Cryopreservation method, resuscitation method and culture method of cells

Abstract

The invention relates to the field of cell culture, in particular to a cryopreservation method, a resuscitation method and a culture method of cells. The invention provides a cell cryopreservation method which comprises the following steps: mixing cells to be cryopreserved with a cryopreservation protective agent, pre-cryopreserving, and cryopreserving to obtain cryopreserved cells, the temperature of the pre-cryopreservation is-86 DEG C to-70 DEG C, and the time of the pre-cryopreservation is 1 hour to 1 minute; the step of pre-cryopreservation and the step of cryopreservation do not comprise a gradient cooling step or a programmed cooling step. The invention mainly provides a rapid cell cryopreservation and resuscitation method which is simple to operate, low in cost and capable of being applied on a large scale. After cell cryopreservation and resuscitation, the cell survival rate, the stability and the cell function of the technology are not lower than those of traditional program cryopreservation, and the technology shows more superior cryopreservation effects in immune cells, genetically modified immune cells and stem cell types compared with the traditional program cryopreservation.

Description

Technical Field

[0001] The present invention relates to the field of cell culture, and in particular to a cell freezing method, a cell recovery method and a cell culture method. Background Art

[0002] With the application and development of cell therapy, cell therapy has become one of the most promising treatment methods in clinical practice, with the potential to cure a variety of intractable diseases. Cell therapy drugs have been widely used in clinical research on genetic diseases, advanced blood diseases, cancer, immune diseases, infectious diseases and other diseases, and have shown considerable clinical application value. Cell freezing and thawing are the key links in the production of cell therapy drugs.

[0003] Cryopreserved cell products are stored in liquid nitrogen for a long time, which is convenient for long-distance transportation and meets clinical applications in various situations. The vitality of cells after recovery is an important indicator for maintaining the activity and function of cell products.

[0004] The existing cell freezing technology resuspends the cells in a cryopreservation solution containing cryoprotectants such as DMSO and performs a step-by-step gradient cooling method for freezing. The traditional gradient cooling method places the cells in a refrigerator at 4℃-30min, 20℃-2h, and -80℃ overnight. This method is complex to operate, time-consuming, and requires at least three storage devices with different temperatures. With the improvement of cryopreservation technology, the gradient cooling step controls the cells to be frozen at a cooling rate of 1℃ / min by using a program cooling box or a program-controlled cooling instrument. The program gradient cooling cell freezing scheme has certain advantages in the freezing effect of various cell lines and has always been a classic method for cell freezing. However, there is not enough data to support whether this method is suitable for freezing immune cells or genetically modified immune cells. At the same time, the program cooling box can only freeze a small number of small-volume cryopreservation tubes. Even if the more advanced program-controlled cooling instrument on the market is used for freezing, the requirements for the freezing equipment are high, and the freezing program needs to be set specifically. The operation is complicated, and the freezing volume of the existing equipment is still limited by the equipment space. How to establish a simplified, efficient, stable and scalable method for cryopreservation of immune cells and stem cells, ensure that the frozen cells have stable biological activity, improve cell viability after recovery and the stability of cell products during clinical use, is a technical difficulty currently faced in cell biology research and industrial production of cell therapy drugs. Summary of the invention

[0005] In view of this, the present invention provides a cell freezing method, a recovery method and a culture method. The present invention mainly provides a cell rapid freezing and recovery method that is simple to operate, economical in cost and can be applied on a large scale. The cell survival rate, stability and cell function after cell freezing and recovery by this technology are not lower than those of traditional cryopreservation procedures, and the freezing effect is more advantageous than that of traditional cryopreservation procedures in immune cells, genetically modified immune cells and stem cell types.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a cell freezing method, which comprises mixing cells to be frozen with a cryoprotectant, pre-freezing, and freezing to obtain frozen cells;

[0008] The pre-freezing temperature is -86°C to -70°C, and the time is 1 hour to 1 month;

[0009] The pre-cryostorage and the cryopreservation do not include a gradient cooling step or a programmed cooling step.

[0010] In some embodiments of the present invention, in the above-mentioned freezing method, the pre-freezing time is 1 hour to 24 hours or 7 days or 1 month.

[0011] In some embodiments of the present invention, in the above-mentioned freezing method, the pre-freezing time is 1 hour, 3 hours, 24 hours, 7 days or 1 month.

