Bioinspired synthetic hydrogel for gastrointestinal cancer organoid modeling

Abstract

Hydrogel compositions for use in generating organoid models of gastrointestinal cancer are disclosed herein. The hydrogel compositions include: a polyethylene glycol functionalized with norbornene, a degradable crosslinking peptide, a non-degradable crosslinking peptide, a cell adhesion peptide and a photoinitiator. The organoids are made by contacting cells, in particular cancer cells of gastrointestinal origin with the hydrogel compositions to generate organoids. The organoids can be used to study cellular properties and the effects of agents on the cells and organoids.

Description

[0001] BIOINSPIRED SYNTHETIC HYDROGEL FOR GASTROINTESTINAL CANCER ORGANOID MODELING

[0002] CROSS REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to U.S. Provisional Application Nos. 63 / 626,180 filed January 29, 2024, and 63 / 575,265 filed April 5, 2024, which are hereby incorporated by reference in its entirety.

[0004] INCORPORATION OF SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (96029604682.xml; Size: 43,100 bytes; and Date of Creation: January 28, 2025) is herein incorporated by reference in its entirety.

[0006] BACKGROUND OF THE DISCLOSURE

[0007] The present disclosure relates generally to the field of tissue engineering for the generation and expansion of patient-derived models of gastrointestinal cancers. More particularly, the present disclosure provides an in vitro application for modeling the physical properties of the desmoplastic stroma and hepatic environment specific in the modeling of gastrointestinal cancer niches. This technical environment is generated to improve the reproducibility in models of gastrointestinal cancer.

[0008] Pancreatic cancer is a representative gastrointestinal cancer as a leading cause of cancer mortality. The most aggressive histology of adenocarcinoma develops through the acquisition of pathologic variants in pro-oncogenes or the loss of tumor suppressor genes to yield uncontrolled growth from tissues of the pancreatic duct (ductal adenocarcinoma). The pancreatic cancer microenvironment includes cancer cells as well as fibroblasts, endothelial cells, immune cells, and endocrine cells that interact with each other and the cancer cells in a complex fashion. With the development of malignancy, a complex interplay of the tissue microenvironment fosters cancer growth, invasion, metastases, as well as local angiogenesis and unique immunologic recruitment. Excessive desmoplasia is a classical feature of pancreatic cancer whereby the cancer produces several components of extracellular matrix (ECM) including fibrillary collagen (type-1, type 2) and fibronectin. Distinct associated cancer associated fibroblasts activate in the tissue microenvironment of pancreatic cancer, resulting in excessive production of extracellular matrix. Together, individual contributions from this model influence the growth, differentiation, and stem like properties of pancreatic cancer.

[0009] A modem standard in tissue engineering is to produce oligoclonal units with intact cancercancer associations termed organoids as representative subclonal units of pancreatic cancer. These units have the ability to expand in populations with different phenotypic characteristics including either infiltratively dense or cystic morphologies. The pathologic driver genomic features of pancreatic cancer are largely preserved under these models. There remains significant risk for clonal selection with traditional culture methods whereby the addition of exogeneous growth factors used in the generation and proliferation of the pancreatic cancer models promote a classical transcriptional expression profile. Growth factors supported in these models are derived from Engelbreth-Holm- Swarm (EHS) tumors, an animal product with significant batch-to-batch diversity in growth factors from commercially available products including Matrigel (Corning Life Sciences). Geltrex (Thermo Fisher Scientific, or CultrexBME (Bio-Teche, catalog no. 3432-010- 01). Batch variation is a significant barrier to clinical translation due to thousands of individual elements. Modern screening applications with EHS are performed at 2% v / v, which fail to maintain semi-solid properties, suggestive that the primary benefit of EHS is its driving role in providing growth factor support. The existing materials cannot be adapted to the standards needed for clinical validation.

[0010] New data has revealed pancreatic cancer biology is modulated as a function of the physical properties in the tissue microenvironment. Prior patent applications (EP3575391B) have used hydrogel materials with final shear modulus of <500 Pa. Herein, the present disclosure describes a first in class material designed to increase the shear modulus to >4000 Pa in order to mimic the hallmark contributions of the physical properties of the hepatic and desmoplastic stroma as distinct, common microenvironments for studying these cancer types.

[0011] SUMMARY

[0012] Generally, the present disclosure relates to hydrogel compositions. In one aspect, hydrogel compositions of the present disclosure can be made and used to alter (e.g., enhance, inhibit and change) cell function, and in particular, cellular expansion, maturation and differentiation, particularly in cancer cells, and more particularly, in pancreatic cancer cells.

[0013] In one aspect, the present disclosure is directed to a hydrogel composition comprising: a polyethylene glycol functionalized with norbornene, a degradable crosslinking peptide, a non- degradable crosslinking peptide, a cell adhesion peptide and a photoinitiator.

[0014] In another aspect, the present disclosure is directed to a method of promoting cellular expansion, the method comprising: preparing a hydrogel composition, wherein the hydrogel composition comprises a polyethylene glycol functionalized with norbornene, a degradable crosslinking peptide, a non-degradable crosslinking peptide, a cell adhesion peptide and a photoinitiator; contacting a cell with the hydrogel composition; and culturing the cell to allow cellular expansion.

[0015] In yet another aspect, the present disclosure is directed to a method of promoting cellular differentiation, the method comprising: preparing a hydrogel composition, wherein the hydrogel composition comprises a polyethylene glycol functionalized with norbornene, a degradable crosslinking peptide, a non-degradable crosslinking peptide, a cell adhesion peptide and a photoinitiator; contacting a cell with the hydrogel composition; and culturing the cell under conditions to promote cellular differentiation.

[0016] In yet another aspect, methods of preparing an organoid model are provided herein. The methods include preparing the hydrogel composition as described above and contacting the hydrogel with at least one cell to prepare the organoid model. The cells may be cancer cells. The organoid model can be a cancer model such as a pancreatic, liver or other gastrointestinal organoid model. The organoid model may be used for screening agents. In methods of screening the organoid model is contacted with at least one agent and at least one property of the organoid or the cells within the organoid is measured. The property may be compared to a control organoid not treated with the agent. Agents that change at least one property of the organoid or the cell measured are screened as active agents.

