Cryopreservation agent and method suitable for cell-hydrogel precursor system

Abstract

The invention provides a cryopreservation agent and method suitable for a cell-hydrogel precursor system, the cryopreservation agent comprises 2 vol%-11 vol% of a permeable cryopreservation agent, a non-permeable cryopreservation agent, 1 vol%-3 vol% of a hydrogel precursor and a solvent, and does not comprise dimethyl sulfoxide and serum; the permeable cryoprotectant is at least two of glycerol, propylene glycol, ethylene glycol and 3-OMG, and the volume fraction of each of the glycerol, the propylene glycol, the ethylene glycol and the 3-OMG is less than 6%; the non-permeable cryoprotectant comprises 0.1 to 0.6 M of non-permeable micromolecular carbohydrates and 1 to 2 vol% of non-permeable macromolecular substances; the impermeable high-molecular substance is at least two of dextran, polyethylene glycol, polyvinyl alcohol, hydroxyethyl starch and antifreeze protein, and the volume fraction of each of the dextran, the polyethylene glycol, the polyvinyl alcohol, the hydroxyethyl starch and the antifreeze protein is less than 1.5%; the cryopreservation agent is suitable for cryopreservation of a cell-hydrogel precursor system and can be directly injected into gel after resuscitation.

Description

Technical Field

[0001] The present invention relates to the field of cell preservation and biomaterial preparation, and in particular to a cryopreservative and a method suitable for a cell-hydrogel precursor system. Background Art

[0002] With the rapid development of cell therapy, personalized medicine, immunotherapy, and tissue engineering, mammalian cells (such as mesenchymal stem cells, immune cells, and primary cells) have been widely used in clinical treatment, drug screening, disease modeling, and tissue repair. However, the large-scale promotion of cell-based products still faces many challenges, among which long-term storage, safe transportation, and ready-to-use are core issues in the preparation and clinical translation process.

[0003] In recent years, cell-loaded injectable hydrogels have become an important vehicle for cell delivery, local regeneration, and tissue repair due to their excellent biocompatibility, controllable physicochemical properties, and adaptability to the local microenvironment. With their widespread application, the overall demand for cryopreservation of cell-hydrogel precursor systems has become increasingly prominent, especially in scenarios requiring long-distance transportation and immediate injection, which places higher demands on the gelation compatibility and cell activity of the preservation system.

[0004] However, existing cryopreservation methods are unlikely to meet the special needs of this emerging system. Injectable hydrogels are typically soft three-dimensional network structures with a low degree of cross-linking. They are extremely sensitive to ice crystal formation and are prone to irreversible collapse of the pore structure, decreased gelation performance, and impaired injectability during the freezing process. In addition, traditional methods rely on the rapid penetration and uniform elution of cryoprotectants, while hydrogel precursors often contain large molecular polymers that easily form local concentrations and diffusion barriers, thereby hindering CPA penetration and exacerbating cell frostbite. This limits its promotion on a macroscale and is only applicable to microscale structures such as microspheres and microcapsules, making it difficult to meet the needs of large-scale clinical applications.

[0005] On the other hand, some protocols utilize cryopreservation of cells prior to thawing and then mixing with hydrogel precursors to form a gel to circumvent the freezing issue. However, this method is cumbersome, involving multiple steps such as centrifugation, thawing, and gelation. This not only prolongs treatment preparation time but also increases the risk of mechanical damage and contamination, making it difficult to achieve the clinical need for a "one-step, ready-to-use" solution. Furthermore, traditional methods generally rely on cryoprotectant systems containing dimethyl sulfoxide (DMSO) and serum. DMSO is cytotoxic, causing cell membrane damage and activation of apoptotic pathways, while serum, with its complex composition, may trigger immune or inflammatory responses, compromising the safety and consistency of treatment. Therefore, a post-thawing washing step is required to reduce residual components, but this further reduces operational efficiency and reproducibility, and increases costs. Although vitrification can prevent ice crystal formation through ultra-rapid cooling, its reliance on high concentrations of permeating cryoprotectants and extreme cooling rates carries significant toxicity and process complexity, making it difficult to scale to clinical scale and address diverse hydrogel materials.

[0006] In summary, there is currently a lack of a cryopreservation technology specifically designed for cell-hydrogel precursor systems. This system must maintain cell viability and function while also being compatible with the hydrogel's gelation mechanism and injectability. Therefore, there is an urgent need to develop a DMSO-free, serum-free, and highly compatible cryopreservation strategy for ready-to-use cell-hydrogel precursor systems to overcome existing technological bottlenecks and promote the clinical translation and application of injectable cell materials in the field of regenerative medicine. Summary of the Invention

[0007] In order to solve the problems existing in the prior art, the present invention provides a cryopreservative and method suitable for a cell-hydrogel precursor system. The cryopreservative is suitable for the cryopreservation of the cell-hydrogel precursor system and can be directly injected into a gel after thawing.