[0012] In some embodiments of the present invention, in the above-mentioned cryopreservation method, the cryopreservation is carried out at -86°C to -70°C or in liquid nitrogen for a period of not less than 3 months.

[0013] In some embodiments of the present invention, in the above-mentioned freezing method, the temperature of the liquid nitrogen is ≤-150°C.

[0014] In some embodiments of the present invention, in the above freezing method, the mixing time is 10 minutes to 120 minutes.

[0015] In some embodiments of the present invention, in the above-mentioned cryopreservation method, the cryoprotectant includes: compound electrolytes, human serum albumin and DMSO.

[0016] In some embodiments of the present invention, in the above-mentioned cryopreservation method, the cryoprotectant further comprises: glucose and dextran 40.

[0017] In some embodiments of the present invention, in the above freezing method, the compound electrolyte, the human serum albumin, the glucose, and the dextran 40 are all added in the form of injection.

[0018] In some embodiments of the present invention, in the above-mentioned cryopreservation method, the cryoprotectant comprises:

[0019] Compound electrolyte injection 20%~40% (v / v)

[0020] Glucose and sodium chloride injection 10%~30% (v / v)

[0021] Human albumin injection 10%~30% (v / v)

[0022] Dextran 40 glucose injection 10%~20% (v / v)

[0023] DMSO 5%~10% (v / v)

[0024] Glucose injection 5%~10% (v / v).

[0025] In some embodiments of the present invention, in the above-mentioned cryopreservation method, the cryoprotectant comprises:

[0026] Compound electrolyte injection 31.25% (v / v)

[0027] Glucose and Sodium Chloride Injection 15.63% (v / v)

[0028] Human albumin injection 20.00% (v / v)

[0029] Dextran 40 Glucose Injection 16.67% (v / v)

[0030] DMSO 7.5%(v / v)

[0031] Glucose injection 8.95% (v / v).

[0032] In some embodiments of the present invention, in the above-mentioned freezing method, the pre-freezing carrier includes: one or more of: a PP material freezing tube, a glass material vial and / or an EVA material freezing bag.

[0033] In some embodiments of the present invention, in the above-mentioned cryopreservation method, the cells include: immune cells and / or stem cells.

[0034] In some embodiments of the present invention, in the above-mentioned freezing method, the immune cells include: one or more of PBMC cells, DC cells, αβT cells, γδT cells, NK cells, CAR-T cells and / or CAR-NK cells.

[0035] In some embodiments of the present invention, in the above-mentioned cryopreservation method, the stem cells include: mesenchymal stem cells.

[0036] In some embodiments of the present invention, in the above freezing method, the density of the cells is: 5×10 5 / mL~1×10 8 / mL.

[0037] In some embodiments of the present invention, in the above freezing method, the density of the cells is: 5×10 5 / mL, 1×10 6 / mL, 1×10 7 / mL or 1×10 8 / mL.

[0038] In some embodiments of the present invention, in the above-mentioned freezing method, the freezing volume is: 1 mL to 70 mL.

[0039] In some embodiments of the present invention, in the above-mentioned freezing method, the freezing volume is: 1 mL, 5 mL, 25 mL, 50 mL or 70 mL.

[0040] The present invention also provides a cell resuscitation method, which comprises taking the cryopreserved cells obtained by the above-mentioned cryopreservation method, resuscitating them, and obtaining resuscitated cells.

[0041] In some embodiments of the present invention, in the above-mentioned resuscitation method, the resuscitation temperature is: 15°C to 37°C.

[0042] In some embodiments of the present invention, in the above-mentioned resuscitation method, the resuscitation temperature is 37°C or 15°C to 37°C.

[0043] The present invention also provides a cell culture method, comprising the above-mentioned freezing method and / or the above-mentioned resuscitation method.

[0044] In some embodiments of the present invention, in the above-mentioned culture method, the cells obtained by the resuscitation method are cultured; and the culture time is 1 hour to 72 hours.

[0045] In some embodiments of the present invention, in the above-mentioned culture method, the culture time is: 1 hour, 3 hours, 5 hours, 24 hours, 48 ​​hours or 72 hours.