[0017] In yet another aspect, the hydrogel composition provides differential expression of genes involved in cancer resistance. Further, in yet another embodiment, the hydrogel composition provides upregulation in regulatory pathways, and thus, the hydrogel composition can be adapted to discovery methods for therapeutic drug screening.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The disclosure will be better understood, and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings, wherein:

[0020] FIG. 1 is an overview of passages from hydrogel materials in organoid applications as analyzed which maintain exogenous serum free conditions in Example 1.

[0021] FIG. 2 depicts physical properties with digestion of hydrogel matrix materials. Comparison of hydrogel digestion at 45 minutes across hydrogel preparations. Digestion was achieved as shown from pellet formation after digestion (black arrow), versus residual hydrogel after digestion (white arrows) which was dependent on hydrogel condition.

[0022] FIG. 3 depicts the durability in hydrogel droplet across timecourse imaging. Representative file of hydrogel across time with dissociation from hydrogel -plate interface (arrows).

[0023] FIG. 4 shows a comparison of elastic modulus between matrix materials. Biomechanical properties of elastic modulus (kPa) across n = 3 independent photoactivations between synthetic hydrogel SP139 and Cultrex. Rheometry was measured with 3.9% strain and constant frequency (1 Hz).

[0024] FIG. 5 is a representative growth profile of pancreatic cancer organoids across hydrogel materials. Representative culture (Pan33) was evaluated every 48 hours using high content imaging in brightfield. The Growth rate compared across populations or organoids is influenced by the functional design of a hydrogel derived from an immediate passage of patient-derived xenograft digestion.

[0025] FIG. 6 is high content imaging of organoid growth across cancer types including pancreatic ductal adenocarcinoma (PAN), high grade neuroendocrine carcinoma (NET), intrahepatic cholangiocarcinoma (CCA), and murine engineered pancreatic cancer model (KPC). Representative growth profiling of a diverse set of organoid cancer models. Shown is brightfield imaging at plating (Day 0) and after culture propagation (Day 7) with corresponding Calcein AM staining (5 pM).

[0026] FIG. 7A is a comparison of growth timecourse in pancreatic organoids. Shown are interval timecourse compared between SP139 (first set of 5 bars on left) and reduced growth factor Cultrex (Cultrex; second set of 5 bars). Shown are representative % changes in size with bar height representing mean and error bar showing SEM across the population.

[0027] FIG.7B depicts normalized change in diameter to PDAC PCO1 (PAN21) grown in SP139 for 144 hours across multiple passages.

[0028] FIG. 7C depicts normalized change in diameter to PDAC PCO1 (PAN21) grown in Cultrex (1) and SP139 (2) compared at 48 hour intervals across 144 hours.

[0029] FIGS. 8A & 8B depict representative imaging of growth between matrix materials. Brightfield imaging showing growth across time between matrix environments. FIG. 8A shows Cultrex matrix yielding both cystic and solid cancer organoids. FIG. 8B shows SP139 matrix with surrounding cellular expansion including infiltration of single cell populations. Size bar represents 1000 pm.

[0030] FIG. 9 depicts representative cell viability assay of pancreatic cancer organoids to chemotherapy with gemcitabine (Gem). Shown is media control versus gemcitabine chemotherapy at 48 hours after therapeutic treatment. Viability staining performed utilizing 33.3% v / v CTG incubated for 30 minutes.

[0031] FIG. 10 shows the principal component analysis between three representative tumors (patient-derived xenografts, 3) and cultures expanded between commercially available matrix (Cultrex, 1) and synthetic hydrogel SP139 (2).

[0032] FIG. 11 depicts a heatmap of gene expression from epoxygenase p450 and MAPK pathways that were shown to be significantly altered by pathway analysis in the different culture environments. Fold change is calculated from PDX (tumor) gene counts. FIG. 12 shows enzymatic levels in cytochrome p450 enzyme CYP3A4 assayed using bioluminescent assay by P450-Glo including ovarian cancer control (black) and comparison for two unique normal liver (NL) cultures grown in cultrex (gray) or SP139 (green).

[0033] DETAILED DESCRIPTION

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs.

[0035] In accordance with the present disclosure, tissue engineering for the generation and expansion of patient-derived models of gastrointestinal cancers, and in particularly, niches of the pancreatic and hepatic microenvironment are disclosed. More particularly, the present disclosure provides an in vitro application for modeling the physical properties of the desmoplastic stroma in the pancreatic cancer microenvironment. Generally, the present disclosure relates to hydrogel compositions. In one aspect, hydrogel compositions of the present disclosure can be made and used to alter (e.g., enhance, inhibit and change) cell function, and in particular, cellular expansion, maturation and differentiation, particularly in cancer cells, and more particularly, in pancreatic cancer cells.

[0036] As known by those skilled in the art, a hydrogel composition is a network of polymer chains that are hydrophilic in which a polymeric material and water are in an equilibrated form. The hydrogel composition is formed using unpolymerized starting components. The polymeric material can be, for example, a natural polymer material, a synthetic polymer material and combinations thereof.

[0037] The methods for preparing hydrogel compositions of the present disclosure advantageously allows for the direct incorporation of peptides into the hydrogel network during polymerization by including a cysteine in the amino acid sequence during synthesis, which allows for eliminating the need for post-synthetic modifications. In this way, peptides can be utilized as crosslinkers by including cysteine on each end or they can be incorporated as pendant groups, which can be precoupled to the polymer backbone and mixed in varying combinations or incorporated during polymerization for simplicity. The hydrogel compositions of the present disclosure have advantages over previously used materials like Cultrex matrix. Particularly, the hydrogel compositions of the present disclosure are well defined, have no exogenous growth factor, are highly tunable, and have an increased elastic modulus that mimics a tumor microenvironment. In contrast, Cultrex matrix has standardized use, batch-to-batch variation, and a low elastic modulus that does not mimic a tumor microenvironment.

[0038] Hydrogel Compositions and Methods for Preparing Hydrogel Compositions

[0039] The present disclosure is generally directed to methods for preparing a hydrogel composition and use of the resulting compositions. Generally, the methods for preparing the hydrogel composition include polymerizing a hydrogel precursor solution resulting in the formation of a hydrogel.

