Chitosan-based hydrogels for 3D cell culture

Abstract

Compositions of chitosan-based synthetic hydrogels and methods of using the same are provided. The chitosan-based hydrogel is prepared from functionalized chitosan, multi-arm polyethylene glycol vinyl sulfone, RGD peptide and VPM peptide. The chitosan-based synthetic hydrogel can be used for three-dimensional cell culture and can be dissolved. These chitosan-based synthetic hydrogels have at least the following advantages: their mechanical properties are similar to those of non-synthetic Matrigol, but the definition is clearer.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 543,808, filed October 12, 2023, pursuant to 35 USC §119, the contents of which are incorporated herein by reference in their entirety.

[0003] Reference to electronic sequence listing

[0004] The contents of the electronic sequence list, named Sequence_Listing_SP23-255.xml (11,704 bytes) and created on September 10, 2024, are incorporated herein by reference in full. Technical Field

[0005] This disclosure relates to chitosan-based synthetic polymeric hydrogels, methods for preparing chitosan-based synthetic polymeric hydrogels, methods for performing biomolecular analysis using said hydrogels, and kits for said hydrogels, particularly for advanced three-dimensional cell culture, such as spheroid and organoid culture. Background Technology

[0006] Organoids are three-dimensional, cell-based in vitro models that mimic corresponding in vivo organs, possessing many of the structural and functional characteristics of those organs. The similarity between organoids and natural organs can play a crucial role in medical applications such as preclinical drug development, toxicity screening, regenerative medicine, and studying the mechanisms of organ development. In organoid culture, hydrogels can be used as scaffolds to support cell growth, proliferation, and differentiation. Hydrogels typically contain hydrophilic homopolymers / copolymers, which are cross-linked using natural, synthetic, or hybrid polymers through physical or chemical means. Hydrogels can be cast into various shapes or sizes and possess network properties such as swelling capacity, stiffness, permeability, and porosity. These network properties can have a profound impact on cell viability and cell fate.

[0007] Matrigel, a natural basement membrane derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma. ® It is the most commonly used scaffold for supporting 3D organoid culture. Matrigel ®Highly mimicking the natural extracellular matrix (ECM), it contains hundreds of proteins (>1800), including laminin (approximately 60%), type IV collagen (approximately 30%), enactin (approximately 8%), and heparin sulfate proteoglycan perlecan (approximately 2% to 3%). ® Gelation occurs above 20°C, during which nestin acts as a cross-linking agent to connect laminin and collagen, thus forming a gel. Although Matrigel... ® It is the preferred hydrogel for organoid culture, but batch-to-batch variations in protein concentration make it difficult to control its network properties (e.g., stiffness). This can lead to inconsistent cell culture results and cause problems.

[0008] Therefore, there is a need for synthetic hydrogels with well-defined chemical and mechanical properties that can also construct advanced three-dimensional cellular structures, such as organoids. Summary of the Invention

[0009] This disclosure provides the following: Matrigel with animal-derived ingredients. ® Compositions of chitosan-based synthetic hydrogels with similar mechanical properties, methods for preparing chitosan-based synthetic hydrogels, methods for using chitosan-based synthetic hydrogels, and kits for preparing chitosan-based synthetic hydrogels.

[0010] According to some aspects of this disclosure, a composition for synthesizing a hydrogel is provided, comprising: chitosan-thioglycolic acid; a multi-arm 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 located at or near the end of said peptide; and a second peptide comprising a valine-proline-methionine (VPM) amino acid sequence and a cysteine ​​amino acid located at or near the end of each of said peptides. The chitosan-thioglycolic acid of the synthetic hydrogel may have a molecular weight of about 50 kDa to about 200 kDa. The multi-arm 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 of about 2 kDa to about 20 kDa. The first peptide may be selected from GRGDSPC (SEQ ID NO: 1), GRGDSPCx (SEQ ID NO: 2) or RGDC (SEQ ID NO: 3), wherein "x" is selected 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 selected from GCRDVPMSMRGGD RCG (SEQ ID NO: 4), xCRDVPMSMRGGDRCx (SEQ ID NO: 5), CRDVPMSMRGGDRC (SEQ ID NO: 6), CRDVPMSMRGGDRCG (SEQ ID NO: 7), CRDVPMSMRGGDRCx (SEQ ID NO: 8), GCRDVPMSMRGGDRC (SEQ ID NO: 9), or xCRDVPMSMRGGDRC (SEQ ID NO: 10), wherein "x" is selected from alanine (A), valine (V), leucine (L), serine (S), threonine (T), aspartic acid (D), or glutamic acid (E), and one of the "x" may be glycine (G). The synthetic hydrogel may have a storage modulus between approximately 125 Pa and approximately 175 Pa. After swelling in aqueous solution for approximately 4.5 days, the synthetic hydrogel may have a storage modulus between approximately 25 Pa and approximately 55 Pa.

[0011] In a particular 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-arm polyethylene glycol ethylene sulfone is a four-arm polyethylene glycol ethylene sulfone having an average molecular weight of about 20 kDa.

[0012] According to another aspect of this disclosure, a method for preparing a synthetic hydrogel is provided, comprising the steps of: (a) providing a thiolated chitosan; (b) providing a multi-arm polyethylene glycol sulfone; (c) providing a first peptide comprising an arginine-glycine-aspartic acid sequence and a cysteine ​​amino acid located at or near the end of the peptide; (d) providing a second peptide comprising a valine-proline-methionine amino acid sequence and a cysteine ​​amino acid located at or near the end of each of the peptides; (e) mixing the thiolated chitosan with the multi-arm polyethylene glycol 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, these steps may be performed in a different order. The thiolated chitosan may be chitosan-thioglycolic acid, and chitosan-thioglycolic acid may have a molecular weight of about 50 kDa to about 200 kDa. The multi-arm polyethylene glycol ethylene sulfone can be a four-arm polyethylene glycol ethylene sulfone, and the four-arm polyethylene glycol ethylene sulfone can further have an average molecular weight of about 2 kDa to about 20 kDa. The first peptide can be selected from GRGDSPC (SEQ ID NO: 1), GRGDSPCx (SEQ ID NO: 2) or RGDC (SEQ ID NO: 3), wherein "x" is selected 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 selected from GCRDVPMSMRGGD RCG (SEQ ID NO: 4), xCRDVPMSMRGGDRCx (SEQ ID NO: 5), CRDVPMSMRGGDRC (SEQ ID NO: 6), CRDVPMSMRGGDRCG (SEQ ID NO: 7), CRDVPMSMRGGDRCx (SEQ ID NO: 8), GCRDVPMSMRGGDRC (SEQ ID NO: 9), or xCRDVPMSMRGGDRC (SEQ ID NO: 10), wherein "x" is selected from alanine (A), leucine (L), serine (S), threonine (T), aspartic acid (D), or glutamic acid (E), and one of the "x" may be glycine (G).

[0013] In a particular 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-arm polyethylene glycol ethylene sulfone is a four-arm polyethylene glycol ethylene sulfone having an average molecular weight of about 20 kDa.

[0014] According to other aspects of this disclosure, a method for 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 at least one enzyme to the synthetic hydrogel, the enzyme cleaving a second peptide at a valine-proline-methionine amino acid sequence; and (c) culturing the combination produced in steps (a) and (b). The enzyme cleaving the second peptide may be selected from collagenase, dispase, protease from Streptomyces griseus, or trypsin-substitute enzyme (e.g., Accutase). ® (or a combination thereof). The ratio of the added hydrogel volume to the volume of the enzyme cleaving the second peptide may be approximately 1:4 for trypsin substitutes, approximately 1:3 for collagenases, approximately 1:3 for dispersants, and approximately 1:3 for proteases from *Streptomyces griseus*, and the concentration of said enzyme may be approximately 10 mg / mL. In a particular embodiment of this aspect, the enzyme cleaving the second peptide may be one of collagenase-1 from *Clostridium histolyticum*, a dispersant from *Bacillus polymyxa*, or a protease from *Streptomyces griseus*, and the incubation process, after incubation at 37°C for approximately 25 minutes, allows the synthetic hydrogel to dissolve.

[0015] According to other aspects of this disclosure, a method for 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 a cell type; (d) combining the conjugate of step (a) with the peptide of step (b) and the cell type of step (c); (e) dispensing a precursor hydrogel from the combination of step (d) onto a matrix in discrete volumes; (f) forming a hydrogel from the dispensed discrete volumes; (g) surrounding the formed hydrogel with discrete volumes of cell culture medium; and (h) culturing the surrounded hydrogel. However, these steps may be performed in a different order. The chitosan-PEG-VS-RGD conjugate may comprise chitosan-thioglycolic acid, a four-arm polyethylene glycol sulfone, and an amino acid sequence comprising arginine-glycine-aspartic acid. The uncoupled form of the amino acid sequence containing arginine-glycine-aspartic acid can be GRGDSPC (SEQ ID NO: 1), and the VPM peptide can be GCRDVPMSMRGGDRCG (SEQ ID NO: 4). The cells can be spheroid- or organoid-forming cells. In a specific embodiment of this aspect, the cells can be MDCK cells or Chinese hamster ovary cells. The matrix can be a microplate. The cell culture method can prepare synthetic hydrogels that do not show significant degradation after 15 days of culture. The cell culture method can prepare synthetic hydrogels that do not show significant degradation after 28 days or longer of culture.

[0016] According to other aspects of this disclosure, a kit is provided comprising a composition for preparing a synthetic hydrogel. The kit includes a first container and a second container, wherein the first container contains a chitosan-PEG-VS-RGD conjugate, and the second container contains a VPM peptide. The chitosan-PEG-VS-RGD conjugate may contain chitosan-thioglycolic acid, a multi-arm polyethylene glycol sulfone having 3 to 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.

[0017] Further features and advantages will be set forth in the following detailed description, and in part will become apparent to those skilled in the art from the description, or will be recognized by practice of the embodiments described herein, including the following detailed description, the claims, and the drawings.