[0008] The present invention is achieved through the following technical solutions: In a first aspect, the present invention provides a cryopreservative suitable for a cell-hydrogel precursor system, comprising 2 vol% to 11 vol% of a permeable cryoprotectant, a non-permeable cryoprotectant, 1 vol% to 3 vol% of a hydrogel precursor, and a solvent, excluding dimethyl sulfoxide and serum; the permeable cryoprotectant is at least two of glycerol, propylene glycol, ethylene glycol, and 3-OMG, with the volume fraction of each being less than 6%; the non-permeable cryoprotectant comprises 0.1 to 0.6 M of a non-permeable small molecule carbohydrate and 1 vol% to 2 vol% of a non-permeable polymer; the non-permeable polymer is at least two of dextran, polyethylene glycol, polyvinyl alcohol, hydroxyethyl starch, and antifreeze protein, with the volume fraction of each being less than 1.5%; The units vol% and M are based on the total volume of the permeable cryoprotectant, the non-permeable polymer, the hydrogel precursor, and the solvent.

[0009] Preferably, the permeable cryoprotectant is 1 vol% to 6 vol% of glycerol and 1 vol% to 5 vol% of propylene glycol, wherein the unit vol% is based on the total volume of the permeable cryoprotectant, the non-permeable polymer substance, the hydrogel precursor and the solvent.

[0010] Preferably, the permeable cryoprotectant is 1 vol% to 6 vol% of glycerol and 1 vol% to 5 vol% of 3-OMG, wherein the unit vol% is based on the total volume of the permeable cryoprotectant, the non-permeable polymer substance, the hydrogel precursor and the solvent.

[0011] Preferably, the non-permeable small molecule sugar substance is one or more of trehalose, fructose, glucose, mannitol and sucrose.

[0012] Preferably, the non-permeable polymer substance is 0.5 vol% to 1.5 vol% of polyethylene glycol and 0.5 vol% to 1.5 vol% of dextran, wherein the unit vol% is based on the total volume of the permeable cryoprotectant, the non-permeable polymer substance, the hydrogel precursor and the solvent.

[0013] Preferably, the non-permeable polymer substance is 0.5 vol% to 1.5 vol% of hydroxyethyl starch and 0.5 vol% to 1.5 vol% of dextran, wherein the unit vol% is based on the total volume of the permeable cryoprotectant, the non-permeable polymer substance, the hydrogel precursor and the solvent.

[0014] Preferably, the hydrogel precursor is selected from hyaluronic acid or its derivatives, alginate or its derivatives, gelatin or its derivatives, chitosan or its derivatives, chondroitin sulfate, heparin, polyglutamic acid, polyvinyl alcohol, polyethylene glycol and its functionalized derivatives, polyamino acids and polylactic acid-glycolic acid copolymers.

[0015] Furthermore, the derivative of hyaluronic acid is carboxymethyl hyaluronic acid, sulfated hyaluronic acid or phenylboronic acid modified hyaluronic acid; the derivative of alginate is acylated alginate, esterified alginate or phenylboronic acid modified alginate; the derivative of gelatin is methacryloyl gelatin or phenylboronic acid gelatin.

[0016] In a second aspect, the present invention provides a cryopreservation method applicable to a cell-hydrogel precursor system, comprising: S1, resuspending the cells in the cryopreservative according to any one of claims 1 to 8 to obtain a cell solution; S2, the cell solution was placed at refrigerated temperature and allowed to stand; S3, cooling the cell solution after standing to a freezing temperature and freezing it to obtain a frozen cell solution; S4, thawing the frozen cell solution, and then adding a substance that can form a gel with the hydrogel precursor, and mixing to form a hydrogel.

[0017] Preferably, in S2, the refrigeration temperature is 4°C and the standing time is 10 to 40 minutes; in S3, the freezing temperature is -80°C or -196°C.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The cryopreservative of the present invention contains a hydrogel precursor and is free of dimethyl sulfoxide (DMSO) and serum. Cells are cryopreserved in the cryopreservative. When needed, after thawing, a gel-forming substance can be added directly to the hydrogel precursor to form a gel. The cryopreservative can be loaded into prefilled syringes, lyophilization ampoules, or cryopreservation vials for "thaw-and-use" clinical applications. Elution and centrifugation are not required, resulting in a simple procedure. Cell viability can reach 80%-90% after freezing, while stemness and immune function are not compromised. This combination of cytoprotective properties and clinical readiness makes it suitable for clinically ready-to-use injectable products. The non-permeable small molecule carbohydrate stabilizes cell membrane structure, inhibits ice crystal formation, and reduces osmotic damage to cells. At appropriate concentrations, the non-permeable polymer helps create a uniform and stable microenvironment, alleviating the permeability barriers to the cryoprotectant caused by the weak fluidity and complex composition of the hydrogel precursor system. At least two non-permeable polymers are used, each at a low concentration to avoid high viscosity resulting from high concentrations. The combined use of the two can achieve dual protection of cell activity and gelation system structure without significantly increasing the viscosity of the system, effectively improving the consistency of cryopreservation, gelation efficiency after recovery, and injection compatibility. The present invention is suitable for the cryopreservation of biological preparations such as cell-hydrogel precursors, significantly improving the recovery activity and functional retention rate of stem cells, immune cells, etc., without affecting the gelation ability and mechanical properties of the hydrogel. It can be used in multiple application scenarios such as tissue engineering, regenerative medicine, immune cell therapy, biomanufacturing, and high-throughput organ-on-a-chip systems.

