Freezing medium and freezing method for universal CAR (chimeric antigen receptor) cells

Abstract

The invention relates to a freezing medium and a freezing method for universal CAR (chimeric antigen receptor) cells. The cryopreservation liquid comprises a compound electrolyte injection, a human serum albumin injection, a sodium chloride injection, a dextran 40 glucose injection, a glucose injection, dimethyl sulfoxide and trehalose. The cell cryopreservation process is deeply analyzed, the specific cell cryopreservation liquid and the cryopreservation method are designed, all the components of the cryopreservation liquid are synergistic, the cryopreservation liquid can adapt to the specific cooling process, cell damage in the cryopreservation process is effectively reduced, the activity of the cryopreserved cells is effectively kept, the cryopreservation liquid can be directly used for reinfusion of patients, a specific sectional programmed cooling program is adopted, and the cell cryopreservation process is simplified. The cell cryopreservation device can further adapt to different cryopreservation volumes, so that the cryopreservation effect is further improved, the survival rate of cells is effectively guaranteed, and the cryopreserved cells still keep good cell activity.

Description

Technical Field

[0001] This invention belongs to the field of cell cryopreservation technology, and relates to a universal cryopreservation solution and cryopreservation method for CAR cells. Background Technology

[0002] Chimeric antigen receptor T-cell immunotherapy (CAR-T) refers to the use of genetic engineering techniques to modify and enhance the cells in vitro, enabling them to recognize and efficiently kill tumor cells. These modified cells are then reinfused into the patient to treat malignant tumors. CAR-T therapy not only has applications in cancer treatment but also shows great promise in areas such as autoimmune diseases, chronic infections, heart disease, and age-related diseases.

[0003] Universal CAR-T cell therapy (also known as UCAR-T, "off-the-shelf" CAR-T, or Allogeneic CAR-T) is an important development direction of CAR-T technology, aiming to solve the bottlenecks of traditional autologous CAR-T (using the patient's own T cells), such as long individualized preparation cycles, high costs, and poor T cell quality in some patients. Its core idea is to utilize healthy donor T cells to prepare "off-the-shelf" CAR-T products in advance, which can be used in multiple patients to eliminate cancer cells.

[0004] UCAR-T immunotherapy shows immense promise in the treatment of hematological malignancies, but its application is limited by several objective factors: large-scale production and multi-dose formulations are required, but infusion times vary among patients, necessitating cryopreservation to ensure product usability; most hematological malignancy patients require UCAR-T infusion within 1-7 days after lymphocyte ablation chemotherapy, making complete synchronization of production cycles difficult; and stringent release testing further complicates matters. Consequently, it is not possible to immediately infuse UCAR-T cells into patients after production. The usual practice is to cryopreserve the prepared UCAR-T cells, transport them via cold chain to the hospital for thawing, and then infuse them. The cryopreservation-thawing system for UCAR-T is the core link between production and clinical application; therefore, ensuring the quality of cryopreserved UCAR-T cells and maintaining high activity for a period after thawing is crucial.

[0005] Cryopreservation solutions used for cell therapy need to be directly reinfused into patients, therefore the requirements for cryopreservation excipients are relatively high. Generally, GMP-grade or higher excipients are required, preferably pharmaceutical-grade for injection. Furthermore, since UCAR-T therapy is tailored to different patients with varying dosage requirements, the cell cryopreservation procedure generally has a direct impact on the cryopreservation effect. However, current methods typically use a uniform cooling procedure when preparing different volumes of UCAR-T cells, without considering the impact of different preparation volumes. This often results in incompatibility with different volumes of cryopreservation solution, leading to significant loss of cell viability after thawing and substantial impact on cell proliferation.

[0006] In conclusion, developing cell cryopreservation solutions suitable for UCAR-T and adapting cryopreservation methods for different preparation volumes is of great significance to the UCAR-T therapeutic field. Summary of the Invention

[0007] To address the shortcomings of existing technologies and practical needs, this invention provides a universal CAR cell cryopreservation solution and cryopreservation method, and develops a universal CAR cell cryopreservation solution and cryopreservation method adapted to different preparation volumes, in order to promote the application of universal CAR cell therapy.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a universal CAR cell cryopreservation solution, the cryopreservation solution comprising compound electrolyte injection, human serum albumin injection, dextran 40 glucose injection, glucose injection, dimethyl sulfoxide and trehalose.

