Cryopreservation protective agent, preparation method and application thereof, and T lymphocyte cryopreservation and resuscitation method

Abstract

The invention belongs to the field of cell cryopreservation, and particularly relates to a cryopreservation protective agent, a preparation method and application thereof and a T lymphocyte cryopreservation and recovery method. The cryopreservation protective agent comprises an anti-freezing agent and a protective solution, the antifreeze agent is a mixture of zwitterionic magnetic nanoparticles and betaine. The zwitterionic magnetic nanoparticles comprise ferroferric oxide on a core layer and a zwitterionic polymer on a shell layer, preferably, the zwitterionic polymer is one of poly (sulphobetaine methacrylate), poly (2-(methacryloyloxy) ethyl) phosphorylcholine or poly (carboxyl betaine methacrylate), and the zwitterionic polymer is one of poly (sulphobetaine methacrylate), poly (2-(methacryloyloxy) ethyl) phosphorylcholine and poly (carboxyl betaine methacrylate). The cryopreservation protective agent is used for preserving the T lymphocytes in liquid nitrogen, the result shows that the survival rate of the recovered T lymphocytes is 80% or above, the immune functions of proliferation, differentiation, tumor killing and the like are reserved, and the effect is better than that of a traditional 10% DMSO cryopreservation protective agent.

Description

Technical Field

[0001] The invention belongs to the field of cell cryopreservation, and specifically relates to a cryoprotectant and a preparation method and application thereof, as well as a T lymphocyte cryopreservation and resuscitation method. Background Art

[0002] Chimeric antigen receptor (CAR) T cell therapy has cured previously incurable blood cancers and changed the field of oncology. In the United States, at least 60,000 blood cancer patients each year do not respond to conventional treatments. Currently, a number of CAR-T cell therapy products have been approved for marketing, suitable for blood cancers such as leukemia, lymphoma and myeloma. This treatment method has brought significant therapeutic effects to many patients. T cells are the cellular basis of CAR-T cell therapy. In order for CAR-T cells to meet clinical needs, a stable, sufficient and quality-controlled supply of T cells is required. Therefore, cryopreservation technology is required to maintain the activity of T lymphocytes. In addition, it also fills the gap between the manufacture of cell therapy drugs and clinical administration, realizing a centralized manufacturing model.

[0003] Cryopreservation technology can slow down or stop the metabolic process of cells and maintain the activity and function of cells for a long time. However, cryopreservation also has some challenges, such as cell dehydration and ice crystal formation during the freezing process, which can cause mechanical damage and osmotic pressure damage to cells and lead to cell death. In particular, T cells, which are composed of many heterogeneous populations, are very sensitive to temperature changes. To date, the most commonly used cryoprotectant for T cell products is dimethyl sulfoxide (DMSO), which can minimize the formation of ice during cryopreservation. However, according to clinical reports, DMSO can produce toxic side effects, causing patients to have neurological, gastrointestinal, allergic, and cardiovascular side effects. In addition, DMSO can also impair the immune function of T cells, including inhibited proliferation and reduced production of proinflammatory cytokines. Secondly, the inevitable ice formation during freezing and thawing can harm T cells, and because the traditional water bath rewarming rate is slow, as the scale of cryopreservation is expanded, the recrystallization of ice will be aggravated, and increasing the rewarming rate to reduce the recrystallization of ice is also challenging. Therefore, there is an urgent need to develop a safer and more effective DMSO-free T cell cryoprotectant. Summary of the invention

[0004] In order to solve the problems existing in the above technologies, the present invention provides a cryoprotectant and a preparation method and application thereof, as well as a T lymphocyte cryopreservation and resuscitation method.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] A cryoprotectant comprises an antifreeze agent and a protective solution; the antifreeze agent is a mixture of zwitterionic magnetic nanoparticles (ZMNPs) and betaine.