[0046] The present invention also provides the use of the cells obtained by the above freezing method, the cells obtained by the above thawing method and / or the cells obtained by the above culturing method in any of the following items:

[0047] (I), improving the survival rate of the cells; and / or

[0048] (II), improving the stability of the cell; and / or

[0049] (III), improving the tolerance of the cells; and / or

[0050] (IV) increasing the number of the cells.

[0051] The present invention also provides the use of the cells obtained by the above freezing method, the cells obtained by the above resuscitation method and / or the cells obtained by the above culture method in any of the following items:

[0052] (I) improving the tumor killing ability of immune cells; and / or

[0053] (II), improving the secretion capacity of cytokines of immune cells; and / or

[0054] (III) Increase the proliferation multiple of stem cells.

[0055] In some embodiments of the present invention, in the above application, the cytokine includes: IFN-γ.

[0056] The present invention provides a cell freezing method, which comprises mixing cells to be frozen with a cryoprotectant, pre-freezing, and freezing to obtain frozen cells;

[0057] The pre-freezing temperature is -86°C to -70°C, and the time is 1 hour to 1 month;

[0058] The pre-cryostorage and the cryopreservation do not include a gradient cooling step or a programmed cooling step.

[0059] The present invention precools the cavity temperature of the cell freezing equipment to -78°C ± 8°C to create an ultra-low temperature environment, triggering the cells to cool down rapidly and stably to reduce the damage to the cells caused by the rise in latent hot spots. The method is simple to operate, economical in cost, and the cell survival rate, stability and anti-tumor function after cell freezing and resuscitation are better than those of traditional programmed freezing schemes. The rapid freezing scheme is suitable for the freezing of cell products with different cell concentrations, different packaging materials and different packaging specifications, and is better than traditional programmed freezing schemes. The present invention simplifies the freezing mode to get rid of the dependence on programmed cryopreservation instruments and the limitation of freezing space, and can be applied to the freezing of cells produced on a large scale and cell products in a variety of packaging methods. At the same time, rapidly frozen cells can achieve room temperature resuscitation and maintain a good cell viability, which has a wider application value in the field of cell therapy drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0061] Figure 1 Shows the trend of sample temperature changing with freezing time;

[0062] Figure 2 The figure shows the viability and stability of cells recovered after rapid freezing at different transfer times; A shows the stability of cell recovery viability of rapidly frozen cell samples at different transfer times over time; B shows the viability of cells recovered after rapid freezing at different transfer times; C shows the 24-hour survival rate of cells recovered after rapid freezing and programmed freezing at different transfer times;

[0063] Figure 3 The figure shows the comparison of cell viability and stability of immune cells frozen by two freezing schemes; A shows PBMC cells; B shows αβT cells; C shows NK cells; D shows γδT cells; E shows DC cells; F shows the summary comparison of cell viability after recovery of immune cells frozen by two freezing schemes; G shows the summary comparison of 24-hour survival rate after recovery of immune cells frozen by two freezing schemes;

[0064] Figure 4 The figures show the comparison of the viability and stability of chimeric antigen-modified immune cells frozen in two freezing schemes; A shows the summary comparison of the viability of chimeric antigen-modified CAR-T and CAR-NK cells after recovery after freezing in two freezing schemes; B shows the summary comparison of the 24-hour survival rate of chimeric antigen-modified CAR-T and CAR-NK cells frozen in two freezing schemes after recovery;

[0065] Figure 5 The figures show the comparison of the target cell killing ability and IFN-γ secretion level of CAR-T cells frozen by two freezing schemes; A shows the comparison of the target cell killing ability of CAR-T cells frozen by two freezing schemes; B shows the comparison of the IFN-γ secretion level of CAR-T cells frozen by two freezing schemes;

[0066] Figure 6 The results show the comparison of the viability of CAR-T cells frozen at different densities under two cryopreservation schemes;

[0067] Figure 7 The results show the comparison of the target cell killing ability and IFN-γ secretion level of CAR-T cells frozen at different densities under two cryopreservation schemes; A shows the target cell killing ability of CAR-T cells frozen at different densities under two cryopreservation schemes; B shows the IFN-γ secretion level of CAR-T cells frozen at different densities under two cryopreservation schemes;

[0068] Figure 8 The figures show the viability and stability of CAR-T cells frozen in different cryopreservation protection solutions; A shows the trend of viability over time before and after recovery of CAR-T cells frozen in different cryopreservation protection solutions; B shows the survival rate of CAR-T cells frozen in different cryopreservation protection solutions 24 hours after recovery;