[0018] It should be understood that the foregoing general description and the following detailed description are merely exemplary and intended to provide an overview or framework for understanding the nature and characteristics of the claims. The accompanying drawings are included to provide further understanding and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiments and, together with this specification, serve to explain the principles and operation of various embodiments. Attached Figure Description

[0019] The following is a description of the diagrams in the accompanying drawings, given purely by way of non-limiting example. The diagrams are not necessarily drawn to scale, and for the purposes of clarity and conciseness, some features and views may be shown enlarged or schematically.

[0020] Figure 1 This is a schematic diagram of a general reaction for the synthesis of chitosan-based hydrogels according to some aspects of this disclosure.

[0021] Figures 2A to 2D This is a graph showing the swelling ratio and solvent fraction of chitosan-based hydrogels prepared according to some aspects of this disclosure. Figure 2A This is a graph showing the average swelling ratio (Q) of chitosan hydrogels in human intestinal organoid cell culture medium over time. Figure 2B This is a graph showing the average swelling ratio (Q) of chitosan hydrogels in HEPES buffer over time. Figure 2C This is a graph showing the solvent fraction of chitosan hydrogels over time in human intestinal organoid cell culture medium. Figure 2D This is a graph showing the solvent fraction of chitosan hydrogel in HEPES buffer over time.

[0022] Figure 3 Based on some aspects of this disclosure, Matrigel ® Figures showing the energy loss modulus of TrueGel-1 or chitosan-PEG-RGD-VPM hydrogels after solidification in human intestinal organoid culture medium from 0 to 120 hours.

[0023] Figure 4 A to Figure 4 D illustrates some aspects of this disclosure, in the treatment of tissue cultures ( Figure 4 A, Figure 4 B) or ultra-low binding ( Figure 4 C Figure 4 Matrigel grown on a 24-well plate (D) ® ( Figure 4 A, Figure 4 C) and chitosan-based gels ( Figure 4 B Figure 4 D) Optical micrograph at 20x magnification after 6 days of cultivation.

[0024] Figure 5 A to Figure 5 F illustrates some aspects of this disclosure regarding untreated 12-well polystyrene plates ( Figure 5 A to Figure 5 C) or 24-well plates cultured in tissue culture ( Figure 5 D to Figure 5 In F), the chitosan-based hydrogel containing MDCK cells was magnified 4 times after 8 days of incubation. Figure 5 A, Figure 5 D) 10x magnification ( Figure 5 B Figure 5 E) and 20x magnification ( Figure 5 C Figure 5 Optical image under F).

[0025] Figure 6 This demonstrates, according to some aspects of the present disclosure, the chitosan-based hydrogel and Matrigel during 25 days after the hydrogel was surrounded by organoid culture medium. ® A graph showing the stability data of the hydrogel.

[0026] Figure 7 Photographs are shown of chitosan-PEG-RGD-VPM hydrogels according to aspects of this disclosure, the hydrogels being exposed to the enzyme Accutase. ® (trypsin substitute enzyme), collagenase, dispersase, or protease from Streptomyces griseus (Sigma-Aldrich) ® It dissolves when exposed to enzyme solutions, but not when exposed to non-enzymatic solutions (Corning). ® It does not dissolve during cell recovery solution. Detailed Implementation

[0027] Various aspects and embodiments will now be fully described herein. However, these aspects and embodiments may be implemented in many different forms and should not be construed as limiting; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the subject matter of the invention to those skilled in the art. All publications, patents, and patent applications cited herein, both above and below, are incorporated herein by reference in their entirety.

[0028] Modifications to this disclosure will arise in the minds of those skilled in the art and those who have prepared or used this disclosure. Therefore, it should be understood that the embodiments shown in the accompanying drawings and described above are for illustrative purposes only and are not intended to limit the scope of this disclosure, which is defined by the appended claims and interpreted in accordance with the principles of patent law, including the doctrine of equivalents.

[0029] A. Definition

[0030] Unless otherwise defined, all terms and phrases used herein include their meanings as they have been acquired in the art, unless the context in which the term or phrase is used explicitly indicates or it is obvious to the contrary. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, specific methods and materials are described here.

[0031] Unless otherwise stated, the use of a single numerical value is as an approximation, as if the value were preceded by the words “about” or “approximately”. Similarly, unless otherwise explicitly stated, numerical values ​​within the various ranges specified in this application are as approximations, as if the minimum and maximum values ​​within the stated ranges were preceded by the words “about” or “approximately”. In this way, variations above and below the stated ranges can be used to obtain substantially the same results as values ​​within the stated ranges. As used herein, when referring to numerical values, the terms “about” and “approximately” should have their common and general meaning to those skilled in the art in the art most closely related to the disclosed subject matter or to the scope or element discussed. The degree of deviation from strict numerical boundaries depends on a number of factors. For example, some factors that may be considered include the criticality of the element, and / or the impact of variations in a given quantity on the performance of the claimed subject matter, and other considerations known to those skilled in the art. As used herein, the use of different numbers of significant figures for different numerical values ​​does not imply a limitation on the use of the words “about” or “approximately” to broaden a specific numerical value or range. Therefore, in general, “about” or “approximately” is used to broaden numerical values. Furthermore, the disclosed range is intended to be a continuous range, including every value between the minimum and maximum values, plus the range broadened by the use of the terms "about" and "approximately". Therefore, the description of the range of values ​​herein is intended only as a shorthand method for individually referring to each individual value falling within the stated range, and each individual value is incorporated into the specification as if it were described separately herein.

[0032] As used herein, when used in a list of two or more items, the term "and / or" means that any one of the listed items may be used alone, or any combination of two or more of the listed items may be used. For example, if a composition is described as containing components A, B, and / or C, then the composition may contain A alone; contain B alone; contain C alone; contain a combination of A and B; contain a combination of A and C; contain a combination of B and C; or contain a combination of A, B, and C.

[0033] As used in this article, “has,” “have,” “having,” “include,” “including,” “comprise,” and “comprising” are used in an open-ended sense and usually mean “including (but not limited to).”

[0034] For the purposes of this disclosure, the term "near-terminus" generally refers to the last residue preceding the N-terminal or C-terminal amino acid (or both) of an amino acid sequence.

[0035] "Optional" or "optionally" means that the elements, components, or situations described below may or may not occur, and therefore the description includes both the occurrence and non-occurrence of the elements, components, or situations.

[0036] As used herein, the terms “the” and “a / an” mean “at least one” and should not be limited to “only one” unless explicitly indicated otherwise. Thus, for example, unless the context explicitly indicates otherwise, references to “component” include embodiments having two or more of the said components.

[0037] In this document, relational terms (such as first and second, top and bottom, etc.) are used only to distinguish one entity or action from another entity or action, and do not necessarily require or imply any actual relationship or order between such entities or actions.

[0038] Unless otherwise stated, all scientific and technical terms used herein have their common meaning in the art. The definitions provided herein are for ease of understanding of certain terms used frequently and are not intended to limit the scope of this disclosure.

[0039] B. Introduction

[0040] This disclosure relates to the field of synthetic hydrogels. This disclosure also relates to synthetic hydrogel compositions; methods for preparing said hydrogel compositions; methods for using said hydrogels; and kits for culturing at least Chinese hamster ovary (CHO) cells, human fibroblasts, endothelial cells, Madin-Darby canine kidney (MDCK) cells, spheroids, and organoids using said hydrogels. These hydrogels are synthetic, but still provide animal-derived standards such as Matrigel. ® Quite mechanical features.

[0041] The hydrogel of this invention has the following advantages: it is synthetic (unlike Matrigel derived from mouse tumors). ®It uses water-soluble precursors and forms gels at common reaction temperatures (such as room temperature and 37°C) (without requiring curing, catalysts, or irradiation); gel formation is rapid (e.g., 5 to 30 minutes); it enables 3D dome-shaped cell culture of MDCK and organoid cells; it is optically transparent, suitable for cell imaging analysis; and it can be rapidly degraded in various enzyme solutions (e.g., degraded within 30 minutes using dispersing enzymes), thus achieving efficient cell recovery. Furthermore, the hydrogel maintains a stable 3D dome structure on various types of culture plates (including tissue culture-treated, untreated, and low-binding well plates).

[0042] Additional features and advantages will be set forth in the following detailed description, and will become apparent to those skilled in the art from the description, or will be recognized by practice of the embodiments described below, in conjunction with the claims and drawings.

[0043] C. Hydrogel compositions

[0044] As disclosed herein, a novel multifunctional chitosan-based hydrogel is provided, incorporating cell-adhesive and hydrogel-degradable peptides for 3D cell culture, including organoid culture. The synthetic hydrogel may comprise: thiol-functionalized chitosan (CS-SH), multi-arm polyethylene glycol sulfone (e.g., PEG-4-VS), a peptide comprising an arginine-glycine-aspartic acid motif and at least one cysteine ​​amino acid located at or near the end of the peptide (RGD peptide) (e.g., GRGDSPC (SEQ ID NO: 1)), and a peptide comprising a valine-proline-methionine motif and at least one cysteine ​​amino acid located at or near the end of each of the peptides (VPM peptide) (e.g., GCRDVPMSMRGGDRCG (SEQ ID NO: 2)). Systematic studies of the hydrogel (including swelling behavior, rheological properties, and MDCK cell culture) have shown that the hydrogel exhibits strong swelling properties (e.g., water content in the swollen matrix can reach up to 98%) and a hardness of approximately 40-160 Pa. Furthermore, the hydrogel is suitable for 3D dome culture on various culture plates and exhibits excellent MDCK cell vesicle formation ability after 2-6 days of culture. The results obtained using the synthetic chitosan-based hydrogel are comparable to those obtained using Matrigel. ® Exhibiting a striking similarity, it provides an alternative to naturally sourced Matrigel. ® The synthetic hydrogel scheme is described herein. Similarly, as described herein, the network properties of the disclosed synthetic chitosan-based hydrogels (such as hardness, swelling, permeability, cell adhesion, and degradability) are tunable (Matrigel). ® (Then it does not possess this adjustability).

[0045] The following section describes the thiol-functionalized chitosan, multi-arm PEG-VS, RGD peptide, and VPM peptide in chitosan-based synthetic hydrogels.

