Cryopreservation method based on ultrasonic ice planting

Abstract

The invention belongs to the technical field of cell cryopreservation, and relates to a cryopreservation method based on ultrasonic ice planting. In the method, the nucleation temperature of ice planting is less than-9 DEG C, and the cooling rate from nucleation to icing is less than 5 DEG C / min. Compared with the prior art, the method has the advantages that the time required for cryopreservation of the cells is remarkably shortened, meanwhile, damage to the cells caused by ice crystals generated in the cooling process is reduced, and the problem that an existing cell cryopreservation cooling method cannot meet the nucleation and freezing uniformity of a cell solution in the cooling process is solved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell cryopreservation, and relates to a freezing method based on ultrasonic ice planting. BACKGROUND

[0002] The low-temperature preservation technology of cells is widely used in the fields of modern regenerative medicine, organ transplantation and assisted reproduction, and has attracted widespread attention. The low-temperature preservation of cells is generally achieved by adding a cell protective solution and external cooling to freeze the cells, slow down the cell metabolism, and thus realize the long-term preservation of cells. However, the cells will be damaged to different degrees during the low-temperature preservation process, mainly including the toxic damage of low-temperature protective agents and the physical damage caused by the formation of ice crystals in the cells during the cooling process.

[0003] Peripheral blood mononuclear cells (PBMCs) are cells with single nuclei in peripheral blood, including lymphocytes and monocytes. The lymphocytes are derived from hemopoietic stem cells (HSCs). The hemopoietic stem cells can differentiate into multipotent progenitor cells (MPCs), and then differentiate into lymphoid stem cells and myeloid stem cells. The lymphoid stem cells can continue to develop into mature T lymphocytes, B lymphocytes and NK cells, etc. At present, the freezing of such cells is carried out by using a programmed cooling method. However, the existing cooling program has a long freezing time, and cannot effectively control the ice crystal formation time of the cells during the cooling process. Part of the cell solution still does not form nucleation and freeze when the temperature is lower than the crystallization temperature, causing the cell solution to be supercooled. When the coldness is too high, the cell solution will suddenly form nucleation, resulting in large ice crystals and causing physical damage to the cells. This affects the subsequent cell differentiation and cannot meet the production needs. SUMMARY

[0004] Based on the above problems, the present application provides a freezing method based on ultrasonic ice planting, which can significantly shorten the freezing time and reduce the damage to the cells caused by the ice crystals generated during the cooling process, and solve the problem that the existing cell freezing and cooling method cannot meet the uniformity of nucleation and freezing of the cell solution during the cooling process.

[0005] The specific technical solutions are as follows:

[0006] A freezing method based on ultrasonic ice planting, in which the nucleation temperature of the ice planting is <-9℃.

[0007] In some specific embodiments, the nucleation temperature of the ice planting is preferably -10℃.

[0008] In some embodiments, the cooling rate during the ultrasonic nucleation and solution freezing process is <5℃ / min.

[0009] In some specific embodiments, the cooling rate of the ultrasonic nucleation in the solution freezing process is preferably 1°C / min.

[0010] In some embodiments, the isothermal time of the ultrasonic nucleation is > 20 min.

[0011] In some specific embodiments, the isothermal time of the ultrasonic nucleation is preferably 20 min.

[0012] The steps of the freezing method of the present application are as follows:

[0013] 1) reducing the cells containing the freezing solution from the initial temperature to the nucleation temperature; preferably, the initial temperature is 4-25°C;

[0014] 2) ultrasonic nucleation;

[0015] 3) ice formation at a cooling rate after successful nucleation;

[0016] 4) cooling to the freezing temperature after ice formation.

[0017] In some specific embodiments, the cooling rate of step 1) from the initial temperature to the nucleation temperature is 10°C / min.

[0018] In some specific embodiments, the ice formation temperature of step 3) is -40°C;

[0019] In some specific embodiments, the freezing temperature of step 4) is -90°C, and the cooling rate is 10°C / min;

[0020] In some specific embodiments, the ultrasonic nucleation uses an ultrasonic frequency of 40 KHz and a power of 400 W.

[0021] In some specific embodiments, the freezing method uses an instrument of Thermo Fisher program cooling instrument, model 7453.

[0022] The present application also provides the use of the freezing method as described above in freezing cells.

