Polyampholyte cell cryoprotectants, methods of making and polyampholyte cell cryopreservation solutions

Abstract

This invention provides a polyamplifier-based cell cryopreservation protectant and a method for preparing a polyamplifier-based cell cryopreservation solution, as well as their applications. It relates to biomedical materials technology and the field of cell cryopreservation, aiming to solve problems such as low cell recovery rates and short cryogenic storage times in existing cell cryopreservation technologies. The polyamplifier-based cell cryopreservation protectant comprises polyacrylic acid, betaine, and graphene oxide linked by chemical bonds. The mass ratio of polyacrylic acid to betaine is 10:1–2:1, and the total mass of polyacrylic acid and betaine to the mass of graphene is 1:2–10:1. The polyamplifier-based cell cryopreservation protectant provided by this invention can not only reduce cell permeability damage by regulating osmotic pressure, but also improve cryopreservation efficiency, effectively inhibit ice crystal formation and growth, and increase cell survival rate. Due to its excellent cell protection properties and good stability, this protectant can also be used in the cryopreservation of stem cells, immune cells, and other cells, possessing significant socio-economic value.

Description

Technical Field

[0001] This invention relates to the fields of biomedical materials technology and cell cryopreservation, and in particular to a polyamplifier cell cryopreservation protectant, its preparation method, and a polyamplifier cell cryopreservation solution. Background Technology

[0002] Existing cell cryopreservation agents typically suffer from several problems during cryopreservation, including high genotoxicity, low cell recovery rates, and short cryopreservation times. These issues significantly reduce the efficiency of cell cryopreservation and adversely affect the application of thawed cells. Genotoxicity can damage cellular genetic material, low recovery rates affect sample quality, and short cryopreservation times limit long-term cell preservation. These shortcomings severely impact the effectiveness, reliability, and validity of cell cryopreservation in practical applications.

[0003] With the rapid development of CAR cell therapy in clinical applications, cell cryopreservation technology has become a crucial step in ensuring the quality and stability of CAR cells. However, traditional cryopreservation agents present significant challenges in protecting the integrity and functionality of CAR cells, such as potentially causing genetic material damage, affecting cell recovery rates, and possibly leaving harmful cryopreservation media residues within the cells. These issues have hindered the further development and application of CAR cell therapy in clinical cancer treatment.

[0004] Therefore, there is an urgent need for a new type of cell cryopreservation agent to overcome the limitations of traditional cryopreservation agents, improve cell cryopreservation efficiency and survival rate, and at the same time ensure the safety and effectiveness of cells in the treatment process.

[0005] In view of this, an invention is proposed. Summary of the Invention

[0006] One of the objectives of this invention is to provide a polyamplifier-based cell cryopreservation protectant to address the technical problems that existing cell cryopreservation agents (such as dimethyl sulfoxide and glycerol) have high cytotoxicity and short cell cryopreservation time, which limit their application in the field of cell cryopreservation.

[0007] The present invention provides a polyamplifier cell cryopreservation protectant comprising polyacrylic acid, betaine, and graphene oxide linked by chemical bonds, wherein the mass ratio of polyacrylic acid to betaine is 10:1-2:1, and the mass ratio of the total mass of polyacrylic acid and betaine to the mass of graphene is 1:2-10:1.

[0008] Furthermore, in this polyampholyte cell cryopreservation protectant, polyacrylic acid and graphene oxide are linked by diisocyanate compounds, and betaine and polyacrylic acid are linked by chemical bonds.

[0009] Among them, diisocyanate compounds include at least one of trimethylhexane diisocyanate, dicyclohexylmethane diisocyanate, toluene diisocyanate, and hexamethylene diisocyanate.

[0010] Furthermore, the molar mass of the polyacrylic acid block is 2000-10000 g / mol, preferably 4000-6000 g / mol.

[0011] The second objective of this invention is to provide a method for preparing a polyampholyte cell cryopreservation protectant, comprising the following steps:

[0012] In step S1, a diisocyanate compound is first modified onto the surface of graphene oxide to obtain activated graphene oxide. Then, the activated graphene oxide is mixed with polyacrylic acid to carry out a grafting reaction to obtain graphene oxide-polyacrylic acid.

[0013] Further, in step S1, graphene oxide is dispersed in a first organic solvent, and then a diisocyanate compound is added and mixed, so that the cyanate groups in the diisocyanate compound react with the carboxyl groups or hydroxyl groups on the graphene oxide to obtain activated graphene oxide.

[0014] Furthermore, the activation reaction is carried out at a temperature of 70-90℃ for 20-30 hours.

[0015] Furthermore, the diisocyanate compounds include at least one of trimethylhexane diisocyanate, dicyclohexylmethane diisocyanate, toluene diisocyanate, and hexamethylene diisocyanate;

[0016] Furthermore, the first organic solvent includes N,N-dimethylformamide;

[0017] Furthermore, the grafting temperature for activated graphene oxide and polyacrylic acid is 50-160℃.

[0018] Step S2 involves mixing graphene oxide-polyacrylic acid and betaine to react and obtain a polyampholyte cell cryopreservation protectant (i.e., graphene oxide-polyacrylic acid-betaine).

[0019] Further, in step S2, graphene oxide-polyacrylic acid is dispersed in a second organic solvent, and then betaine is added to mix and carry out acylation and ring-opening reactions to obtain graphene oxide-polyacrylic acid-betaine.

[0020] Furthermore, the second organic solvent comprises dichloromethane and N,N-dimethylformamide, with a mass ratio of 1-2:1.

[0021] Furthermore, the acylation and ring-opening reactions are carried out at a temperature of 20-25℃ for 12-20 hours.

[0022] A third objective of this invention is to provide another method for preparing a polyampholyte cell cryopreservation protectant, comprising the following steps:

[0023] Step A1: Polyacrylic acid is dispersed in a third organic solvent, and betaine is added to mix and carry out acylation and ring-opening reactions to obtain polyacrylic acid-betaine;

[0024] Step A2 involves mixing activated graphene oxide with polyacrylic acid-betaine for a grafting reaction to obtain the polyampholyte cell cryopreservation protectant.

[0025] Furthermore, in step A1, the acylation reaction and ring-opening reaction are carried out at a temperature of 20-25°C for 12-20 hours.

[0026] Preferably, in step A2, the grafting reaction temperature is 50-160℃.

[0027] Preferably, the third organic solvent includes dichloromethane.

[0028] The fourth objective of this invention is to provide the application of the above-mentioned polyampholyte cell cryopreservation protectant in the cryopreservation of L929 cells, CAR cells, mesenchymal stem cells, neural stem cells, and erythrocytes.