[0007] The zwitterionic magnetic nanoparticles include ferroferric oxide in the core layer and a zwitterionic polymer in the shell layer; preferably, the zwitterionic polymer is one of polymethacrylate sulfobetaine, poly(2-(methacryloyloxy)ethyl)phosphorylcholine or polymethacrylate carboxybetaine;

[0008] Preferably, the zwitterionic magnetic nanoparticles are prepared by the following method: 1) grafting a silane coupling agent on the surface of ferroferric oxide magnetic nanoparticles; 2) adding 2-bromoisobutyryl bromide for bromination; 3) adding a zwitterionic monomer for polymerization reaction;

[0009] The mass concentration of the zwitterionic magnetic nanoparticles in the cryoprotectant is 0.1 mg / mL-2 mg / mL; preferably 0.5 mg / mL-5 mg / mL.

[0010] The ferroferric oxide magnetic nanoparticles (MNPs) described in step 1) are prepared by a solvothermal method; specifically comprising the following steps: first, dissolving FeCl3 and sodium citrate in ethylene glycol and ultrasonically mixing. Then, sodium acetate is added to the mixture and stirred until it becomes a transparent oil. The mixture is heated at 180-220° C., preferably 200° C. for 8-16 hours, preferably 10 hours. After the reaction is completed, the black product is washed with ethanol and water respectively to obtain MNPs.

[0011] The specific steps of grafting silane coupling agent on the surface of ferroferric oxide magnetic nanoparticles are as follows: MNPs are dispersed in a mixed solvent of ethanol and water, and then NH3·H2O is added and ultrasonically mixed. Then, a silane coupling agent, preferably 3-aminopropyltriethoxysilane (APTES), is slowly added to the above mixture under vigorous stirring, and reacted in a nitrogen environment at 40-80°C, preferably 60°C. After the reaction is completed, the obtained product is washed with ethanol to obtain APTES-MNPs nanoparticles.

[0012] The specific steps of step 2) are as follows: APTES-MNPs are dispersed in N,N-dimethylformamide (DMF), and triethylamine is added under vigorous stirring in an ice bath. Then, 2-bromoisobutyryl bromide (BB) is slowly added to the above mixture and reacted at room temperature. The product is washed with water three times to obtain BB-APTES-MNPs.

[0013] The specific steps of step 3) are: BB-APTES-MNPs, zwitterionic monomers SBMA, MPC or CBMA and 2,2'-bipyridine are dispersed in deoxygenated methanol and deoxygenated deionized water. Under nitrogen protection, the above mixture is rapidly stirred at 40°C-80°C. Then, CuBr is added to the above solution and the reaction is continued. Finally, centrifugation and water washing are performed to obtain the final products SB-ZMNPs, MPC-ZMNPs and CB-ZMNPs.

[0014] Preferably, the mass ratio of the BB-APTES-MNPs, the zwitterionic monomer, 2,2'-bipyridine, and CuBr is 0.05:1:0.03:0.014.

[0015] The mass proportion of the betaine in the cryoprotectant is 2-10%, preferably 6-8%, and more preferably 6%.

[0016] The protective solution is one of PBS buffer or DPBS.

[0017] The present invention also includes a method for preparing the cryoprotectant, comprising the following steps: dispersing zwitterionic magnetic nanoparticles ZMNPs and betaine into a protective solution.

[0018] The present invention also includes an application of the cryoprotectant, which is applied to cryopreserved cells; preferably immune cells, wherein the immune cells are one of T lymphocytes, CAR-T cells, Jurkat cells, and NK cells; preferably T lymphocytes.

[0019] The present invention also includes a T lymphocyte cryopreservation method, comprising the following steps: fully mixing the cryopreservation protective agent with the T lymphocytes and putting them into a cryopreservation tube, and cryopreserving the T lymphocytes by rapid freezing with liquid nitrogen.

[0020] The cryopreservation density of the T lymphocytes is 10 6 cells / mL; the cryopreservation volume is 1-50mL. Preferably, when the cryopreservation volume is >3mL, the mass concentration of zwitterionic magnetic nanoparticles is >3mg / mL, more preferably 5mg / mL; when the cryopreservation volume is <3mL, the mass concentration of zwitterionic magnetic nanoparticles is 0.5-2mg / mL; more preferably 1mg / mL.