[0019] The present invention is applicable to a cryopreservation method for a cell-hydrogel precursor system, wherein cells are added to a cryopreservant for cryopreservation. When needed, after thawing, a substance that forms a gel with the hydrogel precursor is directly added and mixed to form a gel. The system can be loaded into a prefilled syringe, a freeze-dried ampoule or a cryopreservation bottle to achieve a "thaw-and-use" clinical application form, without the need for elution and centrifugation operations. The operation process is simple and suitable for the preparation of clinical ready-to-use injection products. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 This is a schematic diagram of the hydrogel formation principle of Example 1 of the present invention; Figure 2 The microstructure of the stem cell hydrogel before and after cryopreservation in Example 1; Figure 3 is the cell survival rate after cryopreservation of Example 1, Comparative Example 1, and Comparative Example 2; Figure 4 Live / dead cell staining images (a) and cell survival rate (b) of the cell-hydrogel precursor system containing the cryoprotectant of Example 1 prepared into hydrogel after culturing for 6 h and 72 h; Figure 5 Metabolic function changes before and after cryopreservation in Example 1: (a) osmotic pressure, (b) glucose consumption, (c) lactic acid production, and (d) pH value. Figure 6 The injectable properties of the hydrogel prepared after cryopreservation in Example 1; (a) shear thinning properties of the hydrogel, (b) letter patterns formed by squeezing the hydrogel out of the syringe; Figure 7 H&E staining (a) and OARSI score (b) of rats with osteoarthritis treated with Example 1; Figure 8 is the cell survival rate after cryopreservation in Example 2 and Comparative Example 3; Figure 9 This is a live / dead cell staining image of the hydrogel prepared in Example 2. DETAILED DESCRIPTION

[0022] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0023] It should be noted that the process equipment or devices not specifically specified in the following embodiments are all conventional equipment or devices in the art.

[0024] It should be noted that the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product, or apparatus comprising a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to these processes, methods, products, or apparatuses. Furthermore, unless otherwise specified, the numbering of each method step is merely a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to define the scope of the invention. Changes or adjustments to their relative relationships, without substantially changing the technical content, should also be considered within the scope of the invention.

[0025] The present invention is suitable for a cryopreservative for a cell-hydrogel precursor system, comprising: 2 vol% to 11 vol% of a permeable cryoprotectant, a non-permeable cryoprotectant, 1 vol% to 3 vol% of a hydrogel precursor, and a solvent; the permeable cryoprotectant is at least two of glycerol, propylene glycol (PROH), ethylene glycol, and 3-OMG (3-O-methyl-D-glucose), with the volume fraction of each being less than 6%; the non-permeable cryoprotectant comprises 0.1 to 0.6 M of a non-permeable small molecule sugar and 1 vol% to 2 vol% of a non-permeable polymer; the non-permeable polymer is at least two of dextran, polyethylene glycol (PEG), polyvinyl alcohol (PVA), hydroxyethyl starch (HES), and antifreeze protein (AFP), with the volume fraction of each being less than 1.5%; The units vol% and M are based on the total volume of the permeable cryoprotectant, the non-permeable polymer, the hydrogel precursor, and the solvent.

[0026] The cryopreservative of the present invention is added with a hydrogel precursor and does not contain dimethyl sulfoxide and serum. Cells are added to the cryopreservative for cryopreservation. When needed, after recovery, a substance that can be directly added to form a gel with the hydrogel precursor can be mixed into a gel. The cell is loaded into a prefilled syringe, a freeze-dried ampoule or a cryopreservation bottle to achieve a "melt-and-use" clinical application form. There is no need for complex centrifugation operations, the operation process is simple, and it has both cell protection, material compatibility and clinical operation simplicity. Among them, non-permeable small molecule sugar substances can stabilize the cell membrane structure, inhibit ice crystal formation, and reduce cell osmotic pressure damage. Non-permeable high molecular substances help to build a uniform and stable microenvironment at appropriate concentrations, and alleviate the cryoprotectant penetration barrier caused by the weak fluidity and complex composition of the hydrogel precursor system. The two are used in combination to achieve dual protection of cell activity and gel system structure without significantly increasing the viscosity of the system, effectively improving the consistency of cryopreservation, the gel efficiency after recovery and the injection compatibility.

[0027] The total concentration of the cryopreservative of the present invention is controlled within an acceptable osmotic pressure and cell tolerance range. During use, each component is maintained within a safe concentration range without toxic side effects on cells after dilution with a substance that forms a gel with the hydrogel precursor, and does not affect the rheological properties and gelation performance of the hydrogel. After resuscitation, the cryopreservative can be mixed and diluted in an appropriate proportion with a substance that does not contain a permeating cryoprotectant or a non-permeating cryoprotectant (i.e., a substance that forms a gel with the hydrogel precursor) to maintain good injectability and gelation structural integrity, while reducing the cryoprotectant concentration to a safe concentration.

[0028] The cryopreservative of the present invention optimizes composition and concentration, takes into account ice crystal inhibition inside and outside cells, osmotic balance and protein stabilization, significantly improves the recovery activity and functional maintenance of different cell types, and is applicable to a variety of injectable hydrogel systems.

[0029] Specifically, in some embodiments of the present invention, the permeable cryoprotectant is 1 vol% to 6 vol% of glycerol and 1 vol% to 5 vol% of propylene glycol, wherein the unit vol% is based on the total volume of the permeable cryoprotectant, the non-permeable polymer, the hydrogel precursor and the solvent.

