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CN-122003256-A - Chitosan-based hydrogels for 3D cell culture

CN122003256ACN 122003256 ACN122003256 ACN 122003256ACN-122003256-A

Abstract

The present invention provides compositions of chitosan-based synthetic hydrogels and methods of use thereof. The chitosan-based hydrogels comprise 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 solubilized. These chitosan-based synthetic hydrogels have at least the advantage that their mechanical properties are similar to those of the non-synthetic Matrigel ® , but are more well defined.

Inventors

  • A. B. Bergeron
  • P. Qinna Yankannan

Assignees

  • 康宁股份有限公司

Dates

Publication Date
20260508
Application Date
20241001
Priority Date
20231012

Claims (20)

  1. 1. A synthetic hydrogel, comprising: Chitosan-thioglycollic acid; Multi-arm polyethylene glycol vinyl sulfone; a first peptide comprising an arginine-glycine-aspartic acid amino acid sequence and a cysteine amino acid at or near the terminus of the peptide, and A second peptide comprising a valine-proline-methionine amino acid sequence and a cysteine amino acid located at or near each terminus of the peptide.
  2. 2. The synthetic hydrogel of claim 1, wherein the chitosan-thioglycollic acid has a molecular weight from about 50 kDa to about 200 kDa.
  3. 3. The synthetic hydrogel of claim 1, wherein the multi-arm polyethylene glycol vinyl sulfone is a four-arm polyethylene glycol vinyl sulfone.
  4. 4. The synthetic hydrogel of claim 3, wherein the four-arm polyethylene glycol vinyl sulfone has an average molecular weight of about 2 kDa to about 20 kDa.
  5. 5. The synthetic hydrogel of 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), Wherein "x" is selected from glycine (G), alanine (a), leucine (L), valine (V), serine (S), threonine (S), aspartic acid (D) or glutamic acid (E).
  6. 6. The synthetic hydrogel of 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) or CRDVPMSMRGGDRCG (SEQ ID NO: 7), CRDVPMSMRGGDRCx (SEQ ID NO: 8), GCRDVPMSMRGGDRC (SEQ ID NO: 9) or xCRDVPMSMRGGDRC (SEQ ID NO: 10), Wherein "x" is selected from alanine (a), valine (V), leucine (L), serine (S), threonine (T), aspartic acid (D) or glutamic acid (E), and wherein one "x" may be glycine (G).
  7. 7. The synthetic hydrogel of claim 6, wherein the first peptide is GRGDSPC (SEQ ID NO: 1), Wherein the second peptide is GCRDVPMSMRGGDRCG (SEQ ID NO: 4) and Wherein the multi-arm polyethylene glycol vinyl sulfone is a four-arm polyethylene glycol having an average molecular weight of about 20 kDa.
  8. 8. The synthetic hydrogel of claim 1, wherein the synthetic hydrogel has a storage modulus of between about 125 Pa and about 175 Pa.
  9. 9. The synthetic hydrogel of claim 1, wherein the synthetic hydrogel has a storage modulus of between about 25 Pa and about 55 Pa after swelling in aqueous solution for about 4.5 days.
  10. 10. A method of preparing a synthetic hydrogel comprising the steps of: (a) Providing a thiolated chitosan; (b) Providing a multi-arm polyethylene glycol vinyl sulfone; (c) Providing a first peptide comprising an arginine-glycine-aspartic acid amino acid sequence and a cysteine amino acid located at or near the terminus 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 each terminus of the peptide; (e) Mixing the sulfhydrylation chitosan with multi-arm polyethylene glycol vinyl sulfone; (f) Adding the first peptide to the mixture of step (d), and (G) Adding the second peptide to the mixture of step (f).
  11. 11. The method of claim 10, wherein the thiolated chitosan is chitosan-thioglycollic acid.
  12. 12. The method of claim 11, wherein the chitosan-thioglycollic acid has a molecular weight from about 50 kDa to about 200 kDa.
  13. 13. The method of claim 10, wherein the multi-arm polyethylene glycol vinyl sulfone is a four-arm polyethylene glycol vinyl sulfone.
  14. 14. The method of claim 13, wherein the four-arm polyethylene glycol vinyl sulfone has an average molecular weight of about 2 kDa to about 20 kDa.
  15. 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), Wherein "x" is selected from glycine (G), alanine (a), leucine (L), valine (V), serine (S), threonine (S), aspartic acid (D) or glutamic acid (E).
  16. 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), Wherein "x" is selected from alanine (a), valine (V), leucine (L), serine (S), threonine (T), aspartic acid (D) or glutamic acid (E), and wherein one "x" may be glycine (G).
  17. 17. The method of claim 16, wherein the first peptide is GRGDSPC (SEQ ID NO: 1), Wherein the second peptide is GCRDVPMSMRGGDRCG (SEQ ID NO: 4) and Wherein the multi-arm polyethylene glycol vinyl sulfone is a four-arm polyethylene glycol vinyl sulfone having an average molecular weight of about 20 kDa.
  18. 18. A method of dissolving a synthetic hydrogel comprising the steps of: (a) Providing a synthetic hydrogel comprising cells cultured in the synthetic hydrogel, Wherein the synthetic hydrogel comprises the composition of claim 1; (b) Adding at least one enzyme to said synthetic hydrogel, said at least one enzyme cleaving said second peptide, and (C) Incubating the combination produced in steps (a) and (b).
  19. 19. The method of claim 18, wherein the at least one enzyme is selected from collagenase (collagenase), dispase, protease from streptomyces griseus (Streptomyces griseus), or trypsin-replacing enzyme (trypsin-substitute enzyme), or a combination thereof.
  20. 20. The method of claim 19, wherein the ratio of the volume of the hydrogel added to the volume of the enzyme is about 1:4 for trypsin-replacing enzyme, about 1:3 for collagenase, about 1:3 for dispase, and about 1:3 for protease from streptomyces griseus, and wherein the concentration of the enzyme added is about 10 mg/mL.

