 5. PEG-peptide gel samples with an initial (pre-polymerization and pre-swelling) volume of 50 L each were prepared in the manner reported by Hernandez-Gordillo et al. https: / / doi.org / 10. 1016 / j.biomaterials.2020.120125 with modification. Briefly, PEG-VS was diluted from a 10% w / v stock solution with

[0254] HEPES / PBS and ultrapure Water (Invitrogen) in a microtube and vortexed to mix. Then, stock solutions of pendant peptides PHSRN-K-RGD, GFOGER, BM-binder, and FN -binder were added to the PEG-VS / buffer mixture and vortexed, then allowed to sit at room temperature for 30 min to allow the PEG-VS macromers to become functionalized with the pendant peptides via thiol-ene Michael addition reaction to form a PEG-peptide macromer. Then, the LW peptide was added to the PEG-peptide macromer solution to the final concentration, the solution was mixed via vortex for 10-15 s, and 50 pL of the pre-gel solution was immediately added to an 8mm diameter, 0.5 mm thick round mold made from silicone sheet (McMaster Carr) pressed onto a SIGMACOTE®-treated glass microscopy slide. The pre-gel solution in the molds was allowed to polymerize into a gel for 30 min in a humidified chamber at 37°C and 5% CO2. After polymerization, the gel samples were removed from the molds and placed in PBS, pH 7.4, to swell.

[0255] Table 5. Detailed concentrations of each component in PEG-peptide gel formulations

[0256] Three PEG-HA gel formulations were used for characterization with varying concentrations of HA-SH, the details of which are included in Table 5. For subsequent cell culture experiments, the 0.25% w / v HA-SH formulation was selected as the “soft” PEG-HA formulation and the 0.38% w / v HA-SH formulation was selected as the “stiff’ PEG-HA formulation. Preparation of PEG-HA gel samples is summarized in FIG. 1, and was performed as follows: First, HA-SH stock solution was thawed on a heat block at 37°C for at least 1 h but no more than 2 h before addition to the pregel solution. Meanwhile, PEG-VS in a 20% w / v stock solution in ultrapure water was added to a microtube with HEPES / PBS, the pendant peptide stock solutions, and ultrapure water added to a volume sufficient to dilute all components to double the concentration in the final gel (Table 6). As the concentration of PEG-VS, HEPES / PBS, and the monofunctional pendant peptides are the same in all three PEG-HA formulations, the solution consisted of 5% w / v PEG-VS, 200 mM HEPES / 2X PBS, 3.0 mM PHSRN- K-RGD, 3.0 mM GFOGER, 1.0 mM BM -binder. and 1.0 mM FN -binder. This mixture was vortexed to mix and allowed to react at room temperature for 30 min to form the pendant peptide-functionalized PEG-peptide macromer. After this, the HA-SH stock solution was diluted in PBS to double the final concentration in the gel (e.g., 0.5% w / v for the formulation with a final concentration of 0.25% w / v in the gel) and was vortexed 20-30 s to mix.

[0257] The PEG-peptide macromer solution in HEPES / PBS was then added 1: 1 by volume to the diluted HA-SH solution to form the pre-gel solution. The pre-gel solution was immediately vortexed 10-15 s to mix and 50 uL was added to round molds identical to those used for preparing the PEG- peptide gel samples. The PEG-HA gel samples in molds were allowed to polymerized for 30 min in a humidified chamber at 37C and 5% CO2. The gels were then removed from the molds and placed in PBS to swell.

[0258] Table 6. Detailed concentrations of each component in PEG-HA gel formulations

[0259] Volumetric Swelling Characterization. Post-polymerization, gel samples were allowed to swell to equilibrium (no net change in sample mass / volume) in divalent cation-free Phosphate Buffered Saline (PBS), pH 7.4 for 18-24 h at 37°C and 5% CO2, to approximate cell culture conditions. To assess volumetric swelling, the samples were removed from the

[0260] PBS, excess moisture on the surface of the gel was removed, and the sample was moved to a glass microscopy slide surface-treated with SIGMACOTE® to prevent sticking and damage to the gel sample. The diameter and height in mm of the swollen gel sample was measured and used to calculate the volume in pL of the swollen gel at equilibrium (i.e., maximally swollen). To calculate the volumetric swelling factor (VSF), the volume (uL) of the equilibrium-swollen gel sample was divided by the initial volume of the sample (50 uL).

[0261] Characterization of Gel Mechanical Properties. The mechanical properties of the gel were assessed via shear oscillatory rheology using an Anton Paar MCR 302 rheometer with an 8 mm parallel plate measuring geometry and a Peltier plate with an air-controlled temperature regulation system. Equilibrium-swollen gel samples were placed on the Peltier plate sample stage, pre-warmed to 37°C, and the measuring geometry was lowered to 70-80% of the height of the equilibrium swollen sample. This compression of the sample prevented sample slippage during measurement. The sample was subject to 180 s time sweep at a frequency of 1 Hz and strain of 5% at 37°C. The storage and loss moduli were averaged over the time sweep for each sample. Those values for each sample were averaged to yield the average for the formulation (n = 3), reported with the standard deviation. Frequency and amplitude sweeps were performed to verify that the testing parameters were within the linear viscoelastic regime (not reported).

[0262] Organoid culture. Endometrial epithelial cells (EEC) were isolated from patient biopsy samples and as described by Gnecco et al. Med. 2023 Aug ll;4(8):554-579.e9. doi: 10.1016 / j.medj.2023.07.004. PMID: 375726 1. After isolation, EECs were expanded as organoids in growth factor-reduced MATRIGEL® using methods detailed by Gnecco et al.