[0029] Furthermore, CAR cells include one of the following: CAR-T cells, CAR-NK cells, and CAR-Macrophage cells.

[0030] Furthermore, CAR cells are CAR-T cells.

[0031] Furthermore, when using the polyampholyte CAR cell cryopreservative to cryopreserve CAR-T cells, the number of CAR-T cells was 5 x 10. 6 mL -1 -1 x 10 7 mL -1 .

[0032] The fifth objective of this invention is to provide a polyamplifier CAR cell cryopreservation solution, comprising the polyamplifier cell cryopreservation protectant (graphene oxide-polyacrylic acid-betaine) and cell culture medium provided in one objective of this invention.

[0033] In some embodiments, the preparation method of the polyamplifier CAR cell cryopreservation solution is as follows: graphene oxide-polyacrylic acid-betaine is added to the cell culture medium and mixed evenly to obtain the polyamplifier CAR cell cryopreservation solution.

[0034] Furthermore, the cell culture medium includes one of RPMI 1640 medium, DMEM medium, and X-VIVO15 medium.

[0035] Specifically, the manufacturer of RPMI 1640 medium is Thermo Fisher Scientific, product number 11875093; the manufacturer of DMEM medium is Thermo Fisher Scientific, product number 11965118; and the manufacturer of X-VIVO15 medium is Lonza Biologics Plc, product number BE02-060Q.

[0036] Furthermore, the cell culture medium is X-VIVO15 medium;

[0037] In some embodiments, the preparation method of polyampholyte CAR cell cryopreservation solution includes the following steps:

[0038] The polyamphoteric electrolyte cell cryopreservation protectant graphene oxide-polyacrylic acid-betaine was added to X-VIVO15 medium and mixed evenly to obtain the polyamphoteric electrolyte CAR cell cryopreservation solution.

[0039] This invention provides a polyamplifier-based cell cryopreservative and a polyamplifier-based cell cryopreservation solution, which have at least the following beneficial effects:

[0040] The polyampholyte cell cryopreservation protectant provided by this invention can overcome the problems of high cytotoxicity and low cell recovery rate in existing cell cryopreservation technologies. This protectant can reduce cell damage by regulating the osmotic pressure inside and outside the cell; at the same time, it can effectively inhibit ice crystal growth, has low cytotoxicity, high recovery rate, and effectively ensures the integrity of cell structure and function.

[0041] The polyamplified electrolyte CAR cell cryopreservation solution provided by this invention effectively solves the problems of high genotoxicity, low cell recovery rate, short low-temperature storage time, and residual cryopreservation media in the cryopreservation of CAR-T cells by traditional cryopreservation agents. This cell cryopreservation solution aims to improve cell cryopreservation efficiency, ensure the integrity and functionality of CAR-T cells, and guarantee a high survival rate after thawing and recovery. Ultimately, it aims to provide a higher-quality cryopreservation solution for the clinical application of CAR-T cell therapy, further promoting innovation and development in fields such as cancer treatment.

[0042] The polyampholyte cell cryopreservation agent provided by this invention, due to its excellent cell protection performance and good stability, can also be used in the field of cell cryopreservation such as stem cells and immune cells, and has good socio-economic value.

[0043] The polyamplifier-electrolyte cell cryopreservation protectant and polyamplifier-electrolyte cell cryopreservation solution provided by this invention can not only regulate the osmotic pressure of the cryopreservation solution and reduce osmotic damage to cells during cryopreservation and thawing, but also improve cryopreservation efficiency, increase cell survival rate, and maintain cell activity and therapeutic effect after thawing. Due to its excellent cell protection properties, it has broad application prospects in the fields of cell cryopreservation and cell therapy. Detailed Implementation

[0044] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] According to one aspect of the present invention, a polyampholyte cell cryopreservation protectant is provided, comprising polyacrylic acid, betaine and graphene oxide linked by chemical bonds, wherein the mass ratio of polyacrylic acid and betaine is 10:1-2:1, and the mass ratio of the total mass of polyacrylic acid and betaine to the mass of graphene is 1:2-10:1.

[0046] Typically, but not limitingly, in the polyampholyte cell cryopreservation protectant provided by the present invention, the mass percentages of polyacrylic acid and betaine are, for example, a range of values ​​consisting of 10 / 1, 8 / 1, 5 / 1, 2 / 1, or any two of these values.

[0047] Typically, but not limitingly, in the polyampholyte cell cryopreservation protectant provided by the present invention, the total mass ratio of polyacrylic acid and betaine to graphene is, for example, 1 / 2, 3 / 1, 5 / 1, 8 / 1, 10 / 1, or any range of two of these values.

[0048] The polyamplifier cell cryopreservation protectant provided by this invention comprises polyacrylic acid and graphene oxide linked by diisocyanate compounds, and betaine linked to the polyacrylic acid by chemical bonds, thereby forming a polyamplifier. This protectant not only reduces osmotic damage to cells during cryopreservation and thawing by regulating osmotic pressure, but also improves cryopreservation efficiency, effectively inhibits ice crystal formation and growth, increases cell viability, and enhances cell survival rate after thawing. Due to its excellent cell protection properties and good stability, this protectant can also be used in the cryopreservation of stem cells, immune cells, and other cells, possessing significant socio-economic value.

[0049] In a preferred embodiment of the present invention, the polyampholyte is prepared according to the following steps:

[0050] Step S1: Graphene oxide is dispersed in a first organic solvent, and then a diisocyanate compound is added and mixed to activate the cyanate groups in the diisocyanate compound with the carboxyl groups on the graphene oxide to obtain activated graphene oxide.

[0051] Preferably, the activation reaction temperature is 70-90℃ and the time is 20-30h. Specifically, the activation reaction temperature is 70℃, 75℃, 80℃, 85℃, 90℃ or any range of two values; the activation reaction time is 20h, 22h, 25h, 28h, 30h or any range of two values.

[0052] The above-mentioned diisocyanate compounds are commonly used compounds in the art, including but not limited to at least one of trimethylhexane diisocyanate, dicyclohexylmethane diisocyanate, toluene diisocyanate, and hexamethylene diisocyanate.

[0053] The first organic solvent mentioned above is a commonly used organic solvent in the art, including but not limited to N,N-dimethylformamide suspension. To further improve the activation efficiency, the preferred temperature for the grafting reaction of activated graphene oxide and polyacrylic acid is 50-160℃, such as 50℃, 60℃, 80℃, 100℃, 120℃, 140℃, 160℃, or any range of two values.