[0023] In some embodiments, the cells are animal cells, preferably human cells, for example, selected from any one or more of germ cells, nerve cells, endocrine cells, phagocytes, white blood cells, red blood cells, epithelial cells, myocardial cells, stem cells.

[0024] In some specific embodiments, the cells are peripheral blood mononuclear cells or cells derived from apheresis.

[0025] Another object of the present application is to provide PBMC or T cells obtained after resuscitation and sorting of cells derived from apheresis, which are cryopreserved by the above-mentioned cryopreservation method.

[0026] Another object of the present application is to provide a method for preparing engineered T cells, comprising transfecting nucleic acid into the above-mentioned T cells.

[0027] In some embodiments, the nucleic acid contains a polypeptide coding sequence, preferably a coding sequence of a CAR.

[0028] The present application has the following beneficial effects:

[0029] Compared with the existing cell cryopreservation cooling method, the present application significantly shortens the time required for cryopreservation of cells. The most commonly used Thermo cooling program in the industry usually takes about 76 minutes, while the present application can shorten the cryopreservation time to about 60 minutes, greatly improving the cryopreservation efficiency of the cell solution. At the same time, the cumulative expansion and stemness protection rate of PBMC sorted as T cells by the above-mentioned cryopreservation method are superior to those of the prior art, effectively solving the problem that the existing cell cryopreservation cooling program cannot meet the uniformity of nucleation and ice formation of the cell solution during the cooling process. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The figure shows the cell viability curve of PBMC sorted cells resuscitated after cryopreservation at different cooling rates, and the cells were cultured for 2 days after activation.

[0031] Figure 2 The figure shows the cell viability curve of PBMC sorted cells resuscitated after cryopreservation by the Thermo cooling program, and the cells were cultured for 10 days after activation.

[0032] Figure 3 The figure shows the cell expansion effect of PBMC sorted cells resuscitated after cryopreservation by the Thermo cooling program, and the cells were cultured for 10 days after activation.

[0033] Figure 4 The figure shows the cell viability curve of PBMC sorted cells resuscitated after cryopreservation by the ultrasonic ice-planting cryopreservation method of the present application, and the cells were cultured for 10 days after activation.

[0034] Figure 5 The figure shows the cell expansion effect of PBMC sorted cells resuscitated after cryopreservation by the ultrasonic ice-planting cryopreservation method of the present application, and the cells were cultured for 10 days after activation. DETAILED DESCRIPTION

[0035] The present application will be described in detail below in conjunction with the drawings and specific examples. The present embodiment is implemented on the basis of the technical solution of the present application, and detailed implementation methods and specific operation processes are given, but the protection scope of the present application is not limited to the following examples.

[0036] The present application aims to provide a cryopreservation method based on ultrasonic ice seeding, in which the ice seeding nucleation temperature is <-9℃.

[0037] Ice nuclei are solid particles that can induce water vapor to undergo desublimation or supercooled water droplets to freeze to form ice crystals. "Ultrasonic ice seeding" refers to a method of inducing ice nuclei in a cell solution by ultrasonic shock; "nucleation" refers to the step of inducing ice nuclei in a solution.

[0038] In some specific embodiments, the ice seeding nucleation temperature is preferably -10℃.

[0039] In some embodiments, the cooling rate during ultrasonic nucleation to solution freezing is <5℃ / min.

[0040] The term "freezing" refers to the process of a liquid becoming a solid in a low temperature environment.

[0041] In some specific embodiments, the cooling rate during ultrasonic nucleation to solution freezing is preferably 1℃ / min.

[0042] In some embodiments, the isothermal time of ultrasonic nucleation is ≥20min.

[0043] In some specific embodiments, the isothermal time of ultrasonic nucleation is preferably 20min, i.e. the solution is kept at the nucleation temperature for 20min.

[0044] The steps of the cryopreservation method of the present application are as follows:

[0045] 1) Reduce the temperature of cells containing a cryopreservation solution from a starting temperature to a nucleation temperature; preferably, the starting temperature is 4-25℃;

[0046] 2) Perform ultrasonic nucleation;

[0047] 3) After successful nucleation, freeze at a cooling rate;

[0048] 4) After freezing, reduce the temperature to a cryopreservation temperature.