[0263] Organoid expansion in PEG-peptide and PEG-HA gels. After organoids were visible and had attained a maximum diameter of approximately 250 pm, EEGs were extracted from MATRIGEL® and dissociated into a single-cell suspension using cold (4°C) Cell Recovery Solution (CRS, Gibco) for approximately 30 min. That cell suspension was centrifuged at 350 ref for 5 min at 4°C to pellet the cells, then the supernatant was aspirated and the cells were further treated with TrypLE Express supplemented with DNAse to dissociate any remaining clusters of cells completely into a homogenous single-cell suspension.

[0264] The EECs in suspension were counted, then divided into aliquots of -500 cells / uL of gel in microtubes and centrifuged to pellet the cells. The cell pellets were kept on ice until use.

[0265] To encapsulate the EECs in soft and stiff PEG-peptide hydrogels for encapsulation into EEOs, the same general method was used for the two formulations, detailed in Table 6. First, PEG-VS, HEPES / PBS, PHSRN-K- RGD, GFOGER, BM-binder, and FN-binder stock solutions were added to a microtube and vortexed to mix. The mixture was allowed to react at room temperature for 30 min to form the PEG-peptide macromer. After 30 min, the PEG-peptide macromer solution was moved to ice until further use.

[0266] The supernatant was then carefully and completely removed from one EEC pellet. The cells were resuspended in the PEG-peptide macromer solution. The LW stock solution was added to the mixture and pipetted up and down repeatedly to mix and form the pre-gel mixture ("pre-gel mixture") referring to a mixture that includes all of the components necessary to form the final hydrogel sample before the components have polymerized / reacted to form the gel. “Mixture” includes cells and / or components that are not soluble in water. “Pre-gel solution” contains all the components necessary to form the final hydrogel but has not polymerized to form the gel, and all components are completely dissolved in water.) 3 LI L droplets of pre-gel mixture were deposited per well in a 96- well, non-tissue culture-treated polystyrene plate. The well plate was moved to a humidified incubator at 37°C and 5% CO2 for 30 min to polymerize. 100 pL of EEO expansion media was then added to each gel sample and then the well plate was returned to the incubator.

[0267] To encapsulate the EECs in soft and stiff PEG-HA hydrogels for encapsulation into EEOs, the same general method was used for the two formulations. The “soft” PEG-HA gel formulation is listed in Table 6 as formulation 1: 2.5% w / v PEG- VS, 100 mM HEPES / 1X PBS, 1.5 mM PHSRN-K-RGD. 1.5 mM GFOGER, 0.5 mM BM-binder, 0.5 mM FN- binder, and 0.25% w / v HA-SH. The “stiff’ PEG-HA gel formulation is listed in Table 6 as formulation 2: 2.5% w / v PEG- VS, 100 mM HEPES / 1X PBS, 1.5 mM PHSRN-K-RGD, 1.5 mM GFOGER, 0.5 mM BM-binder, 0.5 mM FN-binder, and 0.38% w / v HA-SH. First, the reaction mixture for a concentrated peptide-functionalized PEG macromer was made. 5% w / v PEG- VS, 200 mM HEPES / 2X PBS, 3.0 mM PHSRN-K-RGD, 3.0 mM GFOGER, 1.0 mM BM-binder, and 1.0 mM FN-binder were combined in a microtube and vortexed to mix. The mixture was allowed to react at room temperature for 30 min to form the PEG-peptide macromer. After 30 min, the concentrated PEG-peptide macromer solution was moved to ice until further use. The supernatant was then carefully and completely removed from one EEC pellet. The cells were resuspended in sufficient sterile PBS to achieve the desired final volume of the mixture after adding the PEG-peptide macromer solution and the HA-SH stock solution. The concentrated PEG- peptide macromer solution was then added to the cell suspension so that the PEG-peptide macromer solution will be diluted 2-fold in the final pre-gel mixture. Pipette the mixture up and down repeatedly to mix. The HA-SH stock solution was added to the mixture with the cells and pipetted up and down repeatedly to form the pre-gel mixture. Immediately after, 3 uL droplets of pre-gel mixture were deposited per well in a 96-well, non-tissue culture-treated polystyrene plate. The well plate was moved to a humidified incubator at 37°C and 5% CO2 for 30 min to polymerize. 100 pL of EEO expansion media was then added to each gel sample and then the well plate was returned to the incubator.

[0268] All samples were kept in culture for 14 days, with media changes every 3 days. Images of the samples were taken every day via brightfield microscopy to monitor EEO expansion. EEO staining for polarization. After 14 days, gel samples were fixed in 4% paraformaldehyde and stained with DAPI and rhodamine phalloidin to identify cell nuclei and f-actin, respectively.

[0269] Results

[0270] FIG. 2A is a graph of the gel shear storage modulus, G' (Pa), and shear loss modulus, G” (Pa) of acellular gel samples, showing that both G’ and G” increase with increasing concentrations of HA-SH in the gel, from 0.25% w / v, 0.38% w / v, to 0.5% w / v. FIG. 2B-2E are micrographs of the cells in culture, showing morphology as a function of HA concentration. Though both G’ and G” increase with increasing HA-SH content, the ratio of G’7G’, also expressed as the loss factor, tan(5), remains relatively constant between the formulations, indicating a similar degree of viscoelasticity between the formulations.