[0054] In step S2, graphene oxide-polyacrylic acid is dispersed in a second organic solvent, and then betaine is added to mix and carry out acylation and ring-opening reactions to obtain graphene oxide-polyacrylic acid-betaine, which is a polyamplifier CAR cell cryopreservative.

[0055] In this application, the polyampholyte CAR cell cryopreservative is graphene oxide-polyacrylic acid-betaine.

[0056] The specific type of the second organic solvent is not limited, and any commonly used organic solvent in the art may be used. To further improve the dispersion efficiency, the second organic solvent preferably includes dichloromethane and N,N-dimethylformamide, and the mass ratio of the two is 1-2:1, such as 1:1, 1.2:1, 1.5:1, 1.8:1, 2:1, or any range of two values.

[0057] To further improve the preparation efficiency of the polyampholyte cell cryopreservation agent, the preferred temperatures for the acylation and ring-opening reactions are 20-25°C and the preferred times are 12-20 h. Specifically, the temperatures for the acylation and ring-opening reactions can be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, or any combination of two of these values, and the preferred times for the acylation and ring-opening reactions can be 12 h, 14 h, 15 h, 16 h, 18 h, 20 h, or any combination of two of these values.

[0058] In some instances, the molar mass of the aforementioned polyacrylic acid is 2000-10000 g / mol. In particular, when the molar mass of polyacrylic acid is 4000-6000 g / mol, the resulting polyampholyte cell cryopreservation agent is more conducive to regulating intracellular and extracellular osmotic pressure and improving the recovery rate.

[0059] Typical, but not limiting, molar masses of polyacrylic acid include 2000 g / mol, 3000 g / mol, 4000 g / mol, 5000 g / mol, 8000 g / mol, 10000 g / mol, or any range of two values.

[0060] In another preferred embodiment of the present invention, the preparation method of the polyampholyte cell cryopreservation protectant includes the following steps:

[0061] Step A1: Polyacrylic acid is dispersed in a third organic solvent, and betaine is added to mix and carry out acylation and ring-opening reactions to obtain polyacrylic acid-betaine;

[0062] Step A2 involves mixing activated graphene oxide with polyacrylic acid-betaine for a grafting reaction to obtain the polyampholyte cell cryopreservation protectant.

[0063] It should be noted that in step A2, the activation of graphene oxide has the same meaning as the activation of graphene oxide in step S1, and will not be repeated here.

[0064] In some embodiments, in step A1, the temperature of the acylation reaction and the ring-opening reaction is 20-25°C, and the time is 12-20 hours. Specifically, the temperature of the acylation reaction and the ring-opening reaction is such as 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, or any combination of two values; the time of the acylation reaction and the ring-opening reaction is such as 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, or any combination of two values.

[0065] In some embodiments, in step A2, in order to further improve the grafting efficiency, the grafting reaction temperature is preferably 50-160°C. Specifically, the grafting reaction temperature is a range of 50°C, 60°C, 80°C, 100°C, 120°C, 150°C, 160°C, or any two of these values.

[0066] The specific types of the aforementioned third organic solvent are not limited, including but not limited to dichloromethane.

[0067] According to another aspect of the present invention, the present invention provides the application of a polyamplifier-based cell cryopreservation protectant in the cryopreservation of L929 cells, CAR cells, mesenchymal stem cells, neural stem cells, and erythrocytes.

[0068] The aforementioned CAR cells include one of CAR-T cells, CAR-NK cells, or CAR-Macrophage cells. The effects are particularly beneficial when the CAR cells are CAR-T cells.

[0069] Specifically, when cryopreserving the CAR-T cells using the polyampholyte CAR cell cryopreservation agent, the preferred number of CAR-T cells is 5 x 10. 6 mL -1 -1 x 10 7 mL -1 .

[0070] Typical, but not limiting, the number of CAR-T cells in a polyampholyte CAR cell cryopreservative is 5 x 10. 6 6 x 10 6 7 x 10 6 8x10 6 Or 9 x 10 6 mL -1 .

[0071] According to a fourth aspect of the present invention, the present invention provides a polyamplifier CAR cell cryopreservation solution, comprising a cell culture medium and a polyamplifier cell cryopreservation protectant graphene oxide-polyacrylic acid-betaine provided in the first aspect of the present invention, wherein the preparation method is to add graphene oxide-polyacrylic acid-betaine to the cell culture medium and mix them evenly to obtain the polyamplifier cell cryopreservation solution.

[0072] The polyamplifier-based CAR cell cryopreservation solution provided by this invention incorporates a polyamplifier-based cell cryoprotectant into the cell culture medium. This polyamplifier-based CAR cell cryopreservation solution effectively solves the problems of high genotoxicity, low cell recovery rate, short low-temperature storage time, and residual cryopreservation media that exist in traditional cryopreservation solutions during CAR cell cryopreservation. This cryopreservation solution aims to improve cell cryopreservation efficiency, ensure the integrity and functionality of CAR cells, and guarantee a high survival rate after thawing and recovery. The ultimate goal is to provide a higher-quality cryopreservation solution for the clinical application of CAR cell therapy, further promoting innovation and development in fields such as cancer treatment.

[0073] The cell culture media mentioned above include, but are not limited to, one of RPMI 1640 medium, DMEM medium, and X-VIVO15 medium.

[0074] Specifically, the manufacturer of RPMI 1640 culture medium is Thermo Fisher Scientific, and the product code is 11875093.

[0075] Specifically, the DMEM culture medium is manufactured by Thermo Fisher Scientific, and its product designation is 11965118.

[0076] Specifically, the X-VIVO15 culture medium is manufactured by Lonza Biologics Plc, and its product brand is BE02-060Q.

[0077] Specifically, the cell culture medium is X-VIVO15 medium.

[0078] Specifically, flow cytometry was used to assess the survival rate of CAR-T cells after cryopreservation.

[0079] Specifically, the cell viability of CAR-T cells before and after cryopreservation was evaluated using the CCK-8 assay, and the metabolic activity of cells before and after cryopreservation was compared.

[0080] According to a fifth aspect of the present invention, the present invention provides a method for preparing a polyamplifier-based CAR cell cryopreservation solution, comprising the following steps:

[0081] The polyamphoteric electrolyte cell cryopreservation protectant graphene oxide-polyacrylic acid-betaine was added to X-VIVO15 medium and mixed evenly to obtain the polyamphoteric electrolyte CAR cell cryopreservation solution.

[0082] To facilitate understanding by those skilled in the art, the technical solutions provided by the present invention will be further described below in conjunction with embodiments and comparative examples.