[0040] Suitable polymers for use in the hydrogel precursor solution are known by those skilled in the art and can include, for example, poly(ethylene glycol), hyaluronic acid, gelatin, collagen, MATRIGEL®, dithiol polymers (e.g., acrylamide), click-based composite hydrogels (as discussed in Polizzotti et al. Biomacromolecules 2008, 9: 1084-1087, which is hereby incorporated by reference to the extent its disclosure is consistent with the present disclosure), polyethylene glycol)-diacrylate, poly(ethylene glycol)-vinyl sulfone, and the like. Particularly suitable polymers can be, for example, poly(ethylene glycol). Particularly suitable polymers can be, for example, functionalized polymers. Functionalization of the polymer can be confirmed with 'H nuclear magnetic resonance spectroscopy, mass spectroscopy, Elman’s reagent, UV-Vis spectroscopy, infrared spectroscopy, and other methods known to those skilled in the art, for example.

[0041] A particularly suitable functionalized polymer can be, for example, eight-arm poly(ethylene glycol) with terminal hydroxyl (-OH) groups (commercially available from JenKem Technology USA, Allen, TX) that is functionalized with norbornene. Eight-arm poly(ethylene glycol) can be functionalized with norbornene as described in Fairbanks et al. (Adv. Mater. 2009, 21 :5005-5010). Other particularly suitable functionalized polymer can be, for example, six-arm, four-arm, or three-arm poly(ethylene glycol) with terminal hydroxyl (-OH) groups. Other particularly suitable polymers are poly(ethylene glycols) that may be functionalized using click chemistry. “Click” chemistry is an extremely versatile method for chemically attaching biomolecules, which is used to describe the [3+2] cycloaddition between alkyne and azide functional groups. Azides and alkynes are largely inert towards biological molecules and aqueous environments, which allows the use of the Huisgen 1,3-dipolar cycloaddition to yield stable triazoles that are very difficult to oxidize or reduce. Both the copper(I)-catalyzed and copper-free strained-alkyne variant reactions are mild and very efficient. These reactions can also be performed in small volumes of aqueous solutions, are insensitive to oxygen and water, and robust to functional groups on peptides. Click chemistry allows for selectivity in conjugation reactions in biological samples such as, for example, oligonucleotides and proteins. Particularly suitable reagents for click chemistry are commercially available from Laysan Bio Inc. (Arab, AL).

[0042] Generally, the hydrogel precursor solutions include concentrations of polymer of up to, and including, 200 mg / mL, including from about 15 mg / mL to about 160 mg / mL, including from about 20 mg / mL to about 80 mg / mL, and including about 80 mg / mL.

[0043] Suitable polymer crosslinkers for use in the hydrogel precursor solution are known by those skilled in the art. In suitable embodiments, degradable crosslinking peptides and non-degradable crosslinking peptides are used in combination. It has been found that the degradable crosslinking peptide and the non-degradable crosslinking peptide are beneficially used in a ratio of degradable crosslinking peptidemon-degradable crosslinking peptide about 4: 1. This ratio provides opportunities for cancer remodeling and intact matrix as a physical stressor on the cancer for expansion with cellular replication. This is particularly true in the pancreatic cancer desmoplastic stroma and hepatic parenchyma as hallmark niches in gastrointestinal cancers. Other suitable ratios of degradable crosslinking peptidemon-degradable crosslinking peptide include about 1:1, about 2: 1, about 3: 1, about 5:1, about 6:1 or about 7: 1. The optimal ratio would be dependent on the level of matrix metalo proteinases (MMPs) that the cells are secreting, for example, if a particular cancer isolate secreted a lot of MMPs, it may be beneficial to maintain the hydrogel structure to have more non-degradable crosslinker.

[0044] Particularly suitable non-degradable crosslinking peptides can be, for example, polyethylene glycol dithiol (PEG-DT), non-protease-degradable peptide crosslinkers and multi- arm poly(ethylene glycol) terminated with thiol (e.g., 4-arm PEG terminated with thiol). Suitable protease-degradable crosslinking peptides can be, for example, matrix metalloproteinase (MMP)- degradable crosslinkers as described in Nagase and Fields (Biopolymers 1996, 40:399-416, which is hereby incorporated by reference to the extent it is consistent with the present disclosure). More particularly, suitable MMP-degradable crosslinking peptides for use in the hydrogel precursor solution include KCGGPQGIWGQGCK (SEQ ID NO:27) and KCGGPQGIAGQGCK (SEQ ID NO:28). Other particularly suitable crosslinking peptides include, for example, thiolated hyaluronic acid and thiolated dextrans.

[0045] The hydrogel precursor solution can further include an initiator. An initiator can induce polymerization and / or decrease the polymerization rate. Suitable initiators are known to those skilled in the art and can be, for example, chemical initiators and photoinitiators. Particularly suitable photoinitiators can be, for example, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), IRGACURE 2959 photoinitiator (commercially available from Ciba / BASF, Ludwigshafen, Germany) and Eosin Y. In one embodiment, LAP was used as the photoinitiator in an amount of approximately 1.825 mM. The use of these photoinitators generates free radicals which drives the polymerization process. This results in step-growth polymerization which is more consistent and controlled. The use of LAP photoinitiators provides activation via crosslinking. LAP is activated with non-UV light exposure which is of particular importance when considering mechanistic studies in investigations of malignancy (i.e. studies of DNA damaging chemotherapy or radiation).

[0046] The hydrogel precursor solution can be stored with or without an initiator. In some embodiments, the initiator is added at the time of use. In other embodiments, the initiator is added to the hydrogel precursor solution prior to used and stored. In some embodiments, the hydrogel precursor solution and initiator stored frozen.

[0047] In another aspect, the hydrogel precursor solution can include a cell adhesion peptide. As used herein, a “cell adhesion peptide” refers to an amino acid sequence obtained from an adhesion protein to which cells bind via a receptor-ligand interaction. Varying the cell adhesion peptide and concentrations thereof in the solution allow for the ability to control the stability of the cellular attachment to the resulting hydrogel composition. Suitable cell adhesion peptides include, for example, RGD, RGDS (SEQ ID NO:1), CRGDS (SEQ ID NO:2), CRGDSP (SEQ ID NO:3), PHSRN (SEQ ID NO:4), GWGGRGDSP (SEQ ID NO:5), SIDQVEPYSSTAQ (SEQ ID NO:6), GRNIAEIIKDI (SEQ ID NO: 7), DITYVRLKF (SEQ ID NO: 8), DITVTLNRL (SEQ ID NO: 9), GRYVVLPR (SEQ ID NO: 10), GNRWHSIYITRFG (SEQ ID NO: 11), GASIKVAVSADR (SEQ ID NO: 12), GTTVKYIFR (SEQ ID NO: 13), GSIKIRGTYS (SEQ ID NO: 14), GSINNNR (SEQ ID NO: 15), SDPGYIGSR (SEQ ID NO: 16), YIGSR (SEQ ID NO: 17), GTPGPQGIAGQGVV (SEQ ID NO:18), GTPGPQGIAGQRVV (SEQ ID NO: 19), MNYYSNS (SEQ ID NO:20), KKQRFRJTRNRKG (SEQ ID NO:21), XBBXBX, wherein B = basic residue (such as arginine, lysine, or histidine) and X = hydropathic residue (such as alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, or cysteine), XBBBXXBX, wherein B = basic residue (such as arginine, lysine, or histidine) and X = hydropathic residue (such as alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, or cysteine), and RGDSP (SEQ ID NO:44).