[0010] In this invention, through in-depth analysis of the cell cryopreservation process, a cryopreservation solution was designed for universal CAR cells (especially universal CAR-T cells). The components work synergistically to adapt to specific cooling processes, effectively reduce cell damage during cryopreservation, effectively maintain the activity of cryopreserved cells, and can be directly used for patient reinfusion.

[0011] Preferably, the concentrations of the compound electrolyte injection in the cryopreservation solution are 10-40% v / v, the concentration of the human serum albumin injection is 20-40% v / v (e.g., 21%, 22%, 23%, 25%, 30%, 35%, 36%, 37%, 38%, or 39%), the concentration of the dextran 40 glucose injection is 5-20% v / v (e.g., 6%, 7%, 8%, 9%, 10%, 15%, 16%, 17%, 18%, or 19%), the concentration of the glucose injection is 5-20% v / v (e.g., 6%, 7%, 8%, 9%, 10%, 15%, 16%, 17%, 18%, or 19%), the concentration of the 0.9% sodium chloride injection is 5-20% v / v (e.g., 6%, 7%, 8%, 9%, 10%, 15%, 16%, 17%, 18%, or 19%), and the concentration of dimethyl sulfoxide is 5-15%. v / v (e.g., 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13% or 14%, etc.), and trehalose concentration of 1~5% w / v (e.g., 2%, 3% or 4%, etc.).

[0012] Preferably, the glucose concentration in the glucose injection solution is 5-25% w / v, for example, it can be 10% or 20%, etc.

[0013] Secondly, the present invention provides the application of the cryopreservation solution for universal CAR cells described in the first aspect in the cryopreservation of universal CAR cells.

[0014] Preferably, the universal CAR cells include universal CAR-T cells.

[0015] Thirdly, the present invention provides a method for cryopreserving universal CAR cells, the method comprising: mixing universal CAR cells with cryopreservation solution to obtain a sample to be cryopreserved, pre-cooling the sample to be cryopreserved and performing programmed cooling treatment, and designing different cooling programs for samples of different volumes to be cryopreserved.

[0016] Preferably, the cryopreservation solution includes the cryopreservation solution for universal CAR cells described in the first aspect.

[0017] Preferably, the pre-cooling process includes: placing the sample to be frozen at 2~8℃ (e.g., in a refrigerator) for pre-cooling, and then pre-cooling it to 3~5℃ in a programmed cooling device.

[0018] Preferably, for samples to be frozen with a volume ≤30 mL, the cooling program is: cooling to -80 °C at a rate of -1.5~-1 °C / min.

[0019] For samples with a volume > 30 mL to be frozen, the cooling program is as follows: reduce the temperature to -6 to -4℃ at -1.5 to -1℃ / min, then reduce it to -46 to -39℃ at -26 to -19℃ / min (e.g., -25, -24, -23, -22, -21, or -20℃ / min, etc.) (e.g., -45, -44, -43, -42, -41, or -40℃, etc.), hold at 0℃ / min for 0 to 3 min, increase the temperature to -31 to -19℃ at 7 to 16℃ / min (e.g., -30, -29, -25, -24, -23, or -20℃, etc.), and reduce the temperature to -80℃ at -1.5 to -1℃ / min.

[0020] This invention designs different cooling programs for different cryopreservation solution volumes, which can further adapt to cell cryopreservation solutions, thereby further improving the cryopreservation effect and effectively ensuring cell survival rate. Cells that have been cryopreserved still maintain good cell activity.

[0021] Preferably, the universal CAR cells include universal CAR-T cells.

[0022] Preferably, after the cooling treatment, the sample is stored in liquid nitrogen.

[0023] Preferably, for samples to be frozen (30 mL < volume ≤ 50 mL), the cooling program is as follows: decrease to -6 to -4℃ at -1.5 to -1℃ / min, decrease to -41 to -39℃ at -21 to -19℃ / min, hold at 0℃ / min for 1 to 3 min, increase to -26 to -24℃ at 14 to 16℃ / min, and decrease to -80℃ at -1.5 to -1℃ / min.

[0024] Preferably, for samples to be frozen (50 mL < volume ≤ 70 mL), the cooling program is as follows: decrease to -6 to -4℃ at -1.5 to -1℃ / min, decrease to -46 to -44℃ at -26 to -24℃ / min, increase to -31 to -29℃ at 7 to 8℃ / min, increase to -21 to -19℃ at 9 to 11℃ / min, and decrease to -80℃ at -1.5 to -1℃ / min.