[0069] Fig. 9The effectiveness of different cryopreservation protection solutions for freezing CAR-T cells is verified;

[0070] Fig.10 The viability of CAR-T cells cryopreserved with different cryopreservation materials and packaging is shown; A shows the viability of CAR-T cells cryopreserved with different cryopreservation materials and packaging; B shows the viability of CAR-T cells cryopreserved with three cryopreservation materials and packaging, and compares the viability of CAR-T cells cryopreserved with two cryopreservation schemes;

[0071] Fig.11 The figures show the viability and stability of CAR-T cells frozen in different cryopreservation specifications; A shows the trend of viability over time before and after recovery of CAR-T cells frozen in different cryopreservation specifications; B shows the summary of the survival rate of CAR-T cells frozen in different cryopreservation specifications 24 hours after recovery;

[0072] Fig.12 The viability of cells frozen at different cryopreservation solution contact times is shown; wherein: A shows the viability of cells frozen at different cryopreservation solution contact times; B shows a summary comparison of the viability of cells frozen at different cryopreservation solution contact times;

[0073] Fig.13 The figures show the cell viability and stability under different freezing and storage schemes; A shows the change trend of cell viability over time before and after 3-month recovery of CAR-T cells frozen by two freezing procedures; B shows the cell viability after 3-month recovery of cells frozen by two freezing schemes;

[0074] Fig.14 The cell viability of rapid freezing solution in water bath and room temperature recovery is shown;

[0075] Fig.15 The figures show the viability and cell number of stem cells frozen by rapid freezing and programmed freezing; wherein: A shows the comparison of the viability of stem cells frozen by the two freezing procedures after recovery; B shows the comparison of the cell proliferation times of stem cells frozen by the two freezing procedures after recovery;

[0076] Fig.16 Shows the viability and cell number of non-immune cells after cryopreservation and resuscitation; among them: A shows the comparison of the viability of 293T cells frozen and revived by two freezing procedures; B shows the comparison of the resuscitation culture or cell number of 293T cells frozen and revived by two freezing procedures; C shows the comparison of the viability of MRC-5 cells frozen and revived by two freezing procedures; D shows the comparison of the cell number of MRC-5 cells frozen and revived by two freezing procedures. DETAILED DESCRIPTION

[0077] The invention discloses a cell freezing method, a cell resuscitation method and a cell culture method.

[0078] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0079] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0080] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0081] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0082] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0083] The present invention mainly provides a method for rapid cell freezing and thawing that is simple to operate, economical in cost, and can be applied on a large scale. The cell survival rate, stability, and cell function after cell freezing and thawing by this technology are not lower than those of traditional cryopreservation procedures, and it shows a more advantageous cryopreservation effect in immune cells, genetically modified immune cells, and stem cells than traditional cryopreservation procedures.

[0084] The present invention provides a simplified and rapid method for freezing and reviving immune cells and stem cells, mainly by precooling the cavity temperature of the cell freezing equipment to -78°C ± 8°C to create an ultra-low temperature environment to trigger the cells to cool down rapidly and stably to reduce the damage to the cells caused by the rise in temperature of latent hot spots. The viability, function and stability of the cells after recovery are maintained and improved, while simplifying the freezing mode, getting rid of the dependence on programmable cooling devices and the limitation of freezing space, and can be applied to the freezing of cells produced on a large scale and in various packaging methods. At the same time, the cells frozen based on this scheme can be revived at room temperature, and there is no equipment requirement for recovery, the operation is simple, and the cell recovery effect is consistent with the traditional recovery (37°C water bath).

[0085] The cryopreservation and resuscitation scheme protected by the present invention:

[0086] (1) The cells to be frozen are resuspended in a cryoprotectant and dispensed into cryotubes or cryobags, wherein the cryoprotectant is a commercial cryoprotectant or a cryoprotectant containing compound electrolytes, glucose, dextran 40, human serum albumin and DMSO. The volume of the cryoprotectant is based on the cryocontainer, 1 mL to 2 mL for cryotubes and 5 mL to 70 mL for cryobags.