[0048] The concentration of cell adhesion peptide in the hydrogel precursor solution will depend on the specific cell adhesion peptide being used as well as the other components in the hydrogel precursor solution. Typically, however, the hydrogel precursor solution includes from about 0.125 mM to about 4 mM cell adhesion peptide, including from about 0.25 mM to about 2 mM cell adhesion peptide. In one suitable embodiment, the cell adhesion peptide is CRGDS (SEQ ID NO:2), and the hydrogel precursor solution includes from about 0.25 mM to about 4 mM CRGDS (SEQ ID NO:2). In another suitable embodiment, the cell adhesion peptide is a cyclic RGD, and the hydrogel precursor solution includes from about 0.125 mM to about 2 mM cyclic RGD, particularly cyclic RGD{Fd}C (SEQ ID NO:33).

[0049] In another aspect, the hydrogel precursor solution can include a soluble factor binder. In one aspect, a peptide for binding a soluble factor contained in a cell culture medium is included in the hydrogel precursor solution. The density (concentration) of the soluble factor binder in a hydrogel composition can be controlled by altering the concentration of the soluble factor binder in the hydrogel precursor solution. Examples of particularly suitable soluble factor binders are provided in Table 1, below. Table 1. Soluble factor binder peptide sequences for hydrogel compositions. The concentration of soluble factor binder in the hydrogel precursor solution will depend on the specific soluble factor binder being used as well as the other components in the hydrogel precursor solution.

[0050] In another aspect, the hydrogel precursor solution can further include a cell. Suitable cells are known to those skilled in the art and can include, for example, a cancer cell, a cancer cell spheroid, a cancer organoid, a tumor fragment, a biopsy fragment, a patient-derived tumor model, a hepatocyte, a pancreatic beta cell, a pancreatic islet cell and combinations thereof. The cell may be a cancer cell selected from the group consisting of pancreatic cancer cell, colon cancer cell, liver cancer cell, gastrointestinal tract cancer cell, breast cancer cell, brain cancer cell, and metastatic tumor. In some aspects, suitable cells include genetically modified T cells, such as chimeric antigen receptor T cells, and lymphocyte cells, such as Natural Killer cells.

[0051] In another aspect, the hydrogel precursor solution can further include an additional agent selected from the group consisting of collagen, fibronectin, hyaluronic acid, and combinations thereof. An additional agent may be a microsphere carrier (i.e., microcarrier) or added with a microsphere carrier. Microsphere carriers can contain molecules such as, for example, cells, biomolecules, dyes and other molecules known to those skilled in the art. Microspheres can be degradable microspheres that dissolve or degrade to release the contents of the microsphere.

[0052] Once prepared, the hydrogel precursor solution may be contacted with a substrate (e.g., a patterned surface-modified substrate, surface of a cell culture plate, etc.). When used on a patterned surface-modified substrate, the surface-modified substrate can be, for example, mica, glass, silicon, diamond and metal oxide surfaces. The surface-modified substrate can be prepared, for example, by functionalizing a surface such as a glass coverslip having a silane monolayer. A particularly suitable surface-modified substrate can be, for example, a glass slide. A particularly suitable method for functionalizing the substrate can be, for example, silanization. The substrate can be surface-modified by activating both sides of the surface in oxygen plasma treatment. Oxygen plasma treatment can increase the number of activated hydroxyl groups on the surface of the substrate. As known by those skilled in the art, a silane monolayer can be prepared with an alkoxysilane that is dissolved in an anhydrous organic solvent such as, for example, toluene. Other suitable alkoxysilanes can be for example, aminosilanes, glycidoxysilanes and mercaptosilanes. Particularly suitable aminosilanes can be, for example, (3-aminopropyl)-trriethoxysilane, (3- aminopropyl)-diethoxy-methylsilane, (3-aminopropyl)-dimethyl-ethoxysilane and (3- aminopropyl)-trimethoxy silane. Particularly suitable glycidoxy silanes can be, for example, (3- glycidoxypropyl)-dimethyl-ethoxysilane. Particularly suitable mercaptosilanes can be, for example, (3 -mercaptopropyl)-trimethoxy silane and (3 -mercaptopropyl)-methyl-dimethoxy silane. Other suitable silanes are commercially available (Sigma Aldrich, St. Louis, MO). Preparation of a surface-modified silane substrate can be performed using any silane having a terminal functional group that can participate in click chemistry as described herein. For example, mercaptosilane contains a terminal thiol that can react with the norbornene of the PEG-norbornene. Other suitable functional surface-modified silane substrates can be, for example, acrylates and methacrylates. Following surface-modification of the substrate, non-adhesive self-assembled monolayers are formed on the surface-modified substrate.

[0053] After contacting the substrate with the hydrogel precursor solution, the method includes polymerizing the hydrogel precursor solution such that polymerized hydrogel attaches (i.e., is coupled) to the substrate.

[0054] Hydrogel compositions having variable elastic modulus, variable shear modulus, variable ligand identity, variable ligand density and combinations thereof can be prepared according to the methods described herein above. Suitable ligands are known to those skilled in the art and can be, for example, any biomolecule containing a cysteine and / or functionalized with a thiol. Thiol- functionalizing of ligands can be performed using commercially available kits (e.g., Traut’s Reagent (2-iminothiolane*HCl), Thermo Fischer Scientific, Rockford, IL). Suitable ligands can be, for example, proteins, peptides, nucleic acids, polysaccharides, lipids, biomimetic materials and other molecules, and combinations thereof. Particularly suitable proteins can be, for example, adhesion proteins. Particularly suitable adhesion proteins can be, for example, fibronectin, cadherin and combinations thereof. Particularly suitable peptides can be, for example, cell adhesion peptides and / or soluble factor binders, as described herein above.