[0025] Compared with the prior art, the present invention has at least the following beneficial effects:

[0026] This invention provides an in-depth analysis of the cell cryopreservation process, designs a specific cell cryopreservation solution, and a cryopreservation method. The components of the cryopreservation solution work synergistically to adapt to a specific cooling process, effectively reducing cell damage during cryopreservation, effectively maintaining the viability of cryopreserved cells, and allowing for direct reinfusion into patients. A specific segmented cooling program is used to further adapt to different cryopreservation volumes, thereby further improving the cryopreservation effect and effectively ensuring cell survival rate. The cryopreserved cells still maintain good cell activity. Attached Figure Description

[0027] Figure 1 This is a cryopreservation curve for cryopreservation procedure A.

[0028] Figure 2 This is a cryopreservation curve for cryopreservation procedure B.

[0029] Figure 3 This is a cryopreservation curve for cryopreservation procedure C.

[0030] Figure 4 The graph shows the cell viability results for a 30 mL cryopreservation system.

[0031] Figure 5 The graph shows the cell viability results for a 50 mL cryopreservation system.

[0032] Figure 6 The graph shows the cell viability results for a 70 mL cryopreservation system.

[0033] Figure 7 This is a graph showing the viability results of cryopreserved cells. Detailed Implementation

[0034] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0035] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0036] This application addresses the field of universal CAR cell therapy by designing a CAR cell cryopreservation solution that can be directly used for patient reinfusion. In universal CAR cell therapy, CAR cells generally require cryopreservation, and different patients have different dosage (volume) requirements, necessitating the preparation of different doses of CAR cells. This invention reveals that the cryopreservation effects of samples of different volumes under the same cooling procedure can vary significantly. That is, a matching cooling procedure needs to be designed for samples of different volumes, while current technologies typically use a uniform cooling procedure. Therefore, this invention deeply analyzes the impact of different sample volumes on cell viability and activity after cryopreservation and develops a cryopreservation method that can effectively match samples of different volumes.

[0037] In one specific embodiment of the present invention, a universal CAR cell cryopreservation solution is provided, the cryopreservation solution comprising compound electrolyte injection, human serum albumin injection, dextran 40 glucose injection, glucose injection, dimethyl sulfoxide and trehalose.

[0038] Furthermore, the concentrations of the compound electrolyte injection in the cryopreservation solution are 10-40% v / v, the concentration of human serum albumin injection is 20-40% v / v, the concentration of dextran 40 glucose injection is 5-20% v / v, the concentration of glucose injection is 5-20% v / v, the concentration of 0.9% sodium chloride injection is 5-20% v / v, the concentration of dimethyl sulfoxide is 5-15% v / v, and the concentration of trehalose is 1-5% w / v.

[0039] In another specific embodiment of the present invention, a method for cryopreserving universal CAR cells is provided. The cryopreservation method includes: mixing universal CAR cells with cryopreservation solution to obtain a sample to be cryopreserved; pre-cooling the sample to be cryopreserved and performing programmed cooling treatment; and designing different cooling programs for samples of different volumes to be cryopreserved.

[0040] Furthermore, the pre-cooling process includes: pre-cooling the sample to be frozen from 2~8℃ to 3~5℃.

[0041] Furthermore, for samples with a volume ≤30 mL to be cryopreserved, the cooling program is as follows: decrease to -80 °C at a rate of -1.5 to -1 °C / min. For samples with a volume >30 mL to be cryopreserved, the cooling program is as follows: decrease to -6 to -4 °C at a rate of -1.5 to -1 °C / min, then decrease to -46 to -39 °C at a rate of -26 to -19 °C / min, hold at 0 °C / min for 0 to 3 min, increase to -31 to -19 °C at a rate of 7 to 16 °C / min, and decrease to -80 °C at a rate of -1.5 to -1 °C / min.

[0042] Furthermore, for samples to be frozen (30 mL < volume ≤ 50 mL), the cooling program is as follows: reduce to -6 to -4℃ at -1.5 to -1℃ / min, reduce to -41 to -39℃ at -21 to -19℃ / min, hold at 0℃ / min for 1 to 3 min, increase to -26 to -24℃ at 14 to 16℃ / min, and reduce to -80℃ at -1.5 to -1℃ / min.