[0083] Example 1

[0084] This embodiment provides a polyampholyte cell cryopreservation protectant, which is prepared according to the following steps:

[0085] (1) Mix 10g of hexamethylene diisocyanate (sufficient excess) with 3.0g of graphene oxide in anhydrous N,N-dimethylformamide (DMF) and react at 80°C for 25h. The isocyanate group at one end of the hexamethylene diisocyanate reacts with the carboxyl or hydroxyl group on the surface of the graphene oxide and attaches to the surface of the graphene oxide. Wash the product several times with anhydrous DMF to remove unreacted diisocyanate and obtain activated graphene oxide. Then add 4g of polyacrylic acid (the number average molecular weight of polyacrylic acid is 5000g / mol) and react at 80°C for 25h to allow the carboxyl group in the polyacrylic acid to react with the isocyanate group at the other end of the diisocyanate to obtain graphene oxide-polyacrylic acid.

[0086] (2) Dissolve the prepared graphene oxide-polyacrylic acid (4.25 g) and N,N-dimethylethylenediamine (3 mL) in a 1:1 (mass ratio) mixture of dichloromethane:N,N-dimethylformamide (50 mL) and cool the solution to 0 °C. After stirring for 5 minutes, 1-hydroxybenzotriazole hydrate (amidation reaction protectant) (6.5 g), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (crosslinking agent) (8.5 g), and diisopropylethylamine (28 mL) were added sequentially to the solution. The mixture was heated to room temperature overnight and stirred for 48 h. The solution was diluted with dichloromethane and extracted with water and brine. The extract was dissolved in anhydrous tetrahydrofuran (50 mL), and the solution was cooled to 0 °C. β-propiolactone (0.625 mL) was added dropwise, and the solution was stirred and reacted at 23 °C for 20 h. The mixture was filtered, washed with acetone (3 × 50 mL), and dried under high vacuum to obtain the polyampholyte cell cryopreservation protectant. In this polyampholyte cell cryopreservation protectant, the mass ratio of polyacrylic acid to betaine was 5:1, and the mass ratio of the total mass of polyacrylic acid and betaine to the mass of graphene was 1:2.

[0087] Example 2

[0088] This embodiment provides a polyampholyte cell cryopreservation protectant, which is prepared according to the following steps:

[0089] Polyacrylic acid-betaine was synthesized using dichloromethane as a solvent via the acylation reaction of polyacrylic acid with N,N-dimethylethylenediamine and the ring-opening reaction of β-propiolactone. 10 g of hexamethylene diisocyanate (sufficient excess) and 0.01 g of graphene oxide were mixed in anhydrous N,N-dimethylformamide (DMF) and reacted at 90 °C for 22 h. The isocyanate group at one end of the hexamethylene diisocyanate reacted with the carboxyl or hydroxyl groups on the surface of the graphene oxide, attaching to the graphene oxide surface. The product was washed several times with anhydrous DMF to remove unreacted diisocyanate, yielding activated graphene oxide. Polyacrylic acid-betaine was added to the activated graphene oxide and reacted at 80 °C for 12 h to graft a polyampholyte cell cryopreservation protectant (graphene oxide-polyacrylic acid-betaine).

[0090] Polyacrylic acid-betaine is prepared according to the following steps:

[0091] Polyacrylic acid (1.6 g, number average molecular weight 2000 g / mol) and N,N-dimethylethylenediamine (6 mL) were dissolved in a 1:1 (mass ratio) mixture of dichloromethane:N,N-dimethylformamide (50 mL), and the solution was cooled to 0 °C. After stirring for 5 minutes, 1-hydroxybenzotriazole hydrate (amidation reaction protectant) (6.5 g), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (crosslinking agent) (8.5 g), and diisopropylethylamine (28 mL) were added sequentially to the solution. The mixture was heated to room temperature overnight and stirred for 48 h. The solution was diluted with dichloromethane and extracted with water and brine. The extract was dissolved in anhydrous tetrahydrofuran (50 mL), and the solution was cooled to 0 °C. β-propiolactone (0.41 mL) was added dropwise, the solution was stirred, and the mixture was heated to 23 °C and reacted for 18 h. The mixture was filtered, washed with acetone (3 × 50 mL), and dried under high vacuum to obtain the polyampholyte cell cryopreservation protectant. In this polyampholyte cell cryopreservation protectant, the mass ratio of polyacrylic acid to betaine was 8:1, and the mass ratio of the total mass of polyacrylic acid and betaine to the mass of graphene was 10:1.

[0092] Example 3

[0093] This embodiment provides a polyampholyte cell cryopreservation protectant, which is prepared according to the following steps:

[0094] (1) Mix 10g of hexamethylene diisocyanate (sufficient excess) with 1.0g of graphene oxide in anhydrous N,N-dimethylformamide (DMF) and react at 80°C for 25h. The isocyanate group at one end of the hexamethylene diisocyanate reacts with the carboxyl or hydroxyl group on the surface of the graphene oxide and attaches to the surface of the graphene oxide. Wash the product several times with anhydrous DMF to remove unreacted diisocyanate and obtain activated graphene oxide. Then add 8g of polyacrylic acid (the number average molecular weight of polyacrylic acid is 2000g / mol) and react at 80°C for 25h to allow the carboxyl group in the polyacrylic acid to react with the isocyanate group at the other end of the diisocyanate to obtain graphene oxide-polyacrylic acid.

[0095] (2) The prepared graphene oxide-polyacrylic acid (2.31 g) and N,N-dimethylethylenediamine (3 mL) were dissolved in a 1:1 (mass ratio) mixture of dichloromethane:N,N-dimethylformamide (50 mL), and the solution was cooled to 0 °C. After stirring for 5 minutes, 1-hydroxybenzotriazole hydrate (amidation reaction protectant) (6.5 g), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (crosslinking agent) (8.5 g), and diisopropylethylamine (28 mL) were added sequentially to the solution. The mixture was heated to room temperature overnight and stirred for 48 h. The solution was diluted with dichloromethane and extracted with water and brine. The extract was dissolved in anhydrous tetrahydrofuran (50 mL), and the solution was cooled to 0 °C. β-propiolactone (1.876 mL) was added dropwise, the solution was stirred, and the reaction was carried out at 24 °C for 18 h. The mixture was filtered, washed with acetone (3 × 50 mL), and dried under high vacuum to obtain the polyampholyte cell cryopreservation protectant. In this polyampholyte cell cryopreservation protectant, the mass ratio of polyacrylic acid to betaine was 2:1, and the mass ratio of the total mass of polyacrylic acid and betaine to the mass of graphene was 2:1.