[0055] The elastic modulus, or Young's modulus, measures the hydrogel composition’s resistance to uniform elongation or compression. Elastic modulus quantifies the composition’s ability to return to its original shape after an applied stress, and it is directly related to the material's stiffness. It is common for hydrogels to be soft and highly water-absorbent meaning the elastic modulus can be relatively low compared to more rigid materials like metals or ceramics. Herein, the hydrogel compositions disclosed exhibit a significantly higher elastic modulus than other hydrogels commonly used for similar applications.

[0056] The shear modulus quantifies a hydrogel composition’s resistance to deformation under shear stress, which occurs when forces are applied parallel to the surface. In hydrogels, this is particularly important when considering their behavior under shear forces, such as during compression or bending. The use of material that mimics a distinct microenvironment is of critical importance to understanding the accuracy of biologic function. Specifically, distinct tissue niches including the hepatic, pancreatic, and desmoplastic environments have been demonstrated to drive unique dependencies in biologic signaling. Here, we provide a direct comparison using elastic modulus compared between material sources.

[0057] The inventive hydrogel compositions exhibit significantly higher elastic and shear moduli compared to commonly used hydrogels, such as Cultrex. For example, SP139 75% (4.20 ± 0.64 kPa elastic modulus) and SP139 50% (1.14 ± 0.13 kPa elastic modulus) show greater stiffness and resistance to deformation compared to Cultrex (0.07 ± 0.006 kPa elastic modulus) (FIG. 4). This indicates that the inventive compositions provide enhanced mechanical stability, which is crucial for applications requiring structural integrity and physiological relevance to gastrointestinal tissue microenvironments.

[0058] To calculate the shear modulus (G), we use the relationship G = E I [2(1 + y)], where E / .s the elastic modulus and v is the Poisson’s ratio. For hydrogels, v is typically assumed to be approximately 0.5, reflecting their nearly incompressible nature due to high water content (Javanmardi et al., 2021).

[0059] Additionally, the rheological data for G' (storage modulus) and G" (loss modulus) from strain sweeps at a fixed frequency of 1 Hz confirm the gel-like nature and enhanced mechanical properties of these hydrogel compositions. These results underscore the importance of mechanical stability for their intended applications. For hydrogel compositions herein, these elastic and shear moduli may change with the water content, crosslinking density, or external environmental factors. Understanding the relationship between these moduli is crucial for designing hydrogel compositions for applications such as drug delivery, tissue engineering, or sensors, where mechanical properties like stiffness and shear resistance are critical for performance.

[0060] Suitably, the hydrogel compositions of the present disclosure include combinations of cell adhesion peptides and soluble factor binders that are suspected of binding or interacting with a cell to affect cell attachment, spreading, migration, maturation, proliferation, differentiation, and formation of cellular structures (e.g., organoids).

[0061] In another aspect, a patterned hydrogel array can be further assembled with a microarray add-on whereby the patterned hydrogel array is prepared with dimensions to accommodate addons of any size. Suitable microarray add-ons are commercially available (Grace Bio Labs, Bend, OR). A microarray add-on can allow for the isolation of an individual hydrogel spot and hydrogel- free pool of the hydrogel such that soluble factor presentation can be controlled. As used herein, "hydrogel-free" refers to a portion of substrate that is substantially free, and even completely free of hydrogel. The microarray add-on can include the same number of openings as the number of individual hydrogel spots and hydrogel-free pools of the hydrogel such that each hydrogel spot and hydrogel-free pool can be independently interrogated with soluble factor presentation. Alternatively, the microarray add-on can have larger openings that can accommodate more than one individual hydrogel spot and more than one individual hydrogel-free pool. For example, a microarray add-on can have openings large enough to accommodate a single hydrogel spot or a single hydrogel-free pool.

[0062] Methods of Using the Hydrogel Compositions

[0063] In another aspect, the present disclosure is directed to methods of using the hydrogel compositions to promote cellular expansion, maturation and cellular differentiation. In some embodiments, the hydrogel compositions can be used for therapeutic drug screening. Generally, the methods include preparing the hydrogel compositions; contacting a cell with the hydrogel compositions; and culturing the cells. The hydrogel compositions are prepared as described above and typically include a polymer (e.g., a polyethylene glycol functionalized with norbomene), a multifunctional polymer crosslinker (e.g., MMP-degradable crosslinking peptide, non-degradable PEG-dithiol crosslinker), and a cell adhesion peptide as described more fully above. The method further includes contacting a cell with the hydrogel composition. As used herein, “contacting a cell” refers to seeding the cells with the purpose of culturing the cells. As known by those skilled in the art a cell suspension is typically transferred to a substrate and cells are given sufficient time to adhere to the substrate.

[0064] In another embodiment, cells can be incorporated into the hydrogel of the hydrogel compositions using a hydrogel precursor solution that includes the polymer, the crosslinker, the cell adhesion peptide, and the cell.

[0065] The cells are then cultured for a desired time such as, for example, about one hour to about 30 days. After the desired time, cells can be analyzed by microscopy such as, for example, immunofluorescence microscopy, phase contrast microscopy, light microscopy, electron microscopy and combinations thereof. Cells can be analyzed for cell attachment, cell spreading, cell morphology, cell proliferation, cell migration, cell expansion, cell differentiation, protein expression, cell-to-cell contact formation, sprouting, tubulogenesis, formation of structures, and combinations thereof.

[0066] Suitable cells can be any cell known by those skilled in the art. Particularly suitable cells can include, for example, a cancer cell, a hepatocyte, a pancreatic beta cell, a pancreatic islet cell, pancreatic cancer cell, colon cancer cell, liver cancer cell, breast cancer cell, brain cancer cell, a cancer cell spheroid, a cancer organoid, a tumor fragment, a biopsy fragment, a patient-derived tumor model, and metastatic tumor and combinations thereof.

[0067] In one particular aspect, the cell is a pancreatic cancer cell. When used with a pancreatic cancer cell, the hydrogel compositions include 8-arm, 20 kDa poly(ethylene glycol) (PEG) functionalized with norbomene, a MMP degradable crosslinking peptide, a non-degradable crosslinking peptide, a photoinitiator, and a cell adhesion peptide. Particularly suitable cell adhesion peptides include immobilized RGD-containing peptides, including CRGDS (SEQ ID NO:2), Acetylated-GCYGRGDSPG (SEQ ID NO:31); cyclic {RGD(Fd)C} (SEQ ID NO:33); CRGD-(G)13-PHSRN (SEQ ID NO:29); and CPHSRN-(SG)5-RGD (SEQ ID NO:30). Suitably, the hydrogel compositions include at least about 1 mM cell adhesion peptide, including from about 1 mM to about 4 mM cell adhesion peptide. Further, the hydrogel compositions may include from about 20 mg / mL to about 100 mg / mL PEG concentration. The non-degradable crosslinking peptide is a non-degradable PEG-dithiol crosslinker. And, the photoinitiator is lithium phenyl- 2,4,6-trimethylbenzoylphosphinate (LAP).

