EP4791452A1

EP4791452A1 - Chitosan-based hydrogels for 3d cell culture

Info

Publication number: EP4791452A1
Application number: EP24794546.2A
Authority: EP
Inventors: Audrey Blanche BERGERON; Pandiyarajan Chinnayan Kannan
Original assignee: Corning Inc
Current assignee: Corning Inc
Priority date: 2023-10-12
Filing date: 2024-10-01
Publication date: 2026-08-19
Also published as: WO2025080462A1; CN122003256A

Abstract

Compositions for chitosan-based synthetic hydrogels and methods of their use are provided. The chitosan-based hydrogels comprise a functionalized chitosan, a multi-arm polyethylene glycol vinyl sulfone, an RGD peptide, and a VPM peptide. The chitosan-based synthetic hydrogels can be employed in three-dimensional cell cultures and can be dissolved. These chitosan-based synthetic hydrogels are advantageous at least because they have similar mechanical properties to non-synthetic Matrigel^® but are well defined.

Description

CHITOSAN-BASED HYDROGELS FOR 3D CELL CULTURE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. §119 of U.S. Provisional Application Serial No. 63/543,808 filed on October 12, 2023, the content of which is relied upon and incorporated herein by reference in its entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing having file name Sequence_Listing_SP23-255.xml (11,704 bytes), created on September 10, 2024, is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0003] The present disclosure is related to synthetic chitosan-based polymeric hydrogels, methods of manufacturing synthetic chitosan-based polymeric hydrogels, methods of using such hydrogels for biomolecular analysis, and kits for such hydrogels, in particular for advanced three- dimensional cell cultures such as spheroids and organoids.

BACKGROUND OF THE INVENTION

[0004] Organoids are three-dimensional, cell-based in vitro models of corresponding in vivo organs having many of the in vivo organ’s structural and functional aspects. An organoid’s resemblance to native organs can play a vital role in medical applications such as preclinical drug development, toxicity screening, regenerative medicine and studying the mechanism of organ development. Organoids can be cultured using hydrogels as a scaffold to support cell growth, proliferation, and differentiation. Hydrogels generally contain hydrophilic homo-/co-polymers crosslinked physically or chemically using natural, synthetic or hybrid polymers. Hydrogels can be cast into different shapes or sizes and have networks properties such as swelling, stiffness, permeability, porosity. These network property attributes can have profound effect on cell activity and on cell fate.

[0005] A natural basement membrane Matrigel® derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma has been the most used scaffold to support 3D organoids culture. Matrigel® closely mimics the natural extracellular matrix (ECM) incorporating hundreds of proteins (>1800) including laminin (-60%), collagen IV (-30%), enactin (-8%) and the heparin sulfate proteoglycan perlecan (-2-3%). It undergoes gelation above 20°C during which the enactin acts as a crosslinker that connects laminin and collagen resulting in a gel. Although Matrigel® is the most preferred hydrogel for organoid cultures, lot-to-lot variations in its protein concentrations cause a lack of control over its network properties (e.g., stiffness). This leads to inconsistent cell culture results, which is problematic.

[0006] Accordingly, a need exists for synthetic hydrogels that are chemically and mechanically well-defined and yet still produce advanced three-dimensional cellular structures, such as organoids.

SUMMARY OF THE DISCLOSURE

[0007] The present disclosure provides compositions for chitosan-based synthetic hydrogels with mechanical properties similar to animal-derived Matrigel®, methods of making chitosan-based synthetic hydrogels, methods of using chitosan-based synthetic hydrogels, and kits to make chitosan-based synthetic hydrogels.

[0008] According to some aspects of the present disclosure, a composition for a synthetic hydrogel is provided, comprising chitosan-thioglycolic acid, multi-armed polyethylene glycol having a vinyl sulfone moiety, a first peptide comprising an Arginine-Glycine-Aspartic Acid (RGD) amino acid sequence and a cysteine amino acid at or near a terminal end of the peptide, and a second peptide comprising a Valine-Proline-Methionine (VPM) amino acid sequence and a cysteine amino acid at or near each terminal end of the peptide. The chitosan-thioglycolic acid of the synthetic hydrogel may have a molecular weight from about 50 kDa to about 200 kDa. The multi-armed polyethylene glycol vinyl sulfone of the synthetic hydrogel may be a four-arm polyethylene glycol vinyl sulfone and may further have an average molecular weight from about 2 kDa to about 20 kDa. The first peptide may be chosen from GRGDSPC (SEQ ID NO: 1), GRGDSPCx (SEQ ID NO: 2), or RGDC (SEQ ID NO: 3), wherein “x” is chosen from glycine (G), alanine (A), leucine (L), valine (V) serine (S), threonine (S), aspartic acid (D), or glutamic acid (E). The second peptide may be chosen from GCRDVPMSMRGGDRCG (SEQ ID NO: 4), xCRDVPMSMRGGDRCx (SEQ ID NO: 5), CRDVPMSMRGGDRC (SEQ ID NO: 6), or CRDVPMSMRGGDRCG (SEQ ID NO: 7), CRDVPMSMRGGDRCx (SEQ ID NO: 8), GCRDVPMSMRGGDRC (SEQ ID NO: 9), or xCRDVPMSMRGGDRC (SEQ ID NO: 10), wherein “x” is chosen from alanine (A), valine (V) leucine (L), serine (S), threonine (T), aspartic acid (D), or glutamic acid (E), and wherein one “x” may be glycine (G). The synthetic hydrogel may have a storage modulus of between about 125 Pa to about 175 Pa. The synthetic hydrogel may have a storage modulus of between about 25 Pa to about 55 Pa after swelling in an aqueous solution for about 4.5 days.

[0009] In one specific embodiment of this aspect, the first peptide is GRGDSPC (SEQ ID NO: 1), the second peptide is GCRDVPMSMRGGDRCG (SEQ ID NO: 4), and the multi-armed polyethylene glycol vinyl sulfone is a four-arm polyethylene glycol vinyl sulfone having an average molecular weight of about 20 kDa.

[0010] According to another aspect of the present disclosure, a method for making a synthetic hydrogel is provided, comprising the steps of: (a) providing a thiolated chitosan, (b) providing a multi-armed polyethylene glycol vinyl sulfone, (c) providing a first peptide comprising an Arginine- Glycine- Aspartic Acid amino acid sequence and a cysteine amino acid at or near a terminal end of the peptide, (d) providing a second peptide comprising a Valine-Proline- Methionine amino acid sequence and a cysteine amino acid at or near each terminal end of the peptide, (e) mixing the thiolated chitosan and multi-armed polyethene glycol vinyl sulfone, (f) adding the first peptide to the mixture of step (d), and (g) adding the second peptide to the mixture of step (f). However, the steps may be performed in other orders. The thiolated chitosan may be chitosan-thioglycolic acid, and the chitosan-thioglycolic acid may have a molecular weight from about 50 kDa to about 200 kDa. The multi-armed polyethylene glycol vinyl sulfone may be a four- arm polyethylene glycol vinyl sulfone and the four-arm polyethylene glycol vinyl sulfone further may have an average molecular weight from about 2 kDa to about 20 kDa. The first peptide may be chosen from GRGDSPC (SEQ ID NO: 1), GRGDSPCx (SEQ ID NO: 2), or RGDC (SEQ ID NO: 3), wherein “x” is chosen from glycine (G), alanine (A), leucine (L), valine (V), serine (S), threonine (S), aspartic acid (D), or glutamic acid (E). The second peptide may be chosen from GCRDVPMSMRGGDRCG (SEQ ID NO: 4), xCRDVPMSMRGGDRCx (SEQ ID NO: 5), CRDVPMSMRGGDRC (SEQ ID NO: 6), or CRDVPMSMRGGDRCG (SEQ ID NO: 7), CRDVPMSMRGGDRCx (SEQ ID NO: 8), GCRDVPMSMRGGDRC (SEQ ID NO: 9), or xCRDVPMSMRGGDRC (SEQ ID NO: 10), wherein “x” is chosen from alanine (A), leucine (L), serine (S), threonine (T), aspartic acid (D), or glutamic acid (E), and wherein one “x” may be glycine (G).

[0011] In one specific embodiment of this aspect, the first peptide is GRGDSPC (SEQ ID NO: 1), the second peptide is GCRDVPMSMRGGDRCG (SEQ ID NO: 4), and the multi-armed polyethylene glycol vinyl sulfone is a four-arm polyethylene glycol vinyl sulfone having an average molecular weight of about 20 kDa.

[0012] According to other aspects of the present disclosure, a method of dissolving a synthetic hydrogel is provided, comprising the steps of (a) providing a synthetic hydrogel comprising cells cultured in the synthetic hydrogel, wherein the synthetic hydrogel comprises any of the compositions described above, (b) adding to the synthetic hydrogel at least one enzyme that cleaves the second peptide at the Valine-Proline Methionine amino acid sequence, and (c) incubating the resultant combination from steps (a) and (b). The enzyme that cleaves the second peptide may be chosen from collagenase, dispase, protease from Streptomyces griseus, or trypsinsubstitute enzyme (for example, Accutase®), or a combination thereof. The addition of volume of the hydrogel to the volume of the enzyme that cleaves the second peptide may be at a ratio of about 1 :4 for trypsin-substitute enzyme, about 1:3 for collagenase, about 1:3 for dispase, and about 1 :3 for protease from Streptomyces griseus, and the concentration of the enzyme may be about 10 mg/mL. In one specific embodiment of this aspect, the enzyme that cleaves the second peptide may be one of collagenase- 1 from Clostridium histolyticum, dispase from Bacillus polymyxa, or protease from Streptomyces griseus, and the incubation may dissolve the synthetic hydrogel after about 25 minutes at an incubation temperature of 37° C.

[0013] According to still other aspects of the present disclosure, a method of growing a three-dimensional cell culture is provided, comprising the steps of: (a) providing a chitosan-PEG- VS-RGD conjugate, (b) providing a VPM peptide, (c) providing cells of a cell type, (d) combining the conjugate of step (a) with the peptide of step (b) and with the cell type of step (c), (e) dispensing a discrete volume of the combined precursor hydrogel from step (d) onto a substrate, (f) forming a hydrogel from the dispensed discrete volume, (g) surrounding the formed hydrogel with a discrete volume of cell culture medium, and (h) incubating the surrounded hydrogel. However, the steps may be performed in other orders. The chitosan-PEG-VS-RGD conjugate may comprise chitosan-thioglycolic acid, four-arm polyethylene glycol vinyl sulfone, and an amino acid sequence comprising Arginine- Glycine- Aspartic Acid. The amino acid sequence comprising Arginine-Glycine- Aspartic Acid may be GRGDSPC (SEQ ID NO: 1) in its unconjugated form and the VPM peptide may be GCRDVPMSMRGGDRCG (SEQ ID NO: 4). The cells may be cells that form spheroids or organoids. In a specific embodiment of this aspect, the cells may be MDCK cells or Chinese Hamster Ovary cells. The substrate may be a microwell plate. The method of culturing cells may yield a synthetic hydrogel that has not significantly degraded after 15 days of incubation. The method of culturing cells may yield a synthetic hydrogel that has not significantly degraded after 28 days or more after incubation.