[0087] (2) Control the temperature of the ultra-low temperature refrigerator cavity to -78±8℃;

[0088] (3) Place the cryopreservation tubes or cryopreservation bags directly in a pre-cooled ultra-low temperature refrigerator, or package the cryopreservation tubes / bags in a cryopreservation box and place them directly in a pre-cooled ultra-low temperature refrigerator;

[0089] (4) Cells can be cryopreserved for 1 h to 24 h or extended to 7 days and then transferred to liquid nitrogen (≤ -150°C) for long-term storage. They can also be directly stored in an ultra-low temperature freezer at -78±8°C for at least 3 months.

[0090] (5) When thawing cells, place them in a 37°C water bath and thaw quickly until there is no residual ice, or place them directly in room temperature until they are completely thawed.

[0091] The conventional freezing program used for comparison during the implementation of the present invention is:

[0092] 1. Use Thermo's programmable cooling instrument model 7451TF to freeze cells:

[0093] (1) Set the equipment cavity to cool down to -45°C at 1°C / min;

[0094] (2) Set the equipment cavity to cool down to -80°C at -10°C / min and end the program;

[0095] (3) After cryopreservation, transfer the cells to a -80°C ultra-low temperature freezer or store in liquid nitrogen.

[0096] 2. Use CoolCell cell program cooling box to freeze cells:

[0097] (1) Place the cells in cryopreservation tubes, arrange them neatly and place them in FTS30 (1°C / min) program cooling box;

[0098] (2) Place the cooling box in a -80°C ultra-low temperature refrigerator and cool it down for at least 4 hours;

[0099] (3) After cooling, transfer the cells to liquid nitrogen for storage.

[0100] In Examples 1 to 14 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0101] The present invention will be further described below in conjunction with embodiments:

[0102] Example 1 Comparison of cooling curves of samples in rapid freezing and programmed freezing

[0103] According to the rapid freezing scheme provided by the present invention, the CAR-T cells are resuspended with a cryoprotectant containing compound electrolytes, human serum albumin and DMSO and then directly placed in an ultra-low temperature refrigerator precooled to -78±8°C for rapid freezing. At the same time, the control sample is set to a freezing program using a programmable cooling instrument: 1) the temperature is cooled to -45°C at 1°C / min; 2) the temperature is cooled to -80°C at -10°C / min for program freezing.

[0104] The results are shown in Table 1 and Figure 1 As shown in Table 1, the sample temperature values ​​corresponding to the rapid freezing scheme and program freezing as the sample temperature drops at different times. Figure 1 The horizontal axis represents the cooling time, and the vertical axis represents the sample temperature. We unexpectedly found that the cooling rate of the sample in the rapid freezing solution was faster than that of the program freezing. It is speculated that because the cavity temperature was pre-cooled in advance, the sample did not release heat from the latent hot spot during the cooling process, which caused the sample temperature to rise again. The temperature fluctuation during the cooling process was smaller than that of the traditional program freezing. The latent hot spot heat release mentioned in this embodiment is the heat released during the process of the freezing liquid changing from liquid to solid when freezing. The sample temperature will rise in a short time, causing the sample temperature to fluctuate greatly and destroy the cell structure, affecting the cell freezing effect. The flatter the latent hot spot temperature curve on the freezing curve, the smaller the temperature fluctuation, the more beneficial it is to the cell freezing effect.

[0105] Table 1 Trend of sample temperature changes with freezing time

[0106]

[0107] It can be seen from the results of Example 1 that pre-freezing cells for 1 hour to 1 month at a temperature of -86°C to -70°C without using gradient cooling or programmed cooling can better ensure the activity of the frozen cells, wherein the freezing solution contains compound electrolytes, human serum albumin and DMSO.

[0108] On this basis, we further compared the effects of different cryopreservation fluid ratios on the viability and function of the above-mentioned non-programmed cryopreserved cells.