[0021] The present invention also includes a method for reviving frozen T lymphocytes, which uses water bath heating or nano heating to revive the frozen T lymphocytes.

[0022] Preferably, if the cryopreservation volume is >3 mL, nano-heating is used for thawing; if the cryopreservation volume is <3 mL, water bath heating is used for thawing.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The immune cell cryoprotectant of the present invention is a mixture of ZMNPs and small molecule betaine, has good biocompatibility, can effectively inhibit the growth and recrystallization of ice crystals, and can utilize the magnetothermal properties of ZMNPs to achieve rapid and uniform thawing of frozen cells. The above effects synergistically protect frozen cells and ensure the vitality and function of cells after recovery.

[0025] The cryoprotectant is DMSO-free and has good biocompatibility. Among them, small molecule betaine, as a natural osmotic pressure regulator, can enter the cell, adjust the osmotic pressure balance, and inhibit the formation and growth of ice crystals in the cell. First, ZMNPs, as an extracellular protective agent, has a strong hydration effect on the zwitterionic polymer on its surface, forming a spherical hydration shell on its surface, inhibiting the combination of water molecules and ice, thereby controlling the growth and recrystallization of extracellular ice crystals. Secondly, combined with the magnetothermal properties of ZMNPs, the frozen cells can be quickly and evenly rewarmed. After rewarming, the cells are quickly collected by magnetic separation to avoid complicated elution steps. The above effects work together to protect the cells from cryopreservation and ensure the vitality and function of the cells after recovery.

[0026] The present invention uses a cryoprotectant to preserve T lymphocytes in liquid nitrogen. The results show that the survival rate of T lymphocytes after recovery is more than 80%, and the tumor immune killing function of T lymphocytes is retained, which is better than the traditional 10% DMSO cryoprotectant. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the preparation route of ZMNPs in Example 1 of the present invention;

[0028] Figure 2 is a transmission electron micrograph of ZMNPs in Example 1 of the present invention;

[0029] Figure 3 is the Fourier infrared spectrum of ZMNPs in Example 1 of the present invention;

[0030] Figure 4 This is a graph showing the ice crystal growth rate of the ZMNPs cryoprotectant in Example 2 of the present invention;

[0031] Figure 5 This is a graph showing the ability of the ZMNPs cryoprotectant to inhibit ice recrystallization in Example 2 of the present invention;

[0032] Figure 6 This is a graph showing the magnetothermal performance results of ZMNPs in Example 3 of the present invention;

[0033] Figure 7 The results of the ZMNPs water bath rewarming and nano rewarming rate in Example 3 of the present invention are shown in FIG.

[0034] Figure 8 This is a graph showing the nano-reheating rate results of different concentrations of ZMNPs in Example 3 of the present invention;

[0035] Fig. 9 This is a graph showing the cryopreservation efficiency of T lymphocytes after cryopreservation in Example 4 of the present invention;

[0036] Fig.10 This is a graph showing the in vitro killing ability of T lymphocytes after cryopreservation in Example 5 of the present invention;

[0037] Fig.11 This is a graph showing the in vivo tumor killing ability of T lymphocytes after cryopreservation in Example 5 of the present invention. DETAILED DESCRIPTION

[0038] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and the best embodiments.

[0039] Example 1

[0040] 1. Preparation of zwitterionic magnetic nanoparticles ( Figure 1 Showing a schematic diagram of the preparation process):

[0041] 1) Magnetic nanoparticles (MNPs) were prepared by solvothermal method. First, 0.65g FeCl3 and 0.10g sodium citrate were dissolved in 20mL ethylene glycol and ultrasonicated for 30min. Then 1.2g sodium acetate was added to the above mixture and stirred for 30min until it became a transparent oil. It was transferred to a 50mL reactor lined with Teflon stainless steel and heated at 200℃ for 10h. After the reaction, the black product was washed three times with ethanol and water respectively to obtain MNPs.