[0014] According to other aspects of the present disclosure, a kit including a composition for making synthetic hydrogel is provided. The kit comprises a first container and a second container, wherein the first container comprises a chitosan-PEG-VS-RGD conjugate, and the second container comprises a VPM peptide. The chitosan-PEG-VS-RGD conjugate may comprise a chitosan-thioglycolic acid, a multi-arm polyethylene glycol vinyl sulfone having between 3 and 6 arms, and an amino acid sequence comprising Arginine- Glycine -Aspartic Acid. The amino acid sequence comprising Arginine-Glycine- Aspartic Acid may be GRGDSPC (SEQ ID NO: 1). The VPM peptide may be GCRDVPMSMRGGDRCG (SEQ ID NO: 4). The first and second containers may be vials.

[0015] Additional features and advantages will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the embodiments as described herein, including the detailed description which follows, the claims, as well as the appended drawings.

[0016] It is to be understood that both the foregoing general description and the following detailed description are merely exemplary, and are intended to provide an overview or framework to understanding the nature and character of the claims. The accompanying drawings are included to provide a further understanding, and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiment(s), and together with the description serve to explain principles and operation of the various embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The following is a description of the figures in the accompanying drawings, given purely by way of non-limiting example. The figures are not necessarily to scale, and certain features and certain views of the figures may be shown exaggerated in scale or in schematic in the interest of clarity and conciseness.

[0018] FIG. 1 is a is a general reaction schematic of chitosan-based hydrogel synthesis according to some aspects of the present disclosure.

[0019] FIGS. 2A-2D are plots of swelling ratios and solvent fractions from chitosan-based hydrogels prepared according to some aspects of the present disclosure. FIG. 2A is a plot of the average swelling ratio (Q) over time of chitosan-based hydrogels with human intestine organoids cell culture medium. FIG. 2B is a plot of the average swelling ratio (Q) over time of chitosan- based hydrogels with HEPES buffer. FIG. 2C is a plot of the solvent fraction over time of chitosan- based hydrogels with human intestine organoids cell culture medium. FIG. 2D is a plot of the solvent fraction over time of chitosan-based hydrogels with HEPES buffer.

[0020] FIG. 3 is a plot of the storage loss modulus from 0 hours to 120 hours after curing Matrigel®, TrueGel-1, or the chitosan-PEG-RGD-VPM hydrogels in human intestine organoids medium, according to some aspects of the present disclosure.

[0021] FIGS. 4A-4D show optical micrographs at 20x magnification after 6 days of incubation for Matrigel® (FIGS. 4A, 4C) and chitosan-based gels (FIGS. 4B, 4D) grown on either tissue culture treated (FIGS. 4A, 4B) or ultra-low binding (FIGS. 4C, 4D) 24-well plates, according to some aspects of the present disclosure.

[0022] FIGS. 5A-5F show optical images of chitosan-based hydrogels with MDCK cells after 8 days of incubation at 4x magnification (FIGS. 5A, 5D), lOx magnification (FIGS. 5B, 5E), and 20x magnification (FIGS 5C, 5F) in either untreated polystyrene 12-well plates (FIGS. 5A- 5C) or tissue-cultured 24-well plates (FIGS. 5D-5F), according to some aspects of the present disclosure. [0023] FIG. 6 shows a plot of stability data of chitosan-based hydrogels versus Matrigel® hydrogels over a 25 day period after the hydrogels were surrounded by organoids medium, according to some aspects of the present disclosure.

[0024] FIG. 7 shows photographs of chitosan-PEG-RGD-VPM hydrogels, according to aspects of the present disclosure, that dissolved when exposed to the enzymes Accutase® (a trypsin-substitute enzyme), collagenase, dispase, or protease from Streptomyces griseus (Sigma- Aldrich®) respectively, but that did not dissolve when exposed to a non-enzymatic solution (Corning® Cell Recover Solution).

DETAILED DESCRIPTION

[0025] The various aspects and embodiments will now be fully described herein. These aspects and embodiments may, however, be embodied in many different forms and should not be construed as limiting; rather, these embodiments are provided so the disclosure will be thorough and complete, and will fully convey the scope of the present subject matter to those skilled in the art. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0026] Modifications of the disclosure will occur to those skilled in the art and to those who make or use the disclosure. Therefore, it is understood that the embodiments shown in the drawings and described above are merely for illustrative purposes and not intended to limit the scope of the disclosure, which is defined by the following claims, as interpreted according to the principles of patent law, including the doctrine of equivalents.

A. Definitions

[0027] Unless defined otherwise, all terms and phrases used herein include the meanings that the terms and phrases have attained in the art, unless the contrary is clearly indicated or clearly apparent from the context in which the term or phrase is used. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, particular methods and materials are now described. [0028] Unless otherwise stated, the use of individual numerical values are stated as approximations as though the values were preceded by the word “about” or “approximately.” Similarly, the numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word “about” or “approximately.” In this manner, variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. As used herein, the terms “about” and “approximately” when referring to a numerical value shall have their plain and ordinary meanings to a person of ordinary skill in the art to which the disclosed subject matter is most closely related or the art relevant to the range or element at issue. The amount of broadening from the strict numerical boundary depends upon many factors. For example, some of the factors which may be considered include the criticality of the element and/or the effect a given amount of variation will have on the performance of the claimed subject matter, as well as other considerations known to those of skill in the art. As used herein, the use of differing amounts of significant digits for different numerical values is not meant to limit how the use of the words “about” or “approximately” will serve to broaden a particular numerical value or range. Thus, as a general matter, “about” or “approximately” broaden the numerical value. Also, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values plus the broadening of the range afforded by the use of the term “about” or “approximately.” Consequently, 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, and each separate value is incorporated into the specification as if it were individually recited herein.

[0029] As used herein, the term “and/or,” when used in a list of two or more items, means that any one of the listed items can be employed by itself, or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing components A, B, and/or C, the composition can contain A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[0030] As used herein, “has,” “have,” “having,” “include,” “including,” “comprise,” “comprising” or the like are used in their open ended sense, and generally mean “including, but not limited to.” [0031] For purposes of this disclosure, the term “near terminal end” generally means the last residue preceding the N-terminal or C-terminal amino acid (or both) of an amino acid sequence.

[0032] “Optional” or “optionally” means that the subsequently described element, component or circumstance may or may not occur, so that the description includes instances where the element, component, or circumstance occurs and instances where it does not.

[0033] As used herein the terms “the,” “a,” or “an,” mean “at least one,” and should not be limited to “only one” unless explicitly indicated to the contrary. Thus, for example, reference to “a component” includes embodiments having two or more such components unless the context clearly indicates otherwise.

[0034] In this document, relational terms, such as first and second, top and bottom, and the like, are used solely to distinguish one entity or action from another entity or action, without necessarily requiring or implying any actual such relationship or order between such entities or actions.

[0035] All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

B. Introduction

[0036] The present disclosure relates to the field of synthetic hydrogels. It also relates to synthetic hydrogel compositions, methods of making such hydrogel compositions, methods of using such hydrogels, and kits for culturing at least Chinese hamster ovary (CHO) cells, human fibroblasts, endothelial, Madin-Darby canine kidney (MDCK) cells, spheroids, and organoids using such hydrogels. These hydrogels are synthetic and yet provide comparable mechanical characteristics to the animal- derived standard, Matrigel®.

[0037] The hydrogels of the present invention are advantageous as they are synthetic (unlike Matrigel®, which is derived from mouse tumors), they use water soluble precursors that form gels at common reaction temperatures such as room temperature and 37°C (no curing, catalysts, or irradiation is needed), the gels form quickly (for example, 5-30 minutes), they enable 3D dome cell culture of MDCK and organoid cells, they are optically transparent making them suitable for cell-imaging analyses, and they are quickly degradable in multiple enzymatic solutions (for example, degradable with dispase within 30 minutes), which allows for efficient cell recovery. Further, the hydrogels are stable with 3D domes on multiple types of culture plates, including tissue-culture treated, non-treated, and low-binding well plates.

[0038] Additional features and advantages will be set forth in the detailed description which follows and will be apparent to those skilled in the art from the description, or recognized by practicing the embodiments as described in the following description, together with the claims and appended drawings.

C. Compositions of Hydrogels

[0039] As disclosed herein, a new multi-functional chitosan-based hydrogel is provided that incorporates cell-adhesive and hydrogel-degradable peptides for 3D cell culture including organoids culture. The synthetic hydrogel may comprise a thiol-functionalized chitosan (CS-SH), a multi-arm polyethylene glycol vinyl sulfone (for example, PEG-4-VS), a peptide comprising an Arginine-Glycine- Aspartic Acid motif (an RGD peptide) and at least one cysteine amino acid at or near a terminal end of the peptide (for example, GRGDSPC (SEQ ID NO: 1), and a peptide comprising a Valine-Proline-Methionine motif (a VPM peptide) and at least one cystine amino acid at or near each terminal end of the peptide (for example, GCRDVPMSMRGGDRCG (SEQ ID NO: 2). The results of systematic investigations on the hydrogels including swelling behavior, rheological properties, and MDCK cell culture reveal that the hydrogels display strong swelling (for example, can hold up to 98% of water in its swollen matrix) with a stiffness of about 40-160 Pa. In addition, the hydrogels are compatible with 3D dome culture on various culture plates and demonstrate excellent MDCK cyst formation after 2-6 days of culture. The results with the synthetic chitosan-based hydrogel are surprisingly comparable with Matrigel®, presenting a synthetic hydrogel that can be used instead of the naturally derived Matrigel®. The disclosed synthetic chitosan-based hydrogel’s network properties such as stiffness, swelling, permeability, cell adhesion and degradation are tunable (Matrigel® is not), as also described herein. [0040] The thiol-functionalized chitosan, the multi-arm PEG-VS, the RGD peptide, and the VPM peptide of the chitosan-based synthetic hydrogel are described below.

Thiol-Functionalized Chitosan

[0041] Chitosan is a polymer of (1 ->4)-D-glucosamine and 0(1 ->4)- V-acetyl-D- glucosamine units. In one embodiment, thioglycolic acid linked to a chitosan via the amino group at the 2-position of chitosan’s glucosamine subunits results in a thiolated-chitosan referred to as chitosan-thioglycolic acid. This thiol-functionalized chitosan polymer can be synthesized in-house by methods known to those skilled in the art, or alternatively, it can be purchased already functionalized from vendors. In one embodiment the molecular weight of the thiol-functionalized chitosan is a low molecular weight thiol-functionalized chitosan. In another embodiment, the molecular weight of the thiol-functionalized chitosan ranges from about 50 kDa to about 200 kDa. In one specific embodiment, the average molecular weight of the thiol-functionalized chitosan is about 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, or 200 kDa, or at any range or value between about 50 kDa and about 200 kDa.