[0096] Example 4

[0097] This embodiment provides a polyampholyte cell cryopreservation protectant, which is prepared according to the following steps:

[0098] (1) Mix 10g of hexamethylene diisocyanate (sufficient excess) with 1.0g of graphene oxide in anhydrous N,N-dimethylformamide (DMF) and react at 80°C for 25h. The isocyanate group at one end of the hexamethylene diisocyanate reacts with the carboxyl or hydroxyl group on the surface of the graphene oxide and attaches to the surface of the graphene oxide. Wash the product several times with anhydrous DMF to remove unreacted diisocyanate and obtain activated graphene oxide. Then add 30g of polyacrylic acid (the number average molecular weight of polyacrylic acid is 3000g / mol) and react at 80°C for 25h to allow the carboxyl group in the polyacrylic acid to react with the isocyanate group at the other end of the diisocyanate to obtain graphene oxide-polyacrylic acid.

[0099] (2) Dissolve the prepared graphene oxide-polyacrylic acid (5.61 g) and N,N-dimethylethylenediamine (3 mL) in a 1:1 (mass ratio) dichloromethane:N,N-dimethylformamide (50 mL) and cool the solution to 0 °C. After stirring for 5 minutes, 1-hydroxybenzotriazole hydrate (amidation reaction protectant) (6.5 g), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (crosslinking agent) (8.5 g), and diisopropylethylamine (28 mL) were added sequentially to the solution. The mixture was heated to room temperature overnight and stirred for 48 h. The solution was diluted with dichloromethane and extracted with water and brine. The extract was dissolved in anhydrous tetrahydrofuran (50 mL), and the solution was cooled to 0 °C. β-propiolactone (2.16 mL) was added dropwise, the solution was stirred, and the reaction was carried out at 20 °C for 12 h. The mixture was filtered, washed with acetone (3 × 50 mL), and dried under high vacuum to obtain the polyampholyte cell cryopreservation protectant. In this polyampholyte cell cryopreservation protectant, the mass ratio of polyacrylic acid to betaine was 10:1, and the mass ratio of the total mass of polyacrylic acid and betaine to graphene was 5:1.

[0100] Example 5

[0101] This embodiment provides a polyampholyte cell cryopreservation protectant, which is prepared according to the following steps:

[0102] (1) 10g of hexamethylene diisocyanate (sufficient excess) and 1.1g of graphene oxide were mixed in anhydrous N,N-dimethylformamide (DMF) and reacted at 80°C for 25h. The isocyanate group at one end of the hexamethylene diisocyanate reacted with the carboxyl or hydroxyl group on the surface of the graphene oxide and attached to the surface of the graphene oxide. The product was washed several times with anhydrous DMF to remove unreacted diisocyanate and obtain activated graphene oxide. Then 60g of polyacrylic acid (the number average molecular weight of polyacrylic acid is 5000g / mol) was added and reacted at 80°C for 25h to allow the carboxyl group in the polyacrylic acid to react with the isocyanate group at the other end of the diisocyanate to obtain graphene oxide-polyacrylic acid.

[0103] (2) Dissolve the prepared graphene oxide-polyacrylic acid (8.69 g) and N,N-dimethylethylenediamine (3 mL) in a 1:1 (mass ratio) mixture of dichloromethane:N,N-dimethylformamide (50 mL) and cool the solution to 0 °C. After stirring for 5 minutes, 1-hydroxybenzotriazole hydrate (amidation reaction protectant) (6.5 g), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (crosslinking agent) (8.5 g), and diisopropylethylamine (28 mL) were added sequentially to the solution. The mixture was heated to room temperature overnight and stirred for 48 h. The solution was diluted with dichloromethane and extracted with water and brine. The extract was dissolved in anhydrous tetrahydrofuran (50 mL), and the solution was cooled to 0 °C. β-propiolactone (6.14 mL) was added dropwise, the solution was stirred, and the reaction was carried out at 22 °C for 14 h. The mixture was filtered, washed with acetone (3 × 50 mL), and dried under high vacuum to obtain the polyampholyte cell cryopreservation protectant. In this polyampholyte cell cryopreservation protectant, the mass ratio of polyacrylic acid to betaine was 8:1, and the mass ratio of the total mass of polyacrylic acid and betaine to graphene was 10:1.

[0104] Example 6

[0105] This embodiment provides a polyampholyte cell cryopreservation protectant, which is prepared according to the following steps:

[0106] (1) 10g of hexamethylene diisocyanate (sufficient excess) and 1.3g of graphene oxide were mixed in anhydrous N,N-dimethylformamide (DMF) and reacted at 80°C for 25h. The isocyanate group at one end of the hexamethylene diisocyanate reacted with the carboxyl or hydroxyl group on the surface of the graphene oxide and attached to the surface of the graphene oxide. The product was washed several times with anhydrous DMF to remove unreacted diisocyanate and obtain activated graphene oxide. Then 100g of polyacrylic acid (the number average molecular weight of polyacrylic acid is 10000g / mol) was added and reacted at 80°C for 25h to allow the carboxyl group in the polyacrylic acid to react with the isocyanate group at the other end of the diisocyanate to obtain graphene oxide-polyacrylic acid.

[0107] (2) The prepared graphene oxide-polyacrylic acid (12.17 g) and N,N-dimethylethylenediamine (3 mL) were dissolved in a 1:1 (mass ratio) mixture of dichloromethane:N,N-dimethylformamide (50 mL), and the solution was cooled to 0 °C. After stirring for 5 minutes, 1-hydroxybenzotriazole hydrate (amidation reaction protectant) (6.5 g), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (crosslinking agent) (8.5 g), and diisopropylethylamine (28 mL) were added sequentially to the solution. The mixture was heated to room temperature overnight and stirred for 48 h. The solution was diluted with dichloromethane and extracted with water and brine. The extract was dissolved in anhydrous tetrahydrofuran (50 mL), and the solution was cooled to 0 °C. β-propiolactone (18.58 mL) was added dropwise, the solution was stirred, and the reaction was carried out at 25 °C for 16 h. The mixture was filtered, washed with acetone (3 × 50 mL), and dried under high vacuum to obtain the polyampholyte cell cryopreservation protectant. In this polyampholyte cell cryopreservation protectant, the mass ratio of polyacrylic acid to betaine was 5:1, and the mass ratio of the total mass of polyacrylic acid and betaine to graphene was 10:1.