[0049] In the present application, the cryopreservation solution can use a cryopreservation solution known in the art, and in some embodiments, the cryopreservation solution described in CN110959606A is used, the entire contents of which are incorporated herein by reference. For example, the cryopreservation solution formula CB BZ2:

[0050]

[0051] In some specific embodiments, the cooling rate from the starting temperature to the nucleation temperature in step 1) is 10℃ / min;

[0052] In some specific embodiments, the ice nucleation temperature of step 3) is -40°C.

[0053] In some specific embodiments, the freezing temperature of step 4) is -90°C, and the cooling rate is 10°C / min.

[0054] In some specific embodiments, the ultrasonic nucleation uses an ultrasonic frequency of 40 KHz and a power of 400 W. The ultrasonic impact is given when the solution is cooled to -10°C to form ice nuclei.

[0055] In some specific embodiments, the freezing instrument used in the freezing method is a Thermo Fisher program cooling instrument, model 7453.

[0056] The present application also provides the use of the freezing method as described above in freezing cells.

[0057] In some embodiments, the cells are animal cells, preferably human cells, such as any one or more of germ cells, neural cells, endocrine cells, phagocytes, white blood cells, red blood cells, epithelial cells, myocardial cells, stem cells.

[0058] In some specific embodiments, the cells are peripheral blood mononuclear cells or cells derived from apheresis.

[0059] The term "apheresis" refers to blood collected by a blood component separation machine, in which mononuclear cells are the main component, and a small amount of red blood cells, granulocytes and plasma components are also included.

[0060] Another object of the application is to provide T cells obtained after thawing and sorting of PBMCs or cells derived from apheresis by the freezing method described above.

[0061] Another object of the application is to provide a method for preparing engineered T cells, comprising transfecting nucleic acids into the T cells described above.

[0062] The physical methods for introducing nucleic acids into the T cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. The biological methods for introducing nucleic acids into the T cells include the use of DNA and RNA vectors. The chemical means for introducing nucleic acids into the T cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes.

[0063] In some embodiments, the nucleic acid contains a polypeptide coding sequence, preferably a coding sequence of a CAR.

[0064] The term "CAR" refers to a chimeric antigen receptor, which can anchor a specific molecule (such as an antibody) that recognizes a tumor cell surface antigen on an immune cell (such as a T cell), so that the immune cell recognizes the tumor antigen or viral antigen and kills the tumor cell or virus-infected cell. The CAR usually comprises, in order, an optional signal peptide, a polypeptide that binds to a tumor cell membrane antigen, a hinge region, a transmembrane region, and an intracellular signaling region. Generally, the polypeptide that binds to the tumor cell membrane antigen can bind to the membrane antigen widely expressed by the tumor cell with moderate affinity. The polypeptide that binds to the tumor cell membrane antigen can be a natural polypeptide or an artificially synthesized polypeptide; preferably, the artificially synthesized polypeptide is a single-chain antibody, a single-domain antibody, a Fab fragment, a F(ab')2 fragment, and a Fv fragment.

[0065] The present application is further illustrated by the following examples, but the present application is not limited to the scope of the examples. The experimental methods in the following examples are not specified, and are selected according to conventional methods and conditions, or according to the instructions of the commercial product.

[0066] Experimental methods:

[0067] I. Cryopreservation and recovery of peripheral blood mononuclear cells (PBMCs)

[0068] 1.1 Preparation of PBMCs

[0069] 15 mL of lymphocyte separation medium was added to 30-35 mL of blood, and centrifuged at 800 x g for 20 min at room temperature. After centrifugation, the sample was divided into red blood cell layer, Ficoll layer, white membrane layer, and dilution layer from bottom to top. The dilution layer was discarded to the 5 ml scale above the white membrane layer, and PBMCs in the white membrane layer were slowly sucked into a new 50 ml centrifuge tube with a pipette, and the volume was adjusted to 50 ml with saline. Centrifugation was performed at 1500 rpm for 5 min at room temperature.

[0070] 1.2 Cryopreservation of PBMCs

[0071] The PBMCs obtained by centrifugation were resuspended in 1 x 10 7 After mixing, the cells were transferred to the CBS cryotube, and the cryopreservation method described in the application was used for cryopreservation.

[0072] 1.3 Recovery of PBMCs

[0073] Prepare AIM-V complete medium containing SR (serum replacement), IL-7 and IL-15, and preheat to 37°C in advance. Prepare two 40 mL of preheated AIM-V complete medium, take the cryopreserved cells from the liquid nitrogen tank, and place them in a preheated water bath at 37°C. Shake the ice completely dissolved. Transfer the dissolved cells to the centrifuge tube containing 40 mL of pre-prepared AIM-V complete medium using a syringe tip, and centrifuge at 300xg for 10 min at room temperature.