Polyethylene Glycol Vinyl Sulfone

[0042] Polyethylene glycol vinyl sulfone (PEG- VS) is a derivative of polyethylene glycol (PEG). Several PEG-VS molecules linked together through a branched center creates a multi-arm PEG-VS. For example, covalently linking four PEG-VS molecules with a pentaerythritol core produces a PEG conjugate known as a four-arm PEG-VS (i.e., PEG-4-VS). In one embodiment, the polyethylene glycol vinyl sulfone may be chosen from two-arm PEG-VS (i.e., PEG-2-VS), four-arm PEG-VS (i.e., PEG-4-VS), and eight-arm PEG-VS (i.e., PEG-8-VS). In one specific embodiment, the polyethylene glycol vinyl sulfone is PEG-2-VS. In another specific embodiment, the polyethylene glycol vinyl sulfone is PEG-4-VS. In yet another specific embodiment, the polyethylene glycol vinyl sulfone is PEG-8-VS. These PEG-VS conjugates can be synthesized inhouse by methods known to those skilled in the art, or alternatively, they can be purchased already functionalized from vendors such as Sigma Aldrich®. In one embodiment, the molecular weight of the polyethylene glycol (including two-arm, four-arm, and eight-arm PEG) ranges from about 2 kDa to about 20 kDa. In another embodiment, the average molecular weight of the PEG-VS conjugate (including two-arm, four-arm, and eightarm PEG) is from about 2 kDa to about 20 kDa. In one specific embodiment, the average molecular weight of the PEG-VS conjugate (including two-arm, four-arm, and eight-arm PEG) is about 2 kDa, 5 kDa, 7.5 kDa, 10 kDa, 12.5 kDa, 15 kDa, 17.5 kDa, or 20 kDa. In one specific embodiment, the average molecular weight of the PEG-VS conjugate (including two-arm, four-arm, and eightarm PEG-VS) is from about 2 kDa to about 7.5 kDa, from about 7.5 kDa to about 15 kDa, from about 15 kDa to about 25 kDa. In another specific embodiment, the average molecular weight of the two-arm PEG-VS is about 2 kDa, 3.5 kDa, or about 5 kDa. In another specific embodiment, the average molecular weight of the four-arm PEG-VS is about 2 kDa, 5 kDa, 10 kDa or about 20 kDa. In yet another specific embodiment, the average molecular weight of the eight-arm PEG-VS is about 5 kDa, 10 kDa or about 20 kDa.

RGD Peptide

[0043] Peptides comprising an arginine-glycine-aspartic acid amino acid motif (Arg-Gly- Asp) are referred to as RGD peptides. The RGD motif is a type of cell attachment motif, motifs that promote the binding of cells. In one aspect of the invention, the RGD peptides can be conjugated with two-arm, four-arm, or eight-arm PEG-VS when the terminal or near-terminal end of the RGD peptide has at least one cysteine amino acid. A near-terminal cysteine amino acid is a cysteine that is positioned at the first, second, or third amino acid from the C-terminal and/or N- terminal end in a peptide sequence. In one embodiment, the RGD peptide comprises an arginine- glycine-aspartic acid amino acid triad and one cysteine amino acid at or near the C-terminal end. In one specific embodiment, the RGD peptide sequence is GRGDSPC (SEQ ID NO: 1). In this embodiment, the cystine amino acid is located at the C-terminal end of the peptide. In another embodiment, the cysteine residue is located at the near-C-terminal end of the peptide. In one specific embodiment, the RGD peptide sequence is GRGDSPCx (SEQ ID NO: 2), wherein “x” is any of glycine (G), alanine (A), leucine (L), valine (V) serine (S), threonine (S), aspartic acid (D), or glutamic acid (E). In another specific embodiment, the RGD peptide sequence is RGDC (SEQ ID NO: 3). VPM Peptide

[0044] Peptides comprising a valine-proline-methionine amino acid motif (Vai -Pro-Met) are referred to as VPM peptides. The VPM motif is a protease recognition site, a site where certain proteases can cleave the peptide at the VPM amino acids. In one aspect of the invention, a VPM peptide comprises one cystine residue near the C-terminal end of the peptide and one cysteine residue near the N-terminal end of the peptide. In one specific embodiment, the VPM peptide is GCRDVPMSMRGGDRCG (SEQ ID NO: 4). In another aspect of the invention, the N-terminal and/or C-terminal glycine (G) residues of SEQ ID. NO: 4 are substituted with preferably one of alanine (A), valine (V), leucine (L), serine (S), threonine (T), aspartic acid (D), or glutamic acid (E). Said another way, the VPM peptide can be xCRDVPMSMRGGDRCx (SEQ ID NO: 5), wherein “x” may preferably be chosen from alanine (A), valine (V), leucine (L), serine (S), threonine (T), aspartic acid (D), glutamic acid (E), or any combination thereof, and wherein one “x” may be glycine (G). In another aspect of the invention, a VPM peptide comprises one cysteine residue at the C-terminal end of the peptide, and one cysteine residue at the N-terminal end of the peptide. In one specific embodiment, the VPM peptide is CRDVPMSMRGGDRC (SEQ ID NO: 6). In another aspect of the invention, a VPM peptide comprises one near- terminal cysteine residue at the C-terminal end of the peptide, and one cysteine residue at the N-terminal end of the peptide. In one specific embodiment, the VPM peptide is CRDVPMSMRGGDRCG (SEQ ID NO: 7). Alternatively, the C-terminal glycine (G) may have an amino acid substitution, such that the VPM peptide sequence is CRDVPMSMRGGDRCx (SEQ ID NO: 8), wherein “x” is preferably chosen from one of alanine (A), valine (V), leucine (L), serine (S), threonine (T), aspartic acid (D), or glutamic acid. (E). In yet another aspect of the invention, a VPM peptide comprises one nearterminal cysteine residue at the N-terminal end of the peptide and one cysteine at the C-terminal end of the peptide. In one specific embodiment, the VPM peptide is GCRDVPMSMRGGDRC (SEQ ID NO: 9). Alternatively, the C-terminal glycine (G) may have an amino acid substitution, such that the VPM peptide sequence is xCRDVPMSMRGGDRC (SEQ ID NO: 10), wherein “x” is preferably chosen from one of alanine (A), valine (V), leucine (L), serine (S), threonine (T), aspartic acid (D), or glutamic acid (E). For any of the aforementioned embodiments of the VPM peptide, although certain amino acids are preferred, “x” may be any non-polar, uncharged, or charged amino acid. D. Process for Making Hydrogels

[0045] Generally, there are three stages for the synthesis of chitosan-based hydrogels having the mechanical characteristics as contemplated by the present invention:

Stage 1 : Conjugating thiolated-chitosan with PEG- VS

Stage 2: Synthesizing a multi-functional conjugate Chitosan-PEG-VS-RGD

Stage 3: Synthesizing a hydrogel from Chitosan-PEG-VS-RGD and a VPM peptide

[0046] Referring to FIG. 1, a general reaction scheme of a chitosan-based gel synthesis incorporating RGD and VPM peptides is provided, describing some aspects of the present disclosure. A thiolated chitosan is first reacted with a PEG- VS (represented by a squiggle line in FIG. 1 ), then subsequently covalently coupled with an RGD peptide (represented by a rectangle in FIG. 1), and then crosslinked by reacting the chitosan-PEG-VS-RGD conjugate with a difunctionalized VPM peptide crosslinker (represented by a star) at 37 °C to generate a chitosan- based hydrogel (represented by a network schematic on the far right side of FIG. 1).

[0047] The amount of each of thiolated-chitosan, PEG-VS, RGD peptide, and VPM peptide to add to the reaction may vary. For every 10 mmol of vinyl sulfone in PEG-VS, a total of 10 mmol of the thiol groups on thiolated-chitosan, RGD peptide and VPM peptide combined may be added. In one embodiment, the ratio of thiolated chitosan, RGD peptide and VPM peptide to PEG-VS is from about 1 mmol to about 3 mmol of thiol groups on thiolated-chitosan , about 1 mmol to about 3 mmol of thiol groups on the RGD peptide, and from about 4 mmol to about 8 mmol of thiol groups on the VPM peptide may be added (up to a total of about 10 mmol combined) for every approximately 10 mmol of vinyl sulfone in the PEG-VS.

[0048] In one non-limiting embodiment, 3 mmol of thiol groups on thiolated-chitosan, 3 mmol of thiol groups on the RGD peptide, and 4 mmol of thiol groups on the VPM peptide are combined with 10 mmol of vinyl sulfone groups on the PEG-VS to make the chitosan-PEG-VS- RGD-VPM hydrogel. In another non-limiting embodiment, 1 mmol of thiol groups on thiolated- chitosan, 1 mmol of thiol groups on the RGD peptide, and 8 mmol of thiol groups on the VPM peptide are combined with 10 mmol of vinyl sulfone groups on the PEG- VS to make the chitosan- PEG-VS-RGD-VPM hydrogel. In yet another non-limiting embodiment, 2 mmol of thiol groups on thiolated-chitosan, 3 mmol of thiol groups on the RGD peptide, and 5 mmol of thiol groups on the VPM peptide are combined with 10 mmol of vinyl sulfone groups on the PEG- VS to make the chitosan-PEG-VS-RGD-VPM hydrogel. It should be understood that these non-limiting embodiments are exemplary only and that any numerical combination up to a total of about 10 mmol combined for thiol groups on thiolated chitosan, RGD peptide, and VPM peptide may be used for about 10 mmol of vinyl sulfone of the PEG-VS.

Stage 1: Synthesizing a Chitosan-PEG-VS Conjugate

[0049] In one embodiment, a chitosan-PEG-VS conjugate is formed by reacting a thiolated-chitosan with a polyethylene glycol vinyl sulfone (PEG-VS) in an aqueous solution. The reaction may be conducted at a temperature from about 20° C to about 45° C. The reaction time may range from about 1 minute to about 900 minutes. In one embodiment, the reaction is from about 2 minutes to about 60 minutes, from about 2 minutes to about 5 minutes, from about 5 minutes to about 10 minutes, from about 10 minutes to about 15 minutes, from about 15 minutes to about 20 minutes, from about 20 minutes to about 25 minutes, from about 25 minutes to about 30 minutes, from about 30 minutes to about 35 minutes, from about 35 minutes to about 40 minutes, from about 40 minutes to about 45 minutes, from about 40 minutes to about 45 minutes, from about 45 minutes to about 50 minutes, from about 50 minutes to about 55 minutes, from about 55 minutes to about 60 minutes. In another embodiment, the reaction time is from about 1 hour to about 3 hours, from about 3 hours to about 5 hours, from about 5 hours to about 7 hours, from about 7 hours to about 9 hours, from about 9 hours to about 11 hours, from about 11 hours to about 13 hours, or from about 13 hours to about 15 hours.

[0050] The multi-armed PEG vinyl sulfone may be PEG-2-VS, PEG-4-VS, or PEG-8-VS. In one embodiment, the functionalized chitosan selected for the reaction is chitosan-thioglycolic acid. In one embodiment the PEG-VS is four-arm polyethylene glycol vinyl sulfone (PEG-4-VS). In one specific embodiment, the thiolated chitosan is chitosan-thioglycolic acid (CS-TGA) and the PEG-VS is PEG-4-VS, which results in the conjugate chitosan-PEG-4-VS. [0051] The temperature of the chitosan-PEG-VS reaction can range from 10° C to about 45°. In one embodiment the temperature during the synthesis of the chitosan-PEG-VS conjugate may be from at about 10° C to about 37° C. In yet another embodiment the temperature during synthesis is at about 25° C or at about 37° C. In one specific embodiment, the temperature during the synthesis is about 20° C, about 21° C, about 22° C, about 23° C, about 24° C, about 25° C, about 26° C, about 27° C, about 28° C, about 29° C, about 30° C, about 31° C, about 32° C, about 33° C, about 34° C, about 35° C, about 36° C, about 37° C, about 38° C, about 39° C, about 40° C, about 41° C, about 42° C, about 43° C, about 44° C, or about 45° C.