[0108] Example 7

[0109] This embodiment provides a polyampholyte cell cryopreservation protectant, which is prepared according to the following steps:

[0110] Polyacrylic acid-betaine was synthesized using dichloromethane as a solvent via the acylation reaction of polyacrylic acid with N,N-dimethylethylenediamine and the ring-opening reaction of β-propiolactone. 10 g of hexamethylene diisocyanate (sufficient excess) and 0.05 g of graphene oxide were mixed in anhydrous N,N-dimethylformamide (DMF) and reacted at 90 °C for 22 h. The isocyanate group at one end of the hexamethylene diisocyanate reacted with the carboxyl or hydroxyl groups on the surface of the graphene oxide, attaching to the graphene oxide surface. The product was washed several times with anhydrous DMF to remove unreacted diisocyanate, yielding activated graphene oxide. Polyacrylic acid-betaine was added to the activated graphene oxide and reacted at 80 °C for 12 h to graft a polyampholyte cell cryopreservation protectant (graphene oxide-polyacrylic acid-betaine).

[0111] Polyacrylic acid-betaine is prepared according to the following steps:

[0112] Polyacrylic acid (1.8 g, number average molecular weight 3000 g / mol) and N,N-dimethylethylenediamine (6 mL) were dissolved in a 1:1 (mass ratio) mixture of dichloromethane:N,N-dimethylformamide (50 mL), and the solution was cooled to 0 °C. After stirring for 5 minutes, 1-hydroxybenzotriazole hydrate (amidation reaction protectant) (6.5 g), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (crosslinking agent) (8.5 g), and diisopropylethylamine (28 mL) were added sequentially to the solution. The mixture was heated to room temperature overnight and stirred for 48 h. The solution was diluted with dichloromethane and extracted with water and brine. The extract was dissolved in anhydrous tetrahydrofuran (50 mL), and the solution was cooled to 0 °C. β-propiolactone (0.80 mL) was added dropwise, the solution was stirred, and the mixture was heated to 23 °C and reacted for 18 h. The mixture was filtered, washed with acetone (3 × 50 mL), and dried under high vacuum to obtain the polyampholyte cell cryopreservation protectant. In this polyampholyte cell cryopreservation protectant, the mass ratio of polyacrylic acid to betaine was 5.5:1, and the mass ratio of the total mass of polyacrylic acid and betaine to the mass of graphene was 8:1.

[0113] Example 8

[0114] This embodiment provides a polyampholyte cell cryopreservation protectant, which is prepared according to the following steps:

[0115] Polyacrylic acid-betaine was synthesized using dichloromethane as a solvent via the acylation reaction of polyacrylic acid with N,N-dimethylethylenediamine and the ring-opening reaction of β-propiolactone. 10 g of hexamethylene diisocyanate (sufficient excess) and 0.18 g of graphene oxide were mixed in anhydrous N,N-dimethylformamide (DMF) and reacted at 90 °C for 22 h. The isocyanate group at one end of the hexamethylene diisocyanate reacted with the carboxyl or hydroxyl groups on the surface of the graphene oxide, attaching to the graphene oxide surface. The product was washed several times with anhydrous DMF to remove unreacted diisocyanate, yielding activated graphene oxide. Polyacrylic acid-betaine was added to the activated graphene oxide and reacted at 80 °C for 12 h to graft a polyampholyte cell cryopreservation protectant (graphene oxide-polyacrylic acid-betaine).

[0116] Polyacrylic acid-betaine is prepared according to the following steps:

[0117] Polyacrylic acid (2.0 g, number average molecular weight 5000 g / mol) and N,N-dimethylethylenediamine (6 mL) were dissolved in a 1:1 (mass ratio) mixture of dichloromethane:N,N-dimethylformamide (50 mL), and the solution was cooled to 0 °C. After stirring for 5 minutes, 1-hydroxybenzotriazole hydrate (amidation reaction protectant) (6.5 g), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (crosslinking agent) (8.5 g), and diisopropylethylamine (28 mL) were added sequentially to the solution. The mixture was heated to room temperature overnight and stirred for 48 h. The solution was diluted with dichloromethane and extracted with water and brine. The extract was dissolved in anhydrous tetrahydrofuran (50 mL), and the solution was cooled to 0 °C. β-propiolactone (1.15 mL) was added dropwise, the solution was stirred, and the mixture was heated to 23 °C and reacted for 18 h. The mixture was filtered, washed with acetone (3 × 50 mL), and dried under high vacuum to obtain the polyampholyte cell cryopreservation protectant. In this polyampholyte cell cryopreservation protectant, the mass ratio of polyacrylic acid to betaine was 5.3:1, and the mass ratio of the total mass of polyacrylic acid and betaine to the mass of graphene was 5:1.

[0118] Example 9

[0119] This embodiment provides a polyampholyte cell cryopreservation protectant, which is prepared according to the following steps:

[0120] Polyacrylic acid-betaine was synthesized using dichloromethane as a solvent via the acylation reaction of polyacrylic acid with N,N-dimethylethylenediamine and the ring-opening reaction of β-propiolactone. 10 g of hexamethylene diisocyanate (sufficient excess) and 0.60 g of graphene oxide were mixed in anhydrous N,N-dimethylformamide (DMF) and reacted at 90 °C for 22 h. The isocyanate group at one end of the hexamethylene diisocyanate reacted with the carboxyl or hydroxyl groups on the surface of the graphene oxide, attaching to the graphene oxide surface. The product was washed several times with anhydrous DMF to remove unreacted diisocyanate, yielding activated graphene oxide. Polyacrylic acid-betaine was added to the activated graphene oxide and reacted at 80 °C for 12 h to graft a polyampholyte cell cryopreservation protectant (graphene oxide-polyacrylic acid-betaine).

[0121] Polyacrylic acid-betaine is prepared according to the following steps:

[0122] Polyacrylic acid (2.4 g, number average molecular weight 10000 g / mol) and N,N-dimethylethylenediamine (6 mL) were dissolved in a 1:1 (mass ratio) mixture of dichloromethane:N,N-dimethylformamide (50 mL), and the solution was cooled to 0 °C. After stirring for 5 minutes, 1-hydroxybenzotriazole hydrate (amidation reaction protectant) (6.5 g), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (crosslinking agent) (8.5 g), and diisopropylethylamine (28 mL) were added sequentially to the solution. The mixture was heated to room temperature overnight and stirred for 48 h. The solution was diluted with dichloromethane and extracted with water and brine. The extract was dissolved in anhydrous tetrahydrofuran (50 mL), and the solution was cooled to 0 °C. β-propiolactone (1.60 mL) was added dropwise, the solution was stirred, and the mixture was heated to 23 °C and reacted for 18 h. The mixture was filtered, washed with acetone (3 × 50 mL), and dried under high vacuum to obtain the polyampholyte cell cryopreservation protectant. In this polyampholyte cell cryopreservation protectant, the mass ratio of polyacrylic acid to betaine was 5.2:1, and the mass ratio of the total mass of polyacrylic acid and betaine to the mass of graphene was 1:2.