[0030] In some embodiments of the present invention, the permeable cryoprotectant is 1 vol% to 6 vol% of glycerol and 1 vol% to 5 vol% of 3-OMG, wherein the unit vol% is based on the total volume of the permeable cryoprotectant, the non-permeable polymer substance, the hydrogel precursor and the solvent.

[0031] In the embodiment of the present invention, glycerol and propylene glycol or 3-OMG are used in combination, and the content of each substance is relatively low, which can not only play a role in osmotic protection of cells, but also will not produce adverse side effects due to direct gelation for human body after resuscitation.

[0032] In some preferred embodiments of the present invention, the non-permeable small molecule sugar substance is one or more of trehalose, fructose, glucose, mannitol and sucrose.

[0033] In some preferred embodiments of the present invention, the non-permeable polymer substance is 0.5 vol% to 1.5 vol% of polyethylene glycol and 0.5 vol% to 1.5 vol% of dextran, wherein the unit vol% is based on the total volume of the permeable cryoprotectant, the non-permeable polymer substance, the hydrogel precursor and the solvent.

[0034] In some preferred embodiments of the present invention, the non-permeable polymer substance is 0.5 vol% to 1.5 vol% of hydroxyethyl starch and 0.5 vol% to 1.5 vol% of dextran, wherein the unit vol% is based on the total volume of the permeable cryoprotectant, the non-permeable polymer substance, the hydrogel precursor and the solvent.

[0035] In some preferred embodiments of the present invention, the hydrogel precursor may include a natural or synthetic polymer material system to support cell embedding, protection, and subsequent gelation. Preferably, the hydrogel precursor is selected from hyaluronic acid or its derivatives, alginate or its derivatives, gelatin or its derivatives, chitosan or its derivatives, chondroitin sulfate, heparin, polyglutamic acid, polyamino acids, and polylactic-co-glycolic acid (PLGA), etc., and can form good biocompatibility with cells. After thawing, the hydrogel can achieve injectable gelation through physical crosslinking (such as ionic crosslinking, thermosensitive gelation, pH induction, etc.) or chemical crosslinking (such as Schiff base reaction, Michael addition, ester bond, borate ester bond, photoinitiated crosslinking, etc.), maintaining the mechanical properties and biological functions of the gel.

[0036] Among them, the derivatives of hyaluronic acid are carboxymethyl hyaluronic acid, sulfated hyaluronic acid or phenylboronic acid modified hyaluronic acid (hyaluronic acid-phenylboronic acid, HA-PBA); the derivatives of alginate are acylated alginate, esterified alginate or phenylboronic acid modified alginate; the derivatives of gelatin are methacryloyl gelatin GelMA or phenylboronic acid gelatin.

[0037] In some preferred embodiments of the present invention, the solvent is serum-free culture medium, PBS or physiological saline.

[0038] In addition, for specific highly sensitive cells (such as hematopoietic stem cells, primary DCs, etc.), optional auxiliary components, such as antifreeze protein mimics, anti-apoptotic molecules, and antioxidants (such as glutathione and NAC), can be added to the cryopreservative of the present invention to further improve cell survival rate.

[0039] The present invention is applicable to a method for cryopreservation of a cell-hydrogel precursor system, comprising: S1, resuspending the cells in the cryopreservative to obtain a cell solution; S2, placing the cell solution at a refrigerated temperature to allow the osmotic protectant to be evenly distributed and the non-osmotic components to complete cell dehydration and osmotic pressure balance; S3, cooling the rested cell solution to a freezing temperature at a rate of 0.5-5°C / min for cryopreservation to obtain a frozen cell solution; S4, thawing the frozen cell solution, and then adding a substance that can form a gel with the hydrogel precursor, and mixing to form a hydrogel.

[0040] The cryopreservation method of the present invention is applicable to the cell-hydrogel precursor system. The cell density should be optimized before cryopreservation. The recommended density for different applications is 1×10 6 ~2×10 7 cells / mL. Too high a density may increase the risk of ice crystal aggregation and affect the resuscitation activity.

[0041] In S2, the refrigeration temperature is preferably 4°C, and the standing time is preferably 10 to 40 minutes; in S3, the freezing temperature is -80°C; and in S4, the thawing is preferably performed in a 37°C water bath for 1 to 3 minutes.

[0042] The present invention is compatible with injection operations and can optimize the packaging form of the system after cryopreservation, such as packaging it in a prefilled syringe, a freeze-dried ampoule or a low-temperature frozen vial, so as to facilitate "thaw and use".

[0043] In the cryopreservation method applicable to the cell-hydrogel precursor system of the present invention, in S4, the gelation method is selected from physical crosslinking (including ionic crosslinking, thermosensitive gelation, pH induction) or chemical crosslinking (including Schiff base reaction, Michael addition, borate bond formation, photoinitiated crosslinking, etc.).

[0044] To reduce cell stress response and residual toxicity of cryoprotectants after recovery, it is recommended to complete the gelation operation within 5 minutes after thawing to avoid long-term exposure of cells to the permeable cryopreservative environment.