[0074] II. PBMC T cell sorting and T cell culture

[0075] Prepare the sorting buffer (0.5% HSA, 2 mM EDTA in PBS solution), and preheat to 37°C in advance. Take the recovered PBMC cells, add sorting buffer to 50 mL, mix well, and centrifuge at 300xg for 10 min at room temperature. According to the cell amount, add 1x10 7 Add 80 μL of sorting buffer to resuspend the cells, and add 1x10 7 Add 20 μL of CD4 and CD8 magnetic beads to the cells, respectively, as needed. Mix well and incubate in the dark for 30 min, and mix the cell suspension every 3 min. After the magnetic bead incubation, add 1x10 7 Add 1-2 mL of sorting buffer to mix and wash once, centrifuge at 300xg for 10 min at room temperature, discard the supernatant, and resuspend the cells to 1x10 8 Add 500 μL of sorting buffer to resuspend the cells and blow them off completely.

[0076] Install the LS sorting column, rinse with 3 mL of sorting buffer, and slowly add the cell suspension to the LS sorting column when the liquid in the column is about to flow out. Add 3 mL of sorting buffer to wash the LS sorting column after the solution passes through the column, and repeat once. Remove the sorting column from the separator and place it in the appropriate collection tube. Add 5 mL of sorting buffer, push the plunger firmly into the column, and immediately wash out the magnetically labeled cells. Repeat once.

[0077] Take the sorted T cells and resuspend them in AIM-V complete medium containing SR, IL-7, and IL-15, according to 1x10 6 Add 5 μL of Enceed (T cell activation / amplification kit) to activate the culture. Place the culture bottle in the incubator at 37±1.0°C and 5.0±0.2% CO2 concentration for 48 h. After 48 h of activation culture, collect the cells into a 50 mL centrifuge tube, centrifuge at 300xg for 10 min at room temperature, discard the supernatant, and resuspend the cells to 5x10 5The cells / mL were continuously cultured. The density and viability of the cells were detected every other day during the culture process from Day 2 to Day 10. After the cells were mixed, a small amount of cells was taken and subjected to AO / PI staining to detect the cell density and viability.

[0078] III. Detection of the density and viability of the cells

[0079] The cell suspension 20 ul was mixed with an equal volume of AO / PI [acridine orange (AO) and propidium iodide (PI)] and subjected to cell counting using a full-automatic fluorescence cell counter (JSY-FL-049) to detect the cell density and viability.

[0080] Example 1: Exploration of the nucleation temperature and cooling rate for ultrasonic ice-planting

[0081] 180 mL of peripheral blood was collected from a healthy volunteer, and the peripheral blood mononuclear cells (PBMCs) were separated according to the procedure described in 1.1. The cells were divided into 6 portions according to the experimental requirements, 3 portions for the -10°C nucleation group and 3 portions for the -9°C nucleation group. Each group was further divided into a 1°C / min cooling rate group (1°C / min Frozen group), a 10°C / min cooling rate group (10°C / min Frozen group) and a 20°C / min cooling rate group (20°C / min Frozen group) according to the cooling rate during the process from nucleation to solution freezing. The ultrasonic ice-planting freezing procedure is shown in Table 1. The freezing device used was a Thermo Fisher program cooling instrument, model 7453, with an ultrasonic frequency of 40 KHz and a power of 400 W.

[0082] During the ultrasonic ice-planting process, it was found that the nucleation success rate at a nucleation temperature of -9°C was significantly lower than that at -10°C when other parameters were kept unchanged, as shown in Table 2. Therefore, -10°C was used for nucleation freezing in the subsequent experiments.

[0083] The cells were recovered and sorted 48 h after freezing, and the cell viability was detected by cell counting. After 48 h of activation culture, the cell viability was detected by cell counting. The results are shown in Table 3. Figure 1 As shown in Table 3, there was no significant difference in the cell viability before and after sorting in each group, and the cell viability in the 1°C / min Frozen group was better than that in the 20°C / min Frozen group and significantly better than that in the 10°C / min Frozen group after 48 h of activation culture.