[0043] For samples to be frozen (50 mL < volume ≤ 70 mL), the cooling program is as follows: reduce to -6 to -4℃ at -1.5 to -1℃ / min, reduce to -46 to -44℃ at -26 to -24℃ / min, increase to -31 to -29℃ at 7 to 8℃ / min, increase to -21 to -19℃ at 9 to 11℃ / min, and reduce to -80℃ at -1.5 to -1℃ / min.

[0044] In this specific embodiment of the invention, the compound electrolyte injection was purchased from Sichuan Kelun Pharmaceutical Co., Ltd. under the product name "Compound Electrolyte Injection"; the human serum albumin injection was purchased from Octapharma under the product name "Human Serum Albumin (25% mass concentration)"; the dextran 40 glucose injection was purchased from Sichuan Kelun Pharmaceutical Co., Ltd. under the product name "Dextran 40 Glucose Injection"; the 10% glucose injection was purchased from Anhui Fengyuan Pharmaceutical Co., Ltd. Huaihai Pharmaceutical Factory under the product name "Glucose Injection"; the 0.9% sodium chloride injection was purchased from Anhui Fengyuan Pharmaceutical Co., Ltd. Huaihai Pharmaceutical Factory under the product name "Sodium Chloride Injection"; and the dimethyl sulfoxide was purchased from OriGen Biomedical Inc. under the product name "CryoPur". TM 70 mL of product.

[0045] Example 1

[0046] This embodiment tests the freezing effect of samples of different volumes during the freezing process.

[0047] CAR-T cells were mixed with cryopreservation solution to prepare 30 mL, 50 mL, and 70 mL samples for cryopreservation. The CAR-T cell concentration in each sample was 5 × 10⁻⁶. 6 The cryopreservation solution was formulated as follows: compound electrolyte injection at a concentration of 30% v / v, human serum albumin injection at a concentration of 20% v / v, dextran 40 glucose injection at a concentration of 10% v / v, 10% glucose injection at a concentration of 15% v / v, 0.9% sodium chloride injection at a concentration of 15% v / v, dimethyl sulfoxide at a concentration of 8% v / v, and trehalose at a concentration of 2% w / v.

[0048] First, pre-cool each sample at 2℃~8℃, then freeze according to the newly established freezing curve (starting at 4℃, decreasing to -80℃ at a rate of 1℃ / min). The freezing procedure is shown in Table 1. A repeat experiment was designed for each sample to detect the temperature of the cooling instrument chamber and the sample's own temperature during the cooling process. The results are as follows: Figure 1 As shown, when the chamber temperature reaches approximately -15.9℃, the temperature of a 30 mL cryopreservation solution rises rapidly, and the result is consistent when repeated. When the chamber temperature reaches approximately -19.2℃, the temperature of a 50 mL cryopreservation solution rises rapidly, and the result is consistent when repeated. When the chamber temperature reaches approximately -20.1℃, the temperature of a 70 mL cryopreservation solution rises rapidly, and the result is consistent when repeated. It can be seen that the temperature changes of samples of different volumes are inconsistent during cryopreservation, resulting in different degrees of mechanical damage to ice crystals and varying degrees of disruption to cell structure.

[0049] Table 1

[0050]

[0051] Example 2

[0052] This embodiment designs different cooling programs, as shown in Tables 2-4.

[0053] Table 2

[0054]

[0055] Table 3

[0056]

[0057] Table 4

[0058]

[0059] Samples of different volumes were subjected to freezing treatment using cryopreservation procedures A through C (sample preparation is as described in Example 1). A repeat experiment was designed for each sample, and the freezing curves are shown below. Figures 1-3 As shown.

[0060] Cell viability was measured before cryopreservation, after thawing, and 24 h after thawing. The detection methods were as follows:

[0061] 1. Cell counting before cryopreservation:

[0062] Before cryopreservation, cells were counted by mixing them with AO / PI and then loading the sample for counting.

[0063] 2. Cell counting after cryopreservation and resuscitation:

[0064] (1) Remove the cells from the liquid nitrogen and quickly place them in a 37°C water bath to thaw them rapidly. Transfer the thawed cells to a sterile centrifuge tube containing PBS, centrifuge at 1000 rpm for 5 min, and discard the supernatant.

[0065] (2) Add 5 mL of culture medium to resuspend the cells, take the cells and mix them with AO / PI, load the sample and count them;

[0066] 3. Cell count 24 h post-resuscitation:

[0067] (1) Adjust the cell density to 2×10 6 Cells were seeded at a rate of 100 cells / mL into well plates and cultured. After 24 h of recovery, the cells were mixed with AO / PI and loaded for cell counting.