[0271] FIG. 3A shows the stiffness in shear storage modulus, G’ (Pa), of the soft PEG-peptide (Table 5, “Soft”) and PEG-HA (Table 6, “Formulation 1”, 0.25%w / v HA-SH) gel formulations and stiff PEG-peptide (Table 5, “Stiff”) and PEG-HA (Table 6, “Formulation 2”, 0.38%w / v HA-SH) gel formulations selected for comparison. Both peptide-crosslinked and HA- crosslinked PEG gels supported the expansion of human EEOs over the course of 14 days in both soft (-200 Pa G’) and stiff (-1000 Pa G’) formulations. FIG. 3B-E are representative fluorescent micrographs of the EEOs expanded for 14 days in culture and stained for f-actin (green) and cell nuclei (blue) in each of the four formulations: Soft PEG-peptide (FIG. 3B), Soft PEG-HA (FIG. 3C), Stiff PEG-peptide (FIG. 3D), and Stiff PEG-HA (FIG. 3E).

[0272] Remarkably, the soft and stiff PEG-peptide-HA conditions both displayed similar spherical and oblong organoid morphology, with luminal localization of f-actin staining indicative of characteristic epithelial polarization. In contrast, EEOs cultured in stiff (-1000 Pa G’) PEG-peptide gels without HA exhibited a disorganized morphology. Analysis of f-actin polarization images at day 14 showed that the F- actin was polarized at the apical side in both the soft and stiff PEG-peptide gels as well as the soft and stiff PEG-HA gels.

[0273] However, PEG-peptide gels and PEG-HA exhibited differences in adhesion ligand distribution. PEG-peptide hydrogels show a homogenous distribution of FL-PHSRN-K-RGD PEG-peptide + HA hydrogels show a micron-scale heterogeneity of F -PHSRN-K-RGD distribution.

[0274] Cells were treated over a period of nine days for the effect on morphology of hormones. The results showed that gel selection (PEG- peptide or PEG-HA) had more influence on cell morphology than hormone. ESCs exhibit greater spreading in PEG-peptide hydrogels after nine days in culture, as compared to ESCs in PEG-HA. Analysis of f-actin polarization images at day 14 showed that the F-actin was polarized at the apical side in both the soft and stiff PEG-peptide gels as well as the soft and stiff PEG-HA gels. This is characteristic of endometrial epithelial cells, which exhibit polarization not only in their eutopic site in the endometrium, but also in ectopic lesions in endometriosis and adenomyosis.

[0275] Materials. 20 kDa, 8-arm poly(ethylene glycol)-vinyl sulfone (PEG- VS), HEPES and PBS buffers components, and synthetic peptides were obtained as described in EXAMPLE 2. Thiolated 10 kDa (“HA-10”, 30% molar degree of substitution), 50 kDa (“HA-50”, 10% molar degree of substitution), and 1500 kDa (“HA- 1500”, 5% molar degree of substitution) HA was manufactured by and purchased from Creative PEGWorks. Stock solutions of HEPES / PBS buffer, PEG-VS, and peptide components were prepared as detailed in EXAMPLE 2. SIGMACOTE® (a chlorinated organopolysiloxane, Sigma-Aldrich) was purchased and used according to the manufacturer’s specifications to treat glass surfaces to make them more hydrophobic.

[0276] HA-SH was reconstituted at 2% w / v for HA-10 and HA- 1500 and at 2.5% w / v for HA-50 in sterile phosphate buffered saline (PBS), pH 7.4 and stored in aliquots at -20°C until use, without repeated freeze-thaw cycles. When stored at -20°C, the maximum shelf life of the frozen stock solutions was found to be approximately 2 months.

[0277] Preparation of Gel Samples for Characterization. Acellular gel samples were prepared to evaluate the mechanical properties and swelling of formulations with varied molecular weights and concentrations of HA. The detailed composition of formulations tested is given in Table 5 and 6. Hydrogel samples with an initial (pre-polymerization and preswelling) volume of 50 pL each were prepared in the manner reported in EXAMPLE 2 above. First, a 2X concentrated solution of PEG-binder adduct was prepared. PEG-VS was diluted from a 20% w / v stock solution to 5% w / v with 200 mM HEPES / 2X PBS and ultrapure Water (Invitrogen) in a microtube and vortexed to mix. Then, stock solutions of peptide binders were added to the following concentrations: 3.0 mM PHSRN-K-RGD, 3.0 mM GFOGER, 1.0 BM-binder, and 1.0 FN-binder. The mixture was allowed to sit at room temperature for 30 min to allow the PEG-VS macromers to become functionalized with the pendant peptides via thiol-ene Michael addition reaction to form a PEG-binder adduct. After this, the HASH stock solution was diluted in PBS to double the final concentration in the gel, if needed (e.g., 0.5% w / v for the formulation with a final concentration of 0.25% w / v in the gel) and was vortexed 20-30 s to mix.

[0278] The PEG-binder adduct solution in HEPES / PBS was then added 1: 1 by volume to the diluted HA-SH solution to form the pre-gel solution. The pre-gel solution was immediately vortexed 10-15 s to mix and 50 uL was added to round molds identical to those used for preparing the PEG-peptide gel samples. The hydrogel samples in molds were allowed to polymerize for 30 min in a humidified chamber at 37C and 5% CO2. The gels were then removed from the molds and placed in PBS to swell. Example 3: Modifications to include alternative molecular weights of HA

[0279] Hyaluronic acid (HA) notably has physiochemical and biological properties which change with its molecular weight. Hydrogel formulation optimization was performed to identify what gel compositions could be utilized with higher and lower molecular weight HA compared to the preferred formulation (-300 kDa).