[0042] 200 mg of MNPs were dispersed in a mixed solvent of 35 mL of ethanol and 5 mL of water, and then 1 mL of NH3·H2O was added and ultrasonicated for 30 min. Then 300 mg of 3-aminopropyltriethoxysilane (APTES) was slowly added to the above mixture under vigorous stirring and reacted in a nitrogen environment at 60 ° C for 10 h. Finally, the obtained product was washed three times with ethanol to obtain APTES-MNPs nanoparticles.

[0043] 2) Disperse 100 mg of APTES-MNPs in 5 mL of anhydrous DMF and add 200 μL of triethylamine under vigorous stirring at 4°C. Then, slowly add 90 μL of 2-bromoisobutyryl bromide (BB) to the above mixture and react at room temperature for 12 h. Wash the product three times with water to obtain BB-APTES-MNPs.

[0044] 3) Disperse 50 mg BB-APTES-MNPs, 1.0 g zwitterionic monomers (SBMA, MPC and CBMA, respectively) and 30 mg 2,2'-bipyridine (BPY) in 5 mL deoxygenated methanol and 5 mL deoxygenated deionized water. Under nitrogen protection, the above mixture was rapidly stirred at 60 ° C for 10 min. Then, 14 mg CuBr was added to the above solution and the reaction lasted for 8 h. Finally, centrifugation and water washing were performed three times to obtain the final products SB-ZMNPs, MPC-ZMNPs and CB-ZMNPs.

[0045] like Figure 2 Transmission electron microscopy images show that the three ZMNPs are spherical and have uniform particle sizes. Figure 3 As shown, after the three zwitterionic polymers were modified on the surface of MNPs, their characteristic peaks appeared respectively, proving the successful preparation of ZMNPs.

[0046] 2. Test the ability of ZMNPs to inhibit ice growth and recrystallization.

[0047] (1) Ice crystal growth rate test: SB-ZMNP, MPC-ZMNP, and CB-ZMNP were taken and water was added to prepare dispersions with concentrations of 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, and 2.0 mg / mL, respectively. Ice growth and recrystallization ability tests were performed.

[0048] The above cryoprotectants were tested for ice crystal growth rate using a nanoliter osmometer. A microinjector was used to take 10 nL of sample and add it to the circular hole of a copper sheet filled with silicone oil. The temperature of the cold stage was lowered to -20°C, and the droplets quickly froze. The temperature was then slowly raised, and the ice crystals gradually melted until a single ice crystal appeared in the field of view, and the size of the single ice crystal did not change for 20 seconds. This temperature was recorded as the melting temperature (T m ). Then, it is lowered to a certain temperature to allow ice crystals to grow, and this temperature is recorded as the freezing temperature (T f ). f -T m The supercooling temperature (ΔT) is called supercooling temperature. The growth process of a single ice crystal was recorded using an optical microscope. The growth rate of an ice crystal is the ratio of the elongation of a single ice crystal to the time required for the growth process. The growth of a single ice crystal under different ΔT was tested.

[0049] like Figure 4 The effects of different cryoprotectants on the growth rate of ice crystals are shown. PSB, PMPC, and PCB are used as control examples and are prepared in the following manner.

[0050] Preparation steps of PSB: 5g of SBMA monomer, 0.8 chain transfer agent 2,2[(thioxomethylene)disulfonyl]bis(2-methylpropionic acid) and 0.12g initiator 4,4'-azo(4-cyanovaleric acid) were added to a 100mL round-bottom flask. Then methanol and water (1:1) were added to make a 20% w / w solution. The reaction system was purged with nitrogen for 30 minutes. The flask was immersed in a 70°C oil bath and the polymerization reaction was carried out under nitrogen for 12 hours. After the reaction, a white powder was obtained by dialysis and freeze-drying.

[0051] Preparation steps of PMPC: First, methanol and water were added to a Schlenk flask in a ratio of 1:1 and deoxygenated with nitrogen for 30 minutes. Then 8g of MPC monomer, 0.2g of initiator 4,4'-azo(4-cyanovaleric acid), 1g of 4-cyano-4-(thiobenzoyl)valeric acid and 4g of NaHCO3 were added to the above mixed solvent. The Schlenk flask was immersed in an oil bath at 70°C and the polymerization reaction was carried out in a nitrogen atmosphere for 10 hours.