[0123] Example 10

[0124] This embodiment provides a polyampholyte cell cryopreservation solution, which is prepared according to the following steps:

[0125] Add 3 mL of the polyampholyte cell cryopreservative provided in Example 1 to 97 mL of DMEM culture medium and mix well to obtain the polyampholyte cell cryopreservation solution.

[0126] Example 11

[0127] This embodiment provides a polyampholyte CAR-T cell cryopreservation solution, which is prepared according to the following steps:

[0128] Add 3 mL of the polyampholyte cell cryopreservation agent provided in Example 2 to 97 mL of X-VIVO15 culture medium and mix well to obtain the polyampholyte CAR-T cell cryopreservation solution.

[0129] Example 12

[0130] This embodiment provides a polyampholyte CAR-T cell cryopreservation solution, which is prepared according to the following steps:

[0131] Add 3 mL of the polyampholyte cell cryopreservation agent provided in Example 3 to 97 mL of DMEM culture medium and mix well to obtain the polyampholyte CAR-T cell cryopreservation solution.

[0132] Example 13

[0133] This embodiment provides a polyampholyte CAR-T cell cryopreservation solution, which is prepared according to the following steps:

[0134] Add 3 mL of the polyampholyte cell cryopreservative provided in Example 5 to 97 mL of DMEM culture medium and mix well to obtain the polyampholyte CAR-T cell cryopreservation solution.

[0135] Example 14

[0136] This embodiment provides a polyamplifier CAR-T cell cryopreservation solution, which differs from Example 10 in that the amount of polyamplifier cell cryopreservation protectant graphene oxide-polyacrylic acid-betaine added is 2 mL, and the amount of cell culture medium is 98 mL.

[0137] Example 15

[0138] This embodiment provides a polyamplifier CAR-T cell cryopreservation solution, which differs from Example 10 in that the amount of the polyamplifier cell cryopreservation protectant graphene oxide-polyacrylic acid-betaine added is 1 mL, and the amount of cell culture medium is 99 mL.

[0139] Example 16

[0140] This embodiment provides a polyampholyte CAR-T cell cryopreservation solution, which is prepared according to the following steps:

[0141] Add 3 mL of the obtained polyampholyte cell cryopreservation protectant graphene oxide-polyacrylic acid-betaine provided in Example 6 to 97 mL of DMEM culture medium and mix well to obtain polyampholyte CAR-T cell cryopreservation solution.

[0142] Example 17

[0143] This embodiment provides a polyampholyte CAR-T cell cryopreservation solution, which is prepared according to the following steps:

[0144] Add 3 mL of the obtained polyampholyte cell cryopreservation protectant graphene oxide-polyacrylic acid-betaine from Example 8 to 97 mL of DMEM culture medium and mix well to obtain polyampholyte CAR-T cell cryopreservation solution.

[0145] Example 18

[0146] This embodiment provides a polyampholyte cell cryopreservation solution, which is prepared according to the following steps:

[0147] Add 0.5 mL of the polyampholyte cell cryopreservative provided in Example 1 to 99.5 mL of DMEM culture medium and mix well to obtain the polyampholyte cell cryopreservation solution.

[0148] Comparative Example 1

[0149] This comparative example provides a universal serum-type cell cryopreservation solution, which is prepared according to the following steps:

[0150] Add 7 mL of fetal bovine serum and 3 mL of DMSO (dimethyl sulfoxide) to 90 mL of basal culture medium (DMEM) and mix well.

[0151] Comparative Example 2

[0152] This comparative example provides a serum-free cell cryopreservation solution, which is prepared according to the following steps:

[0153] Add 3 mL of DMSO (dimethyl sulfoxide) to 97 mL of X-VIVO15 medium and mix well.

[0154] Experimental Example 1

[0155] The cell cryopreservation solutions provided in Example 10 and Comparative Example 1 were used to evaluate the cell viability of L929 cells before and after cryopreservation using the CCK-8 assay. The results showed that the L929 cells in Example 1 had 8% higher viability than the L929 cells in Comparative Example 1.

[0156] Experimental Example 2

[0157] The cell cryopreservation solutions provided in Example 10 and Comparative Example 1 were used to detect the survival rate of L929 cells before and after cryopreservation by flow cytometry. The results showed that the survival rate of L929 cells in Example 10 was 80%, while the survival rate of L929 cells in Comparative Example 1 was 72%. The survival rate of L929 cells in Example 10 was 8% higher than that in Comparative Example 1.

[0158] Experimental Example 3

[0159] The cell cryopreservation solutions provided in Example 10 and Comparative Example 1 were evaluated using the CCK-8 assay to assess the cell viability of neural stem cells before and after cryopreservation. The results showed that the neural stem cell viability of Example 10 was 6% higher than that of Comparative Example 1.

[0160] Test Example 4

[0161] The cell cryopreservation solutions provided in Example 10 and Comparative Example 1 were used to detect the survival rate of neural stem cells before and after cryopreservation by flow cytometry. The results showed that the survival rate of neural stem cells in Example 10 was 75%, while the survival rate of neural stem cells in Comparative Example 1 was 68%. The survival rate of neural stem cells in Example 10 was 7% higher than that in Comparative Example 1.

[0162] Experimental Example 5

[0163] The cell cryopreservation solutions provided in Example 11 and Comparative Example 2 were evaluated using the CCK-8 assay to assess the cell viability of CAR-T cells before and after cryopreservation. The results showed that the CAR-T cell viability of Example 11 was 5% higher than that of Comparative Example 2.

[0164] Experimental Example 6

[0165] The cell cryopreservation solutions provided in Example 11 and Comparative Example 2 were used to detect the survival rate of CAR-T cells before and after cryopreservation by flow cytometry. The results showed that the survival rate of CAR-T cells in Example 11 was 50%, while the survival rate of CAR-T cells in Comparative Example 2 was 43%. The survival rate of CAR-T cells in Example 11 was 7% higher than that of CAR-T cells in Comparative Example 2.

[0166] Experimental Example 7

[0167] The cell cryopreservation solutions provided in Examples 14-15 and Comparative Example 2 were used to evaluate the cell viability of CAR-T cells before and after cryopreservation using the CCK-8 assay. The results showed that the CAR-T cell viability in Example 14 was 4% higher than that in Comparative Example 2, and the CAR-T cell viability in Example 15 was 3% higher than that in Comparative Example 2.