Description

Chitosan-based hydrogels for 3D cell culture Cross Reference to Related Applications The present application claims priority from U.S. c. ≡119, U.S. provisional application No. 63/543,808 filed on day 2023, month 10, 12, the contents of which are hereby incorporated by reference in their entirety. Reference to electronic sequence Listing The contents of the electronic Sequence list created at 2024, 9, 10, and file name sequence_listing_sp23-255.Xml (11,704 bytes) are incorporated herein by reference in their entirety. Technical Field The present disclosure relates to chitosan-based synthetic polymeric hydrogels, methods of preparing chitosan-based synthetic polymeric hydrogels, methods of using the hydrogels for biomolecular analysis, and kits of the hydrogels, particularly for advanced three-dimensional cell culture such as spheroid and organoid culture. Background Organoids are three-dimensional, cell-based in vitro models that mimic corresponding in vivo organs, with many of the structural and functional features of the in vivo organs. Organoids' similarity to natural organs can play a key 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 homo-/copolymers crosslinked by physical or chemical means using natural, synthetic or hybrid polymers. Hydrogels can be cast in different shapes or sizes and have network properties such as swelling, hardness, permeability, porosity. These reticulation properties may have profound effects on cell activity and cell fate. Natural basement membrane Matrigel ® derived from Engelbres-Holm-Schwann (Engelbreth-Holm-Swarm, EHS) mouse sarcoma is the most commonly used scaffold to support 3D organoid culture. Matrigel ® highly mimics the natural extracellular matrix (ECM), with hundreds of proteins (> 1800) bound, including laminin (about 60%), collagen type IV (about 30%), entactin (enactin) (about 8%), and heparan sulfate proteoglycan monioglycan (heparin sulfate proteoglycan perlecan) (about 2% to 3%). Matrigel ® gelled above 20 ℃, during which time the entactin acts as a cross-linker to link the laminin with the collagen, forming a gel. Although Matrigel ® is the most preferred hydrogel in organoid culture, the batch-to-batch variation in its protein concentration results in difficult control of its reticulation properties (e.g., stiffness). This causes inconsistent results in cell culture, which creates problems. Thus, there is a need for synthetic hydrogels that have well-defined chemical and mechanical properties, and yet are capable of building advanced three-dimensional cellular structures, such as organoids. Disclosure of Invention The present disclosure provides compositions of chitosan-based synthetic hydrogels having similar mechanical properties to Matrigel ® of animal origin, methods of preparing chitosan-based synthetic hydrogels, methods of using chitosan-based synthetic hydrogels, and kits for preparing chitosan-based synthetic hydrogels. According to some aspects of the present disclosure, there is provided a composition of a synthetic hydrogel comprising chitosan-thioglycollic 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 at or near the terminus of the peptide, and a second peptide comprising a valine-proline-methionine (VPM) amino acid sequence and a cysteine amino acid at or near each terminus of the peptide. The chitosan-thioglycollic acid of the synthetic hydrogel may have a molecular weight of from 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) or CRDVPMSMRGGDRCG (SEQ ID NO: 7), CRDVPMSMRGGDRCx (SEQ ID NO: 8), GCRDVPMSMRGGDRC (SEQ ID NO: 9) or xCRDVPMSMRGGDRC (SEQ ID NO: 10), wherein "x" is selected from alanine (A), valine (V), leucine (L), serine (S), threonine (T), aspartic acid (D) or glutamic acid (E), and wherein one "x" may be glycine (G). The synthetic hydrogel may have a storage modulus of between about 125 Pa and about 175 Pa. The synthetic hydrogel may have a storage modulus of between about 25 Pa and about 55 Pa after swelling in aqueous solution for about 4.5 days. In a pa