[0068] It has been found that as the hydrogel composition provides differential expression of genes involved in cancer resistance including cytochrome p450 expression and expression markers involving in MAP kinase signaling, hallmark pathways involving in therapeutic resistance across gastrointestinal cancers.

[0069] As this material provides upregulation in regulatory pathways of MAP kinase, it could be adapted to discovery methods for therapeutic drug screening. Given unique impact on the expression of p450 enzymes, this material could be adapted for screening applications for hepatocyte toxicity assays.

[0070] In some aspects, the hydrogel compositions are prepared to include crosslinking to an extent of at least 35%, including at least 45%, and including from about 35% to about 75%, and including from about 45% to about 50%.

[0071] Suitably, the hydrogel compositions for use with pancreatic cancer cells include a shear modulus in the range of greater than 3 kPa, including from about 3 kPa to about 12 kPa. This is aligned with the physical properties of common tissues including the microenvironments of liver and the desmoplastic stroma, both key components of pancreatic cancer.

[0072] The method may further include contacting the cell with a soluble molecule by including the soluble molecule in the culture medium in which the cells are cultured. Particularly suitable soluble molecules can be growth factors and proteoglycans. Suitable growth factors can be, for example, proteins from the transforming growth factor beta superfamily, fibroblast growth factor family of growth factors, platelet derived growth factor family of growth factors and combinations thereof. Particularly suitable growth factors can be, for example, vascular endothelial growth factor, bone morphogenetic proteins, fibroblast growth factor, insulin-like growth factor and combinations thereof. Suitable proteoglycans and be, for example, proteoglycans with heparin, heparin sulfate, and / or chondroitin glycosaminoglycan side chains.

[0073] The disclosure will be more fully understood upon consideration of the following nonlimiting Examples. The utility of a synthetic defined hydrogel includes maintenance of cultures in fully defined conditions. This provides feasibility to propagate cultures without animal derived contributions to limit off target immunogenicity and cross reactivity for downstream molecular biologic assays. Batch-to-batch variation between alternative matrices and animal derived serum remains a critical uncontrolled component in tissue culture assays.

[0074] Additional Definitions

[0075] The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and / or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements.

[0076] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.” As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0077] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0078] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0079] The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims. EXAMPLES

[0080] Material Design

[0081] The properties of hydrogel can be functionally modulated to optimize physical properties, including cell plate adhesion, physical matrix stiffness (elastic modulus), and applications for combined mechanical and enzymatic digestion. In this Example, a panel of five independent hydrogel formulations was prepared to evaluate optimized properties. This included cross titration of cross linker contributions and non-degradable polyethylene glycol (PEG-DT). These materials could be produced with appropriate Ellman’s QC as outlined.

[0082] Table 2. Overview of hydrogel materials cross titrated by cross linker utilized in organoid generation Method of Propagation

[0083] A method was generated for serial passaging of organoids to compare between individual hydrogel materials (see FIG. 1). Technical details provided include the concentration of hydrogel contents (% v / v). Photoactivation was achieved using Lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP) 1.875mM in bright light wavelength 395 nm for 2 minutes. The durability of hydrogel formation was evaluated every 72 hours and digested using traditional methods with collagenase (Sigma- Aldrich) Img / mL, Dispase II (Sigma-Aldrich) 0.125mg / mL, and ROCK inhibitor Y-27632 lOpM (MedChemExpress).

[0084] Material Digestion

[0085] The physical properties were subsequently evaluated for the success of mechanical digestion (FIG. 2). Increasing concentration of PEG-DT >50% v / v was found to predict failure of enzymatic digestion using traditional methods (conditions 4 & 5 in FIG. 2). Digestion was achieved in a timeline appropriate for downstream cell culture applications (<1 hour) in conditions 1, 2, and SP139.

[0086] Durability of Hydrogel in Culture

[0087] Next, the durability in hydrogel formation was evaluated across time. Formulations 1 & 2 were found to have loss of adhesion at the droplet-plate interface. This was visualized between 72 and 144 hours which was a functional barrier to visual alignment for organoid growth evaluation (FIG. 3).

[0088] Evaluation by Rheometry

[0089] To measure bulk biomechanical properties of SP139 hydrogels, 75% (v / v) and 50% (v / v) hydrogels were mixed with LAP (1 ,875mM) and base media mixture. The mixture was loaded into a 12 mm (diameter) x 2 mm (thickness) plastic round washer to form cylinder shape matrix gels. The matrix was moved to a 12-well plate, and the Light activated for 120 seconds using Waveform lighting, 395 nm LED (without lid) and keeping the light at the center of the plate at 2-3 cm above and washed the dome using warm PBS twice and added ImL growth media in the 12-well plate. The plate was incubated for 24-72 hours in a humidified incubator at 37C, 95% air, and 5% CO2 to allow equilibrium swelling to occur before rheological characterization with the preparation of hydrogel discs of 1.5 mm.

[0090] For rheometry, the discs were placed between an 8 mm sandblasted parallel plate geometry and sandblasted base. The shear modulus was determined by performing small-strain oscillatory shear measurements on an ARES Rheometer. The mechanical response was recorded by performing dynamic strain sweep test (0.001%-100%) at a constant frequency (1Hz) at 25°C. The elastic modulus (E) is reported as a measure of bulk matrix mechanical properties derived from the recorded storage modulus (GO) in the linear elastic regime.

[0091] SP139 had increased elastic modulus when compared to Cultrex materials (FIG. 4). The proportion of SP139 (v / v) relative to carrier media enhanced this physical property at >4 kPa at 75% v / v when compared to Culture at 100% v / v (p<0.001).

[0092] Comparison of Organoid Growth Parameters

[0093] All materials were compared for growth profiling across time and in comparison to reduced growth factor CultrexBME (Trevigene; AMS Biotechnology). The growth of individual organoids was maintained across time points in this defined hydrogel material (FIG. 5).