[0046] Thiol-functionalized chitosan

[0047] Chitosan is a polymer of β(1→4)-D-glucosamine and β(1→4)-N-acetyl-D-glucosamine units. In one embodiment, thioglycolic acid is attached to chitosan via an amino group at the 2-position of the glucosamine subunit, producing a thiolated chitosan, referred to as chitosan-thioglycolic acid. This thiofunctionalized chitosan polymer can be synthesized in a laboratory by methods known to those skilled in the art, or it can be purchased from a supplier as a pre-functionalized product. In one embodiment, the thiofunctionalized chitosan has a low molecular weight. In another embodiment, the thiofunctionalized chitosan has a molecular weight in the range of about 50 kDa to about 200 kDa. In one particular 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 any range or value between about 50 kDa and about 200 kDa.

[0048] polyethylene glycol ethylene sulfone

[0049] Polyethylene glycol vinyl sulfone (PEG-VS) is a derivative of polyethylene glycol (PEG). Multiple PEG-VS molecules are linked together via branching centers to create multi-arm PEG-VS. For example, covalently linking four PEG-VS molecules via a pentaerythritol core produces a PEG conjugate called a four-arm PEG-VS (i.e., PEG-4-VS). In one embodiment, polyethylene glycol vinyl sulfone can be selected 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 particular embodiment, polyethylene glycol vinyl sulfone is PEG-2-VS. In another particular embodiment, polyethylene glycol vinyl sulfone is PEG-4-VS. In yet another particular embodiment, polyethylene glycol vinyl sulfone is PEG-8-VS. These PEG-VS conjugates can be synthesized in a laboratory using methods known to those skilled in the art, or they can be obtained from suppliers such as Sigma Aldrich. ® Purchase products that have been functionalized.

[0050] In one embodiment, the molecular weight of polyethylene glycol (including biarmed, tetraarmed, and octaarmed PEG) is in the range of about 2 kDa to about 20 kDa. In another embodiment, the average molecular weight of the PEG-VS conjugate (including biarmed, tetraarmed, and octaarmed PEG) is about 2 kDa to about 20 kDa. In a particular embodiment, the average molecular weight of the PEG-VS conjugate (including biarmed, tetraarmed, and octaarmed PEG) is about 2 kDa, 5 kDa, 7.5 kDa, 10 kDa, 12.5 kDa, 15 kDa, 17.5 kDa, or 20 kDa. In a particular embodiment, the average molecular weight of the PEG-VS conjugate (including biarmed, tetraarmed, and octaarmed PEG-VS) is about 2 kDa to about 7.5 kDa, about 7.5 kDa to about 15 kDa, or about 15 kDa to about 25 kDa. In another particular embodiment, the average molecular weight of the biarmed PEG-VS is about 2 kDa, 3.5 kDa, or about 5 kDa. In another specific embodiment, the average molecular weight of the four-armed 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-armed PEG-VS is about 5 kDa, 10 kDa, or about 20 kDa.

[0051] RGD peptide

[0052] A peptide containing the arginine-glycine-aspartic acid motif (Arg-Gly-Asp) is called an RGD peptide. RGD motifs are a class of cell attachment motifs, i.e., motifs that promote cell binding. In one aspect of the invention, when the RGD peptide has at least one cysteine ​​amino acid at or near its terminal end, the RGD peptide can be coupled to a two-, four-, or eight-armed PEG-VS. The proximal cysteine ​​amino acid is the cysteine ​​residue located at the first, second, or third amino acid from the C-terminus and / or N-terminus in the peptide sequence. In one embodiment, the RGD peptide comprises an arginine-glycine-aspartic acid triplet and contains one cysteine ​​amino acid at or near its C-terminus. In a particular embodiment, the RGD peptide sequence is GRGDSPC (SEQ ID NO: 1). In this embodiment, the cysteine ​​amino acid is located at the C-terminus of the peptide. In another embodiment, the cysteine ​​residue is located near the C-terminus of the peptide. In one specific embodiment, the RGD peptide sequence is GRGDSPCx (SEQ ID NO: 2), wherein "x" is any one 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).

[0053] VPM peptides

[0054] A peptide containing the valine-proline-methionine amino acid motif (Val-Pro-Met) is called a VPM peptide. The VPM motif is a protease recognition site, i.e., a site where certain proteases can cleave the peptide at the VPM amino acid. In one aspect of the invention, the VPM peptide contains a cysteine ​​residue near its C-terminus and a cysteine ​​residue near its N-terminus. In a particular embodiment, the VPM peptide is GCRDVPMSMRGGDRCG (SEQ ID NO: 4). In another aspect of the invention, the glycine (G) residues at the N-terminus and / or C-terminus of SEQ ID NO: 4 are preferably substituted with one of alanine (A), valine (V), leucine (L), serine (S), threonine (T), aspartic acid (D), or glutamic acid (E). In other words, the VPM peptide can be xCRDVPMSMRGGDRCx (SEQ ID NO: 5), wherein "x" can preferably be selected from alanine (A), valine (V), leucine (L), serine (S), threonine (T), aspartic acid (D), glutamic acid (E), or any combination thereof, and one of the "x" can be glycine (G). In another aspect of the invention, the VPM peptide comprises a cysteine ​​residue at the C-terminus of the peptide and a cysteine ​​residue at the N-terminus of the peptide. In a particular embodiment, the VPM peptide is CRDVPMSMRGGDRC (SEQ ID NO: 6). In another aspect of the invention, the VPM peptide comprises a proximal cysteine ​​residue at the C-terminus of the peptide and a cysteine ​​residue at the N-terminus of the peptide. In a particular embodiment, the VPM peptide is CRDVPMSMRGGD RCG (SEQ ID NO: 7). Alternatively, the C-terminal glycine (G) may be substituted with an amino acid, such that the VPM peptide sequence is CRDVPMSMRGGDRCx (SEQ ID NO: 8), wherein "x" is preferably selected from alanine (A), valine (V), leucine (L), serine (S), threonine (T), aspartic acid (D), or glutamic acid (E). In another aspect of the invention, the VPM peptide comprises a proximal cysteine ​​residue at the N-terminus of the peptide and a cysteine ​​residue at the C-terminus of the peptide. In a particular embodiment, the VPM peptide is GCRDVPMSMRGGDRC (SEQ ID NO: 9). Alternatively, the C-terminal glycine (G) may be substituted with an amino acid, such that the VPM peptide sequence is xCRDVPMSMRGGDRC (SEQ ID NO: 10), wherein "x" is preferably selected from alanine (A), valine (V), leucine (L), serine (S), threonine (T), aspartic acid (D), or glutamic acid (E). In any of the above embodiments of the VPM peptide, although certain amino acids are preferred, "x" can be any nonpolar, uncharged, or charged amino acid.

[0055] D. Methods for preparing hydrogels

[0056] Typically, the synthesis of chitosan-based hydrogels with the mechanical characteristics considered in this invention involves the following three stages:

[0057] Phase 1: Coupling thiolated chitosan with PEG-VS

[0058] Phase 2: Synthesis of multifunctional conjugates, namely chitosan-PEG-VS-RGD

[0059] Phase 3: Synthesis of hydrogel from chitosan-PEG-VS-RGD and VPM peptides

[0060] refer to Figure 1 This disclosure provides a general reaction scheme for the synthesis of chitosan-based gels incorporating RGD and VPM peptides, and describes some aspects of this disclosure. First, thiolated chitosan is combined with PEG-VS (in... Figure 1 The reaction (represented by wavy lines) followed by the reaction with RGD peptide (in... Figure 1 The chitosan-PEG-VS-RGD conjugate was covalently coupled with a bifunctionalized VPM peptide crosslinking agent (represented by a rectangle) and then crosslinked with the conjugate at 37°C to produce a chitosan-based hydrogel. Figure 1 (The diagram on the far right shows the mesh pattern).

[0061] The amounts of thiolated chitosan, PEG-VS, RGD peptide, and VPM peptide added to the reaction can vary. For every 10 mmol of vinyl sulfone in PEG-VS, a total of 10 mmol of thiolated chitosan, RGD peptide, and VPM peptide thiols can be added. In one embodiment, the ratio of thiolated chitosan, RGD peptide, and VPM peptide to PEG-VS is: for every approximately 10 mmol of vinyl sulfone in PEG-VS, approximately 1 to approximately 3 mmol of thiolated chitosan, approximately 1 to approximately 3 mmol of RGD peptide, and approximately 4 to approximately 8 mmol of VPM peptide thiols can be added (to a maximum of approximately 10 mmol).

[0062] In one non-limiting embodiment, 3 mmol of thiol groups on thiolized chitosan, 3 mmol of thiol groups on RGD peptide, and 4 mmol of thiol groups on VPM peptide were combined with 10 mmol of vinyl sulfone groups on PEG-VS to prepare a chitosan-PEG-VS-RGD-VPM hydrogel. In another non-limiting embodiment, 1 mmol of thiol groups on thiolized chitosan, 1 mmol of thiol groups on RGD peptide, and 8 mmol of thiol groups on VPM peptide were combined with 10 mmol of vinyl sulfone groups on PEG-VS to prepare a chitosan-PEG-VS-RGD-VPM hydrogel. In yet another non-limiting embodiment, 2 mmol of thiol groups on thiolized chitosan, 3 mmol of thiol groups on RGD peptide, and 5 mmol of thiol groups on VPM peptide were combined with 10 mmol of vinyl sulfone groups on PEG-VS to prepare a chitosan-PEG-VS-RGD-VPM hydrogel. It should be understood that these non-limiting embodiments are merely exemplary, and for approximately 10 mmol of vinyl sulfone groups in PEG-VS, any numerical combination of thiol groups on chitosan, RGD peptides, and VPM peptides can be used, as long as their total does not exceed approximately 10 mmol.