[0084] Therefore, -10°C nucleation freezing is better than -9°C nucleation, the optimal nucleation temperature is -10°C, and it is preliminarily inferred that low cooling rate freezing is better than high cooling rate freezing,

[0085] Table 1: Ultrasonic ice-planting freezing procedure and time required for freezing cells

[0086]

[0087] Table 2 Nucleation success rate at nucleation temperature of -9°C and -10°C

[0088] Nucleation temperature Nucleation success tube number Nucleation failure tube number Total tube number Nucleation success rate -9℃ 9 9 18 50％ -10℃ 9 1 10 90％

[0089] Example 2 Optimization of ice-planting cooling rate

[0090] Peripheral blood 180 mL of healthy volunteers was collected, and peripheral blood mononuclear cells (PBMC) were separated according to the steps described in 1.1. The cells were evenly divided into three parts, and the cooling rates during the process of nucleation success to solution ice formation were 1°C / min cooling rate group (1°C / min Frozen group), 5°C / min cooling rate group (5°C / min Frozen group) and Thermo program cooling group (Thermo Frozen group). The Thermo Frozen group was stored according to the procedure shown in Table 3. This procedure is used with the Thermo program cooling instrument, which is the most commonly used cooling program in the art.

[0091] After 48 hours of cell freezing, the cells were recovered and sorted. Cell density and viability were detected by cell counting, and the proportion of stem cells was detected by flow cytometry. After 10 days of culture, cell counting was performed on D2, D4, D6, D8 and D10 to detect cell density and viability; flow cytometry was performed on D2 and D10 to detect the proportion of stem cells.

[0092] The results are shown in Figure 2 、 Figure 3 Table 4 and Table 5, there was no significant difference in cell viability between each freezing group, the cumulative expansion of the 1°C / min Frozen group and the Thermo Frozen group remained basically consistent, and both were significantly better than the 5°C / min Frozen group. In the early culture period, the proportion of stem cells in the 5°C / min Frozen group was higher than that in the 1°C / min Frozen group and the Thermo Frozen group, and in the late culture period, the proportion of stem cells in the 1°C / min Frozen group and the Thermo Frozen group was higher than that in the 5°C / min Frozen group. Overall, the freezing effect of the 1°C / min Frozen group and the Thermo Frozen group was similar, and significantly better than that of the 5°C / min Frozen group, and it was again confirmed that the best cooling rate was 1°C / min.

[0093] Table 3 Thermo cooling program and time required for frozen cells

[0094]

[0095] Table 4 Flow cytometry analysis results after 2 days of culture

[0096] 1 °C / min Frozen 5 °C / min Frozen Thermo Frozen Tn (Tnaive + Tscm) % 1.42 5.87 0.89 TCM % 5.83 9.13 2.63 Tn + TCM % 7.25 15 3.52

[0097] Table 5 Flow cytometry detection results after 10 days of culture

[0098]

[0099]

[0100] Example 3 Cryopreservation procedure based on ultrasonic ice planting

[0101] 180 mL of peripheral blood was collected from healthy volunteers, and peripheral blood mononuclear cells (PBMCs) were isolated according to the procedure described in 1.1. The cells were evenly divided into three parts, and the cooling rates during the process of successfully nucleating into ice solution were different. The cooling rates were 1°C / min (the cooling procedure described in the application, New 1°C / min Frozen group), 1°C / min (the cooling procedure described in Table 1, Old 1°C / min Frozen group), and the procedure cooling group (Thermo Frozen group). The cryopreservation procedure of the New 1°C / min Frozen group is shown in Table 6. After 48 hours of cryopreservation, the cells were recovered and sorted. Cell density and viability were detected by cell counting, and the proportion of stem cells was detected by flow cytometry.

[0102] After 10 days of culture, cell counting was performed on D2, D4, D6, D8, and D10 to detect cell density and viability. Flow cytometry was performed on D2, D6, and D10 to detect the proportion of stem cells.

[0103] The results are shown in Tables 7-10. Figure 4 and Figure 5 There was no significant difference in cell viability between the groups. The cumulative expansion of the New 1°C / min Frozen group was slightly better than that of the Thermo Frozen group and significantly better than that of the Old 1°C / min Frozen group. Based on the overall experimental data of stem cell protection rate (Tables 7-10), the New 1°C / min Frozen group had a slightly better stem cell protection rate than the Thermo Frozen group. Based on the cell culture data and flow cytometry data, the cumulative expansion and stem cell protection rate of the New 1°C / min Frozen group were slightly better than those of the Thermo Frozen group. Moreover, the time required for cryopreservation of cells was significantly shortened, and the efficiency of cell cryopreservation was significantly improved.