[0045] The cryopreservatives and methods of this invention are suitable for a variety of mammalian cell types, including but not limited to: mesenchymal stem cells (MSCs, including those derived from bone marrow, adipose tissue, and umbilical cord); primary cells (such as chondrocytes, keratinocytes, osteoblasts, and endothelial cells); immune cells (such as T cells, dendritic cells, and macrophages); iPS cells, induced differentiated cells, and tumor cells. The cryopreservatives and methods of this invention can be applied in the following fields: tissue engineering; regenerative medicine; cell therapy; injectable hydrogel formulations; high-throughput organ-on-a-chip construction; and the development or treatment of localized disease models. Applications include osteoarthritis treatment, hydrogel-encapsulated wound repair, immune cell therapy, and organ-on-a-chips.

[0046] If cells are stored in a gel system containing active proteins, gene delivery systems or drug-loaded structures, the cryopreservative of the present invention does not significantly affect their structural activity and can be expanded to low-temperature storage of composite delivery systems or local controlled release devices.

[0047] Comparative Example 1 1. Composition of cryopreservatives The cryopreservative components are as follows: Osmotic protectant: DMSO 10 vol%; Non-permeable protective agent: trehalose 0.5M, dextran 1vol%, polyethylene glycol 1vol%; Hydrogel precursor: hyaluronic acid-phenylboronic acid (HA-PBA) 2 vol%; Solvent: PBS buffer; The unit vol% is based on the total volume of DMSO, dextran, polyethylene glycol, hyaluronic acid-phenylboronic acid and PBS buffer.

[0048] 2. Operation steps (1) Cell preparation: P5 human umbilical cord mesenchymal stem cells were cultured to approximately 80% confluence, collected after trypsin digestion, and resuspended in the above-mentioned cryopreservative at a final concentration of 3×10 6 cells / mL to prepare a cell-hydrogel precursor system.

[0049] (2) Pre-cooling treatment: The cell-hydrogel precursor system is placed in a cryovial and the cryovial is placed at 4°C for 30 min to facilitate the permeability of the protective agent to fully enter the cells and the non-permeability of the protective agent to induce pre-dehydration of the cells.

[0050] (3) Programmed cooling: Place the cryovial in a programmed cooling apparatus and cool it to -80°C at a rate of -1°C / min, then transfer it to liquid nitrogen for long-term storage.

[0051] (4) Recovery and gelation: The thawing step was carried out in a 37°C water bath for 2 minutes. The thawed cell-hydrogel precursor system was mixed with a 2% alginate-dopamine (Alg-DA) solution at a volume ratio of 1:2 and cross-linked through boronate bonds to form an injectable hydrogel.

[0052] Comparative Example 2 1. Composition of cryopreservatives The cryopreservative components are as follows: Osmotic protective agent: glycerol 10vol%; Non-permeable protective agent: trehalose 0.5M, dextran 1vol%, polyethylene glycol 1vol%; Hydrogel precursor: hyaluronic acid-phenylboronic acid (HA-PBA) 2 vol%; Solvent: PBS buffer; The unit vol% is based on the total volume of glycerol, dextran, polyethylene glycol, hyaluronic acid-phenylboronic acid and PBS buffer.

[0053] 2. Operation steps (1) Cell preparation: P5 human umbilical cord mesenchymal stem cells were cultured to approximately 80% confluence, collected after trypsin digestion, and resuspended in the above-mentioned cryopreservative at a final concentration of 3×10 6cells / mL to prepare a cell-hydrogel precursor system.

[0054] (2) Pre-cooling treatment: The cell-hydrogel precursor system is placed in a cryovial and the cryovial is placed at 4°C for 30 min to facilitate the permeability of the protective agent to fully enter the cells and the non-permeability of the protective agent to induce pre-dehydration of the cells.

[0055] (3) Programmed cooling: Place the cryovial in a programmed cooling apparatus and cool it to -80°C at a rate of -1°C / min, then transfer it to liquid nitrogen for long-term storage.

[0056] (4) Recovery and gelation: The thawing step was carried out in a 37°C water bath for 2 minutes. The thawed cell-hydrogel precursor system was mixed with a 2% alginate-dopamine (Alg-DA) solution at a volume ratio of 1:2 and cross-linked through boronate bonds to form an injectable hydrogel.

[0057] Comparative Example 3 1. Composition of cryopreservatives The cryopreservative components are as follows: Osmotic protectant: glycerol 4 vol%, 3-OMG 3 vol%; Non-permeable protective agent: trehalose 0.4M, hydroxyethyl starch 2vol%; Hydrogel precursor: hyaluronic acid (HA) 1.5 vol%; Solvent: PBS buffer.

[0058] The unit vol% is based on the total volume of glycerol, 3-OMG, hydroxyethyl starch, hyaluronic acid and PBS buffer.

[0059] 2. Operation steps (1) Cell preparation: P4 rat adipose-derived mesenchymal stem cells were cultured to approximately 80% confluence, collected after trypsin digestion, and resuspended in the above-mentioned cryopreservative at a final concentration of 2×10 6 cells / mL to prepare a cell-hydrogel precursor system.

[0060] (2) Pre-cooling treatment: The cell-hydrogel precursor system is placed in a cryovial and the cryovial is placed at 4°C for 20 min to facilitate the permeability of the protective agent to fully enter the cells and the non-permeability of the protective agent to induce pre-dehydration of the cells.