[0063] Phase 1: Synthesis of chitosan-PEG-VS conjugates

[0064] In one embodiment, a chitosan-PEG-VS conjugate is formed by reacting thiolated chitosan with polyethylene glycol sulfone (PEG-VS) in an aqueous solution. The reaction can be carried out at a temperature of about 20°C to about 45°C. The reaction time can range from about 1 minute to about 900 minutes. In one embodiment, the reaction is carried out for about 2 minutes to about 60 minutes, about 2 minutes to about 5 minutes, about 5 minutes to about 10 minutes, about 10 minutes to about 15 minutes, about 15 minutes to about 20 minutes, about 20 minutes to about 25 minutes, about 25 minutes to about 30 minutes, about 30 minutes to about 35 minutes, about 35 minutes to about 40 minutes, about 40 minutes to about 45 minutes, about 40 minutes to about 45 minutes, about 45 minutes to about 50 minutes, about 50 minutes to about 55 minutes, and about 55 minutes to about 60 minutes. In another embodiment, the reaction time is about 1 hour to about 3 hours, about 3 hours to about 5 hours, about 5 hours to about 7 hours, about 7 hours to about 9 hours, about 9 hours to about 11 hours, about 11 hours to about 13 hours, or about 13 hours to about 15 hours.

[0065] The multi-armed PEG vinyl sulfone can 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 a four-armed polyethylene glycol vinyl sulfone (PEG-4-VS). In a particular embodiment, the thiolated chitosan is chitosan-thioglycolic acid (CS-TGA), and the PEG-VS is PEG-4-VS, thereby producing the conjugate chitosan-PEG-4-VS.

[0066] The temperature for the chitosan-PEG-VS reaction can range from 10°C to about 45°C. In one embodiment, the temperature during the synthesis of the chitosan-PEG-VS conjugate can be from about 10°C to about 37°C. In yet another embodiment, the temperature during synthesis is about 25°C or about 37°C. In a particular embodiment, the temperature during 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.

[0067] The synthesis of chitosan-PEG-VS conjugates can be carried out in water, buffer solutions, cell culture media, or combinations thereof. In one embodiment, the aqueous solution used for synthesizing the chitosan-PEG-VS conjugates comprises at least one of water, phosphate-buffered saline (PBS) buffer, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer, or cell culture media. In another embodiment, the cell culture medium is one of organoid cell culture medium, MDCK cell culture medium, or Chinese hamster ovary (CHO) cell culture medium.

[0068] Phase 2: Synthesis of multifunctional conjugates, chitosan-PEG-VS-RGD

[0069] A multifunctional chitosan-PEG-VS-RGD 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 conjugate obtained in the synthesis of stage 1. In a particular embodiment, the chitosan-PEG-VS conjugate is a conjugate of chitosan-thioglycolic acid and a four-arm PEG-VS (i.e., chitosan-PEG-4-VS produced by the synthesis of stage 1). In another embodiment, the reaction may be carried out at a temperature of about room temperature to about 37°C. In yet another embodiment, the reaction time may be about 5 minutes to about 60 minutes.

[0070] An RGD peptide can be any peptide containing an RGD cell attachment motif and at least one cysteine ​​residue. Specific RGD peptides described elsewhere above may also be used.

[0071] In one embodiment, the temperature during the synthesis of the chitosan-PEG-VS-RGD conjugate is from about 10°C to about 45°C, or any range or value therebetween. In another embodiment, the temperature during the synthesis of the chitosan-PEG-VS-RGD conjugate is from about 20°C to about 40°C. In yet another embodiment, the temperature during the synthesis of the chitosan-PEG-VS-RGD conjugate is about 25°C or about 37°C. In one particular 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.

[0072] In one embodiment, the reaction time for synthesizing the chitosan-PEG-VS-RGD conjugate is from about 2 minutes to about 60 minutes, or any range or 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 a particular embodiment, the reaction time for synthesizing the chitosan-PEG-VS-RGD conjugate is from about 3 minutes, from about 5 minutes, from about 10 minutes, from about 15 minutes, from about 20 minutes, from about 25 minutes, from about 30 minutes, from about 35 minutes, from about 40 minutes, from about 45 minutes, from about 50 minutes, from about 55 minutes, or from about 60 minutes.

[0073] The synthesis of the chitosan-PEG-VS-RGD conjugate can be carried out in water, a buffer solution, a cell culture medium, or a combination thereof. In one embodiment, the aqueous solution used in the synthesis of the chitosan-PEG-VS-RGD conjugate comprises at least one of water, phosphate-buffered saline (PBS) buffer, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer, or a cell culture medium. In another embodiment, the cell culture medium is one of organoid cell culture medium, MDCK cell culture medium, or Chinese hamster ovary (CHO) cell culture medium.

[0074] Phase 3: Synthesis of hydrogel from chitosan-PEG-VS-RGD and VPM peptides

[0075] 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 can be carried out at a temperature of about room temperature to about 37°C. In another embodiment, the reaction time can be about 5 minutes to about 30 minutes.

[0076] The chitosan-PEG-VS-RGD conjugate can be any chitosan-PEG-VS-RGD conjugate described in Stage 2 above. In one specific embodiment, the chitosan-PEG-VS-RGD conjugate is formed by conjugating chitosan-thioglycolic acid with a four-arm PEG-VS (i.e., chitosan-PEG-4-VS), and then further conjugating it with an RGD peptide having the amino acid sequence GRGDSPC (SEQ ID NO: 1). In another specific embodiment, the chitosan-PEG-VS-RGD conjugate is formed by conjugating chitosan-thioglycolic acid with a four-arm PEG-VS (i.e., chitosan-PEG-4-VS), and then further conjugating it with an RGD peptide having the amino acid sequence RGDC (SEQ ID NO: 4).

[0077] VPM peptides can be any peptide containing a VPM motif and at least two cysteine ​​residues. Any VPM peptide described in the section on VPM peptides above can be used to synthesize chitosan-PEG-RGD-VPM hydrogels. When VPM peptides are mixed with chitosan-PEG-VS-RGD conjugates, the addition ratio is the molar ratio of vinyl sulfone groups available in chitosan-PEG-VS-RGD to mercapto groups (SH groups) available in the VPM peptide, wherein the ratio of available vinyl sulfone to available mercapto groups is preferably about 1:1. Other ratios of available vinyl sulfone groups in chitosan-PEG-RGD conjugates to available mercapto groups in VPM peptides can 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 any range or value between about 1:0.5 and about 1:1.5.

[0078] The synthesis temperature of chitosan-PEG-RGD-VPM can be from about 10°C to about 45°C, or any range or value therebetween. In another embodiment, the temperature during the synthesis of the chitosan-PEG-RGD-VPM conjugate is from about 25°C to about 37°C. In yet another embodiment, the temperature during the synthesis of the chitosan-PEG-VS-RGD conjugate is either about 25°C or about 37°C. In one particular 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.

[0079] The reaction time for synthesizing the chitosan-PEG-RGD-VPM conjugate is between about 2 minutes and about 60 minutes, or any range or value thereof. In one embodiment, the reaction time is about 2 minutes to about 3 minutes, about 5 minutes to about 30 minutes, or about 30 minutes to 60 minutes. In a particular embodiment, the reaction time for synthesizing 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 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes.

[0080] The synthesis of the chitosan-PEG-RGD-VPM conjugate can be carried out in water, a buffer solution, a cell culture medium, or a combination thereof. In one embodiment, the aqueous solution used in the synthesis of the chitosan-PEG-RGD-VPM conjugate comprises at least one of water, phosphate-buffered saline (PBS) buffer, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer, or a cell culture medium. In another embodiment, the cell culture medium is one of organoid cell culture medium, MDCK cell culture medium, or Chinese hamster ovary (CHO) cell culture medium.

[0081] The synthesis of chitosan-PEG-RGD-VPM results in a shaped (cured) hydrogel. The aqueous, uncured chitosan-PEG-RGD-VPM mixture is the uncured precursor hydrogel. The uncured precursor hydrogel can be dispensed into droplets to prepare dome-shaped gels, injected into the wells of a plate to form a hydrogel layer, spread onto an existing layer or matrix to form a hydrogel layer, or used in any other way. The hydrogel can be used for two-dimensional or three-dimensional cell culture.

[0082] E. Properties of hydrogels

[0083] One way to measure the properties of hydrogels is through their swelling ratio (Q) and solvent fraction (φ). s The relationship between the swelling ratio and the solvent fraction is as follows:

[0084]

[0085] Where φ p It is the solid fraction, and among which

[0086]

[0087] And among them

[0088]

[0089] The swelling ratio (Q) is the ratio of the wet mass to the dry mass of the hydrogel. Wet mass is the mass of the hydrogel when it contains liquid. Dry mass is the mass of the hydrogel after rapid freezing.

[0090] 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 about 35 to about 60 over a time period of about 5 to about 13 days after the cured hydrogel dome is surrounded by an aqueous solution. In one embodiment, the chitosan-based hydrogel has a swelling ratio of about 35 after about 5, 6, 7, 8, 9, 10, 11, 12, or 13 days after the cured hydrogel dome is surrounded by an aqueous solution. In another embodiment, the chitosan-based hydrogel has a swelling ratio of about 40 after about 5, 6, 7, 8, 9, 10, 11, 12, or 13 days after the cured hydrogel dome is surrounded by an aqueous solution. In another embodiment, the chitosan-based hydrogel has a swelling ratio of about 45 after approximately 5, 6, 7, 8, 9, 10, 11, 12, or 13 days following the curing of the hydrogel dome being surrounded by an aqueous solution. In yet another embodiment, the chitosan-based hydrogel has a swelling ratio of about 50 after approximately 5, 6, 7, 8, 9, 10, 11, 12, or 13 days following the curing of the hydrogel dome being surrounded by an aqueous solution. In yet another embodiment, the chitosan-based hydrogel has a swelling ratio of about 55 after approximately 5, 6, 7, 8, 9, 10, 11, 12, or 13 days following the curing of the hydrogel dome being surrounded by an aqueous solution. In another embodiment, the chitosan-based hydrogel has a swelling ratio of about 60 after approximately 5, 6, 7, 8, 9, 10, 11, 12, or 13 days following the curing of the hydrogel dome being surrounded by an aqueous solution.