[0280] Materials and Methods

[0281] Materials. 20 kDa, 8-arm poly(ethylene glycol)-vinyl sulfone (PEG- VS), HEPES and PBS buffers components, and synthetic peptides were obtained as described in EXAMPLE 2. Thiolated 10 kDa (“HA-10”, 30% molar degree of substitution), 50 kDa (“HA-50”, 10% molar degree of substitution), and 1500 kDa (“HA- 1500”, 5% molar degree of substitution) HA was manufactured by and purchased from Creative PEGWorks. Stock solutions of HEPES / PBS buffer, PEG-VS, and peptide components were prepared as detailed in EXAMPLE 2. S1GMACOTE® (a chlorinated organopolysiloxane, Sigma-Aldrich) was purchased and used according to the manufacturer’s specifications to treat glass surfaces to make them more hydrophobic.

[0282] HA-SH was reconstituted at 2% w / v for HA-10 and HA- 1500 and at 2.5% w / v for HA-50 in sterile phosphate buffered saline (PBS), pH 7.4 and stored in aliquots at -20°C until use, without repeated freeze-thaw cycles. When stored at -20°C, the maximum shelf life of the frozen stock solutions was found to be approximately 2 months.

[0283] Preparation of Gel Samples for Characterization.

[0284] Acellular gel samples were prepared to evaluate the mechanical properties and swelling of formulations with varied molecular weights and concentrations of HA. The detailed composition of formulations tested is given in Table 6. Hydrogel samples with an initial (pre-polymerization and pre-swelling) volume of 50 pL each were prepared in the manner reported in EXAMPLE 2 above. First, a 2X concentrated solution of PEG-binder adduct was prepared. PEG-VS was diluted from a 20% w / v stock solution to 5% w / v with 200 mM HEPES / 2X PBS and ultrapure Water (Invitrogen) in a microtube and vortexed to mix. Then, stock solutions of peptide binders were added to the following concentrations: 3.0 mM PHSRN-K-RGD, 3.0 m GFOGER, 1.0 BM-binder, and 1.0 FN-binder. The mixture was allowed to sit at room temperature for 30 min to allow the PEG-VS macromers to become functionalized with the pendant peptides via thiol-ene Michael addition reaction to form a PEG-binder adduct. After this, the HASH stock solution was diluted in PBS to double the final concentration in the gel, if needed (e.g., 0.5% w / v for the formulation with a final concentration of 0.25% w / v in the gel) and was vortexed 20-30 s to mix.

[0285] The PEG-binder adduct solution in HEPES / PBS was then added 1: 1 by volume to the diluted HA-SH solution to form the pre-gel solution. The pre-gel solution was immediately vortexed 10-15 s to mix and 50 uL was added to round molds identical to those used for preparing the PEG-peptide gel samples. The hydrogel samples in molds were allowed to polymerize for 30 min in a humidified chamber at 37C and 5% CO2. The gels were then removed from the molds and placed in PBS to swell.

[0286]

[0287] Volumetric Swelling Characterization. Post-polymerization, gel samples were allowed to swell to equilibrium (no net change in sample mass / volume) in divalent cation-free Phosphate Buffered Saline (PBS), pH 7.4 for 18-24 h at 37°C and 5% CO?, to approximate cell culture conditions.

[0288] To assess volumetric swelling, the samples were removed from the PBS, excess moisture on the surface of the gel was removed, and the sample was moved to a glass microscopy slide surface-treated with SIGMACOTE® to prevent sticking and damage to the gel sample. The diameter and height in mm of the swollen gel sample was measured and used to calculate the volume in pL of the swollen gel at equilibrium (i.e., maximally swollen). To calculate the volumetric swelling factor (VSF), the volume (uL) of the equilibrium-swollen gel sample was divided by the initial volume of the sample (50 uL).

[0289] Characterization of Gel Mechanical Properties. The mechanical properties of the gel were assessed via shear oscillatory rheology using an Anton Paar MCR 302 rheometer with an 8 mm parallel plate measuring geometry and a Peltier plate with an air-controlled temperature regulation system. Equilibrium-swollen gel samples were placed on the Peltier plate sample stage, pre-warmed to 37°C, and the measuring geometry was lowered to 70-80% of the height of the equilibrium swollen sample. This compression of the sample prevented sample slippage during measurement. The sample was subject to 180 s time sweep at a frequency of 1 Hz and strain of 5% at 37°C. The storage and loss moduli were averaged over the time sweep for each sample. Those values for each sample were averaged to yield the average for the formulation (n = 3), reported with the standard deviation. Frequency and amplitude sweeps were performed to verify that the testing parameters were within the linear viscoelastic regime (not reported).

[0290] Results

[0291] Of the formulations tested (A-H, listed in Table 7), two formulations, C and G, did not gel and were not subject to further characterization. The volumetric swelling factor, the ratio of the volume of the fully swollen gel at equilibrium to the starting volume of the gel immediately after polymerization, and the shear storage modulus of the remaining formulations were evaluated. The results are shown in FIG. 4A-B.