[0052] The synthesis of the chitosan-PEG-VS conjugate can occur in water, buffer solutions, cell culture mediums, or a combination thereof. In one embodiment, the aqueous solution for the synthesis of the chitosan-PEG-VS conjugate includes at least one of water, phosphate-buffered saline (PBS) buffer, 4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid (HEPES) buffer, or cell media. In another embodiment, the cell media is one of organoids cell culture medium, MDCK cell culture medium, or Chinese hamster ovary (CHO) cell culture medium.

Stage 2: Synthesizing a Multifunctional Conjugate, Chitosan -PEG- VS-RGD

[0053] A chitosan-PEG-VS -RGD multifunctional conjugate is formed by reacting a chitosan-PEG-VS conjugate with an RGD peptide in an aqueous solution. The aqueous solution may be water. In one embodiment, the chitosan-PEG-VS conjugate is the chitosan-PEG-VS conjugated from the synthesis in Stage 1. In one specific embodiment, the chitosan-PEG-VS conjugate is the conjugate of chitosan-thioglycolic acid and four-arm PEG-VS (i.e., chitosan-PEG- 4-VS resulting from the synthesis in Stage 1). In another embodiment, the reaction may be conducted at a temperature from about room temperature to about 37° C. In yet another embodiment, the reaction time may be from about 5 minutes to about 60 minutes.

[0054] The RGD peptide may be any peptide containing the RGD cell attachment motif and at least one cysteine residue. The specific RGD peptides described elsewhere above may also be used. [0055] In one embodiment, the temperature during the synthesis of the chitosan-PEG-VS- RGD conjugate is from at about 10° C to about 45° C, or in any range or at any value therebetween. In another embodiment, the temperature during the synthesis of the chitosan-PEG-VS-RGD conjugate is from at about 20° C to about 40° C. In yet another embodiment the temperature during synthesis of the chitosan-PEG-VS-RGD conjugate is at about 25° C or is at about 37° C. In one specific embodiment, the temperature during the synthesis of the chitosan-PEG-VS-RGD conjugate is about 20° C, about 21° C, about 22° C, about 23° C, about 24° C, about 25° C, about 26° C, about 27° C, about 28° C, about 29° C, about 30° C, about 31° C, about 32° C, about 33° C, about 34° C, about 35° C, about 36° C, about 37° C, about 38° C, about 39° C, about 40° C, about 41° C, about 42° C, about 43° C, about 44° C, or about 45° C.

[0056] In one embodiment, the reaction time for the synthesis of the chitosan-PEG-VS- RGD conjugate is from about 2 minutes to about 60 minutes, or in any range or at any value therebetween. In another embodiment, the reaction time for synthesis is from about 2 minutes to about 5 minutes, from about 5 minutes to 15 minutes, from about 15 minutes to about 30 minutes, from about 30 minutes to about 45 minutes, or from about 45 minutes to 60 minutes. In one specific embodiment, the reaction time for the synthesis of the chitosan-PEG-VS-RGD conjugate is about 3 minutes, is about 5 minutes, is about 10 minutes, is about 15 minutes, is about 20 minutes, is about 25 minutes, is about 30 minutes, is about 35 minutes, is about 40 minutes, is about 45 minutes, is about 50 minutes, is about 55 minutes, or is about 60 minutes.

[0057] The synthesis of the chitosan-PEG-VS-RGD conjugate can be run in water, buffer solutions, cell culture mediums, or a combination thereof. In one embodiment, the aqueous solution in the synthesis of the chitosan-PEG-VS-RGD conjugate includes at least one of water, phosphate-buffered saline (PBS) buffer, 4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid (HEPES) buffer, or cell media. In another embodiment, the cell media is one of organoids cell culture medium, MDCK cell culture medium, or Chinese hamster ovary (CHO) cell culture medium.

Stage 3: Synthesizing a Hydrogel from Chitosan-PEG-VS-RGD and a VPM peptide

[0058] A chitosan-PEG-VS-RGD- VPM hydrogel can be formed by reacting a chitosan- PEG-VS-RGD conjugate with a VPM peptide in an aqueous solution. In one embodiment, the reaction may be conducted at a temperature from about room temperature to about 37 C. In another embodiment, the reaction time may be from about 5 minutes to about 30 minutes.

[0059] The chitosan-PEG-VS-RGD conjugate may be any of the chitosan-PEG-VS-RGD conjugates described in Stage 2 above. In one specific embodiment, the chitosan-PEG-VS-RGD conjugate is chitosan-thioglycolic acid conjugated to four-arm PEG-VS (i.e., chitosan-PEG-4-VS) further conjugated with an RGD peptide having amino acid sequence GRGDSPC (SEQ ID NO: 1). In another specific embodiment, the chitosan-PEG-VS-RGD conjugate is chitosan-thioglycolic acid conjugated to four-arm PEG-VS (i.e., chitosan-PEG-4-VS) further conjugated with an RGD peptide having amino acid sequence RGDC (SEQ ID NO: 4).

[0060] The VPM peptide may be any peptide containing the VPM motif and at least two cysteine residues. Any of the VPM peptides described in the section above about VPM peptides can be used for synthesizing a chitosan-PEG-RGD-VPM hydrogel. The ratio of VPM peptide to chitosan-PEG-VS-RGD conjugate added together is a molar ratio of available vinyl sulfone groups in the chitosan-PEG-VS-RGD to available sulfhydryl groups (SH groups) in the VPM peptide, wherein the available vinyl sulfone to available sulfhydryl groups is preferably about 1: 1. Other ratios of available vinyl sulfone groups in the chitosan-PEG-RGD conjugate to available sulfhydryl groups in the VPM peptide may be used, such as about 1:0.5, 1 :0.6, 1:0.7, 1 :0.8, 1:0.9, 1 :1.1, 1: 1.2, 1: 1.3, 1: 1.4, or 1: 1.5, or in any range or at any value between about 1:0.5 and about 1 :1.5.

[0061] The temperature for the chitosan-PEG-RGD-VPM synthesis may be from about 10° C to about 45° C, or in any range or at any value therebetween. In another embodiment the temperature during the synthesis of the chitosan-PEG-RGD-VPM conjugate is from at about 25° C to about 37° C. In yet another embodiment the temperature during synthesis of the chitosan- PEG-VS-RGD conjugate at about 25° C or at about 37° C. In one specific embodiment, the temperature during the synthesis of the chitosan-PEG-VS-RGD conjugate is about 20° C, about

21° C, about 22° C, about 23° C, about 24° C, about 25° C, about 26° C, about 27° C, about 28° C, about 29° C, about 30° C, about 31° C, about 32° C, about 33° C, about 34° C, about 35° C, about

36° C, about 37° C, about 38° C, about 39° C, about 40° C, about 41° C, about 42° C, about 43° C, about 44° C, or about 45° C. [0062] The reaction time for the synthesis of the chitosan-PEG-RGD-VPM conjugate is between about 2 minutes and about 60 minutes, or in any range or at any value therebetween. In one embodiment, the reaction time is from about 2 minutes to about 3 minutes, from about 5 minutes to about 30 minutes, or from about 30 minutes to 60 minutes. In one specific embodiment, the reaction time for the synthesis of the chitosan-PEG-VS-RGD-VPM conjugate is about 3 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about or about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes.

[0063] The synthesis of the chitosan-PEG-RGD-VPM conjugate can occur in water, buffer solutions, cell culture mediums, or a combination thereof. In one embodiment, the aqueous solution in the synthesis of the chitosan-PEG-RGD-VPM conjugate includes at least one of water, phosphate-buffered saline (PBS) buffer, 4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid (HEPES) buffer, or cell media. In another embodiment, the cell media is one of organoids cell culture medium, MDCK cell culture medium, or Chinese hamster ovary (CHO) cell culture medium.

[0064] The result of the chitosan-PEG-RGD-VPM synthesis is a set (cured) hydrogel. The aqueous, pre-cured chitosan-PEG-RGD-VPM mixture is a precursor, uncured hydrogel. The uncured precursor hydrogel may be aliquoted into drops to form dome gels, put into wells of a well-plate to create a hydrogel layer, spread across an existing layer or substrate to create a hydrogel layer, or used in any other way. The hydrogels may be used for two or three dimensional cell cultures.

E. Properties of the Hydrogels

[0065] One way hydrogel properties are measured are by their swelling ratio (Q) and their solvent fraction (cp_s). The relationship between the swelling ratio and the solvent fraction is as follows: wherein cp_p is the solids fraction, and wherein (p_p + (p_s = 1 and wherein

The swelling ratio (Q) is the ratio of wet and dry masses of the gels. The wet mass is the mass of the hydrogel when it contains liquid. The dry mass is the mass of the hydrogel after it is flash frozen.

[0066] In one aspect of the invention, the chitosan-based hydrogel (i.e., the cured chitosan- PEG-RGD-VPM hydrogel) has a swelling ratio (Q) of between about 35 and about 60 during the time frame of about 5 days to about 13 days after surrounding a cured hydrogel dome with an aqueous solution. In one embodiment, the chitosan-based hydrogel has a swelling ratio of about 35 at about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, or 13 days after surrounding a cured hydrogel dome with an aqueous solution. In another embodiment, the chitosan-based hydrogel has a swelling ratio of about 40 about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, or 13 days after surrounding a cured hydrogel dome with an aqueous solution. In another embodiment, the chitosan-based hydrogel has a swelling ratio of about 45 about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, or 13 days after surrounding a cured hydrogel dome with an aqueous solution. In yet another embodiment, the chitosan-based hydrogel has a swelling ratio of about 50 about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, or 13 days after surrounding a cured hydrogel dome with an aqueous solution. In another embodiment, the chitosan-based hydrogel has a swelling ratio of about 55 about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, or 13 days after surrounding a cured hydrogel dome with an aqueous solution. In yet another embodiment, the chitosan-based hydrogel has a swelling ratio of about 60 about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, or 13 days after surrounding a cured hydrogel dome with an aqueous solution.

[0067] In another embodiment, the chitosan-based hydrogel has a swelling ratio (Q) of between about 60 and about 95 at about 2 days after surrounding a cured hydrogel dome with an aqueous solution. In another embodiment, the chitosan-based hydrogel has a swelling ratio (Q), at about 2 days after surrounding a cured hydrogel dome with an aqueous solution, from about 60 to about 65, from about 65 to about 70, from about 70 to about 75, from about 75 to about 80, from about 80 to about 85, from about 85 to about 90, or from about 90 to about 95. In one embodiment, the chitosan-based hydrogel has a swelling ratio (Q), at about 2 days after surrounding a cured hydrogel dome with an aqueous solution, of about 60, about 65, about 70, about 75, about 80, about 85, about 90, or about 95.