[0091] In another embodiment, about 2 days after the cured hydrogel dome is surrounded by an aqueous solution, the chitosan-based hydrogel has a swelling ratio (Q) between about 60 and about 95. In another embodiment, about 2 days after the cured hydrogel dome is surrounded by an aqueous solution, the chitosan-based hydrogel has a swelling ratio (Q) of about 60 to about 65, about 65 to about 70, about 70 to about 75, about 75 to about 80, about 80 to about 85, about 85 to about 90, or about 90 to about 95. In one embodiment, about 2 days after the cured hydrogel dome is surrounded by an aqueous solution, the chitosan-based hydrogel has a swelling ratio (Q) of about 60, about 65, about 70, about 75, about 80, about 85, about 90, or about 95.

[0092] In another aspect of the invention, after approximately 2 to approximately 13 days following the curing of the hydrogel dome being surrounded by an aqueous solution, the chitosan-based hydrogel has a solvent fraction (φ) between approximately 0.95 and 0.99. s In one embodiment, the solvent fraction of the chitosan-based hydrogel is about 0.95 after approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or about 13 days following the curing of the hydrogel dome being surrounded by an aqueous solution. In one embodiment, the solvent fraction of the chitosan-based hydrogel is about 0.96 after approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or about 13 days following the curing of the hydrogel dome being surrounded by an aqueous solution. In one embodiment, the solvent fraction of the chitosan-based hydrogel is about 0.97 after approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or about 13 days following the curing of the hydrogel dome being surrounded by an aqueous solution. In one embodiment, the solvent fraction of the chitosan-based hydrogel is about 0.98 after approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or about 13 days following the curing of the hydrogel dome being surrounded by an aqueous solution. In another embodiment, the solvent fraction of the chitosan-based hydrogel is about 0.99 after approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or about 13 days following the curing of the hydrogel dome being surrounded by an aqueous solution.

[0093] In another embodiment, approximately one day after the cured hydrogel dome was surrounded by an aqueous solution, the chitosan-based hydrogel had a solvent fraction (φ) between approximately 0.90 and approximately 0.95. sIn one embodiment, the chitosan-based hydrogel has a solvent fraction of about 0.90 approximately one day after the cured hydrogel dome is surrounded by an aqueous solution. In another embodiment, the chitosan-based hydrogel has a solvent fraction of about 0.91, about 0.92, about 0.93, about 0.94, or about 0.95 approximately one day after the cured hydrogel dome is surrounded by an aqueous solution.

[0094] In another aspect of the invention, after curing the precursor hydrogel at about 37°C for about 30 minutes, the storage modulus (Gʹ) of the chitosan-based hydrogel is any value between about 100 Pa and about 1000 Pa (measured at a frequency of 1 Hz using a rheometer equipped with a parallel plate geometry and a Peltier plate). In one particular embodiment, after curing the precursor hydrogel at about 37°C for about 30 minutes, the chitosan-based hydrogel has a storage modulus (Gʹ) between about 100 Pa and about 500 Pa. In another particular embodiment, after curing the precursor hydrogel at about 37°C for about 30 minutes, the chitosan-based hydrogel has a storage modulus (Gʹ) between about 125 Pa and about 175 Pa. In another embodiment, after the precursor hydrogel is cured at about 37°C for about 30 minutes, 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.

[0095] In another embodiment, after the cured hydrogel dome was surrounded by an aqueous solution at about 37°C for about 4.5 days, the chitosan-based hydrogel had a storage modulus (Gʹ) between about 25 Pa and about 55 Pa (measured at a frequency of 1 Hz using a rheometer equipped with a parallel plate geometry and a Peltier plate). In one embodiment, after the cured hydrogel dome was surrounded by an aqueous solution at about 37°C for about 4.5 days, the chitosan-based hydrogel had a storage modulus (Gʹ) of about 25 Pa, 30 Pa, about 35 Pa, about 40 Pa, about 50 Pa, or about 55 Pa.

[0096] In another aspect of the invention, after the precursor hydrogel is cured at about 37°C for about 30 minutes, the chitosan-based hydrogel has a loss modulus (Gʺ) of about 1 Pa to about 10 Pa (measured at a frequency of 1 Hz using a rheometer equipped with a parallel plate geometry and a Peltier plate). In one embodiment, after the precursor hydrogel is cured at about 37°C for about 30 minutes, the chitosan-based hydrogel has a loss modulus (Gʺ) 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.

[0097] In some embodiments, the aqueous solution used to measure the hydrogel properties is a cell culture medium, a buffer solution, water, or a combination thereof. For the cell culture medium, any cell culture medium sufficient to grow the desired cell type can be used. For example, if growth of human intestinal organoids is desired, then human intestinal organoid cell culture medium can be used. An example composition of human intestinal organoid cell culture medium is: approximately a 1:1 ratio of actin depolymerization factor (ADF) to basal medium, wherein the ADF comprises Dulbecco's Modified Eagle Medium (DMEM), glucose, HEPES, and penicillin-streptomycin, and wherein the basal medium comprises DMEM and fetal bovine serum (FBS). In a particular 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 particular embodiment, the basal culture medium is 450 mL of DMEM (ATCC) and 50 mL of FBS (Corning). ® Any other variations in the composition of the culture medium used for human intestinal organoid cells are also applicable to forming or surrounding the hydrogel of the present invention. Other non-limiting examples include: cell culture media for Martin-Darby canine kidney (MDCK) cells or cell culture media for Chinese hamster ovary (CHO) cells.

[0098] In some embodiments, the chitosan-based hydrogel, after being surrounded by an aqueous solution for approximately 0 to 180 days at a temperature between approximately 4°C and 45°C, showed no significant degradation. In some embodiments, the chitosan-based hydrogel, after being surrounded by an aqueous solution for approximately 0 to 180 days at a temperature between approximately 15°C and 45°C, showed no significant degradation. In some embodiments, the chitosan-based hydrogel, after being surrounded by an aqueous solution for approximately 0 to 180 days at a temperature between approximately 20°C and 37°C, showed no significant degradation. In some embodiments, the chitosan-based hydrogel, after being surrounded by an aqueous solution for approximately 0 to 150 days, approximately 0 to 120 days, approximately 0 to 90 days, approximately 0 to 60 days, approximately 0 to 45 days, approximately 0 to 30 days, approximately 0 to 24 days, or approximately 0 to 15 days at a temperature between approximately 4°C and 45°C, or approximately 15°C and approximately 20°C and 37°C, showed no significant degradation. In one embodiment, the cured chitosan-based hydrogel, after being surrounded by an aqueous solution for approximately 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 24, 30, 45, 60, 90, 120, 150 days, or approximately 180 days, under temperature conditions between approximately 4°C and 45°C, showed no significant degradation. Significant degradation of the hydrogel of the present invention is considered to have occurred if at least 70% or more of the hydrogel is visually observed to have dissolved (liquefied).

[0099] F. Cell culture using hydrogels

[0100] This disclosure also provides a method for synthesizing chitosan-based hydrogels, which further includes providing a cell type. In one aspect of the invention, the method includes the steps of: (a) providing a chitosan-PEG-VS-RGD conjugate, (b) providing a VPM peptide, (c) providing a cell of a cell type, (d) combining the conjugate of step (a) with the peptide of step (b) and the cell of the type of step (c), (e) dispensing a precursor hydrogel from the combination of step (d) onto a matrix in discrete volumes, and (f) forming a hydrogel from the dispensed discrete volumes.

[0101] The step of providing the chitosan-PEG-VS-RGD conjugate is to provide one of the chitosan-PEG-VS-RGD conjugates described above in the method of preparing hydrogels. In some embodiments, the provided chitosan-PEG-VS-RGD conjugate is in an aqueous solution. The aqueous solution includes (but is not limited to) water, a buffer solution, and a cell culture medium. In one particular embodiment, the aqueous solution used for the chitosan-PEG-VS-RGD conjugate is selected from water, HEPES, PBS, human intestinal organoid cell culture medium, MDCK cell culture medium, CHO cell culture medium, or combinations thereof. Alternatively, any other buffer solution or cell culture medium for growing two-dimensional or three-dimensional cells may be used. In other embodiments, the provided chitosan-PEG-VS-RGD conjugate is lyophilized.

[0102] The step of providing the VPM peptide is to provide one of the VPM peptides described above in the method for preparing a hydrogel, or any of the VPM peptides described elsewhere in this disclosure. In one embodiment, the provided VPM peptide may be in an aqueous solution. The aqueous solution includes (but is not limited to) water, a buffer solution, and a cell culture medium. In a particular embodiment, the aqueous solution for the VPM peptide is selected from water, HEPES, PBS, human intestinal organoid cell culture medium, MDCK cell culture medium, CHO cell culture medium, or combinations thereof. Alternatively, any other buffer solution or cell culture medium for growing two-dimensional or three-dimensional cells may be used. In another aspect of the invention, the provided VPM peptide is freeze-dried.

[0103] Any cell type capable of growing in a two-dimensional or three-dimensional hydrogel can be used in the step of providing a cell type. In one embodiment, the cell type may be Martin-Darby dog ​​kidney (MDCK) cells, Chinese hamster ovary (CHO) cells, or patient-derived organoids (cells obtained from human or other animal patients that can be cultured into organoids). In one embodiment, the provided cells are in a cell suspension. The concentration of the cell suspension may be from about 10 cells / μL to about 1000 cells / μL, or any range or value therebetween. In yet another embodiment, the concentration of the cell suspension may be from about 10 cells / μL to about 250 cells / μL, from about 250 cells / μL to about 500 cells / μL, from 400 cells / μL to about 600 cells / μL, from about 500 cells / μL to about 750 cells / μL, or from about 750 cells / μL to about 1000 cells / μL. In one specific embodiment, the concentration of the cell suspension may be approximately 50 cells / μL, 100 cells / μL, 150 cells / μL, 200 cells / μL, 250 cells / μL, 300 cells / μL, 350 cells / μL, 400 cells / μL, approximately 450 cells / μL, approximately 500 cells / μL, approximately 550 cells / μL, approximately 600 cells / μL, approximately 625 cells / μL, approximately 650 cells / μL, 700 cells / μL, 750 cells / μL, 800 cells / μL, 850 cells / μL, 900 cells / μL, 950 cells / μL, or approximately 1000 cells / μL. The aqueous portion of the cell suspension may be any solution in which the desired cell type can grow.