[0052] Preparation steps of PCBMA: 5 mmol CBMA was added to a mixed solvent of DMF and water (1:1), and deoxygenated with nitrogen for 30 min. Then, BIBB, CuBr, Cu2Br and BPY were added to the above mixture in a ratio of 1:4:0.4:8.8. The reaction was carried out under nitrogen protection at 25°C for 24 h. After the reaction, a white powder was obtained by dialysis and freeze drying.

[0053] The test results show that CB-ZMNPs have the strongest ability to inhibit ice crystal growth. When the concentration is 1 mg / mL, the ability to inhibit ice growth is the strongest. Then, as the concentration increases, the ice crystal growth rate no longer decreases.

[0054] (2) Test of ice recrystallization inhibition ability: The ice recrystallization inhibition activity of ZMNPs was evaluated by the "splat" method. Take 10 μL of ZMNPs PBS dispersion and drop it from a height of 1.4 m onto a quartz crucible pre-cooled to -60°C. The droplet quickly freezes to form a thin layer of ice, which is then transferred to a cold stage and heated to -6°C at a rate of 20°C / min. Anneal at -6°C for 30 minutes, and use a camera to record the ice crystal grain size at 0 min and 30 min. The average maximum grain size indicates the ability to inhibit ice crystallization. The larger the size, the weaker the ability to inhibit ice crystallization. Figure 5 The test results of the ability of different cryoprotectants to inhibit ice recrystallization showed that CB-ZMNPs had the strongest activity in inhibiting ice recrystallization. When the concentration was 1 mg / mL, the activity in inhibiting ice recrystallization was the strongest. Subsequently, as the concentration increased, the activity in inhibiting ice recrystallization no longer increased.

[0055] 3. Testing of ZMNPs magnetocaloric properties and nano-reheating rate

[0056] ZMNPs dispersions with concentrations of 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, and 5 mg / mL were prepared, and their magnetocaloric properties and nano-reheating rate were tested using a magnetic induction heating device.

[0057] (1) Magnetothermal performance test: ZMNPs of different concentrations were placed in 1.5 mL pointed centrifuge tubes, placed in an 8-ring coil with a diameter of 2.5 cm, and the power was adjusted to 10 kW. After magnetic induction heating for 1 min, the ZMNPs were taken out and the temperature was recorded using a thermal imager. Figure 6 As shown, ZMNPs exhibit excellent magnetocaloric properties, and the temperature rise increases with the increase in concentration. When the concentration is 5 mg / mL, the temperature rises by 47°C in 1 min.

[0058] (2) Nano-reheating rate test: 20 mL of ZMNPs were placed in a cryotube, thermocouples were placed in the middle and edge of the cryotube, and the tube was rapidly frozen using liquid nitrogen. After it was completely frozen, it was taken out and placed in an 8-ring coil with a diameter of 4 cm. The power was adjusted to 10 kW for nano-heating and the temperature rise was recorded. Figure 7 As shown in the figure, the nano-reheating rate is three times that of the water bath reheating rate, and the temperature in the middle and edge positions is consistent, indicating that the nano-reheating rate is faster and more uniform than the traditional water bath reheating rate. At the same time, the nano-reheating rate of different concentrations of ZMNPs was tested, such as Figure 8 As shown in the figure, with the increase of ZMNPs concentration, the nano-reheating rate is faster, and when the concentration is 5 mg / mL, the reheating rate is the fastest.

[0059] 4. Cryopreservation method of T lymphocytes:

[0060] (1) Cryopreservation of 1 mL volume of T lymphocytes. T lymphocytes were extracted from the spleen of mice, dispersed in a cell protectant, placed in a 1 mL cryotube and directly placed in liquid nitrogen for overnight freezing. After thawing in a water bath, the survival rate of the revived cells was tested using AM / PI cell live-dead staining reagent. Fig. 9 As shown in a, when the concentration of single betaine is 6%, the cell survival rate is up to 40%. Three types of ZMNPs were added to 6% betaine, and the results showed that after adding CB-ZMNPs and freezing, the cell survival rate was up to 80.3% ( Fig. 9 b). At the same time, as the concentration of ZMNPs increased, the cell survival rate gradually increased after freezing. When the concentration exceeded 1 mg / mL, the cell survival rate no longer increased ( Fig. 9 c).