[0094] Growth and viability was confirmed across a diversity of cancer types. Representative cultures included patient-derived cancer organoids of pancreatic ductal adenocarcinoma (PAN21 & PAN33), gastric neuroendocrine carcinoma (NET26), intrahepatic cholangiocarcinoma (CCA), as well as murine models of pancreatic ductal adenocarcinoma (KPC) (see FIG. 6).

[0095] The impact of growth across time was compared after expanded culture at passage 4. It was seen that SP139 and Cultrex both had growth over the timecourse imaging (FIG. 7A). Organoid growth is sustained and remains consistent across multiple passages with SP139 (FIG. 7B). Organoids grown in Cultrex have consistent increase in diameter, and SP139 has nonsignificant change in growth across time (FIG. 7C).

[0096] Growth was also qualitatively compared between organoid cultures. Cultrex induced heterogeneity in organoid growth including both solid and cystic organoid populations as previously described. SP139 matrix material produced extensions from the initial plated organoid culture including release of multiple single cellular populations surrounding the initial plated organoid (FIGS. 8A & 8B).

[0097] This material was also compared for traditional well level assays of cellular viability (see FIG. 9), utilizing 3D Cell Titer Gio 3D (CTG, Promega, Inc). Shown is the representative response to organoids between SP139 and Cultrex as assayed to media control at 48 hours. This is shown as proof of concept for signal to noise ratios achieved with this material integrating a positive control of excessive chemotherapy.

[0098] The impact of cancer signaling was subsequently analyzed using RNA sequencing analysis (RNAseq). Briefly, RNA was extracted using the Qiagen RNease Plus Mini Kit with quality control verified using NanoDrop and Tapestation. The library was prepared using the KAPA mRNA HyperPrep (Roche) with sequencing performed at the University of Wisconsin Center for Human Genomics and Precision Medicine using the NextSeqlOOO (Illumina). Statistical calculations were performed using custom Rmarkdown scripts. The RSEM estimated counts were generated across all samples then mapped to corresponding gene symbols. Shown in FIG. 10 is PCA plot comparing fresh tumor to varied matrix environments. Relative to Cultrex, SP139 causes down regulation of epoxygenase p450, upregulation in genes that negatively regulate the p38 MAPK pathway, and in MAPK pathways through dual specificity phosphates (DPS) (FIG. 11).

[0099] The impact on common enzymatic function in hepatocytes was evaluated using cell luminescence assays by p450-GLO CYP3A4 (p450-GLO, Promega, Inc). Compared to ovarian cancer control, normal liver organoids (NL-141 and NL-158) exhibited significantly higher CYP3A4 expression in the SP139 matrix compared to Cultrex. This observation suggests improved hepatocyte function of drug metabolism, where cytochrome P450 enzymes, particularly CYP3A4, play a central role [1], Liver organoids, derived from hepatocytes, retain key functional characteristics of liver tissue, including the expression of cytochrome P450 enzymes, which are critical for the metabolism of endogenous and exogenous compounds. Matrix stiffness, a hallmark of pancreatic and GI cancers, is known to regulate cellular behavior through mechanotransduction pathways, including YAP / TAZ and integrin signaling, which can impact the expression of drugmetabolizing enzymes like CYP3A4 [2, 3], These mechanotransduction pathways have also been linked to alterations in drug metabolism and resistance, specifically CYP3A4 expression[3]. The increased CYP3A4 expression observed in SP139, compared to Cultrex — a less stiff matrix with exogenous growth factors — further supports the role of mechanical properties in modulating hepatocyte function (FIG. 12).

[0100] Summary

[0101] Functionalized PEG-based synthetic hydrogel matrix materials can be tuned to more accurately mimic cancer tissue microenvironments. SP139, designed to mimic the physical properties of the pancreatic and hepatic microenvironment, has favorable properties including controlled growth rates and durability in passaging for patient-derived cancer organoids. Synthetic hydrogel compositions have compatibility with high content imaging by luminescence and fluorescence. Initial RNA expression data shows distinct expression profding when compared to commercially available materials. Comparing the distinct line specific sensitivities as a function of matrix environment could be used to discover niche specific therapeutic vulnerabilities.

[0102] REFERENCES lavanmardi, Y., Colin-York, H., Szita, N., et al. Quantifying cell-generated forces: Poisso 's ratio matters. Commun Phys, 2021. 4(237).

[0103] Takayama, K., et al., 3D spheroid culture of hESC / hiP SC -derived hepatocyte-like cells for drug toxicity testing. Biomaterials, 2013. 34(7): p. 1781-9.

[0104] Walker, C., E. Mojares, and A. Del Rio Hernandez, Role of Extracellular Matrix in Development and Cancer Progression. Int J Mol Sci, 2018. 19(10).

[0105] Ozkan, A., et al., Tumor Microenvironment Alters Chemoresistance of Hepatocellular Carcinoma Through CYP 3 A4 Metabolic Activity. Frontiers in Oncology, 2021. 11.

Claims

CLAIMSWhat is claimed:

1. A hydrogel composition comprising: a polyethylene glycol functionalized with norbomene, a degradable crosslinking peptide, a non-degradable crosslinking peptide, a cell adhesion peptide and a photoinitiator.

2. The hydrogel composition as set forth in claim 1, wherein the polyethylene glycol functionalized with norbornene comprises an 8-arm, 20 kDa polyethylene glycol functionalized with norbornene.

3. The hydrogel composition as set forth in claim 2, wherein the composition comprises from about 20 mg / ml to about 80 mg / ml of the 8-arm, 20 kDa polyethylene glycol functionalized with norbomene.

4. The hydrogel composition as set forth in claim 2, wherein the composition comprises about 80 mg / ml 8-arm, 20 kDa polyethylene glycol functionalized with norbomene.

5. The hydrogel composition as set forth in any one of claims 1-4, wherein the composition comprises the degradable crosslinking peptide and the non-degradable crosslinking peptide in a ratio of degradable crosslinking peptidemon-degradable crosslinking peptide about 4: 1.

6. The hydrogel composition as set forth in claim 5, wherein the degradable crosslinking peptide is a matrix metalloproteinase (MMP)-degradable crosslinker selected form the group consisting of KCGGPQGIWGQGCK (SEQ ID NO:27) and KCGGPQGIAGQGCK (SEQ ID NO:28).

7. The hydrogel composition as set forth in claim 5, wherein the non-degradable crosslinking peptide is a non-degradable PEG-dithiol crosslinker.