[0068] 4. Cell viability (%) = (Total number of viable cells / Total number of cells) × 100%

[0069] In vitro killing experiments using revived cells specifically include:

[0070] 1. Cell preparation:

[0071] Effector cells: Collect and count effector cells.

[0072] Target cells: Raji-target cells and Raji cells were collected separately and labeled with fluorescent dyes for subsequent differentiation in flow cytometry.

[0073] 2. Co-cultivation:

[0074] Effector cells and target cells were added to the well plate at an effector-to-target ratio of 1:10 and cultured together.

[0075] At the same time, control groups were set up where effector cells and target cells were cultured separately.

[0076] Incubate in an incubator for approximately 20 hours.

[0077] 3. Detection and Analysis:

[0078] After the culture was completed, a portion of the supernatant was collected for cytokine detection.

[0079] Cells were stained using Annexin V-APC and 7-AAD dyes to distinguish between cell viability (early apoptosis and late apoptosis / necrosis).

[0080] Finally, flow cytometry was used for detection.

[0081] 4. Result Calculation:

[0082] The percentage of viable cells in the co-culture group and the target cell culture group were compared by analyzing data from flow cytometry.

[0083] Killing efficiency = (Percentage of viable target cells or control target cells) - (Percentage of viable cells in the co-culture group).

[0084] Cell viability results as follows Figures 4-6 As shown, the tumor killing results are as follows: Figure 7 As shown, cryopreservation program A resulted in better cell viability after thawing in the 30 mL cryopreservation system. However, for the 50 mL and 70 mL cryopreservation systems, the cell viability after thawing significantly decreased, and this was also observed in the cell killing assay. After adjusting the cryopreservation program, a specific cooling procedure was used for both the 50 mL and 70 mL cryopreservation systems, resulting in a significant improvement in both cell viability and cell killing after thawing.

[0085] The above results indicate that the cryopreservation program designed for cryopreservation samples of different volumes in this invention can further improve the cryopreservation effect, effectively ensure the cell survival rate, and maintain good cell activity and strong tumor killing ability after cryopreservation.

[0086] Comparative Example 1

[0087] In this comparative example, cells were cryopreserved using cryopreservation procedure B in Example 2, with 50 mL of sample cryopreserved. The only difference from Example 2 was the use of a different cryopreservation solution. The human serum albumin injection solution in the cryopreservation solution was replaced with an equal amount of compound electrolyte injection solution, while the other components remained the same.

[0088] Comparative Example 2

[0089] In this comparative example, cells were cryopreserved using cryopreservation procedure B from Example 2. The only difference from Example 2 was the use of a different cryopreservation solution. The dextran 40 glucose injection solution in the cryopreservation solution was replaced with an equal amount of compound electrolyte injection solution, while the other components remained the same.

[0090] Comparative Example 3

[0091] In this comparative example, cells were cryopreserved using cryopreservation procedure B in Example 2, with 50 mL of sample cryopreserved. The only difference from Example 2 was the use of a different cryopreservation solution, in which glucose injection was replaced with an equal amount of compound electrolyte injection, while the other components remained the same.

[0092] Comparative Example 4

[0093] In this comparative example, cells were cryopreserved using cryopreservation procedure B in Example 2, with 50 mL of sample cryopreserved. The only difference from Example 2 was the use of a different cryopreservation solution, in which dimethyl sulfoxide was replaced in equal amounts with compound electrolyte injection solution, while the other components remained the same.

[0094] Comparative Example 5

[0095] In this comparative example, cells were cryopreserved using cryopreservation procedure B from Example 2. The only difference from Example 2 was the use of a different cryopreservation solution. Trehalose in the cryopreservation solution was replaced with an equal amount of compound electrolyte injection solution, while the other components remained the same.

[0096] Comparative Example 6

[0097] In this comparative example, cells were cryopreserved using cryopreservation procedure B in Example 2, with 50 mL of sample cryopreserved. The only difference from Example 2 was the use of a different cryopreservation solution, in which trehalose was replaced with an equal amount of fructose, while the other components remained the same.

[0098] Comparative Example 7

[0099] In this comparative example, cells were cryopreserved using cryopreservation procedure B in Example 2, with 50 mL of sample cryopreserved. The only difference from Example 2 was the use of a different cryopreservation solution, in which trehalose was replaced with an equal amount of raffinose, while the other components remained the same.

[0100] The viability and tumor-killing ability of the revived cells after cryopreservation were detected and analyzed, and the results are shown in Table 5.