[0061] (2) Cryopreservation of 20 mL volume of T lymphocytes. T lymphocytes were extracted from mouse spleen, dispersed in a cell protectant, placed in a 20 mL cryotube and directly placed in liquid nitrogen for overnight freezing. After nano-warming, the survival rate of cells was tested using AM / PI cell live-dead staining reagent. Fig. 9 As shown in d, for the cryopreservation of large volumes of T lymphocytes, the cell survival rate after nano-thawing was up to 80%.

[0062] 5. Test the immune function of T lymphocytes after freezing and thawing.

[0063] (1) In vitro tumor killing ability test. EG.7-OVA (a mouse lymphoma cell line) cells were used as target cells. Cryopreserved T lymphocytes were co-cultured with EG.7-OVA cells in a 96-well plate at a ratio of 1:1, 2:1, and 5:1 (T lymphocytes: EG.7-OVA). After co-culture in the incubator for 12 h, cell viability was assessed using CCK-8 staining. Fig.10 As shown, T cells cryopreserved using ZMNPs cryoprotectant retained in vitro tumor killing ability similar to that of fresh cells.

[0064] (2) In vivo tumor killing ability test. A mouse lymphoma model was constructed. When the tumor volume reached about 100 mm 3 The nude mice bearing EG.7-OVA tumors were randomly divided into four groups (5 mice in each group): PBS group was phosphate buffered saline, Fresh group was freshly extracted T lymphocytes, CB-ZMNP group was T lymphocytes cryopreserved by CB-ZMNPs, and DMSO group was T lymphocytes cryopreserved by 10% DMSO. Each of the four groups of mice was infused with 5×10 6 The mental state, tumor volume and body weight of each group of mice were monitored every day for 13 days. Fig.11 As shown, T lymphocytes cryopreserved with CB-ZMNPs retained the tumor-killing ability in vivo.

[0065] The present invention tests the ice growth and recrystallization ability and magnetocaloric properties of the cryoprotectant, compares the ice inhibition effects of three ZMNPs, and comprehensively optimizes the cryoprotectant formula. Specifically, the ice growth and recrystallization inhibition performance of the three ZMNPs were investigated respectively, and it was found that the ice crystal growth rate in CB-ZMNPs was the slowest and the ice recrystallization activity was the strongest. The mass concentration of ZMNPs was investigated from 0.1 mg / mL to 2 mg / mL, and it was found that when the concentration of ZMNPs was 1 mg / mL, the ice crystal growth rate was the slowest and the ice recrystallization activity was the strongest. As the concentration of ZMNPs increased, the ability to inhibit ice growth and recrystallization no longer increased. In addition, the magnetocaloric properties of ZMNPs in the above-mentioned cell cryopreservative were investigated, and the mass concentration was investigated from 1 mg / mL to 5 mg / mL. It was found that as the concentration of ZMNPs increased, the heating rate increased. It was found that when the concentration of ZMNPs was 5 mg / mL, the heating rate was the fastest. Taking all factors into consideration, the optimal mass concentrations of ZMNPs and betaine for small-volume cryopreservation were 1 mg / mL and 6%, respectively, using water bath thawing; the optimal mass concentrations of ZMNPs and betaine for large-volume cryopreservation were 5 mg / mL and 6%, respectively, using nano-thawing.

[0066] The immune cell cryoprotectant of the present invention is a mixture of ZMNPs and small molecule betaine, has good biocompatibility, can effectively inhibit the growth and recrystallization of ice crystals, and can utilize the magnetothermal properties of ZMNPs to achieve rapid and uniform thawing of frozen cells. The above effects synergistically protect frozen cells and ensure the vitality and function of cells after recovery.