[0109] Prepare cryoprotectant according to the following scheme:

[0110] Scenario 0:

[0111]

[0112] Scenario 1:

[0113]

[0114] Scenario 2:

[0115]

[0116] Solution 3:

[0117]

[0118]

[0119] Solution 4:

[0120]

[0121] Solution 5:

[0122]

[0123] The CAR-T cells were resuspended in the above freezing solution and according to the rapid freezing solution provided by the present invention, the frozen samples were directly placed in a pre-cooled -78±8°C ultra-low temperature refrigerator for rapid freezing. The cells were revived after being frozen for at least 7 days (7 days) for viability detection. The detection results are shown in Table 2:

[0124] Table 2 Comparison of cell viability before and after cryopreservation and thawing

[0125]

[0126] The group columns in the above Table 2 represent CAR-T cells with different CAR structures and different freezing times. 104GZ28 is a CAR targeting CD19 in which the intracellular co-stimulatory signal is derived from CD28 and the intracellular primary stimulation signal is derived from CD3ζ. 1025ZB is a CAR targeting CD19 in which the intracellular co-stimulatory signal is derived from 41BB and the intracellular primary stimulation signal is derived from CD3ζ. 815GZ19 is a CAR targeting CD19 in which the intracellular primary stimulation signal is derived from CD3ζ and there is no co-stimulatory signal.

[0127] As shown in Table 2, Scheme 4 has a relatively stable recovery rate in different CAR structures.

[0128] The CAR-T cells 1025ZB resuscitated in the above-mentioned schemes 0, 3, 4 and 5 were further tested for in vitro killing to verify their functions: Raji-Luc-GFP cells were used as positive target cells for killing detection, CAR-T cells and target cells were plated on target cells at a ratio of 1:1, and killing was detected by luciferase after 24 hours, and the results are shown in Table 3. Luciferase principle: During the detection, after the target cells are lysed with lysis buffer, the luciferase therein will decompose the substrate to emit fluorescence, and the fluorescence value analysis result formula is: CAR-T cell killing rate = 1-(fluorescence value of the experimental group ÷ fluorescence value of the blank control group) * 100%.

[0129] Table 3

[0130]

[0131] The results showed that cells frozen using Scheme 4 could recover better killing function in a short period of time after being revived.

[0132] Therefore, under the non-programmed freezing scheme described in the present invention, Scheme 4 is preferred for freezing, and subsequent experimental freezing solutions are all frozen using the scheme described in Scheme 4.

[0133] Example 2 Rapid freezing protocol Cell sample transfer time

[0134] The T cells in the PBMC were activated with antibodies or magnetic beads containing CD3 / CD28, and the lentiviral vector targeting CD19 was transduced to prepare CAR-T cells. The cells were resuspended with freezing solution and placed directly in a pre-cooled -78±8℃ ultra-low temperature refrigerator for rapid freezing according to the rapid freezing scheme provided by the present invention. The cells were frozen and cooled for 1hour, 3hours and 24hours, and then transferred to liquid nitrogen for storage, or the cells were placed in a -78±8℃ environment for 7days and then transferred to liquid nitrogen for storage, or the cells were placed in a -78±8℃ environment for 1month. The viability of the resuscitated cells was tested, and the cells containing the cryoprotectant were placed at room temperature for at least 5hours, during which the cell viability was monitored, and the two freezing schemes were compared to maintain the viability and stability of different immune cells.

[0135] The results are shown in Table 4 and Figure 2 Table 4 shows the viability and stability of cells after rapid freezing and different transfer times. Figure 2 A The horizontal axis represents different test times after cell recovery, and the vertical axis represents cell viability, which represents the stability of cell viability after rapid freezing of cell samples at different transfer times. Figure 2 B The horizontal axis represents the different transfer times of the rapidly frozen cell samples, and the vertical axis represents the cell viability at the time of cell recovery. The results were statistically analyzed using one-way ANOVA. The results showed that the cell viability and stability after cell recovery were consistent when the cell samples were transferred to liquid nitrogen after rapid freezing for 1 hour, 3 hours, 24 hours, 7 days or 1 month, and there was no statistical difference in cell viability after recovery ( Figure 2 B, one-way ANOVA, P = 0.2728).

[0136] Table 4 Viability and stability of cells after rapid cryopreservation and different transfer times

[0137]

[0138]

[0139] CAR-T cells were prepared by transducing CD19-targeted lentiviral vectors and cryopreserved according to the traditional cryopreservation procedure and the rapid cryopreservation scheme described in the present invention. The cells were cryopreserved and cooled for 1 hour, 3 hours, 24 hours, 48 ​​hours and 7 days, and then transferred to liquid nitrogen for storage. After thawing, the cells were cultured in a medium containing FBS for 24 hours and then counted. The effects of different cooling times of the two cryopreservation schemes on cell survival were monitored. The results are shown in Figure 2As shown in C, the horizontal axis represents the transfer of cells into liquid nitrogen at different times of freezing, and the vertical axis represents the survival rate of cells after 24 hours of recovery. The results were statistically analyzed using paired T test.