[0292] Generally, formulations made with HA- 10 exhibited a lower volumetric swelling factor than formulations made with HA-50 or HA- 1500 (FIG. 4A). This is especially notable when comparing the 3 formulations each made with 1.0%w / v HA-SH, but of varying molecular weights: A (HA- 10), E (HA-50), and F (HA-1500). The gel formulations made with HA- 10 also were the stiffest gels, according to their shear storage modulus (FIG. 4B). This is to be expected, as HA- 10 is the most hydrophobic molecule out of the three, and therefore would interact the least with water, leading to less swelling. Surprisingly, the formulations that gelled that were made with HA- 50 and HA- 1500 exhibited similar properties in terms of swelling and shear storage modulus.

[0293] However, the physical properties of the HA are not the only contributing factor in the properties of the gel. The degree of crosslinking, which is related to the concentration of available thiols on the HA molecule, is also one determinant of gel properties. The concentration of thiol groups in the pre-gel solution can be estimated based on the concentration and degree of substitution of the HA, along with the molecular weight of the monomer of HA (one D-glucuronic acid group and one N-acetyl-D-glucosamine group, MW = 379.32) which is constant. Therefore, while the gels made with 1.0% w / v HA all contain approximately 2.64 mM of HA monomers, there are differences in the concentration of thiols between the three groups, with 0.132 mM in the HA-1500 gel, 0.264 mM in the HA-50 gel, and 0.792 mM in the HA- 10 gel Additionally, as a different comparison, formulations B and D are estimated to contain roughly the same amount of thiol groups, 0.316 mM, based on the concentration and degree of substitution of the HA used for each. Example 4: Culture of isolated primary human adipocytes

[0294] Long-term culture of adipocytes, particularly primary human adipocytes, has long been technically challenging. Notably, when conventional adherent cell culture methods are used, adipocytes float, do not adhere to the culture surface, and die. One alternative to using adipocytes has been to use adipocyte-like differentiated cells, including human preadipocytes and murine pre-adipocytes or cell lines such as 3T3-L1 or C3H10T1 / 2 cells, but these cells have limited resemblance to primary, mature adipocytes (Ruiz-Ojeda et al. Int. J. Mol. Sci., 2016) https: / / doi.org / 10.3390 / ijmsl7071040. Alternatively, other culture methods have been developed for primary adipocytes. One such method is adherent ceiling cultures, where cells are ‘floated’ to the top surface of a culture flask filled with liquid media and adhere on that surface, but those cells then dedifferentiate and lose their adipocytic phenotype over the course of 2 weeks (Sugihara et al. Differentiation, https: / / doi.0rg / lO.ll l l / j.1432- 0436.1986. tb00381.x 1986). Recently, Harms et al. have reported another method using membrane mature adipocyte aggregate cultures, but this method is highly technically challenging and difficult to reproduce (Harms et al. Cell Reports, 2019 https: / / doi.Org / 10.1016 / j.celrep.2019.03.026). 3D culture of primary adipocytes via encapsulation in a hydrogel matrix offers an accessible alternative method which would potentially maintain the adipocytic phenotype for time periods on the order of several weeks. The goal of this pilot study was to compare the fitness for primary adipocyte culture of PEG-HA hybrid hydrogels, previously published peptide- crosslinked PEG hydrogels (Hernandez- Gordillo et al., Biomaterials, 2020 https: / / doi.Org / 10.1016 / j.biomaterials.2020.120125), and a commercially available hydrogel for 3D cell culture, HyStem-C.

[0295] Materials and Methods

[0296] Materials. 20kDa, 8-arm PEG-VS and -300 kDa HA-SH with a molar degree of substitution -20-30% were obtained commercially and reconstituted as detailed in Example 2. Concentrated HEPES / PBS stock solutions were prepared as described in Example 2. Custom- synthesized peptides detailed in Table 1 described were ordered and prepared in stock solutions in Example 2. PBS in solution, pH 7.4, was purchased from Gibco (Catalog #10010-023). Krebs buffer was prepared as a 5X stock solution containing 552 mM sodium chloride (Millipore Sigma S5886), 24 rnM potassium chloride (Fisher Scientific, P217), 6 mM potassium phosphate (VWR 7100), 6 mM magnesium sulfate heptahydrate (VWR 0662), and 12.6 mM calcium chloride dihydrate (Millipore Sigma C7902) in MilliQ distilled water. Krebs buffer stock solution was diluted 5 -fold in MilliQ distilled water before use at IX. HYSTEM-C®, a commercially available HA- collagen gel for 3D cell culture, was purchased from Sigma-Aldrich (HYSC020) and used according to manufacturer’s instructions with slight modification, as described in the following section. HYSTEM-C® is packaged as a kit containing three reagents: GLYCOSIL®, a thiolated HA also used in the preparation of the PEG-HA hydrogels, GELIN-S®, a thiolated porcine gelatin, and EXTRALINK®, a PEG-diacrylate. Cell culture media used for the adipocytes consisted of basal medium Dulbecco’s modified Eagle’s medium / Ham’s nutrient mixture F12 (DMEM / F12; Gibco 10565-018), 10% heat-inactivated fetal bovine serum (R&D Systems), and IX penicillin / streptomycin (Gibco 15140-148) / .

[0297] Gel encapsulation of adipocytes. Adipocytes were isolated from tissue samples according to an adaptation of the method published by Alexandersson et al. (J. Vis. Exp., 2020 https: / / dx.doi.org / 10.3791 / 60485) then were washed with Krebs buffer and centrifuged at 50 RCF for 3 min. The centrifuged mixture yielded a non-adipocyte cell pellet at the bottom, an aqueous phase, an oily phase at the top, and a layer of adipocytes between the aqueous and oily phases. As much of the oily phase of free lipids on top of the cells was then aspirated manually with a needle and syringe and disposed. Next, the aqueous phase and the non-adipocyte cell pellet at the bottom of the tube were removed via needle and syringe. The remaining adipocytes are roughly separated by size, with the largest cells at the top and the smallest cells at the bottom. Using a wide-bore pipette tip, cells from the middle were collected and moved to a sterile microtube.