[0104] The steps of combining the chitosan-PEG-VS-RGD conjugate of step (a), the VPM peptide of step (b), and the cell type of step (c) can be performed in any order. For example, a certain amount of an aqueous solution containing the chitosan-PEG-VS-RGD conjugate can be added to any suitable mixing container (such as a test tube or vial), followed by adding a certain amount of an aqueous solution containing the VPM peptide, and then mixing the mixture by any suitable mixing method (e.g., pipetting the mixture until mixed, or using a vortex mixer), followed by adding a certain amount of an aqueous solution containing a cell type (such as MDCK cells), and then mixing the mixture by any suitable method (e.g., stirring with a sterile rod, or pipetting the mixture). In another example, an aqueous solution containing the chitosan-PEG-VS-RGD conjugate is added to any suitable mixing container, followed by an aqueous solution containing a cell type (e.g., Chinese hamster ovary cells), and then an aqueous solution containing the VPM peptide. The mixture is then mixed by any suitable mixing method (e.g., by stirring with a sterile rod or by pipetting the mixture). In some embodiments, step (d) can be broken down into two steps, for example, first combining the PVP-PEG-VS-RGD conjugate with a specific cell type, and then combining the mixture with the VPM peptide.

[0105] The step of dispensing the combined precursor hydrogel into a matrix in discrete volumes is performed using any suitable volume and on any suitable matrix. In one embodiment, the discrete volume can be from about 1 μL to about 1 mL, or any range or value therebetween. In another embodiment, the discrete volume can be from about 2 μL to about 20 μL or from about 8 μL to about 10 μL. In another embodiment, the discrete volumes can be about 4 μL to about 50 μL, 50 μL to about 100 μL, about 100 μL to about 150 μL, about 150 μL to about 200 μL, about 200 μL to about 250 μL, about 250 μL to about 300 μL, about 300 μL to about 350 μL, about 350 μL to about 400 μL, about 400 μL to about 450 μL, about 450 μL to about 500 μL, about 500 μL to about 550 μL, about 550 μL to about 600 μL, about 600 μL to about 650 μL, about 650 μL to about 700 μL, about 700 μL to about 750 μL, about 750 μL to about 800 μL, about 800 μL to about 850 μL, about 850 μL to about 900 μL, about 900 μL... From approximately μL to approximately 950 μL or from approximately 950 μL to approximately 1000 μL. In one particular embodiment, the discrete volumes may be about 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, 15 μL, 16 μL, 17 μL, 18 μL, 19 μL, 20 μL, 21 μL, 22 μL, 23 μL, 24 μL, 25 μL, 26 μL, 27 μL, 28 μL, 29 μL, 30 μL, 31 μL, 32μL, 33 μL, 34 μL, 35 μL, 36 μL, 37 μL, 38 μL, 39 μL, 40 μL, 41 μL, 42 μL, 43 μL, 44 μL, 45μL, 46 μL, 47 μL, 48 μL, 49 μL or 50 μL.

[0106] Discrete volumes can be allocated to any suitable matrix for forming the hydrogel, such as well plates or coverslips. In one embodiment, the matrix can be any number of well plates, including (but not limited to) 4-well, 6-well, 8-well, 12-well, 24-well, 48-well, 96-well, or 384-well plates. The well plates can be treated or untreated. The well plates can be specifically designed for culturing cells. In one embodiment, the well plate is treated with tissue culture. In another embodiment, the well plate is an ultra-low binding well plate. In yet another embodiment, the well plate is untreated. In a particular embodiment, the well plate is a culture plate with 24 wells and is ultra-low binding. In another embodiment, the well plate is a culture plate with 24 wells and is untreated. In one embodiment, the matrix is ​​a coverslip. The coverslip can be a tissue culture-treated, ultra-low binding, or untreated coverslip. In another embodiment, the matrix is ​​a culture dish. The culture dish can be a tissue culture-treated, ultra-low binding, or untreated culture dish.

[0107] The step of forming a hydrogel from the allocated discrete volume can occur within a timeframe of about 1 minute to about 60 minutes. During this step, the precursor hydrogel combined with the cells solidifies into a shaped hydrogel. In one embodiment, the formation of the hydrogel from the allocated discrete volume occurs within a timeframe of about 1 minute to about 5 minutes, or about 5 minutes to about 30 minutes, or about 30 minutes to about 30 minutes. In another embodiment, the formation of the hydrogel from the allocated discrete volume occurs within a timeframe of about 1 minute to about 5 minutes, about 5 minutes to about 15 minutes, about 15 minutes to about 25 minutes, about 25 minutes to about 35 minutes, about 35 minutes to about 45 minutes, or about 45 minutes to about 60 minutes. However, the formation of the hydrogel from the allocated discrete volume may also require longer times, such as between about 60 minutes and about 180 minutes.

[0108] In one embodiment, the matrix containing the dispensed discrete volumes remains upright during hydrogel formation. In another embodiment, the matrix containing the dispensed discrete volumes remains inverted during hydrogel formation. In yet another embodiment, the matrix containing the dispensed discrete volumes remains upright at least once during hydrogel formation and inverted at least once during hydrogel formation.

[0109] This 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 following steps: (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 the cells of the type from step (c); (e) dispensing a precursor hydrogel from the combination of step (d) onto a matrix in discrete volumes; (f) forming a hydrogel from the dispensed discrete volumes; (g) surrounding the formed hydrogel with discrete volumes of cell culture medium; and (h) culturing the surrounded hydrogel. These steps may be performed in other orders as described elsewhere in this disclosure.

[0110] The step of surrounding the formed hydrogel with a culture medium can be performed using any suitable volume of cell culture medium. In one embodiment, surrounding the formed hydrogel with a cell culture medium is achieved by adding a discrete volume of about 3 μL to about 5 mL of any suitable cell culture medium around the formed hydrogel. Suitable cell culture media include those detailed in other sections above. In another embodiment, the discrete volume used to surround the formed hydrogel is about 5 μL to about 50 μL, about 50 μL to about 250 μL, about 250 μL to about 1 mL, about 500 μL to about 1.5 mL, about 1 mL to about 2 mL, about 2 mL to about 3 mL, about 3 mL to about 4 mL, or about 4 mL to about 5 mL. Larger volumes, such as 10 mL, 25 mL, or more, are also suitable for this disclosure. In some embodiments, the formed hydrogel may be immersed in the volume of cell culture medium surrounding it.

[0111] The incubation process of the surrounding hydrogel can be performed under any suitable conditions for any suitable duration to allow the cell culture to grow. For example, incubation can be performed at room temperature (e.g., about 25°C) and atmospheric pressure. As another example, incubation can be performed at a temperature of about 25°C to about 37°C. As another example, incubation can be performed at 37°C. In another example, incubation can 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 higher. In another example, the carbon dioxide content in the air during incubation may be as high as 5%, 2.5%-5%, 5%-7.5%, 7.5%-10%, or higher. In one embodiment, the incubation time is about 2 days to about 6 days. In another embodiment, the incubation time is approximately 1 to approximately 2 days, approximately 2 to approximately 7 days, approximately 7 to approximately 14 days, approximately 14 to approximately 21 days, approximately 21 to approximately 28 days, approximately 28 to approximately 35 days, approximately 35 to approximately 60 days, approximately 60 to approximately 90 days, approximately 90 to approximately 120 days, approximately 120 to approximately 150 days, or approximately 150 to approximately 180 days. When the incubation time is long, the cell culture medium can be replaced or added throughout the incubation period.

[0112] In another embodiment, the cell-containing, solidified chitosan-based hydrogel, after being surrounded by an aqueous solution and incubated at a temperature between about 10°C and 45°C for about 0 to about 180 days, did not undergo significant degradation. Degradation of the hydrogel can be determined visually, as the degraded portion of the gel becomes liquefied. Changes in gel size and the amount of liquefaction can be observed visually. Significant degradation is considered to have occurred when at least 70% of the hydrogel has liquefied. In one embodiment, the cell-containing, solidified chitosan-based hydrogel, after being surrounded by an aqueous solution and incubated at a temperature between about 10°C and 45°C for about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, 10 to 15 days, 15 to 30 days, 30 to 45 days, 45 to 60 days, 60 to 90 days, 90 to 120 days, 120 to 150 days, or 150 to 180 days, did not undergo significant degradation. In some embodiments, the temperature may be 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.

[0113] G. Dissolving chitosan-PEG-RGD-VPM hydrogel

[0114] This disclosure provides a method for dissolving the chitosan-based hydrogel 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 discrete volumes of an aqueous solution comprising an enzyme that cleaves the VPM peptide of the conjugate, and (c) cultivating the combination produced in steps (a) and (b). In some embodiments, the hydrogel comprising the chitosan-PEG-RGD-VPM conjugate is any of the cell-type hydrogels described in this disclosure. In other embodiments, the hydrogel comprising the chitosan-PEG-RGD-VPM conjugate is any of the cell-free hydrogels described in this disclosure.