[0140] The results showed that the cell survival rate of the programmed freezing scheme decreased significantly as the freezing time increased compared with the rapid freezing scheme, and the rapid freezing scheme maintained better cell survival rate stability ( Figure 2 C, Paired test, P=0.0253, P=0.0435, P=0.0329).

[0141] Example 3 Rapid freezing scheme maintains better viability and stability of immune cells after freezing

[0142] Prepare and culture different types of immune cells. The main cell types and culture methods are as follows:

[0143] PBMC: peripheral blood mononuclear cells, peripheral blood was collected from donors and PBMC was obtained by Ficoll density gradient centrifugation;

[0144] DC cells: PBMCs were resuspended in cell culture medium containing 10% FBS for at least 2 h, non-adherent cells were removed, and adherent monocytes were cultured with GM-CSF and IL-4 for 7 days to induce cell differentiation into DC cells;

[0145] αβT cells: T cells in PBMCs are activated with CD3 / CD28 antibodies or magnetic beads, and expanded and cultured for 9 to 11 days in a cell culture medium containing cytokines such as IL-2 / IL-7 / IL-15 / IL-21 to prepare αβT cells.

[0146] γδT cells: T cells in PBMCs were activated with zoledronic acid and expanded and cultured in a cell culture medium containing cytokine IL-2 for 9 to 11 days to prepare γδT cells.

[0147] NK cells: NK cells in PBMCs are activated with NK cell activators and cultured in a culture medium containing cytokines such as IL-2 / IL-15 / IL-21 for 9 to 11 days to obtain NK cells.

[0148] Before freezing the cells, resuspend the cells with a cryoprotectant containing DMSO. The same cell type was cryopreserved according to the traditional freezing program (using Thermo's programmable cooling instrument model 7451TF to freeze cells and using CoolCell cell program cooling box to freeze cells) and the rapid freezing program. After thawing, the cell viability was tested. At the same time, the cells containing the cryoprotectant were placed at room temperature for at least 5 hours, during which the cell viability was monitored to compare the two freezing programs to maintain the viability and stability of different immune cells. The thawing cells were inoculated in a culture medium containing 10% FBS and continued to be cultured for 24 hours, and the cell survival rate after culture was tested.

[0149] The results are shown in Table 5 and Figure 3 As shown in Table 5, the viability stability of different types of immune cells after cryopreservation and thawing and the cell viability after 24 hours of culture are shown in Table 5. Figure 3 A~E show the viability stability of different types of immune cells after cryopreservation and recovery under two cryopreservation schemes. The horizontal axis represents different test times after cell recovery, and the vertical axis represents cell viability. Figure 3 F-G summarize the viability of 16 batches of various types of immune cells including PBMC, αβT, γδT, NK and DC cells after cryopreservation and 24-hour culture. The horizontal axis represents the two cryopreservation schemes, and the vertical axis represents the cell viability. The paired T test was used to perform statistical analysis on the results. The results showed that the rapid cryopreservation cells could maintain a better viability stability of PBMC, DC, αβT, γδT and NK after thawing. At the same time, the cell viability was compared after cell cryopreservation and inoculation and culture for 24 hours. The rapid cryopreservation scheme was significantly better than the traditional cryopreservation procedure ( Figure 3 F, paired t test P = 0.0006; Figure 3 G, Paired t test P = 0.0299).

[0150] Table 5 Comparison of cell viability and stability of cryopreserved immune cells under two cryopreservation schemes

[0151]

[0152]

[0153] Example 4 Rapid freezing scheme to maintain the viability and stability of various chimeric antigen receptor modified immune cells

[0154] Prepare and culture immune cells modified with chimeric antigen receptors of different types and targets. The main cell types and culture methods are as follows:

[0155] CAR-T: T cells in PBMC are activated with antibodies or magnetic beads containing CD3 / CD28, and lentiviral vectors targeting CD19, BCMA or CEA are transduced to prepare CAR-T cells with chimeric antigen receptors targeting CD19, BCMA or CEA. The cells can be collected the next day after viral transduction; or they can be expanded and cultured for 8 to 11 days using cell culture medium containing cytokines such as IL-2 / IL-7IL-15 / IL-21 or a combination thereof, and the CAR-T cells can be harvested.