[0298] To encapsulate the cells in the HyStem-C gel, the contents of the kit (Glycosil, Gelin-S, and Extralink) were reconstituted according to the manufacturer’s instructions. Glycosil and Gelin-S were combined in a 1:1 ratio by volume. Adipocytes were added so that they comprised 15% by volume of the final pre-gel mixture and pipetted up and down several times to mix. 30 uL of Extralink solution per 125 uL of Glycosil / Gelin-S mixture used was added to the center of a well of a 24 well plate. The Glycosil / Gelin- S / adipocyte mixture was then added directly on top of the Extralink solution in the well and was mixed by gently shaking the well plate from side to side. The well plate was incubated at 37C and 5% CO2 in a humidified incubator for 1 h to allow samples to gel. Culture media was then added to the samples and they were moved back to the incubator.

[0299] The PEG-peptide gel formulation used to culture the adipocytes was 3% w / v (1.50 mM) PEG-VS, 100 mM HEPES / 1X PBS, 1.5 mM PHSRN-K- RGD, 1.5 mM GFOGER. 0.5 m BM-binder, 0.5 mM FN-binder, and 2.70 mM LW. To encapsulate the cells in the gel, PEG-VS, HEPES / PBS, PHSRN-K-RGD, GFOGER, BM-binder, and FN -binder stock solutions were added to a microtube and vortexed to mix. The mixture was allowed to react at room temperature for at least 15 min to form the PEG-binder adduct. A volume of ultrapure water was added to the PEG-binder adduct solution sufficient to bring the final pre-gel solution (including adipocytes and LW) to the desired final volume, before adding adipocytes. Adipocytes were added so that they comprised 15% by volume of the final pre-gel mixture. The PEG-binder adduct, ultrapure water, and adipocyte mixture was gently pipetted up and down to mix. Then LW stock solution was added to obtain a final concentration of 2.70 mM in the pre-gel solution (0.45 crosslinking ratio). As used herein, "crosslinking ratio" here is the ratio of the concentration of reactive groups on the bifunctional crosslinking molecule in the mixture to the concentration of total 'arms' of the multiarm PEG in the mixture (i.e., mM concentration of the PEG macromer x 8 for the 8-arm PEG-VS).

[0300] The pre-gel mixture was pipetted up and down to mix, and then 12 uL droplets of pre-gel mixture were deposited per well in a 24- well plate. The well plate was moved to a humidified incubator at 37C and 5% CO2 for 30 min to polymerize. Culture media was added to each gel sample and then the well plate was returned to the incubator.

[0301] The PEG-HA gel formulation used to culture the adipocytes was 1.65% w / v (0.825 mM) PEG-VS, 66.7 mM HEPES / 0.67X PBS, 0.165% HA-SH (GLYCOSIL®), 1 mM PHSRN-K-RGD, 1 mM GFOGER, 0.33 mM BM-binder, and 0.33 mM FN-binder. To encapsulate the adipocytes in the PEG-HA gel, first a solution of 5% w / v PEG-VS, 200 mM HEPES / 2X PBS, 3 mM PHSRN-K-RGD, 3 mM GFOGER, 1 mM BM-binder, and 1 mM FN-binder was prepared in a microtube. This solution was mixed thoroughly via vortex and allowed to react at room temperature for at least 15 min to form the concentrated peptide-functionalized PEG macromer. Meanwhile, the HA-SH was diluted in PBS in a microtube and mixed thoroughly via vortex. Adipocytes were added to the diluted HA-SH so that they comprised 15% by volume of the final pre-gel mixture and were mixed gently by pipetting up and down. The concentrated peptide-functionalized PEG macromer was then added to the HA-SH / adipocyte mixture so that it comprised 33% of the final pre-gel mixture by volume. This was mixed by pipetting up and down several times, and immediately after 12 pL droplets of pre-gel mixture were deposited per well in a 24-well plate. The well plate was moved to a humidified incubator at 37°C and 5% CO2 for 30 min to polymerize. Culture media was added to each gel sample and then the well plate was returned to the incubator.

[0302] For all gel formulations, media was changed every 2 days.

[0303] Live / Dead staining and imaging. Gel samples were removed from the incubator and the culture media was removed. Samples were washed once with excess PBS, and then stained with LIVE / DEAD viability / cytotoxicity stain (Invitrogen, Carlsbad, CA, USA) diluted 1:1000 in PBS for 20 min in the incubator at 37°C. Epifluoresence images were collected at 4X magnification.

[0304] Results

[0305] Initial attempts at 3D culture of primary adipocytes in PEG-HA hydrogels used a formulation consisting of 2.5% w / v PEG-VS + 0.25% w / v HA-SH + 100 mM HEPES / 1X PBS + 1.5 mM PHSRN-K-RGD + 1.5 mM GFOGER + 0.5 mM BM -binder + 0.5 mM FN-binder. However, when the adipocytes were added to the pre-gel solution, the mixture gelled instantaneously and could not be pipetted, which made it impossible to deposit gel-encapsulated cells into the well plate. This rapid gelation was likely due to phase separation induced by free lipids in the liquid containing the cells. A modified, 33% less concentrated formulation was subsequently used, which did not gel immediately upon addition of adipocytes and had a working time (i.e., time before the pre-gel solution became too viscous to pipette) on the order of several minutes, allowing for thorough mixing and the encapsulation of the adipocytes for 3D culture.