[0061] (3) Programmed cooling: Place the cryovial in a programmed cooling apparatus and cool it to -80°C at a rate of -1°C / min, then transfer it to liquid nitrogen for long-term storage.

[0062] (4) Recovery and gelation: The thawing step was carried out in a 37°C water bath for 2 minutes. The thawed cell-hydrogel precursor system was mixed with a 3% methylcellulose (MC) solution at a volume ratio of 1:2, and then the mixed system was incubated at 37°C for 5 minutes to achieve thermosensitive cross-linking and gelation, forming an injection-molded hydrogel.

[0063] Example 1: Cryopreservation and Application of Mesenchymal Stem Cell-Hydrogel Precursor System This example is based on human umbilical cord mesenchymal stem cells (UC-MSCs), uses the cryopreservative of the present invention to construct a cell-hydrogel precursor system, and applies it to the treatment of osteoarthritis. It is one of the representative application examples of the present invention.

[0064] 1. Composition of cryopreservatives The cryopreservative components are as follows: Osmotic protective agent: glycerol 5vol%, propylene glycol 3vol%; Non-permeable protective agent: trehalose 0.5M, dextran 1vol%, polyethylene glycol 1vol%; Hydrogel precursor: hyaluronic acid-phenylboronic acid (HA-PBA) 2 vol%; Solvent: PBS buffer.

[0065] The unit vol% is based on the total volume of glycerol, propylene glycol, dextran, polyethylene glycol, hyaluronic acid-phenylboronic acid and PBS buffer.

[0066] 2. Operation steps (1) Cell preparation: P5 human umbilical cord mesenchymal stem cells were cultured to approximately 80% confluence, collected after trypsin digestion, and resuspended in the above-mentioned cryopreservative at a final concentration of 3×10 6 cells / mL to prepare a cell-hydrogel precursor system.

[0067] (2) Pre-cooling treatment: The cell-hydrogel precursor system is placed in a cryovial and the cryovial is placed at 4°C for 30 min to facilitate the permeability of the protective agent to fully enter the cells and the non-permeability of the protective agent to induce pre-dehydration of the cells.

[0068] (3) Programmed cooling: Place the cryovial in a programmed cooling apparatus and cool it to -80°C at a rate of -1°C / min, then transfer it to liquid nitrogen for long-term storage.

[0069] (4) Recovery and gelation: The thawing step was carried out in a 37°C water bath for 2 minutes. The thawed cell-hydrogel precursor system was mixed with a 2% alginate-dopamine (Alg-DA) solution at a volume ratio of 1:2 and cross-linked through boronate bonds to form an injectable hydrogel (MSC hydrogel), which was immediately used for in vivo injection or in vitro application. The schematic diagram of hydrogel cross-linking is shown in the figure. Figure 1 shown.

[0070] 3. Verification (1) Structural stability Scanning electron microscopy (SEM) was used to observe the hydrogel structures formed by the cell-hydrogel precursor system that was not cryopreserved and the cell-hydrogel precursor system that was cryopreserved and revived. Figure 2 As shown. Figure 2 It can be seen that the hydrogel structure of the cell-hydrogel precursor system after cryopreservation and thawing in this example is basically similar to the hydrogel structure of the cell-hydrogel precursor system that was not cryopreserved, indicating that the cryoprotectant of the present invention did not destroy the hydrogel microstructure.

[0071] (2) Cell survival rate like Figure 3 As shown, the trypan blue staining test after thawing shows that the cell survival rate after recovery in Example 1 is significantly higher than that in Comparative Examples 1 and 2. This is because the DMSO and glycerol concentrations in Comparative Example 1 (10% DMSO) and Comparative Example 2 (10% glycerol) are relatively high, and high concentrations of DMSO and glycerol are toxic to cells without centrifugation elution. However, Example 1 does not contain DMSO and has a low glycerol concentration. Although it has not been centrifuged and eluted, the glycerol concentration in the hydrogel formed is still within the safety threshold, so it will not significantly affect the cell survival rate. This demonstrates the superiority of the cryoprotectant of the present invention. In addition, after the cell-hydrogel precursor system containing the cryoprotectant was prepared into a hydrogel, the effect of the presence of the cryoprotectant on cytotoxicity was observed by live / dead cell staining, and the cell survival rate was quantified. The results are shown in FIG. Figure 4 shown. Figure 4 The results showed that the presence of the cryoprotectant in Example 1 did not cause cell death, indicating that the concentration of the cryoprotectant in this example was below the safety threshold, facilitating clinical translation.

[0072] (3) Maintenance of metabolic function After 24 hours of culture of the cross-linked hydrogel after thawing, blood gas analysis was used to detect osmotic pressure (a), glucose consumption (b), lactate production (c), and pH value (d). The hydrogel formed by cross-linking the cell-hydrogel precursor system without cryopreservation was used as a control. The results are shown in Figure 5. Figure 5 .from Figure 5It can be seen that the hydrogel structure of the cell-hydrogel precursor system after cryopreservation and resuscitation in this example is compared with the hydrogel structure of the cell-hydrogel precursor system that was not cryopreserved. After 24 hours of in vitro culture, the osmotic pressure of the culture medium, the glucose consumption of the cells, the lactic acid production, and the pH did not change significantly, indicating that cryopreservation did not significantly affect the metabolic activity of the cells.