[0115] Any enzyme that recognizes the VPM peptide as a cleavage site can be used to dissolve chitosan-PEG-RGD-VPM hydrogels. In some embodiments, the enzyme is matrix metalloproteinase (MMP)-1 or MMP-2. In one embodiment, the enzyme is selected from collagenases, dispersases, proteases from *Streptomyces griseus*, or trypsin substitutes. Trypsin substitutes can be commercially available, such as Accutase. ® (Innovative Cell Technologies, Inc.) In one particular embodiment, the enzyme may be collagenase-1 (255 U / mg) from Clostridium histolytica. In another particular embodiment, the enzyme is a dispersant enzyme (1.84 U / mg) from Bacillus polymyxa. In yet another particular embodiment, the protease is a protease (4 U / mg) from Streptomyces griseus, which may be available from at least Sigma Aldrich. ® In another embodiment, the enzyme is a trypsin substitute enzyme (such as Accutase). ® The aqueous solution used for the enzyme can be any buffer that will not degrade the enzyme therein. Exemplary aqueous solutions for the enzyme can be PBS or HEPES buffer. It may contain collagenase, dispersant enzyme, protease from *Streptomyces griseus*, or trypsin substitute enzymes (including Accutase). ®The discrete volume of an aqueous solution of at least one of the following can be a hydrogel volume to an aqueous solution volume of enzyme in a ratio of about 1:2 to about 1:5, wherein the concentration of the added enzyme is about 10 mg / mL. For example, for a 100 μL hydrogel, about 200 mL to about 500 mL of an aqueous solution containing a proteolytic enzyme is added to the wells to dissolve the hydrogel. In another embodiment, the hydrogel volume to an aqueous solution volume of enzyme is in a ratio of about 1:2.5 to about 1:4.5, wherein the concentration of the added enzyme is about 10 mg / mL. In a particular embodiment, the hydrogel volume to an aqueous solution containing at least one of collagenase-1, a dispersant enzyme from Bacillus polymyxa, or a protease from Streptomyces griseus is in a ratio of about 1:3, wherein the concentration of the added enzyme is about 10 mg / mL. In another particular embodiment, the hydrogel volume to an aqueous solution containing a trypsin substitute enzyme is in a ratio of about 1:4, wherein the concentration of the added enzyme is about 10 mg / mL.

[0116] In one embodiment, the cultivation of the mixture of steps (a) and (b) is carried out at room temperature. In another embodiment, the cultivation of the mixture of steps (a) and (b) is carried out at a temperature of about 4°C to about 37°C. In another embodiment, the cultivation step is carried out at a temperature of about 20°C to about 30°C. In a particular embodiment, the cultivation step is carried out 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.

[0117] This disclosure further provides a method for harvesting cells from a three-dimensional cell culture grown in a chitosan-based hydrogel 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 discrete volumes of an aqueous solution comprising at least one of MMP-1 or MMP-2; (c) culturing a mixture of steps (a) and (b); and (d) harvesting cells from the dissolved hydrogel. In one embodiment, the hydrogel comprising the chitosan-PEG-RGD-VPM conjugate is any of the hydrogels described herein, and the hydrogel further comprises a cell type. In another embodiment, the cell type can be any cell type described herein. In one embodiment, MMP-1 and / or MMP-2 can be selected from collagenase-1 from Clostridium histolyticum, dispersase from Bacillus polymyxa, or protease from Streptomyces griseus (Sigma-Aldrich). ® The steps of adding discrete volumes of an aqueous solution containing at least one of MMP-1 or MMP-2 and the cultivation steps (a) and (b) of the mixture can both be performed as described above.

[0118] Harvesting cells from a dissolved hydrogel can be done by any suitable method of recovering cells from the dissolved hydrogel.

[0119] H. Kits for preparing and using hydrogels

[0120] Embodiments of this disclosure also relate to kits comprising one or more components for preparing the chitosan-based hydrogels described herein. In one embodiment, the kit may comprise at least two container systems housed together in a single package. In this embodiment, one container contains a polymer-peptide conjugate (i.e., a chitosan-PEG-VS-RGD conjugate), and the other container contains a VPM peptide. In one aspect of the invention, one or both containers may contain an aqueous solution. In another aspect of the invention, one or both containers may contain a lyophilized powder. In some embodiments, the lyophilized powder is a sterile lyophilized powder. In some embodiments, the container is a vial. The vial may have a cap or lid. In some embodiments, the container is a test tube. The test tube may have a cap or lid. Any other suitable container may be used, including (but not limited to) capped flasks or bottles.

[0121] In some embodiments, the kit may include an additional container. The additional container may include an aqueous solution for forming the hydrogel, a protease for dissolving the gel, or a container for 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 still other embodiments, the aqueous solution may be sterile cell culture medium. In one embodiment, the additional container contains at least one protease selected from collagenase, dispersase, and protease from *Streptomyces griseus* (Sigma-Aldrich). ® One or more of the following. The container for the aqueous solution can be a vial, test tube, bottle, or flask. The container for the aqueous solution is fitted with a cap or lid. In another aspect of the invention, the container may contain powder, lyophilized or freeze-dried enzyme, or lyophilized or freeze-dried component, instead of an aqueous solution. For example, in one embodiment, an additional container may include a lyophilized or freeze-dried enzyme selected from collagenase, dispersase, or protease from *Streptomyces griseus* (Sigma-Aldrich). ® (or a combination thereof).

[0122] The kit may further include any culture and growth components described in the embodiments herein, such as well plates, coverslips, culture dishes, or slides. For example, in some embodiments, the kit may include containers for culturing three-dimensional cell cultures. Containers for culturing three-dimensional cell cultures include, for example, microplates with 6, 12, 24, 48, 96, 128, 384, or even more wells; cell culture plates with 6, 12, 24, 48, or even more wells; culture dishes; flasks with microcavities; etc. These examples are not intended to be limiting, but are merely examples. Any container suitable for three-dimensional cell culture can be used.

[0123] Example

[0124] The following examples illustrate certain embodiments of this disclosure. However, those skilled in the art will understand that modifications can be made to the specific embodiments disclosed without departing from the spirit and scope of the invention, and similar results can still be obtained. Therefore, all content set forth herein should be interpreted as illustrative and not restrictive.

[0125] Example 1 - Synthesis of Chitosan-Based Hydrogels

[0126] Chitosan with a molecular weight range of 10-200 kDa, thioglycolated with thioglycolic acid, was purchased from Polymer Source. A 20 kDa vinyl sulfonated four-arm PEG was selected as the polymer. An RGD peptide with the sequence GRGDSPC (SEQ ID NO: 1) was purchased from GenScript. A VPM peptide with the sequence GCRDVPMSMRGGDRCG (SEQ ID NO: 4) was purchased from GenScript.

[0127] 1 μmol of thiolated chitosan was added to 10 μmol of PEG-4-VS, and the mixture was reacted at room temperature for 60 minutes to generate a chitosan-PEG-4-VS conjugate. Subsequently, 1 μmol of RGD peptide was added to the chitosan-PEG-4-VS conjugate, and the mixture was reacted at 37 °C for approximately 60 minutes to generate a chitosan-PEG-4-VS-RGD conjugate. Finally, 7 μmol of VPM peptide was added to the chitosan-PEG-4-VS-RGD conjugate, and the mixture was crosslinked at 37 °C for approximately 60 minutes to generate a cured chitosan-PEG-RGD-VPM hydrogel.

[0128] Example 2 - Hydrogel Properties

[0129] Swelling properties

[0130] The swelling characteristics of chitosan-based hydrogels were investigated. Using the composition from Example 1, approximately 150 μL of chitosan-PEG-RGD-VPM precursor hydrogel was pipetted in a dome shape onto a polystyrene culture dish, and the wet weight of the hydrogel was obtained. After curing at 25°C for 30 minutes, 5 mL of human intestinal organoid cell culture medium or 5 mL of neutral pH (approximately 7.4) HEPES buffer was added to the culture dish, immersing the dome-shaped hydrogel in it. The dome-shaped hydrogel was then allowed to swell for 15 days, during which time the dome-shaped hydrogel was weighed daily or every two days to determine the swelling mass.

[0131] In addition, 150 μL of chitosan-PEG-RGD-VPM precursor hydrogel from Example 1 was dropwise transferred into individual glass vials to form dome shapes. After curing at 25°C for 30 minutes, the dome-shaped hydrogels were freeze-dried, and weighed before and after freeze-drying to accurately determine the dry mass of the dome-shaped hydrogels.

[0132] The swelling ratio (Q) and solvent fraction were calculated using the swelling mass and dry mass of the dome-shaped hydrogel, which respectively reflect the degree of swelling and the water content of the hydrogel. Figures 2A to 2B Demonstrated in human intestinal organoid cell culture medium ( Figure 2A ) and HEPES buffer ( Figure 2B In the figure, the swelling ratio of the hydrogel during the 15-day swelling period. Figures 2C to 2D Demonstrated in human intestinal organoid cell culture medium ( Figure 2C ) and HEPES buffer ( Figure 2D In the figure, the solvent fraction of the hydrogel during the 15-day swelling period is shown. The swelling ratio and solvent fraction increase with time, reaching a plateau after 1-2 days. The hydrogel can retain up to 98% water in its swollen matrix.

[0133] Mechanical properties

[0134] The hardness of the cured hydrogel was investigated. The storage and loss modulus (Gʹ) and loss modulus (Gʺ) of the cured hydrogel at 1 Hz were calculated using a TA Instruments DHR3 rheometer equipped with a parallel plate geometry (20 mm top plate), a Peltier plate (serial number 113258), and a solvent evaporation blocking system. Approximately 250–300 μL of precursor hydrogel using the composition from Example 1 was deposited onto a 20 mm disposable (AI, bottom plate) parallel plate. The precursor hydrogel was cured at 37 °C for 30–60 min. Strain scans (0.01–10%) were performed to determine the working strain (i.e., 1%), followed by frequency scans at 1% strain to determine the storage and loss modulus (Gʹ) at 1 Hz. Strain scans were also performed after hydrogel curing and after incubation in cell culture medium for 0–15 days. Approximately 250–300 μL of Corning ® Matrigel ® and Sigma-Aldrich ® TrueGel3D TRUE1 repeats this process (including curing and swelling of the hydrogel). Figure 3 The storage loss modulus (Gʹ) is shown at certain time points from t=0 to t=120 hours after the hydrogel solidifies and is surrounded by human intestinal organoid culture medium. Table 1 below summarizes the storage loss modulus (Gʹ) and loss modulus (Gʺ) of the hydrogel surrounded by human intestinal organoid culture medium at t=0 and t=4.6 days.

[0135] Table 1

[0136]

[0137] The results showed that the newly cured hydrogel was harder than the swollen hydrogel, and the chitosan-based hydrogel was significantly less hard than the Sigma-Aldrich hydrogel. ® TRUE1 hydrogel.