[0306] Live / Dead staining of primary adipocytes after 7 days in culture indicates a higher viability in the control condition, HYSTEM-C® gel (FIG. 5A), and the PEG-HA condition (FIG. 5C) compared to the PEG-peptide condition (FIG. 5B). This suggests that the PEG-HA hydrogel formulation tested better supports primary human adipocyte viability in 3D culture than the peptide-crosslinked PEG hydrogel formulation. Because cell viability is also high in the HYSTEM-C® hydrogel, the adipocytes may require HA in their microenvironment to survive 7 days in 3D culture.

[0307] Example 5: Culture of human iPSC-derived neurons

[0308] Endometrial ectopic lesions characteristic of endometriosis and adenomyosis exhibit a high degree of innervation with peripheral neurons. The impact of these nerve cells on the growth of the lesion and the pain experienced by patients diagnosed with endometriosis and adenomyosis has yet to be elucidated. In vitro models of endometriotic lesions which include nociceptors, peripheral nerve cells specialized to respond to noxious stimuli, would therefore be a valuable tool to interrogate molecular and cellular mechanisms involved in interactions between endometrial cells and nerve cells. However, developing highly reproducible culture conditions which support the growth and maintenance of both nerve cells and endometrial cells is non-trivial.

[0309] This work details a pilot study conducted using the nerve-on-a-chip model developed by Moore et al. for the culture of induced pluripotent stem cell (iPSC)-derived nociceptors (Khoshakhlagh et al., J. Neural Eng., 2018, DOI: 10.1088 / 174 l-2552 / aael29) (Pollard et al., Sci. Adv. 2021, DOI: 10.1126 / sciadv.abj2899) to test the support of iPSC-nociceptor growth by synthetic PEG-peptide and hybrid PEG-HA gels compared to the current standard, MATRIGEL®.

[0310] Materials and Methods

[0311] Materials. 20kDa, 8-arm PEG-VS and -300 kDa HA-SH with a molar degree of substitution -20-30% were obtained commercially and reconstituted as detailed in Example 2. Concentrated HEPES / PBS stock solutions were prepared as described in Example 2. Custom-synthesized peptides detailed in Table 1 described were ordered and prepared in stock solutions in Example 2.

[0312] Nerve-on-a-chip model. Details of the nerve-on-a-chip model, including the method of fabrication of the growth-impermissive (poly(ethylene glycol)-di-methacrylate, PEGDMA) hydrogel mold used to hold the growth-permissive hydrogel, as well as its dimensions and composition are included in Khoshakhlagh et al., Journal of Neural Engineering, 2018. DOI: 10.1088 / 1741-2552 / aael29. Methods for nerve spheroid formation and implantation of cells in the nerve-on-a-chip constructs as well as details of MATRIGEL preparation are reported by Pollard et al., Science Advances, 2021. DOI: 10. 1126 / sciadv.abj2899. iPSC- derived nociceptors (i.e., peripheral neurons sensitive to noxious signals) were sourced from Anatomic, Inc. and maintained in culture according to the manufacturer’s protocols.

[0313] Cell encapsulation in PEG-peptide and PEG-HA hydrogels. In brief, an acellular pre-gel mixture of a peptide-crosslinked PEG hydrogel (“PEG-peptide”) was prepared of 2.6% w / v PEG-VS + 100 mM HEPES / 1X PBS + 2.81 mM LW (0.54 branching ratio) + 1.5 mM PHSRN-K-RGD + 1.5 mM GFOGER + 0.5 mM BM-binder + 0.5 mM FN-binder according to the method reported in EXAMPLE 2 for preparing gel samples for characterization. Additionally, an acellular pre-gel mixture of an HA- crosslinked hybrid hydrogel (“PEG-HA”) of 2.5% w / v PEG-VS + 0.25% w / v HA-SH + 100 mM HEPES / 1X PBS + 1.5 mM PHSRN-K-RGD + 1.5 mM GFOGER + 0.5 mM BM-binder + 0.5 mM FN-binder was prepared using the method reported in EXAMPLE 2 for preparing gel samples for characterization. However, at the final step, the pre-gel solution was added to the central channel of the PEGDMA hydrogel mold, and then one spheroid per construct was immediately added via pipette to one end of the barbellshaped channel. With the spheroids added, the constructs were moved to the humidified incubator at 37°C and 5% CO2 for 30 min to allow the gel to polymerize. After that, media was added around the constructs by pipetting into the gap between the Transwell plate insert containing the construct and the wall of the well beneath it. Samples were cultured for 37 days before fixing with 4% paraformaldehyde, immunostaining for beta 3 tubulin, and staining for actin using ActinRed.

[0314] Results

[0315] Constructs fixed and stained for beta 3 tubulin (green) and f-actin (red) after 37 days in culture demonstrate cell growth beyond the initially implanted spheroids in all 3 hydrogels. The MATRIGEL® control, shown in FIG. 6A, demonstrated the extension of processes from the nerve cells, identified by beta 3 tubulin-positive staining (green) as opposed to f-actin- positive staining (red), from the spheroid down the length of the central channel of the barbell-shaped growth-permissive gel compartment (boundaries indicated by the white dashed line). Additionally, the MATRIGEL®. condition exhibits some of these processes extending outside the bounds of the central, MATRIGEL®. -filled channel of the construct.