[0068] In another aspect of the invention, the chitosan-based hydrogel has a solvent fraction (q>_s) of between about 0.95 and 0.99 at between about 2 days to about 13 days after surrounding cured hydrogel domes with an aqueous solution. In one embodiment, the solvent fraction of the chitosan-based hydrogel is about 0.95 at about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, or about 13 days, after surrounding cured hydrogel domes with an aqueous solution. In one embodiment the solvent fraction of the chitosan- based hydrogel is about 0.96 at about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, or about 13 days, after surrounding cured hydrogel domes with an aqueous solution. In one embodiment, the solvent fraction of the chitosan-based hydrogel is about 0.97 at about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, or about 13 days, after surrounding cured hydrogel domes with an aqueous solution. In one embodiment, the solvent fraction of the chitosan-based hydrogel is about 0.98 at about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, or about 13 days, after surrounding cured hydrogel domes with an aqueous solution. In one embodiment, the solvent fraction of the chitosan-based hydrogel is about 0.99 at about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, or about 13 days, after surrounding cured hydrogel domes with an aqueous solution.

[0069] In another embodiment, the chitosan-based hydrogel has a solvent fraction (q>_s) of between about 0.90 and about 0.95 about 1 day after surrounding cured hydrogel domes with an aqueous solution. In one embodiment, the chitosan-based hydrogel has a solvent fraction of about 0.90 about 1 day after surrounding cured hydrogel domes with an aqueous solution. In one embodiment, the chitosan-based hydrogels have a solvent fraction of about 0.91, about 0.92, about 0.93, about 0.94, or about 0.95 about 1 day after surrounding cured hydrogel domes with an aqueous solution.

[0070] In another aspect of the invention, the chitosan-based hydrogel has a storage modulus (G') of any value between about 100 Pa to about 1000 Pa (when measured by a rheometer with parallel plate geometry with a Peltier plate at 1 Hz frequency) after a precursor hydrogel is cured for about 30 minutes at about 37° C. In one specific embodiment, the chitosan-based hydrogel has a storage modulus (G') of between about 100 Pa to about 500 Pa after a precursor hydrogel is cured for about 30 minutes at about 37° C. In another specific embodiment, the chitosan-based hydrogel has a storage modulus (G') of between about 125 Pa to about 175 Pa after a precursor hydrogel is cured for about 30 minutes at about 37° C. In another embodiment, the chitosan-based hydrogel has a storage modulus of about 125 Pa, 130 Pa, 135 Pa, 140 Pa, 145 Pa, 150 Pa, 155 Pa, 160 Pa, 165 Pa, 165 Pa, 170 Pa, or about 175 Pa, after a precursor hydrogel is cured for about 30 minutes at about 37° C.

[0071] In another embodiment, the chitosan-based hydrogel has a storage modulus (G') of between about 25 Pa to about 55 Pa (when measured by a rheometer with parallel plate geometry with a Peltier plate at 1 Hz frequency) after a cured hydrogel dome have been surrounded by aqueous solution at about 37° C for about 4.5 days. In one embodiment, the chitosan-based hydrogel has a storage modulus (G') of about 25 Pa, 30 Pa, about 35 Pa, about 40 Pa, about 50 Pa, or about 55 Pa, after a cured hydrogel dome have been surrounded by aqueous solution at about 37° C for about 4.5 days.

[0072] In another aspect of the invention, the chitosan-based hydrogel has a loss modulus (G") of from about 1 Pa to about 10 Pa (when measured by a rheometer with parallel plate geometry with a Peltier plate at 1 Hz frequency) after a precursor hydrogel is cured for about 30 minutes at about 37° C. In one embodiment, the chitosan-based hydrogel has a loss modulus (G") of about 1 Pa, about 2 Pa, about 3 Pa, about 4 Pa, about 5 Pa, about 6 Pa, about 7 Pa, about 8 Pa, about 9 Pa, or about 10 Pa, after a precursor hydrogel is cured for about 30 minutes at about 37° C.

[0073] In some embodiments, the aqueous solution for measuring the hydrogel properties is a cell culture medium, a buffer solution, water, or a combination thereof. For cell culture medias, any cell medium sufficient to grow a desired cell type can be used. As an example, if human 1 intestine organoids are desired to be grown, human intestine organoids cell culture media can be used. One example of a composition of human intestine organoids cell culture media is an approximately 1 : 1 ratio of actin depolymerizing factor (ADF) and base medium, wherein the ADF comprises Dulbecco's Modified Eagle Medium (DMEM), glucose, HEPES and penicillin streptomycin, and wherein the base medium comprises DMEM and fetal bovine serum (FBS). In one specific embodiment, the ADF solution is 500 mL of DMEM/F-12 (Gibco), 5 mL of 20 mM Glutamax (1000X) (Gibco), 5 mL of 1 M HEPES, and 5 mL of Penicillin-Streptomycin (100X) (Corning®). In one specific embodiment, the base medium is 450 mL of DMEM (ATCC) and 50 mL of FBS (Corning®). Any other variation of compositions for human intestine organoids cell culture media also works to form or surround the hydrogel of the present invention. Other nonlimiting examples include cell culture medium for Madin-Darby canine kidney (MDCK) cells or cell culture medium for Chinese hamster ovary (CHO) cells.

[0074] In some embodiments, a cured chitosan-based hydrogel does not significantly degrade after being surrounded by aqueous solution for between about 0-180 days when held at a temperature of between about 4-45° C. In some embodiments, a cured chitosan-based hydrogel does not significantly degrade after being surrounded by aqueous solution for between about 0- 180 days when held at a temperature of between about 15-45° C. In some embodiments, a cured chitosan-based hydrogel does not significantly degrade after being surrounded by aqueous solution for between about 0-180 days when held at a temperature of between about 20-37° C. In some embodiments, a cured chitosan-based hydrogel does not significantly degrade after being surrounded by aqueous solution for between about 0-150 days, between about 0-120 days, between about 0-90 days, between about 0-60 days, between about 0-45 days, between about 0-30 days, between about 0-24 days, or between about 0-15 days, when held at a temperature of between about 4-45 C, or between about 15-45 C, or between about 20-37 C. In one embodiment, a cured chitosan-based hydrogel does not significantly degrade after been surrounded by aqueous solution when held at a temperature of between about 4-45°C at about 0 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 24 days, 30 days, 45 days, 60 days, 90 days, 120 days, 150 days, or about 180 days. A hydrogel of the present invention is significantly degraded when at least 70% or more of the hydrogel is dissolved (liquified) as measured by visual observation. F. Cell Cultures Using Hydrogels

[0075] The present disclosure also provides a method for synthesizing a chitosan-based hydrogel that further comprises providing a cell type. In one aspect of the invention, the method comprises the steps of (a) providing a chitosan-PEG-VS-RGD conjugate, (b) providing a VPM peptide, (c) providing cells of a cell type, (d) combining the conjugate of step (a) with the peptide of step (b) and with the cell type of step (c), (e) dispensing a discrete volume of the combined precursor hydrogel from step (d) onto a substrate, and (f) forming a hydrogel from the dispensed discrete volume.

[0076] The step of providing the chitosan-PEG-VS-RGD conjugate is providing one of the chitosan-PEG-VS-RGD conjugates described above in the process for making the hydrogels. In some embodiments, the provided chitosan-PEG-VS-RGD conjugate is in an aqueous solution. Aqueous solutions include, but are not limited to, water, buffer solutions, and cell culture mediums. In one specific embodiment, the aqueous solution for the chitosan-PEG-VS-RGD conjugate is chosen from water, HEPES, PBS, human intestine organoids cell culture media, MDCK cell media, CHO cell media, or a combination thereof. Alternatively, any other buffer solution or cell culture medium for growing two or three dimensional cells can be used. In other embodiments, the provided chitosan-PEG-VS-RGD conjugate is freeze-dried.

[0077] The step of providing the VPM peptide is providing one of the VPM peptides described above in the process for making hydrogels, or any of the VPM peptides described elsewhere in the present disclosure. In one embodiment, the VPM peptide provided may be in an aqueous solution. Aqueous solutions include, but are not limited to, water, buffer solutions, and cell culture mediums. In one specific embodiment, the aqueous solution for the VPM peptide is chosen from water, HEPES, PBS, human intestine organoids cell culture media, MDCK cell media, CHO cell media, or a combination thereof. Alternatively, any other buffer solution or cell culture medium for growing two or three dimensional cells can be used. In another aspect of the present invention, the provided VPM peptide is freeze-dried.

[0078] Any cell type that grows with two or three dimensional hydrogels may be used for the step of providing cells of a cell type. In one embodiment, the cell type may be Madin-Darby canine kidney (MDCK) cells, Chinese hamster ovary (CHO) cells, or patient derived organoids (cells taken from a human or other animal patient that can be grown into organoids). In one embodiment, the provided cells are in a cell suspension. The cell suspension may be in a concentration from about 10 cells/pL to about 1000 cells/pL, or in any range or at any value therebetween. In yet another embodiment, the cell suspension may be in a concentration from about 10 cells/pL to about 250 cells/pL, from about 250 cells/pL to about 500 cells/pL from 400 cells/pL to about 600 cells/ pL, from about 500 cells/pL to about 750 cells/ pL, or from about 750 cells/pL to about 1000 cells/pL. In one specific embodiment, the cell suspension may be in a concentration of about 50 cells/pL, 100 cells/pL, 150 cells/pL, 200 cells/pL, 250 cells/pL 300 cells/pL, 350 cells/pL, 400 cells/ L, about 450 cells/pL, about 500 cells/pL, about 550 cells/pL, about 600 cells/pL, about 625 cells/pL, about 650 cells/pL, 700 cells/pL,750 cells/pL, 800 cells/pL, 850 cells/pL, 900 cells/pL, 950 cells/pL, or about 1000 cells/pL. The aqueous portion of the cell suspension may be any solution that the desired cell type may grow in.

[0079] The step of combining of the chitosan-PEG-VS-RGD conjugate of step (a) with the VPM peptide of step (b) and with the cell type of step (c) can be performed in any order. For example, an amount of an aqueous solution containing a chitosan-PEG-VS-RGD conjugate may added into any suitable mixing vessel (such as a tube or vial), followed by adding into the mixing vessel an amount of aqueous solution containing a VPM peptide, followed by mixing the mixture by any suitable mixing means (for example, by pipetting the mixture until mixed or by using a vortex machine), followed by adding an amount of aqueous solution containing a cell type (such as MDCK cells), followed by mixing the mixture by any suitable means (for example, by stirring with a sterilized rod or by pipetting the mixture). In another example, an amount of an aqueous solution containing a chitosan-PEG-VS-RGD conjugate is added into any suitable mixing vessel, followed by adding an amount of aqueous solution containing a cell type (for example, Chinese Hamster Ovary cells), followed by addition of an amount of aqueous solution containing a VPM peptide, followed by mixing the mixture by any suitable mixing means (for example, by stirring with a sterilized rod or by pipetting the mixture). In some embodiments, step (d) could be broken into two steps, for example, combining thePVP-PEG-VS-RGD conjugate with cells of a cell type, followed by combining that mixture with the VPM peptide. [0080] The step of dispensing a discrete volume of the combined precursor hydrogel onto a substrate is performed with any suitable volume and on any suitable substrate. In one embodiment, the discrete volume may be from about 1 pL to about 1 mL, or in any range or at any value therebetween. In another embodiment, the discrete volume may be from about 2 pL to about 20 pL or from about 8 pL to about 10 pL. In yet another embodiment, the discrete volume may be from about 4 pL to about 50 pL, 50 pL to about 100 pL, from about 100 pL to about 150 pL, from about 150 pL to about 200 pL, from about 200 pL to about 250 pL, from about 250 pL to about 300 pL, from about 300 pL to about 350 pL, from about 350 pL to about 400 pL, from about 400 pL to about 450 pL, from about 450 pL to about 500 pL, from about 500 pL to about 550 pL, from about 550 pL to about 600 pL, from about 600 pL to about 650 pL, from about 650 pL to about 700 pL, from about 700 pL to about 750 pL, from about 750 pL to about 800 pL, from about 800 pL to about 850 pL, from about 850 pL to about 900 pL, from about 900 pL to about 950 pL, or from about 950 pL to about 1000 pL. In one specific embodiment, the discrete volume may be about 4 pL, 5 pL, 6 pL, 7 pL, 8 pL, 9 pL, 10 pL, 11 pL, 12 pL, 13 pL, 14 pL, 15 pL, 16 pL, 17 pL, 18 pL, 19 pL, 20 pL, 21 pL, 22 pL, 23 pL, 24 pL, 25 pL, 26 pL, 27 pL, 28 pL, 29 pL,

30 pL, 31 pL, 32 pL, 33 pL, 34 pL, 35 pL, 36 pL, 37 pL, 38 pL, 39 pL, 40 pL, 41 pL, 42 pL, 43 pL, 44 pL, 45 pL, 46 pL, 47 pL, 48 pL, 49 pL, or 50 pL.