[0104] Table 6 Cryopreservation procedure based on ultrasonic ice planting and time required for cryopreservation of cells

[0105]

[0106] Table 7 Flow cytometry detection results after sorting

[0107] New 1 °C / min Frozen Thermo Frozen Old 1 °C / min Frozen Tn (Tnaive + Tscm) % 7.13 8.54 4.89 TCM % 4.02 4.34 4.34 Tn + TCM % 11.5 12.88 9.23

[0108] Table 8 Flow cytometry detection results after 2 days of culture

[0109] New 1 °C / min Frozen Thermo Frozen Old 1 °C / min Frozen Tn (Tnaive + Tscm) % 1.42 1.65 0.93 TCM % 2.44 3.66 3.19 Tn + TCM % 3.86 5.31 4.12

[0110] Table 9 Flow cytometry detection results after 6 days of culture

[0111] New 1 °C / min Frozen Thermo Frozen Old 1 °C / min Frozen Tn (Tnaive + Tscm) % 3.89 3.28 1.4 TCM % 6.71 3.17 2.02 Tn + TCM % 10.6 6.45 3.42

[0112] Table 10 Flow cytometry detection results after 10 days of culture

[0113] New 1 °C / min Frozen Thermo Frozen Old 1 °C / min Frozen Tn (Tnaive + Tscm) % 0.68 0.4 0.39 TCM % 1.18 1.12 0.64 Tn + TCM % New 1 °C / min Frozen Thermo Frozen Old 1 °C / min Frozen Tn (Tnaive + Tscm) % TCM % Tn + TCM % 1.86 1.52 1.03

[0114] In summary, the present application provides a cryopreservation method based on ultrasonic ice planting. Compared with the existing Thermo cell cryopreservation cooling program, the time required for cryopreservation cells is significantly shortened, and the damage caused by ice crystals to cells during the cooling process is reduced, solving the problem that the existing cell cryopreservation cooling program cannot meet the uniformity of cell solution nucleation and ice formation during the cooling process.

Claims

1. A cryopreservation method based on ultrasonic ice-coating, characterized in that, In the method, the core temperature of the ice-planting is <-9℃, preferably -10℃.

2. The cryopreservation method as described in claim 1, characterized in that, The cooling rate during the process of ultrasonic placement of the nucleus into the solution freezing is <5℃ / min, preferably 1℃ / min.

3. The cryopreservation method as described in claim 1, characterized in that, The isothermal time for ultrasonic placement is ≥20 min, preferably 20 min.

4. The cryopreservation method as described in claim 1, characterized in that, The steps of the cryopreservation method are as follows: 1) The cells containing cryopreservation solution are cooled from the initial temperature to the nucleation temperature; preferably, the initial temperature is 4-25°C; 2) Perform ultrasonic implantation; 3) After successful core placement, ice will form according to the cooling rate; 4) After freezing, cool to the freezing temperature.

5. The cryopreservation method as described in claim 4, characterized in that, The cryopreservation method has one or more of the following characteristics: 1) The cooling rate from the initial temperature to the core placement temperature in step 1) is 10℃ / min; 2) The ice-forming temperature in step 3) is -40℃; 3) The freezing temperature in step 4) is -90℃, and the cooling rate is 10℃ / min.

6. The application of the cryopreservation method according to any one of claims 1-5 in cryopreserved cells.

7. The application as described in claim 6, characterized in that, The cells are animal cells, preferably human cells, such as any one or more selected from germ cells, nerve cells, endocrine cells, phagocytes, white blood cells, red blood cells, epithelial cells, cardiomyocytes, and stem cells; preferably, the cells are peripheral blood mononuclear cells (PBMCs) or cells derived from apheresis.

8. A PBMC or T cells obtained after cell resuscitation and sorting from apheresis blood, cryopreserved by any one of the cryopreservation methods described in claims 1-7.

9. A method for preparing engineered T cells, characterized in that, This includes transfecting nucleic acids into the T cells described in claim 8.

10. The method as described in claim 9, characterized in that, The nucleic acid contains a polypeptide coding sequence, preferably a CAR coding sequence.

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