8. The hydrogel composition as set forth in any one of claims 1-7, wherein the cell adhesion peptide is selected from the group consisting of CRGDS (SEQ ID NO: 2), Acetylated- GCYGRGDSPG (SEQ ID NO:31), cyclic RGD, CRGD-(G)13-PHSRN (SEQ ID NO:29), and CPHSRN-(SG)5-RGD (SEQ ID NO:30).

9. The hydrogel composition as set forth in any one of claims 1-8, wherein the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).

10. The hydrogel composition as set forth in any one of claims 1-9, further comprising an additional agent selected from the group consisting of collagen, fibronectin, hyaluronic acid, and combinations thereof.

11. A method of preparing an organoid culture, the method comprising:(a) preparing a hydrogel composition, wherein the hydrogel composition comprises a polyethylene glycol functionalized with norbomene, a degradable crosslinking peptide, a non- degradable crosslinking peptide, a cell adhesion peptide and a photoinitiator;(b) contacting a cell with the hydrogel composition; and(c) culturing the cell to prepare the organoid culture.

12. The method as set forth in claim 11, wherein the polyethylene glycol functionalized with norbomene comprises an 8-arm, 20 kDa polyethylene glycol functionalized with norbomene.

13. The method as set forth in claim 11 or 12, wherein the degradable crosslinking peptide and the non-degradable crosslinking peptide are present in the hydrogel composition in a ratio of degradable crosslinking peptidemon-degradable crosslinking peptide about 4: 1.

14. The method as set forth in claim 13, wherein the degradable crosslinking peptide is a matrix metalloproteinase (MMP)-degradable crosslinker selected form the group consisting of KCGGPQGIWGQGCK (SEQ ID NO:27) and KCGGPQGIAGQGCK (SEQ ID NO:28).

15. The method as set forth in claim 13, wherein the non-degradable crosslinking peptide is a non-degradable PEG-dithiol crosslinker.

16. The method as set forth in any one of claims 11-15, wherein the cell adhesion peptide is selected from the group consisting of CRGDS (SEQ ID NO: 2), Acetylated-GCYGRGDSPG (SEQ ID NO:31), cyclic RGD, CRGD-(G)13-PHSRN (SEQ ID NO:29), and CPHSRN-(SG)5-RGD (SEQ ID NO:30).

17. The method as set forth in any one of claims 11-16, wherein the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).

18. The method as set forth in any one of claims 11-17, wherein the cell is a cancer cell selected from the group consisting of pancreatic cancer cell, colon cancer cell, liver cancer cell, breast cancer cell, brain cancer cell, and metastatic tumor.

19. The method of claim 11, wherein the polyethylene glycol functionalized with norbomene is a 8-arm, 20 kDa polyethylene glycol functionalized with norbornene at between 20 mg / mL and 80 mg / mL, wherein the degradable crosslinking peptide is a matrix metalloproteinase (MMP)- degradable crosslinker, wherein the non-degradable crosslinking peptide is a non-degradable PEG- dithiol crosslinker, wherein the degradable crosslinking peptide and the non-degradable crosslinking peptide are present in the hydrogel composition in a ratio of degradable crosslinking peptide:non-degradable crosslinking peptide about 4: 1, wherein the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and wherein the cell is a cancer cell selected from the group consisting of pancreatic cancer cell, colon cancer cell, liver cancer cell, breast cancer cell, brain cancer cell, and metastatic tumor.

20. A method of promoting cellular differentiation or promoting cellular expansion, the method comprising:(a) preparing a hydrogel composition of any one of claims 1-10;(b) contacting a cell with the hydrogel composition; and(c) culturing the cell to promote cellular differentiation or cellular expansion.

21. The method as set forth in claim 20, wherein the polyethylene glycol functionalized with norbornene comprises an 8-arm, 20 kDa polyethylene glycol functionalized with norbornene.

22. The method as set forth in claim 20 or 21, wherein the degradable crosslinking peptide and the non-degradable crosslinking peptide in a ratio of degradable crosslinking peptidemon- degradable crosslinking peptide about 4:1.

23. The method as set forth in claim 22, wherein the degradable crosslinking peptide is a matrix metalloproteinase (MMP)-degradable crosslinker selected form the group consisting of KCGGPQGIWGQGCK (SEQ ID NO:27) and KCGGPQGIAGQGCK (SEQ ID NO 28).

24. The method as set forth in claim 22, wherein the non-degradable crosslinking peptide is a non-degradable PEG-dithiol crosslinker.

25. The method as set forth in any one of claims 20-24, wherein the cell adhesion peptide is selected from the group consisting of CRGDS (SEQ ID NO: 2), Acetylated-GCYGRGDSPG (SEQ ID NO:31), cyclic RGD, CRGD-(G)13-PHSRN (SEQ ID NO:29), and CPHSRN-(SG)5-RGD (SEQ ID NO:30).

26. The method as set forth in any one of claims 20-25, wherein the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).

27. A method of screening an agent for a therapeutic effect, the method comprising:(a) preparing a hydrogel composition, wherein the hydrogel composition comprises a polyethylene glycol functionalized with norbomene, a degradable crosslinking peptide, a non- degradable crosslinking peptide, a cell adhesion peptide and a photoinitiator;(b) contacting one or more cells with the hydrogel composition;(c) culturing the one or more cells into an organoid ;(d) applying the agent to the organoid;(e) measuring at least one property of the organoid, wherein the property is selected from the group consisting of growth, cell viability, protein expression, cellular signaling and / or gene expression; and(f) identifying changes in growth, cell viability, and / or gene expression after application of the agent in step (d) determine if the agent has a therapeutic effect.

28. The method of claim 27, wherein growth of the organoids is measured by change in diameter of the organoid over time as compared to a control organoid to which the agent was not applied.

29. The method of claim 27, wherein cell viability of the cells in the organoid is measured by quantifying live and dead cells within the organoid as compared to a control organoid to which the agent was not applied.

30. The method of claim 27, wherein gene expression of the cells in the organoid is measured by RNA sequencing, in situ hybridization, and / or RT-qPCR as compared to a control organoid to which the agent was not applied.31 . The method of claim 27, wherein the cell type of the one or more cells is selected from the group consisting of pancreatic cancer cell, colon cancer cell, liver cancer cell, breast cancer cell, brain cancer cell, and metastatic tumor cell.

32. An organoid made by the method of any one of claims 11-19.

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