[0073] (4) Injectability The thawed cell-hydrogel precursor system was mixed with 2% Alg-DA solution at a volume ratio of 1:2 to form a hydrogel. Figure 6 Firstly, the shear thinning properties of the hydrogel were analyzed using a rheometer. Figure 6 The results in (a) show that as the shear rate increases, the viscosity of the hydrogel decreases significantly, exhibiting typical shear-thinning behavior, giving it good injectability. The experiment further verified its injectability: under the push of a syringe, the hydrogel can be smoothly injected into a water sample bottle, and its structure remains stable in water without degradation. In addition, the hydrogel can be squeezed out of the syringe to form a letter pattern. After injection, the hydrogel can maintain a clear shape, showing excellent molding retention ability ( Figure 6 These results indicate that the hydrogel possesses excellent fluidity and localized gelation, meeting the requirements for precise injection and molding stability in personalized treatments.

[0074] (5) In vivo therapeutic effect In the rat ACLT osteoarthritis model, PBS buffer (PBS group), cell-free hydrogel (hydrogel group), simple stem cell suspension (MSC suspension group), or MSC hydrogel formed by cryopreservation and thawing according to Example 1 (Example 1 group) were injected at week 1 and week 4 after surgery, and samples were collected and evaluated at week 4 and week 8. Figure 7 As shown in (a), the H&E staining results showed that the cartilage tissue structure in the PBS group was severely damaged, manifested as rough joint surface, extensive cartilage degeneration, disordered chondrocyte arrangement, and loss of structural integrity. The hydrogel group showed a certain cartilage protection effect, but there was still surface erosion and irregular cell arrangement. In contrast, the degree of cartilage degeneration in the MSC suspension group was slightly alleviated, the cartilage surface was relatively intact, and the cell structure tended to be regular. The Example 1 group showed the best tissue repair effect, with a smooth cartilage surface, intact cartilage plates, tight and orderly cell arrangement, and a tissue morphology close to normal cartilage. In addition, from Figure 7 As can be seen in (b), the OARSI score of the Example 1 group was significantly lower than that of the other three groups, further verifying the significant therapeutic effect of the stem cell hydrogel of Example 1 in cartilage repair.

[0075] Example 2 Cell cryopreservation and application based on hyaluronic acid-methylcellulose hydrogel This example constructs a cryopreservation strategy for adipose-derived mesenchymal stem cells based on a hyaluronic acid (HA)-methylcellulose (MC) injectable thermosensitive hydrogel precursor system.

[0076] 1. Composition of cryopreservatives The cryopreservative components are as follows: Osmotic protectant: glycerol 4vol%, 3-OMG 3vol%; Non-permeable protective agent: trehalose 0.4M, dextran 1.5 vol%, hydroxyethyl starch 0.5 vol%; Hydrogel precursor: hyaluronic acid (HA) 1.5 vol%; Solvent: PBS buffer.

[0077] The unit vol% is based on the total volume of glycerol, 3-OMG, dextran, hydroxyethyl starch, hyaluronic acid and PBS buffer.

[0078] 2. Operation steps (1) Cell preparation: P4 rat adipose-derived mesenchymal stem cells were cultured to approximately 80% confluence, collected after trypsin digestion, and resuspended in the above-mentioned cryopreservative at a final concentration of 2×10 6 cells / mL to prepare a cell-hydrogel precursor system.

[0079] (2) Pre-cooling treatment: The cell-hydrogel precursor system is placed in a cryovial and the cryovial is placed at 4°C for 20 min to facilitate the permeability of the protective agent to fully enter the cells and the non-permeability of the protective agent to induce pre-dehydration of the cells.

[0080] (3) Programmed cooling: Place the cryovial in a programmed cooling apparatus and cool it to -80°C at a rate of -1°C / min, then transfer it to liquid nitrogen for long-term storage.

[0081] (4) Recovery and gelation: The thawing step was carried out in a 37°C water bath for 2 minutes. The thawed cell-hydrogel precursor system was mixed with a 3% methylcellulose (MC) solution at a volume ratio of 1:2, and then the mixed system was incubated at 37°C for 5 minutes to achieve thermosensitive cross-linking and gelation, forming an injection-molded hydrogel.

[0082] 3. Verification After thawing, the cell-hydrogel precursor system of Example 2 was directly stained with trypan blue, and it was found that its cell survival rate was better than that of Comparative Example 3. The specific results are as follows Figure 8This may be because, in Comparative Example 3, only hydroxyethyl starch was used, and its concentration was relatively high, which would increase the viscosity to a certain extent, resulting in a weakening of the permeability of the cryoprotectant, thereby affecting the cell protection effect. In addition, after thawing, the cell-hydrogel precursor system of Example 2 was directly mixed with the methylcellulose solution without elution, and no gelation obstacle or flocculation phenomenon was observed. After gelation, the cell-hydrogel was stained for live / dead cells, and it was found that the number of live cells was significantly greater than that of dead cells, confirming its good biocompatibility. The specific results are shown in FIG. Figure 9 shown.