[0081] The discrete volume may be dispensed onto any suitable substrate for forming a hydrogel, such as a well plate or a coverslip. In one embodiment, the substrate may be well plates of any number, including but not limited to a 4 well plate, 6 well pate, 8 well plate, 12 well plate, a 24 well plate, a 48 well plate, a 96 well plate, or a 384 well plate. The well plate may be treated or untreated. The well plate may be specific for culturing cells. In one embodiment, the well plate is tissue culture-treated. In another embodiment, the well plate is an ultra-low binding well plate. In yet another embodiment, the well plate is not treated. In one specific embodiment, the well plate is a culture well plate having 24 wells and is ultra-low binding. In another embodiment, the well plate is a culture well plate having 24 wells and is untreated. In one embodiment the substrate is cover slip. The coverslip may be tissue culture-treated, an ultra-low binding coverslip, or an untreated coverslip. In another embodiment, the substrate is a petri dish. The petri dish may be tissue culture-treated, an ultra-low binding petri dish, or an untreated petri dish. [0082] The step of forming a hydrogel from the dispensed discrete volume may occur at a time between about 1 minute to about 60 minutes. In this step, the combined precursor hydrogel with cells cures into a set hydrogel. In one embodiment, the forming a hydrogel from the dispensed discrete volume occurs at a time from about 1 minute to about 5 minutes, or from about 5 minutes to about 30 minutes, or from about 30 minutes to about 30 minutes. In another embodiment, the forming a hydrogel from the dispensed discrete volume occurs from about 1 to about 5 minutes, from about 5 minutes to about 15 minutes, from about 15 minutes to about 25 minutes, from about 25 minutes to about 35 minutes, from about 35 minutes to about 45 minutes, or from about 45 minutes to about 60 minutes. However, the forming a hydrogel from the dispensed discrete volume may occur over a longer time, for example a time between about 60 minutes and about 180 minutes.

[0083] In one embodiment, a substrate containing the dispensed discrete volume is kept upright during the forming of the hydrogel. In another embodiment, a substate containing the dispensed discrete volume is kept upside down during the formation of the hydrogel. In yet another embodiment, a substrate containing the dispensed discrete volume is kept upright at least once during the formation of the forming of the hydrogel and is kept upside down at least once during the formation of the hydrogel.

[0084] The present disclosure further provides a method for growing three-dimensional cell cultures in a chitosan-based hydrogel. In one aspect of the invention, the method comprises the steps of (a) providing a chitosan-PEG-VS-RGD conjugate, (b) providing a VPM peptide, (c) providing cells of a cell type, (d) combining the conjugate of step (a) with the peptide of step (b) and with the cell type of step (c), (e) dispensing a discrete volume of the combined precursor hydrogel from step (d) onto a substrate, (f) forming a hydrogel from the dispensed discrete volume, (g) surrounding the formed hydrogel with a discrete volume of cell culture medium, and (h) incubating the surrounded hydrogel. The steps may be performed in other orders as described elsewhere in the present disclosure.

[0085] The step of surrounding the formed hydrogel with culture medium can be performed with any suitable volume of cell culture medium. In one embodiment, surrounding the formed hydrogel with cell culture medium is achieved by adding a discrete volume from about 3 pL to about 5 mL of any suitable cell culture medium around the formed hydrogel. Suitable cell culture mediums include those detailed in other sections above. In another embodiment, the discrete volume for surrounding the formed hydrogel is from about 5 pL to about 50 pL, from about 50 pL to about 250 pL, from about 250 pL to about 1 mL, from about 500 pL to about 1.5 mL, from about 1 mL to about 2 mL, from about 2 mL to about 3 mL, from about 3 mL to about 4 mL, or from about 4 mL to about 5 mL. Larger volumes such as 10 mL, 25 mL, or more will also work with the invention of the present disclosure. In some embodiments, the formed hydrogel may be submerged under the volume of cell culture medium that surrounds it.

[0086] The step of incubating the surrounded hydrogel can be done at any suitable conditions for any suitable amount of time to grow the cell cultures. For example, the incubation may occur at room temperature (e.g., about 25° C) and under atmospheric pressure. As another example, the incubation may occur at a temperature from about 25° C to about 37° C. As another example, the incubation may occur at 37° C. In another example, the incubation may be performed at a relative humidity of 10-20%, 20%, 20-30%, 30-40%, 40-50%, 50-60%, 70-80%, 80-90%, 90- 95%, or more. In another example, the carbon dioxide level in the air at incubation may be up to 5%, 2.5%-5%, 5%-7.5%, 7.5%-10%, or more. In one embodiment, the incubation time is from about 2 days to about 6 days. In another embodiment, the incubation time is from about 1 day to about 2 days, from about 2 days to about 7 days, from about 7 days to 14 days, from about 14 days to about 21 days, from about 21 days to about 28 days, from about 28 days to about 35 days, from about 35 days to about 60 days, from about 60 days to about 90 days, from about 90 days to about 120 days, from about 120 days to about 150 days, or from about 150 days to about 180 days. Where incubation times are longer, the cell culture medium may be changed out or added to throughout the incubation period.

[0087] In another embodiment, a cured chitosan-based hydrogel with cells mixed in does not significantly degrade after been surrounded by aqueous solution for a time between about 0 days to about 180 days when incubated at a temperature between about 10-45° C. Degradation of a hydrogel can be visually observed as the degraded parts of the gel become liquid. The change in size of the gel and the amount of liquification can be observed visually. When at least 70% of the hydrogel has liquified, this is considered significant degradation. In one embodiment, a cured chitosan-based hydrogel with cells mixed in does not significantly degrade after been surrounded by aqueous solution when incubated at a temperature between about 10-45° C at about 0 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, 10 days to 15 days, 15 days to 30 days, 30 days to 45 days, 45 days to 60 days, 60 days to 90 days, 90 days to 120 days, 120 days to 150 days, or 150 days to 180 days. In some embodiments the temperature may be between about 10-15° C, about 15-20° C, about 20-25° C, about 25-30° C, about 30-35° C, about 35-40° C, or about 40-45° C.

G. Dissolving Chitosan-PEG-RGD-VPM hydrogels

[0088] The present disclosure provides a method for dissolving the chitosan-based hydrogels of the present invention. In one aspect of the invention, the method comprises the steps of (a) providing a hydrogel comprising a chitosan-PEG-RGD-VPM conjugate, (b) adding a discrete volume of an aqueous solution comprising an enzyme that cleaves the VPM peptide of the conjugate, and (c) incubating the resultant combination from steps (a) and (b). In some embodiments, the hydrogel comprising a chitosan-PEG-RGD-VPM conjugate is any of the hydrogels described in the present disclosure that include a cell type. In other embodiments, the hydrogel comprising a chitosan-PEG-RGD-VPM conjugate is any of the hydrogels described in the present disclosure that has no cell type included in the hydrogel.

[0089] Any enzyme that recognizes the VPM peptide as a cleavage site may be used for dissolving the chitosan-PEG-RGD-VPM hydrogel. In some embodiments, the enzyme is a matrix metalloproteinase (MMP)-l or MMP-2 protease. In one embodiment, the enzyme is chosen from collagenase, dispase, protease from Streptomyces griseus, or a trypsin-substitute enzyme. Trypsinsubstitute enzymes may be commercially purchased, for example, Accutase® (Innovative Cell Technologies, Inc.). In one specific embodiment, the enzyme is collagenase-1 from Clostridium histolyticum (255 U/mg). In another specific embodiment, the enzyme is dispase is from Bacillus polymyxa (1.84 U/mg). In another specific embodiment, the protease is protease from Streptomyces griseus (4 U/mg), which can be purchased from at least Sigma Aldrich®. In another embodiment, the enzyme is a trypsin-substitute enzyme (such as Accutase®). The aqueous solution for the enzyme may be any buffer that does not degrade the enzyme in it. Example aqueous solutions for the enzyme may be PBS or HEPES buffer. The discrete volume of an aqueous solution comprising at least one of collagenase, dispase, protease from Streptomyces griseus, or trypsin-substitute enzyme (including Accutase®) may be at a ratio of hydrogel volume to aqueous solution of enzyme from about 1:2 to about 1:5, wherein the concentration of enzyme being added is about 10 mg/mL. As an example, for 100 pL of hydrogel, anywhere from about 200 mb to about 500 mL of aqueous solution containing the proteolytic enzyme is added to the well to dissolve the hydrogel. In another embodiment, the ratio of hydrogel volume to aqueous solution of enzyme of about 1:2.5 to about 1:4.5, wherein the concentration of enzyme being added is about 10 mg/mL. In a specific embodiment, the ratio of hydrogel volume to aqueous solution comprising at least one of collagenase- 1, dispase from Bacillus polymyxa, or protease from Streptomyces griseus is about 1:3, wherein the concentration of enzyme being added is about 10 mg/mL. In another specific embodiment, the ratio of hydrogel volume to aqueous solution comprising trypsinsubstitute enzyme is about 1:4, wherein the concentration of enzyme being added is about 10 mg/mL.

[0090] In one embodiment, the step of incubating the mixture of steps (a) and (b) is performed at room temperature. In another embodiment, the step of incubating of the mixture of steps (a) and (b) is performed at a temperature from about 4° C to about 37° C. In another embodiment, the step of incubating is performed at a temperature from about 20° C to about 30° C. In one specific embodiment, the step of incubating is performed at a temperature of about 4° C, about 10° C, about 15° C, about 20° C, about 25° C, about 30° C, about 34° C, or about 37° C.

[0091] The present disclosure further provides a method for harvesting cells from three- dimensional cell cultures grown in the chitosan-based hydrogels described herein. In one aspect of the invention, the method comprises the steps of (a) providing a hydrogel comprising a chitosan- PEG-RGD-VPM conjugate and a cell type, (b) adding a discrete volume of an aqueous solution comprising at least one of an MMP-1 or MMP-2, (c) incubating the mixture of steps (a) and (b), and (d) harvesting the cells from the dissolved hydrogel. In one embodiment, the hydrogel comprising a chitosan-PEG-RGD-VPM conjugate is any of the hydrogels described in the present disclosure and the hydrogel further comprises a cell type. In another embodiment, the cell type may be any of the cell types described in the present disclosure. In one embodiment, the MMP-1 and/or MMP-2 may be chosen from collagenase- 1 from Clostridium histolyticum, dispase from Bacillus polymyxa, or protease from Streptomyces griseus (Sigma- Aldrich®). The step of adding a discrete volume of an aqueous solution comprising at least of an MMP-1 or MMP-2, and the step of incubating the mixture of steps (a) and (b) can both be performed as described above.