[0101] Table 5

[0102]

[0103] As shown in Table 5, the present invention designs a universal CAR cell cryopreservation solution with specific components. The components work synergistically to effectively reduce cell damage during cryopreservation and effectively maintain the viability of cells after cryopreservation.

[0104] In summary, this invention, through in-depth analysis of the cell cryopreservation process, designs a specific cell cryopreservation solution and cryopreservation method. The components of the cryopreservation solution work synergistically to adapt to a specific cooling process, effectively reducing cell damage during cryopreservation, effectively maintaining the viability of cryopreserved cells, and allowing for direct reinfusion into patients. The specific segmented cooling program can further adapt to different cryopreservation volumes, thereby further improving the cryopreservation effect and effectively ensuring cell survival rate. The cryopreserved cells still maintain good cell viability.

[0105] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A cryo-preserved solution of a universal CAR cell, characterized in that, The cryopreservation solution includes compound electrolyte injection, human serum albumin injection, dextran 40 glucose injection, glucose injection, dimethyl sulfoxide, and trehalose.

2. The cryo stock of the universal CAR cell according to claim 1, characterized in that, The concentrations of the compound electrolyte injection in the cryopreservation solution are 10-40% v / v, the concentration of human serum albumin injection is 20-40% v / v, the concentration of dextran 40 glucose injection is 5-20% v / v, the concentration of glucose injection is 5-20% v / v, the concentration of 0.9% sodium chloride injection is 5-20% v / v, the concentration of dimethyl sulfoxide is 5-15% v / v, and the concentration of trehalose is 1-5% w / v.

3. The cryo-preserved universal CAR cell of claim 1 or 2, wherein the CAR cell is a T cell. The glucose concentration in the glucose injection solution is 5-25% w / v.

4. The use of the cryopreservation solution for universal CAR cells according to any one of claims 1-3 in the cryopreservation of universal CAR cells.

5. Use according to claim 4, characterized in that, The universal CAR cells include universal CAR-T cells.

6. A method of cryopreservation of a universal CAR cell, characterized in that, The cryopreservation method includes: mixing universal CAR cells with cryopreservation solution to obtain a sample to be cryopreserved; pre-cooling the sample to be cryopreserved and performing programmed cooling treatment; and designing different cooling programs for samples of different volumes to be cryopreserved. The cryopreservation solution includes the cryopreservation solution for universal CAR cells as described in any one of claims 1-3.

7. The method for cryopreserving universal CAR cells according to claim 6, characterized in that, The pre-cooling process includes: placing the sample to be frozen at 2~8℃ for pre-cooling, and then placing it in a programmed cooling instrument to pre-cool to 3~5℃; Preferably, for samples to be frozen with a volume ≤30 mL, the cooling program is: cooling to -80 °C at a rate of -1.5~-1 °C / min; Preferably, for samples with a volume > 30 mL to be frozen, the cooling program is as follows: reduce the temperature to -6 to -4°C at -1.5 to -1°C / min, then reduce it to -46 to -39°C at -26 to -19°C / min, hold at 0°C / min for 0 to 3 min, increase the temperature to -31 to -19°C at 7 to 16°C / min, and reduce the temperature to -80°C at -1.5 to -1°C / min.

8. The method of cryopreservation of universal CAR cells according to claim 6 or 7, characterized in that, The universal CAR cells include universal CAR-T cells; Preferably, after the cooling treatment, the sample is stored in liquid nitrogen.

9. The method for cryopreserving universal CAR cells according to claim 7, characterized in that, For samples to be frozen (30 mL < volume ≤ 50 mL), the cooling program is as follows: reduce to -6 to -4℃ at -1.5 to -1℃ / min, reduce to -41 to -39℃ at -21 to -19℃ / min, hold at 0℃ / min for 1 to 3 min, increase to -26 to -24℃ at 14 to 16℃ / min, and reduce to -80℃ at -1.5 to -1℃ / min.

10. The method for cryopreserving universal CAR cells according to claim 7, characterized in that, For samples to be frozen (50 mL < volume ≤ 70 mL), the cooling program is as follows: reduce to -6 to -4℃ at -1.5 to -1℃ / min, reduce to -46 to -44℃ at -26 to -24℃ / min, increase to -31 to -29℃ at 7 to 8℃ / min, increase to -21 to -19℃ at 9 to 11℃ / min, and reduce to -80℃ at -1.5 to -1℃ / min.

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