[0083] The above examples demonstrate the effectiveness of the cryopreservative of the present invention in terms of cell function protection, hydrogel compatibility and convenience in clinical application. The cryopreservative is not only applicable to MSC-like cells, but can also be extended to other therapeutic cell types such as immune cells and primary cells, and can be applicable to mammalian cells of any origin, including but not limited to human, mouse, porcine, etc. The HA-PBA / Alg-DA hydrogel used is only an exemplary material, and other physical or chemical cross-linked injectable hydrogels can also be substituted, such as natural / synthetic polysaccharides, protein hydrogels and functionally modified derivatives thereof, which will not affect the implementation effect of the present invention. The materials, cells and concentrations in the embodiments of the present invention can be equivalently replaced as needed.

[0084] The applicant states that the present invention uses the above examples to illustrate the detailed process flow of the present invention. The above description is merely a preferred embodiment of the present invention and the present invention is not limited to the above process flow. It will be apparent to those skilled in the art that any improvements to the present invention, equivalent replacements of raw materials in the products of the present invention, and the addition of auxiliary ingredients, without departing from the principles of the present invention, fall within the scope of protection and disclosure of the present invention.

Claims

1. A cryopreservative suitable for a cell-hydrogel precursor system, characterized in that: The invention relates to a method for preparing a cryoprotectant comprising: a permeable cryoprotectant of 2 vol% to 11 vol%, a non-permeable cryoprotectant, a hydrogel precursor of 1 vol% to 3 vol%, and a solvent, excluding dimethyl sulfoxide and serum; wherein the permeable cryoprotectant is at least two of glycerol, propylene glycol, ethylene glycol, and 3-OMG, and the volume fraction of each is less than 6%; wherein the non-permeable cryoprotectant comprises 0.1 to 0.6 M of a non-permeable small molecule carbohydrate and 1 vol% to 2 vol% of a non-permeable polymer; wherein the non-permeable polymer is at least two of dextran, polyethylene glycol, polyvinyl alcohol, hydroxyethyl starch, and antifreeze protein, and the volume fraction of each is less than 1.5%; The units vol% and M are based on the total volume of the permeable cryoprotectant, the non-permeable polymer, the hydrogel precursor, and the solvent.

2. The cryopreservative suitable for the cell-hydrogel precursor system according to claim 1, characterized in that: The permeable cryoprotectant is 1 vol% to 6 vol% of glycerol and 1 vol% to 5 vol% of propylene glycol, wherein the unit vol% is based on the total volume of the permeable cryoprotectant, the non-permeable polymer substance, the hydrogel precursor and the solvent.

3. The cryopreservative suitable for the cell-hydrogel precursor system according to claim 1, characterized in that: The permeable cryoprotectant is 1 vol% to 6 vol% of glycerol and 1 vol% to 5 vol% of 3-OMG, wherein the unit vol% is based on the total volume of the permeable cryoprotectant, the non-permeable polymer substance, the hydrogel precursor and the solvent.

4. The cryopreservative suitable for the cell-hydrogel precursor system according to claim 1, characterized in that: The non-permeable small molecule sugar substance is one or more of trehalose, fructose, glucose, mannitol and sucrose.

5. The cryopreservative suitable for the cell-hydrogel precursor system according to claim 1, characterized in that: The non-permeable polymer substance is 0.5 vol% to 1.5 vol% of polyethylene glycol and 0.5 vol% to 1.5 vol% of dextran, wherein the unit vol% is based on the total volume of the permeable cryoprotectant, the non-permeable polymer substance, the hydrogel precursor and the solvent.

6. The cryopreservative suitable for the cell-hydrogel precursor system according to claim 1, characterized in that: The non-permeable polymer substance is 0.5 vol% to 1.5 vol% of hydroxyethyl starch and 0.5 vol% to 1.5 vol% of dextran, wherein the unit vol% is based on the total volume of the permeable cryoprotectant, the non-permeable polymer substance, the hydrogel precursor and the solvent.

7. The cryopreservative suitable for the cell-hydrogel precursor system according to claim 1, characterized in that: The hydrogel precursor is selected from hyaluronic acid or its derivatives, alginate or its derivatives, gelatin or its derivatives, chitosan or its derivatives, chondroitin sulfate, heparin, polyglutamic acid, polyvinyl alcohol, polyethylene glycol and its functionalized derivatives, polyamino acids and polylactic acid-glycolic acid copolymers.

8. The cryopreservative suitable for the cell-hydrogel precursor system according to claim 5, characterized in that: The derivatives of hyaluronic acid are carboxymethyl hyaluronic acid, sulfated hyaluronic acid or phenylboronic acid modified hyaluronic acid; the derivatives of alginate are acylated alginate, esterified alginate or phenylboronic acid modified alginate; the derivatives of gelatin are methacryloyl gelatin or phenylboronic acid gelatin.

9. A cryopreservation method for a cell-hydrogel precursor system, characterized in that: include: S1, resuspending the cells in the cryopreservative according to any one of claims 1 to 8 to obtain a cell solution; S2, the cell solution was placed at refrigerated temperature and allowed to stand; S3, cooling the cell solution after standing to a freezing temperature and freezing it to obtain a frozen cell solution; S4, thawing the frozen cell solution, and then adding a substance that can form a gel with the hydrogel precursor, and mixing to form a hydrogel.

10. The cryopreservation method for a cell-hydrogel precursor system according to claim 9, characterized in that: In S2, the refrigeration temperature is 4°C and the standing time is 10 to 40 minutes; in S3, the freezing temperature is -80°C or -196°C.

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