[0168] Experimental Example 8

[0169] The cell cryopreservation solutions provided in Examples 14-15 and Comparative Example 2 were used to detect the survival rate of CAR-T cells before and after cryopreservation using flow cytometry. The results showed that the cell survival rate of Comparative Example 2 was 43%, and the CAR-T cell survival rate of Example 14 was 49%, which was 6% higher than that of Comparative Example 2. The CAR-T cell survival rate of Example 15 was 47%, which was 4% higher than that of Comparative Example 2.

[0170] Experimental Example 9

[0171] The cell cryopreservation solutions provided in Examples 12-13 and 16-17, and the cell cryopreservation solution provided in Comparative Example 1, were used to evaluate the cell viability of CAR-T cells before and after cryopreservation using the CCK-8 assay. The results showed that the CAR-T cell viability in Example 12 was 5% higher than that in Comparative Example 1; the CAR-T cell viability in Example 13 was 4% higher than that in Comparative Example 1; the CAR-T cell viability in Example 16 was 5% higher than that in Comparative Example 1; and the CAR-T cell viability in Example 17 was 3% higher than that in Comparative Example 1.

[0172] Experimental Example 10

[0173] The cell cryopreservation solutions provided in Examples 12-13, 16-17, and Comparative Example 1 were used to detect the survival rate of CAR-T cells before and after cryopreservation by flow cytometry. The results showed that the survival rate of CAR-T cells in Comparative Example 1 was 40%, and the survival rate of CAR-T cells in Example 12 was 48%, which was 8% higher than that in Comparative Example 1; the survival rate of CAR-T cells in Example 13 was 46%, which was 6% higher than that in Comparative Example 1; the survival rate of CAR-T cells in Example 16 was 47%, which was 7% higher than that in Comparative Example 1; and the survival rate of CAR-T cells in Example 17 was 45%, which was 5% higher than that in Comparative Example 1.

[0174] Experimental Example 11

[0175] The cell cryopreservation solution provided in Example 18 was used to detect the survival rate of L929 cells before and after cryopreservation by flow cytometry. The results showed that the survival rate of L929 cells in Example 18 was 80%.

[0176] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polyampholyte cell cryopreservation protectant, characterized in that, The polyampholyte cell cryopreservation protectant comprises polyacrylic acid, betaine, and graphene oxide linked by chemical bonds, wherein the mass ratio of polyacrylic acid to betaine is 10:1-2:1, and the mass ratio of the total mass of polyacrylic acid and betaine to the mass of graphene oxide is 1:2-10:

1.

2. The polyampholyte cell cryopreservation protectant according to claim 1, characterized in that, The molar mass of polyacrylic acid is 2000-10000 g / mol, preferably 4000-6000 g / mol.

3. The polyampholyte cell cryopreservation protectant according to claim 1, characterized in that, Polyacrylic acid and graphene oxide are linked by diisocyanate compounds, and betaine is linked to polyacrylic acid by chemical bonds; The diisocyanate compounds include at least one of trimethylhexane diisocyanate, dicyclohexylmethane diisocyanate, toluene diisocyanate, and hexamethylene diisocyanate.

4. The method for preparing the polyampholyte cell cryopreservative according to any one of claims 1-3, characterized in that, The preparation method includes: Step S1: First, a diisocyanate compound is modified onto the surface of graphene oxide to obtain activated graphene oxide. Then, the activated graphene oxide is mixed with polyacrylic acid to carry out a grafting reaction to obtain the graphene oxide-polyacrylic acid. Step S2: The graphene oxide-polyacrylic acid and betaine are mixed and reacted to obtain the polyampholyte cell cryopreservation protectant.

5. The preparation method according to claim 4, characterized in that, In step S1, graphene oxide is dispersed in a first organic solvent, and then a diisocyanate compound is added and mixed, so that the cyanate groups in the diisocyanate compound react with the carboxyl groups or hydroxyl groups on the graphene oxide to obtain the activated graphene oxide. Preferably, the activation reaction is carried out at a temperature of 70-90°C for 20-30 hours. Preferably, the diisocyanate compound includes at least one of trimethylhexane diisocyanate, dicyclohexylmethane diisocyanate, toluene diisocyanate, and hexamethylene diisocyanate; Preferably, the first organic solvent comprises N,N-dimethylformamide; Preferably, the grafting reaction between the activated graphene oxide and polyacrylic acid is carried out at a temperature of 50-160°C.

6. The preparation method according to claim 4, characterized in that, In step S2, the graphene oxide-polyacrylic acid is dispersed in a second organic solvent, and then betaine is added to mix and carry out acylation and ring-opening reactions to obtain the polyampholyte cell cryopreservation protectant. Preferably, the second organic solvent comprises dichloromethane and N,N-dimethylformamide, and the mass ratio of the two is (1-2):1; Preferably, the acylation reaction and the ring-opening reaction are carried out at a temperature of 20-25°C for 12-20 hours.

7. The method for preparing the polyampholyte cell cryopreservative according to any one of claims 1 to 3, characterized in that, The preparation method includes: Step A1: Polyacrylic acid is dispersed in a third organic solvent, and betaine is added to mix and carry out acylation and ring-opening reactions to obtain polyacrylic acid-betaine; Step A2: Activated graphene oxide is mixed with polyacrylic acid-betaine and grafted to obtain the polyampholyte cell cryopreservation protectant. Preferably, in step A1, the acylation reaction and the ring-opening reaction are carried out at a temperature of 20-25°C for 12-20 hours. Preferably, in step A2, the grafting reaction temperature is 50-160°C; Preferably, the third organic solvent includes dichloromethane.

8. The application of the polyampholyte cell cryopreservation agent according to any one of claims 1 to 3 in the cryopreservation of L929 cells, CAR cells, mesenchymal stem cells, neural stem cells, and erythrocytes; Preferably, the CAR cells include one of CAR-T cells, CAR-NK cells, and CAR-Macrophage cells; Preferably, the CAR cells are CAR-T cells; Preferably, when the CAR-T cells are cryopreserved using the polyampholyte CAR cell cryopreservation agent, the number of CAR-T cells is 5 x 10. 6 mL -1 -1 x 10 7 mL -1 .

9. A polyampholyte CAR cell cryopreservation solution, characterized in that, By weight percentage, it comprises 1%-3% of the polyampholyte cell cryopreservative as described in any one of claims 1 to 3 and 97-99% of cell culture medium.

10. The polyampholyte CAR cell cryopreservation solution according to claim 9, characterized in that, The cell culture medium includes one of RPMI 1640 medium, DMEM medium, and X-VIVO 15 medium; Preferably, the cell culture medium is X-VIVO15 medium.