[0067] The cryoprotectant is DMSO-free and has good biocompatibility. Among them, small molecule betaine, as a natural osmotic pressure regulator, can enter the cell, adjust the osmotic pressure balance, and inhibit the formation and growth of ice crystals in the cell. First, ZMNPs, as an extracellular protective agent, has a strong hydration effect on the zwitterionic polymer on its surface, forming a spherical hydration shell on its surface, inhibiting the combination of water molecules and ice, thereby controlling the growth and recrystallization of extracellular ice crystals. Secondly, combined with the magnetothermal properties of ZMNPs, the frozen cells can be quickly and evenly rewarmed. After rewarming, the cells are quickly collected by magnetic separation to avoid complicated elution steps. The above effects work together to protect the cells from cryopreservation and ensure the vitality and function of the cells after recovery.

[0068] The present invention uses a cryoprotectant to preserve T lymphocytes in liquid nitrogen. The results show that the survival rate of T lymphocytes after recovery is more than 80%, and immune functions such as proliferation, differentiation and tumor killing are retained, and the effect is better than the traditional 10% DMSO cryoprotectant.

[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several modifications and improvements without departing from the principle of the present invention. These modifications and improvements should also be regarded as within the scope of protection of the present invention.

Claims

1. A cryoprotectant, characterized in that: It comprises an antifreeze agent and a protective liquid; the antifreeze agent is a mixture of zwitterionic magnetic nanoparticles and betaine.

2. The cryoprotectant according to claim 1, characterized in that The zwitterionic magnetic nanoparticles include ferroferric oxide in the core layer and a zwitterionic polymer in the shell layer; preferably, the zwitterionic polymer is one of polymethacrylate sulfobetaine, poly(2-(methacryloyloxy)ethyl)phosphorylcholine or polymethacrylate carboxybetaine; Preferably, the zwitterionic magnetic nanoparticles are prepared by the following method: 1) grafting a silane coupling agent on the surface of ferroferric oxide magnetic nanoparticles; 2) adding 2-bromoisobutyryl bromide for bromination; 3) adding a zwitterionic monomer for polymerization reaction; The mass concentration of the zwitterionic magnetic nanoparticles in the cryoprotectant is 0.1 mg / mL-5 mg / mL, preferably 0.5 mg / mL-5 mg / mL.

3. The cryoprotectant according to claim 1, characterized in that The mass proportion of the betaine in the cryoprotectant is 2-10%, preferably 6-8%, and more preferably 6%.

4. The cryoprotectant according to claim 1, characterized in that The protective solution is one of PBS buffer or DPBS.

5. A method for preparing the cryoprotectant according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: dispersing zwitterionic magnetic nanoparticles and betaine into a protective solution.

6. A use of the cryoprotectant according to any one of claims 1 to 4, characterized in that: Applicable to cryopreserved cells; preferably immune cells, wherein the immune cells are one of T lymphocytes, CAR-T cells, Jurkat cells, and NK cells; preferably T lymphocytes.

7. A method for freezing T lymphocytes, characterized in that: The method comprises the following steps: fully mixing the cryoprotectant described in any one of claims 1 to 4 with T lymphocytes and placing the mixture into a cryopreservation tube, and cryopreserving the T lymphocytes by rapid freezing with liquid nitrogen.

8. The cryopreservation method according to claim 7, characterized in that: The cryopreservation density of the T lymphocytes is 10 6 cells / mL; the cryopreservation volume is 1-50mL; preferably, the cryopreservation volume>3mL, the mass concentration of zwitterionic magnetic nanoparticles is>3mg / mL, more preferably 5mg / mL; for the cryopreservation volume<3mL, the mass concentration of zwitterionic magnetic nanoparticles is 0.5-2mg / mL; more preferably 1mg / mL.

9. A method for reviving frozen T lymphocytes, characterized in that: Use water bath heating or nanoheating to thaw frozen T lymphocytes.

10. The method for reviving cryopreserved T lymphocytes according to claim 9, characterized in that: If the cryopreserved volume is >3 mL, use nano heating to thaw; if the cryopreserved volume is <3 mL, use water bath heating to thaw.

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