[0138] Example 3 - 3D Cell Culture Using Hydrogels

[0139] Martin-Darby canine kidney (MDCK) cells were used as the cell assay system, and 3D culture was performed using the chitosan-based hydrogel of this disclosure. The precursor hydrogel from Example 1 was mixed with a cell suspension containing approximately 180-240 MDCK cells using a pipette tip. Approximately 8-10 μL of dome-shaped cells were formed in 24-well plates, which were tissue culture-treated plates, ultra-low-binding polystyrene plates, or untreated polystyrene plates. The 24-well plates were inverted, and the hydrogel-cell mixture was cured at 37°C for 30 minutes. Subsequently, the 24-well plates were restored to their upright position, and approximately 1 mL of MDCK cell culture medium was added to each well. The 24-well plates were then incubated at 37°C. Matrigel was used. ® The composition used as an alternative to hydrogel Example 1 repeats this process, except that the mixing step is carried out at 4-10°C and the curing step is carried out at 25-37°C.

[0140] Cell growth was optically monitored at different time intervals using an optical microscope over 6–8 days, with optical images taken periodically throughout the process. Figure 4 A to Figure 4 D shows the results after treatment with tissue culture ( Figure 4 A, Figure 4 B) or ultra-low binding ( Figure 4 C Figure 4 Matrigel grown on a 24-well plate (D) ® ( Figure 4 A, Figure 4 C) and chitosan-based gels ( Figure 4 B Figure 4 D) Optical micrographs at 20x magnification after 6 days of incubation. MDCK cell vesicles were present in all micrographs, indicating that 3D cultures can be generated using these hydrogels.

[0141] Stability was also optically monitored over 6-8 days, with optical images captured periodically throughout the process. The chitosan-based hydrogel dome remained stable throughout the MDCK cell culture. (Matrigel) ® The hydrogel dome tends to degrade slowly after one week. Figure 5 A to Figure 5 F shows an untreated 12-well polystyrene plate ( Figure 5 A to Figure 5 C) or 24-well plates cultured in tissue culture ( Figure 5 D to Figure 5 In F), the chitosan-based hydrogel containing MDCK cells was magnified 4 times after 8 days of incubation. Figure 5 A, Figure 5 D) 10x magnification ( Figure 5 B Figure 5 E) and 20x magnification ( Figure 5 C Figure 5 Optical images under F). MDCK cell vesicles are present in all micrographs, indicating that these hydrogels can be used to generate 3D cultures.

[0142] By analyzing the quality of the gel dome, the stability of the dome after being surrounded by organoid culture medium was further monitored up to 25 days. Figure 6 The chitosan-based hydrogel was shown to remain stable throughout 25 days, while Matrigel... ® The hydrogel began to degrade slowly after one week and continued to degrade throughout the monitoring period.

[0143] Example 4 - Digestion based on chitosan hydrogels

[0144] The effects of several different digestive enzymes on the digestion of chitosan-based hydrogels were investigated. Accutase was the enzyme tested. ® (Innovative Cell Technology Company), type 1 collagenase from Clostridium histolytica (Gibco™), dispersant enzyme from Bacillus polymyxa (Gibco™), and protease from Streptomyces griseus (Sigma-Aldrich). ® Corning ® The cell recovery solution (product number 354253) is a non-enzymatic solution and is therefore used as a control. The amount added depends on the solution used. For collagenase-1, dispersant, and protease from *Streptomyces griseus*, the hydrogel-to-enzyme ratio is 1:3. In other words, for every 100 μL of hydrogel, add 300 μL of enzyme at a concentration of 10 mg / mL. Alternatively, for every 100 μL of hydrogel, add 300 μL of a solution containing approximately 5.5 units of dispersant, 12 units of protease from *Streptomyces griseus*, or 765 units of collagenase-1 from *Clostridium histolyticum*. For Accutase... ® The ratio of hydrogel to enzyme is 1:4. For Corning ® Cell recovery solution was prepared with a hydrogel-to-solution ratio of 1:8. The digestion process was visually monitored using a colored hydrogel. Figure 7 The study demonstrated that the chitosan-based hydrogel was completely dissolved by collagenase within approximately 20 minutes, by dispersant enzyme within approximately 25 minutes, and by protease from *Streptomyces griseus* within approximately 20 minutes. Complete dissolution of the hydrogel was considered achieved when it had liquefied (judged visually). Accutase ® Digestion takes longer, at least 1 to 2 hours. Using Corning... ® Cell recovery solutions cannot digest gels.

[0145] Although the number of embodiments included in this disclosure is limited, those skilled in the art who benefit from this disclosure will understand that other embodiments can be devised without departing from the scope of this disclosure.

Claims

1. A synthetic hydrogel comprising: Chitosan-thioglycolic acid; Multi-arm polyethylene glycol ethylene sulfone; The first peptide comprises an arginine-glycine-aspartic acid sequence and a cysteine ​​amino acid located at or near the end of the peptide; and The second peptide comprises a valine-proline-methionine amino acid sequence and a cysteine ​​amino acid located at or near the end of each of the peptides.

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

3. The synthetic hydrogel according to claim 1, wherein the multi-arm polyethylene glycol ethylene sulfone is a four-arm polyethylene glycol ethylene sulfone.

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

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

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

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

8. The synthetic hydrogel according to claim 1, wherein the synthetic hydrogel has a storage modulus between about 125 Pa and about 175 Pa.

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

10. A method for preparing a synthetic hydrogel, comprising the following steps: (a) Provide thiolated chitosan; (b) Provide multi-arm polyethylene glycol ethylene sulfone; (c) Provide a first peptide comprising an arginine-glycine-aspartic acid sequence and a cysteine ​​amino acid located at or near the end of the peptide; (d) Provide a second peptide comprising a valine-proline-methionine amino acid sequence and a cysteine ​​amino acid located at or near the end of each of the peptides; (e) Mix the thiolized chitosan with multi-arm polyethylene glycol ethylene sulfone; (f) Add the first peptide to the mixture from step (d); and (g) Add the second peptide to the mixture from 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 of about 50 kDa to about 200 kDa.

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

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

15. The method of claim 10, wherein the first peptide is selected from GRGDSPC (SEQ ID NO: 1), GRGDSPCx (SEQ ID NO: 2), or RGDC (SEQ ID NO: 3). Where "x" is selected from 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 selected from GCRDVPMSMR GGDRCG (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). The "x" is selected from alanine (A), valine (V), leucine (L), serine (S), threonine (T), aspartic acid (D), or glutamic acid (E), and one of the "x" can be glycine (G).

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

18. A method for dissolving and synthesizing a hydrogel, comprising the following steps: (a) Providing a synthetic hydrogel containing cells cultured in the synthetic hydrogel, The synthetic hydrogel comprises the composition according to claim 1; (b) Adding at least one enzyme to the synthetic hydrogel, said at least one enzyme cleaving the second peptide; and (c) Combinations produced in cultivation steps (a) and (b).

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

20. The method of claim 19, wherein the volume ratio of the added hydrogel to the enzyme is about 1:4 for trypsin replacement enzyme, about 1:3 for collagenase, about 1:3 for dispersant enzyme, and about 1:3 for protease from Streptomyces griseus, and wherein the concentration of the added enzyme is about 10 mg / mL.

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

22. A method for growing a three-dimensional cell culture, comprising the following steps: (a) Provide chitosan-PEG-VS-RGD conjugates; (b) Provide VPM peptides; (c) A cell type that enables the growth of a three-dimensional cell culture; (d) Combining the conjugate of step (a) with the peptide of step (b) and the type of cell of step (c); (e) Dispense the precursor hydrogel from the combination of steps (d) onto the matrix in discrete volumes; (f) Hydrogels are formed from the distributed discrete volumes; (g) The formed hydrogel is surrounded by discrete volumes of cell culture medium; as well as (h) Cultivate the surrounded hydrogel.

23. The method of claim 22, wherein the chitosan-PEG-VS-RGD conjugate comprises chitosan-thioglycolic acid, a four-arm polyethylene glycol ethylene sulfone, and an amino acid sequence comprising arginine-glycine-aspartic acid.

24. The method of claim 23, wherein the uncoupled form of the amino acid sequence comprising arginine-glycine-aspartic acid is GRGDSPC (SEQ ID NO: 1), and the VPM peptide is GCRDVPMSMRGGDRCG (SEQ ID NO: 4).

25. The method of claim 23, wherein the cell is a cell that forms a sphere or organoid.

26. The method of claim 23, wherein the cell is an MDCK cell or a Chinese hamster ovary cell.

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

28. The method of claim 22, wherein the synthetic hydrogel did not undergo significant degradation after 15 days of cultivation.

29. The method of claim 22, wherein the synthetic hydrogel did not undergo significant degradation after 28 days of cultivation.

30. A kit for forming synthetic hydrogels, comprising: The first container; and Second container; The first container contains a chitosan-PEG-VS-RGD conjugate, and The second container contains the VPM peptide.

31. The kit according to claim 30, wherein the chitosan-PEG-VS-RGD conjugate comprises chitosan-thioglycolic acid, multi-arm polyethylene glycol ethylene sulfone having 3 to 6 arms, and an amino acid sequence comprising arginine-glycine-aspartic acid.

32. The kit according to claim 31, wherein the amino acid sequence comprising arginine-glycine-aspartic acid is GRGDSPC (SEQ ID NO: 1).

33. The kit according to claim 32, wherein the VPM peptide is GCRDVPMS MRGGDRCG (SEQ ID NO:4).

34. The kit according to claim 33, wherein the first container and the second container are vials.

35. A kit for three-dimensional cell cultures, comprising: Chitosan-PEG-VS-RGD conjugate; and VPM peptide; The chitosan-PEG-VS-RGD conjugate and the VPM peptide are in powder form.

36. The kit according to claim 36, wherein the chitosan-PEG-VS-RGD conjugate comprises chitosan-thioglycolic acid, multi-arm polyethylene glycol ethylene sulfone having 3 to 6 arms, and an amino acid sequence comprising arginine-glycine-aspartic acid.

37. The kit according to claim 36, wherein the amino acid sequence comprising arginine-glycine-aspartic acid is GRGDSPC (SEQ ID NO: 1).

38. The kit according to claim 37, wherein the VPM peptide is GCRDVPMS MRGGDRCG (SEQ ID NO:4).

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

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