[0092] The harvesting the cells from the dissolved hydrogel can be performed by any suitable means of recovering cells from dissolved hydrogels.

H. Kits for Making and Using Hydrogels

[0093] Embodiments of the present disclosure also relate to kits that include one or more of the components for making chitosan-based hydrogels as described herein. In one embodiment, a kit may include at least a two container system provided together in a package. In this embodiment, one container contains the polymer-peptide conjugate (i.e., the chitosan-PEG-VS- RGD conjugate) and the other container contains the VPM peptide. In one aspect of the invention, one or both containers may contain aqueous solution. In another aspect of the invention, one or both containers may contain lyophilized powder. In certain embodiments, the lyophilized powder is sterilized lyophilized powder. In some embodiments, the container is a vial. The vials may have a cap or lid. In some embodiments the container is a tube. The tube may have a cap or lid. Any other suitable container may be used, including, but not limited to flasks or bottles having caps.

[0094] In certain embodiments, the kits may include additional containers. The additional containers may include aqueous solutions for forming hydrogels, protease enzymes for dissolving the gels, or containers of each. In some embodiments, the aqueous solution may be water. In other embodiments the aqueous solution may be a buffer solution, such as HEPES buffer or PBS buffer. In yet other embodiments the aqueous solution may be sterilized cell culture medium. In one embodiment, an additional container contains at least one protease enzyme chosen from one or more of collagenase, dispase, and protease from Streptomyces griseus (Sigma-Aldrich®). The containers for aqueous solutions may be a vial, a tube, a bottle, or a flask. The containers for aqueous solutions have caps or lids. In another aspect of the invention, the containers may contain powders, lyophilized or freeze-dried enzymes, or lyophilized or freeze-dried components instead of aqueous solutions. For example, in one embodiment, the additional container may include lyophilized or freeze-dried enzyme chosen from collagenase, dispase, or protease from Streptomyces griseus (Sigma- Aldrich®), or a combination thereof. [0095] The kits may further include any of the culture growing components described in the embodiments herein, such as well plates, coverslips, petri dishes, or slides. For example, in some embodiments, a kit may comprise a vessel for culturing three-dimensional cell culture. Vessels for culturing three-dimensional cell cultures include, for example, 6- well, 12- well, 24- well, 48-well, 96-well, 128-well, 384- well, or even higher well microplates, 6- well, 12-well, 24- well, 48-well, or even higher well cell culture plates, culture dishes, flasks with microcavities, etc. These examples are not intended to be limiting, they are merely examples. Any vessel that can be used for three-dimensional cell cultures may be used.

EXAMPLES

[0096] The following examples are included to demonstrate certain embodiments of the present disclosure. Those of skill in the art should, however, in light of the present disclosure, appreciate that modifications can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Therefore, all matter set forth is to be interpreted as illustrative and not in a limiting sense.

Example 1 - Chitosan-based hydrogel synthesis

[0097] Chitosan, thiolated with thioglycolic acid and having a molecular weight range of 10-200 kDa, was purchased from Polymer Source, Inc. A 20 kDa vinyl sulfonated four-arm PEG was selected as the polymer. An RGD peptide having the sequence GRGDSPC (SEQ ID NO: 1) was purchased from GenScript. A VPM peptide having the sequence GCRDVPMSMRGGDRCG (SEQ ID NO: 4) was purchased from GenScript.

[0098] 1 pmol of thiolated chitosan was added to 10 pmol of PEG-4- VS and then allowed to react at room temperature for 60 minutes to generate a chitosan-PEG-4-VS conjugate. Next, 1 pmol of the RGD peptide was added to the chitosan-PEG-4-VS conjugate and then allowed to react at 37° C for approximately 60 minutes to generate a chitosan-PEG-4-VS-RGD conjugate. Then, 7 pmol of the VPM peptide was added to the chitosan-PEG-4-VS-RGD conjugate and then allowed to crosslink at 37° C for approximately 60 minutes to generate the cured chitosan-PEG- RGD-VPM hydrogel. Example 2 - Hydrogel Properties

Swellins Properties

[0099] The swelling characteristics of the chitosan-based hydrogel was investigated. Approximately 150 pL drops of the chitosan-PEG-RGD-VPM precursor hydrogel using the composition from Example 1 were pipetted as domes onto polystyrene petri dishes and weighed to obtain the wet mass of the hydrogel. After 30 minutes at 25° C for curing, either 5 mL human intestine organoids cell culture medium or 5 mL HEPES buffer at neutral pH (~ 7.4) was added to the petri dishes and submerging the domes. The hydrogel domes were then allowed to swell over 15 days and the domes were weighed every day or two during that time to determine the swollen mass of the hydrogel domes.

[00100] Separately, 150 L drops of the chitosan-PEG-RGD-VPM precursor hydrogel from Example 1 were pipetted into domes in individual glass vials. After 30 minutes at 25° C for curing, the domes were weighed both before and after freeze drying by lyophilization to determine the exact dry mass of the hydrogel domes.

[00101] The swollen and dry masses of the hydrogel domes were used to calculate the swelling ratios (Q) and solvent fractions, which gives the degree of swelling of the hydrogels and water fraction respectively. FIGS. 2A-2B shows swelling ratio of the hydrogels throughout the 15- day swelling period in human intestine organoids cell medium (FIG. 2A) and in HEPES buffer (FIG. 2B). FIGS. 2C-2D shows the solvent fraction of the hydrogels throughout the 15-day swelling period in human intestine organoids cell medium (FIG. 2C) and in HEPES buffer (FIG. 2D). The swelling ratio and solvent fraction increased with time and reached plateau after 1-2 days. The hydrogels were able to hold up to 98% water in their swollen matrix.

Mechanical Properties

[00102] The stiffness of the cured hydrogels was investigated. Storage loss modulus (G') and loss modulus (G") at 1Hz was calculated for cured hydrogels using a TA Instruments DHR3 rheometer equipped with parallel plate geometry (top plate 20 mm), a Peltier plate (S#l 13258) and solvent evaporation blocking system. Approximately 250-300 pL of precursor hydrogel using the composition from Example 1 was deposited on a 20 mm disposable (Al, bottom plate) parallel plate. The precursor hydrogel was cured for 30-60 minutes at 37° C. A strain sweep (0.01-10%) was performed to identify the working strain (i.e., 1%) followed by a frequency sweep at 1% strain to determine the storage modulus (G') and loss modulus (G") at 1Hz. Strain sweeps were also performed after curing and after incubation of the hydrogels in cell culture medium for between 0 days and 15 days. This process (both for cured and swollen hydrogels) was repeated for approximately 250-300 pL of Corning® Matrigel® and Sigma- Aldrich® TrueGel3D TRUE1. FIG. 3 shows the storage loss modulus (G') at certain points from t=0 to t=120 hours after the hydrogels have cured and been surrounded by human intestine organoids medium. A summary of the store loss modulus (G') and the loss modulus (G") of the hydrogels surrounded by human intestine organoids medium at t=0 and t=4.6 days are shown in Table 1 below.

TABLE 1

Storage loss modulus (G') and loss modulus (G") of Chitosan-based, Matrigel®, and TRUE1 hydrogels

Cured Hydroseis (G') (G")

Chitosan-PEG-4-VS-RGD-VPM 151 ± 10 Pa 1.8 ± 0.5 Pa hydrogel

Matrigel® 110 ± 10 Pa 6.4 ± 1.0 Pa

TRUE 1 290 ± 5 Pa 3.7 ± 0.5 Pa

Swollen Hydrosels at 4.6 days

Chitosan-PEG-4-VS-RGD-VPM 40 ± 4 Pa 2.0 ± 0.5 Pa hydrogel

Matrigel® 11 ± 1 Pa 2.4 ± 0.5 Pa

TRUE 1 130 ± 10 Pa 1.9 ± 0.5 Pa

[00103] The results show that the freshly cured hydrogels are stiffer than the swollen hydrogels and the stiffness of the chitosan-based hydrogels are significantly lower than the Sigma- Aldrich® TRUE1 hydrogel.

Example 3 - 3D Cell Culture with Hydrogels

[00104] Madin-Darby canine kidney (MDCK) cells were employed as a test system of cells for 3D culturing with the chitosan-based hydrogels of the present disclosure. The precursor hydrogel from Example 1 was mixed with about 180-240 cells of MDCK cell suspension using a pipet tip. Domes of about 8-10 pL were formed on 24- well plates that were tissue culture- treated plates, ultra-low binding polystyrene plates, or untreated polystyrene plates. The 24-well plates were placed upside down and the hydrogel-cell mixtures were cured for 30 minutes at 37° C. Then, the 24-well plates were placed back upright and approximately 1 mL of MDCK cell culture medium was added to each well. The 24-well plates were then incubated at 37° C. This process was repeated using Matrigel® as the hydrogel instead of the composition of Example 1, with the exception that the mixing step was performed at 4-10° C and the curing step was performed at 25- 37° C.

[00105] Cell growth was optically monitored with an optical microscope at different time intervals over 6-8 days, optical images were taken periodically throughout. FIGS. 4A-4D show optical micrographs at 20x magnification after 6 days of incubation for the Matrigel® (FIGS. 4A, 4C) and chitosan-based gels (FIGS. 4B, 4D) grown on either tissue culture treated (FIGS. 4A, 4B) or ultra-low binding (FIGS. 4C, 4D) 24-well plates. MDCK cysts are present in all the micrographs, indicating that 3D cultures can be generated from those hydrogels.

[00106] Stability was also optically monitored over 6-8 days and optical images were taken periodically throughout. The chitosan-based hydrogel domes were stable during the entire MDCK cell culture. The Matrigel® hydrogel domes tended to degrade slowly after a week. FIGS. 5A-5F show optical images of chitosan-based hydrogels with MDCK cells after 8 days of incubation at 4x magnification (FIGS. 5 A, 5D), lOx magnification (FIGS. 5B, 5E), and 20x magnification (FIGS 5C, 5F) in either untreated polystyrene 12-well plates (FIGS. 5A-5C) or tissue-cultured 24-well plates (FIGS. 5D-5F). MDCK cysts are present in all the micrographs, indicating that 3D cultures can be generated from those hydrogels.

[00107] Stability was further monitored until 25 days after the domes were surrounded by organoids medium by analyzing the mass of the gel domes. FIG. 6 shows that the chitosan-based hydrogels were stable over the entire 25 day period whereas the Matrigel® hydrogels began to slowly degrade after one week and continued to degrade throughout the monitoring period.