[0316] These processes appear in confocal microscopy to be growing over the top of the outer PEGDMA hydrogel. MATRIGEL®. is the current standard growth- permissive gel used in the nerve-on-a-chip model (Pollard el al., Science Advances, 2021. DOI: 10.1 I26 / sciadv.abj2899), and these results in the MATRIGEL® control are in line with previous experiments. In contrast, as shown in representative images in FIG. 6B-6C, spheroids seeded in PEG- peptide gel in the central compartment exhibited beta 3 tubulin-positive processes extending outside the boundaries of the central channel, on top of the surrounding PEGDMA hydrogel, but not within the PEG-peptide gel in the central channel. Furthermore, f-actin-positive (red), beta 3 tubulinnegative (i.e., non-neuronal) cells appeared to cover the bottom of the channel. These cells are assumed to have migrated from the implanted spheroids, but the details of their phenotype are unknown at time of writing. Lastly, spheroids seeded in PEG-HA gels in the central channel exhibited widely divergent morphology among the replicates in the experiment (n =3), as demonstrated in two representative images of fixed and stained constructs in FIG. 6D-6E. Two of the three replicates resembled the image on the left, with an abundant layer of f-actin-positive (red), beta 3 tubulin-negative cells along the bottom of the central channel, with beta 3 tubulin-positive (green) cell processes appearing to extend beyond the bounds of the barbell-shaped growth-permissive gel channel and along the surface of the PEGDMA outer gel, similar to the PEG-peptide gel samples. However, one of the three PEG- HA gel constructs, shown to the right in FIG. 6D-6E, exhibited a high degree of extension of beta 3 tubulin-positive (green) cellular processes along the length of the central gel channel, with relatively few extending on the surface of the PEGDMA gel, outside the boundaries of the central gel channel. Additionally, this sample exhibited few, if any, actin-positive, beta 3 tubulin-negative cells growing along the bottom of the central channel. The reason for the stark differences from sample-to-sample in the PEG-HA gel condition is uncertain, but may be due to incomplete mixing of PEG-rich and HA-rich phases during the preparation of the pre-gel solution, which can be addressed with a longer and more vigorous mixing step in the protocol. The storage conditions of the HA-SH stock solutions may also contribute to incomplete mixing between PEG-rich and HA-rich phases in gel preparation, as HA-SH stock solutions stored at -20C exhibit increasing viscosity when stored for over 2 months, which was the case here. Further testing has demonstrated that the shelf life of the HA-SH stock solutions, without this viscosity increase, is significantly increased by storing the solutions at -80C. Ongoing investigations with an updated pre-gel solution preparation protocol for the PEG-HA gel. Additionally, ongoing experiments are testing the impact of adding 0.5 mM each of two synthetic peptides with cell-adhesive sequences derived from the alpha and beta chains of laminin- 111 into both the PEG-peptide and the PEG-HA hydrogel formulations.

[0317] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0318] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:

1. A formulation for a matrix for culturing cells comprising a branched poly(alkyl oxide) polymers such as an 8-arm, 20kDa PEG macromers (“PEG- VS”) synthesized with a tripentaerythritol core and functionalized with vinyl sulfone terminal groups (molar degree of substitution < 90%), thiol-functionalized extracellular matrix polymer, preferably hyaluronic acid (HA-SH), preferably with a molecular weight of approximately 300 kDa and molar degree of substitution of approximately 20-30, preferably thiolated at the carboxylic acid residues of the D- glucuronic acid portion of the repeating units of the polymer, preferably consisting of one D-glucuronic acid group and one N-acetyl-D-glucosamine group.

2. The formulation of claim 1 further comprising an aqueous solution, preferably sterile phosphate buffered saline (PBS), pH 7.4 or Dulbecco’s phosphate buffered saline and 1 M 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES) pH 7.8-8.2.

3. The formulation comprising monofunctional or bifunctional peptides containing one or two thiol groups or functional groups reactive with or bound to the reactive functional group present on the branched poly(alkyl oxide) species4. The formulation of any of claims 1-3 wherein the peptides are selected from the group consisting of“LW,” a peptide with one cysteine residue at each end to facilitate crosslinking of PEG-VS macromers, containing a MMP-labile domain (GPQGIWGQ) (SEQ ID NO:43) and a Sortase-labile domain (LPRTG) (SEQ ID NO:27).“PHSRN-K-RGD,” a fibronectin-derived peptide containing the RGD motif and the PHSRN (SEQ ID NO:2) synergy site from the 9th fibronectin type III repeat in a branched configuration;“GFOGER,” a collagen I-derived peptide presented as a triple helix;“FN -binder ” a peptide with non-covalent binding affinity for fibronectin; andBM -binder. " a peptide with non-covalent binding affinity for collagen IV and laminin.

5. The formulation of any of claims 1-4 wherein the polymers are crosslinked to form a matrix.

6. The formulation of claim 5 further comprising cells.

7. A method of making the formulation of any of claims 1 -6 comprising providing the polyalkylene oxide and extracellular matrix polymers and adhesion ligand peptides and crosslinking.

8. The method of claim 7 further comprising providing cells.

9. The method of claim 8 wherein the cells are endothelial cells, smooth muscle cells, epithelial cells, and organoid forming cells.

10. The method of claim 9 further comprising testing drugs or hormones on the cells in the matrix.

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