Example 4 - Digestion of chitosan-based hydrogels [00108] Digestion of chitosan-based hydrogels was investigated using several different digestion enzymes. Enzymes tested were Accutase® (Innovative Cell Technologies, Inc.), collagenase type 1 from Clostridium histolyticum (Gibco™), dispase from Bacillus polymyxa (Gibco™), and protease from Streptomyces griseus (Sigma-Aldrich®). Corning® Cell Recovery Solution (product #354253) was used as a control as it is a non-enzymatic solution. The amount added depended on which solution was used. For collagenase- 1, dispase, and protease from Streptomyces griseus, the hydrogel to enzyme ratio was 1:3. In other words, for every 100 pL of hydrogel, 300 pL of enzyme at 10 mg/mL concentration was added. Stated another way, for every 100 pL of hydrogel, 300 pL containing either about 5.5 U of dispase, 12 U of protease from Streptomyces griseus, or 765 U of collagenase-1 from Clostridium histolyticum was added. For Accutase®, the hydrogel to enzyme ratio was 1:4. For Corning® Cell Recovery Solution, the hydrogel to solution ratio was 1:8. Monitoring of the digestion progress was monitored visually using colored hydrogels. FIG. 7 shows that the chitosan-based hydrogels were completely dissolved in about 20 minutes with collagenase, in about 25 minutes with dispase, and in about 20 minutes for protease from Streptomyces griseus. Gels were considered completely dissolved when the hydrogel had liquified (seen by visual inspection). Accutase® took longer, at least 1-2 hours to digest. No digestion occurred with the Corning® Cell Recovery Solution.

[00109] While the present disclosure includes a limited number of embodiments, those skilled in the art, having benefit of this disclosure, will appreciate that other embodiments can be devised which do not depart from the scope of the present disclosure.

Claims

CLAIMS What Is Claimed Is:

1. A synthetic hydrogel, comprising: a chitosan-thioglycolic acid; a multi-armed polyethylene glycol vinyl sulfone; a first peptide comprising an Arginine-Glycine- Aspartic Acid amino acid sequence and a cysteine amino acid at or near a terminal end of the peptide; and a second peptide comprising a Valine-Proline-Methionine amino acid sequence and a cysteine amino acid at or near each terminal end of the peptide.

2. The synthetic hydrogel of claim 1 , wherein the chitosan-thioglycolic acid has a molecular weight from about 50 kDa to about 200 kDa.

3. The synthetic hydrogel of claim 1, wherein the multi-armed polyethylene glycol vinyl sulfone is a four-arm polyethylene glycol vinyl sulfone.

4. The synthetic hydrogel of claim 3, wherein the four-arm polyethylene glycol vinyl sulfone comprises an average molecular weight from about 2 kDa to about 20 kDa.

5. The synthetic hydrogel of claim 1, wherein the first peptide is chosen from GRGDSPC (SEQ ID NO: 1), GRGDSPCx (SEQ ID NO: 2), or RGDC (SEQ ID NO: 3), wherein “x” is chosen from glycine (G), alanine (A), leucine (L), valine (V) serine (S), threonine (S), aspartic acid (D), or glutamic acid (E).

6. The synthetic hydrogel of claim 1 , wherein the second peptide is chosen from GCRDVPMSMRGGDRCG (SEQ ID NO: 4), xCRDVPMSMRGGDRCx (SEQ ID NO: 5), CRDVPMSMRGGDRC (SEQ ID NO: 6), or CRDVPMSMRGGDRCG (SEQ ID NO: 7), CRDVPMSMRGGDRCx (SEQ ID NO: 8), GCRDVPMSMRGGDRC (SEQ ID NO: 9), or xCRDVPMSMRGGDRC (SEQ ID NO: 10), wherein “x” is chosen from alanine (A), valine (V), leucine (L), serine (S), threonine (T), aspartic acid (D), or glutamic acid (E), and wherein one “x” may be glycine (G).

7. The synthetic hydrogel of claim 6, wherein the first peptide is GRGDSPC (SEQ ID NO: 1), wherein the second peptide is GCRDVPMSMRGGDRCG (SEQ ID NO: 4), and wherein the multi-armed polyethylene glycol vinyl sulfone is a four-arm polyethylene glycol comprising an average molecular weight of about 20 kDa.

8. The synthetic hydrogel of claim 1, wherein the synthetic hydrogel comprises a storage modulus of between about 125 Pa to about 175 Pa.

9. The synthetic hydrogel of claim 1, wherein the synthetic hydrogel comprises a storage modulus of between about 25 Pa to about 55 Pa after swelling in an aqueous solution for about 4.5 days.

10. A method of making a synthetic hydrogel, comprising the steps of:

(a) providing a thiolated chitosan;

(b) providing a multi-armed polyethylene glycol vinyl sulfone;

(c) providing a first peptide comprising an Arginine-Glycine- Aspartic Acid amino acid sequence and a cysteine amino acid at or near a terminal end of the peptide;

(d) providing a second peptide comprising a Valine-Proline-Methionine amino acid sequence and a cysteine amino acid at or near each terminal end of the peptide;

(e) mixing the thiolated chitosan and multi-armed polyethene glycol vinyl sulfone;

(f) adding the first peptide to the mixture of step (d); and

(g) adding the second peptide to the mixture of step (f).

11. The method of claim 10, wherein the thiolated chitosan is chitosan-thioglycolic acid.

12. The method of claim 11, wherein the chitosan-thioglycolic acid has a molecular weight from about 50 kDa to about 200 kDa.

13. The method of claim 10, wherein the multi-armed polyethylene glycol vinyl sulfone is a four-arm polyethylene glycol vinyl sulfone.

14. The method of claim 13, wherein the four-arm polyethylene glycol vinyl sulfone has an average molecular weight from about 2 kDa to about 20 kDa.

15. The method of claim 10, wherein the first peptide is chosen from GRGDSPC (SEQ ID NO: 1), GRGDSPCx (SEQ ID NO: 2), or RGDC (SEQ ID NO: 3), wherein “x” is chosen from of glycine (G), alanine (A), leucine (L), valine (V) serine (S), threonine (S), aspartic acid (D), or glutamic acid (E).

16. The method of claim 10, wherein the second peptide is chosen from GCRDVPMSMRGGDRCG (SEQ ID NO: 4), xCRDVPMSMRGGDRCx (SEQ ID NO: 5), CRDVPMSMRGGDRC (SEQ ID NO: 6), or CRDVPMSMRGGDRCG (SEQ ID NO: 7), CRDVPMSMRGGDRCx (SEQ ID NO: 8), GCRDVPMSMRGGDRC (SEQ ID NO: 9), or xCRDVPMSMRGGDRC (SEQ ID NO: 10), wherein “x” is chosen from alanine (A), valine (V), leucine (L), serine (S), threonine (T), aspartic acid (D), or glutamic acid (E), and wherein one “x” may be glycine (G).

17. The method of claim 16, wherein the first peptide is GRGDSPC (SEQ ID NO: 1), wherein the second peptide is GCRDVPMSMRGGDRCG (SEQ ID NO: 4), and wherein the multi-armed polyethylene glycol vinyl sulfone is a four-arm polyethylene glycol vinyl sulfone having an average molecular weight of about 20 kDa.

18. A method of dissolving a synthetic hydrogel, comprising the steps of:

(a) providing a synthetic hydrogel comprising cells cultured in the synthetic hydrogel, wherein the synthetic hydrogel comprises the composition of claim 1 ; (b) adding to the synthetic hydrogel at least one enzyme that cleaves the second peptide; and

(c) incubating the resultant combination from steps (a) and (b).

19. The method of claim 18, wherein the at least one enzyme is chosen from collagenase, dispase, protease from Streptomyces griseus, or a trypsin-substitute enzyme, or a combination thereof.

20. The method of claim 19, wherein the addition of volume of the hydrogel to the volume of the enzyme is at a ratio of about 1 :4 for a trypsin-substitute enzyme, about 1 :3 for collagenase, about 1 :3 for dispase, and about 1:3 for protease from Streptomyces griseus, and wherein the concentration of the enzyme being added is about 10 mg/mL.

21. The method of claim 19, wherein the enzyme is one of collagenase-1 from Clostridium histolyticum, dispase from Bacillus polymyxa, or protease from Streptomyces griseus, and wherein the incubation dissolves the synthetic hydrogel after about 25 minutes at an incubation temperature of 37° C.

22. A method of growing a three-dimensional cell culture, comprising the steps of:

(a) providing a chitosan-PEG-VS-RGD conjugate;

(b) providing a VPM peptide;

(c) providing cells of a cell type, wherein the cell type is capable of growing a three- dimensional cell culture;

(d) combining the conjugate of step (a) with the peptide of step (b) and with the cell type of step (c);

(e) dispensing a discrete volume of the combined precursor hydrogel from step (d) onto a substrate;

(f) forming a hydrogel from the dispensed discrete volume;

(g) surrounding the formed hydrogel with a discrete volume of cell culture medium; and (h) incubating the surrounded hydrogel.

23. The method of claim 22 wherein the chitosan-PEG-VS-RGD conjugate comprises chitosan-thioglycolic acid, four-arm polyethylene glycol vinyl sulfone, and an amino acid sequence comprising Arginine-Glycine-Aspartic Acid.

24. The method of claim 23, wherein the amino acid sequence comprising Arginine-Glycine- Aspartic Acid is GRGDSPC (SEQ ID NO: 1) in its unconjugated form, and the VPM peptide is GCRDVPMSMRGGDRCG (SEQ ID NO: 4).

25. The method of claim 23, wherein the cells are cells that form spheroids or organoids.

26. The method of claim 23, wherein the cells are MDCK cells or Chinese Hamster Ovary cells.

27. The method of claim 23, wherein the substrate is a micro well plate.

28. The method of claim 22, wherein the synthetic hydrogel has not significantly degraded after 15 days of incubation.

29. The method of claim 22, wherein the synthetic hydrogel has not significantly degraded after 28 days of incubation.

30. A kit for forming a synthetic hydrogel, comprising: a first container; and a second container; wherein the first container comprises a chitosan-PEG-VS-RGD conjugate, and wherein the second container comprises a VPM peptide.

31. The kit of claim 30, wherein the chitosan-PEG-VS-RGD conjugate comprises a chitosan- thioglycolic acid, a multi-arm polyethylene glycol vinyl sulfone having between 3 and 6 arms, and an amino acid sequence comprising Arginine-Gly cine- Aspartic Acid.

32. The kit of claim 31, wherein the amino acid sequence comprising Arginine- Glycine- Aspartic Acid is GRGDSPC (SEQ ID NO: 1).

33. The kit of claim 32 wherein the VPM peptide is GCRDVPMSMRGGDRCG (SEQ ID NO: 4).

34. The kit of claim 33, wherein the first and second containers are vials.

35. A kit for a three-dimensional cell culture comprising: a chitosan-PEG-VS-RGD conjugate; and a VPM peptide; wherein the chitosan-PEG-VS-RGD conjugate and the VPM peptide are powders.

36. The kit of claim 36, wherein the chitosan-PEG-VS-RGD conjugate comprises a chitosan- thioglycolic acid, a multi-arm polyethylene glycol vinyl sulfone having between 3 and 6 arms, and an amino acid sequence comprising Arginine-Gly cine- Aspartic Acid.

37. The kit of claim 36, wherein the amino acid sequence comprising Arginine- Glycine- Aspartic Acid is GRGDSPC (SEQ ID NO: 1).

38. The kit of claim 37 wherein the VPM peptide is GCRDVPMSMRGGDRCG (SEQ ID NO: 4).

39. The kit of claim 35, further comprising a vessel for three-dimensional cell culture.