[0156] CAR-NK: Activate NK cells in PBMC with NK cell activators, transduce lentiviral vectors containing CAR, prepare CAR-NK cells, culture them in culture medium containing cytokines such as IL-2 / IL-21 for 8 to 11 days, and harvest CAR-NK cells.

[0157] Before freezing the cells, resuspend the cells with a cryoprotectant containing DMSO. The same cell type was cryopreserved according to the traditional freezing program (using Thermo's programmable cooling instrument model 7451TF to freeze cells and using CoolCell cell program cooling box to freeze cells) and the rapid freezing program. After thawing, the cell viability was tested. At the same time, the cells containing the cryoprotectant were placed at room temperature for at least 5 hours, during which the cell viability was monitored to compare the two freezing programs to maintain the viability and stability of different immune cells. The thawing cells were inoculated in a culture medium containing 10% FBS and continued to be cultured for 24 hours, and the cell survival rate after culture was tested.

[0158] The results are shown in Table 6 and Figure 4 As shown in Table 6, the viability stability of immune cells modified with different types of chimeric antigens after cryopreservation and thawing under two cryopreservation schemes and the cell viability after 24 hours of culture are shown. Figure 4 A to B summarize the cell viability of 28 batches of immune cells modified with various types of chimeric antigens, including CD19 CAR-T, CEA CAR-T, BCMACAR-T and CAR-NK, frozen in two freezing schemes after cryopreservation and 24 hours of culture. The horizontal axis represents the two freezing schemes, and the vertical axis represents the cell viability. The paired T test was used to perform statistical analysis on the results. The results showed that the rapid frozen cells could maintain the better viability stability of CAR-T cells and CAR-NK cells of various targets after recovery. At the same time, the cell viability was compared after cell freezing and inoculation and culture for 24 hours. The rapid freezing scheme was significantly better than the traditional freezing procedure. ( Figure 4 A, Paired t test P = 0.0332; Figure 4 B, Paired t test P<0.0001).

[0159] Table 6 Comparison of the viability and stability of chimeric antigen modified immune cells cryopreserved under two cryopreservation schemes

[0160]

[0161]

[0162]

[0163] Example 5 Rapid freezing scheme improves the anti-tumor function and IFN-γ secretion level of CAR-T cells

[0164] CAR-T cells with chimeric antigen receptors targeting CD19, BCMA or CEA were prepared according to the culture scheme in Example 3. A total of 26 batches of cells were cryopreserved according to traditional procedures (cells were cryopreserved using Thermo's programmable cooling instrument model 7451TF and cells were cryopreserved using CoolCell cell program cooling box) and rapid freezing schemes. The revived CAR-T cells were co-cultured with tumor cell lines expressing CD19, BCMA or CEA antigens for 24 hours, and the tumor killing ability of CAR-T cells was detected. At the same time, 21 batches of culture supernatants were collected, and the IFN-γ secretion level in the supernatant was detected by ELISA.

[0165] The results are shown in Table 7 and Figure 5 As shown in Table 7, the target cell killing ability and IFN-γ secretion level of CAR-T cells frozen by two freezing schemes are compared. Figure 5 A The horizontal axis represents the two freezing schemes, and the vertical axis represents the tumor cell killing ratio. Figure 5 B The horizontal axis is the two freezing schemes, and the vertical axis is the secretion of IFN-γ by CAR-T cells. The paired T test was used to perform statistical analysis on the results, and the results showed that the rapid freezing technology maintained better killing ability and factor secretion ability of CAR-T cells. ( Figure 5 A, Paired ttest P = 0.0038; Figure 5 B, Pairedt test P = 0.0077).

[0166] Table 7 Comparison of the target cell killing ability and IFN-γ secretion level of CAR-T cells frozen by two cryopreservation schemes

[0167]

[0168]

[0169] Example 6 Rapid freezing scheme is suitable for freezing cell products of different densities and is superior to programmed freezing

[0170] CAR-T cells targeting CD19 chimeric antigen receptor were prepared according to the CAR-T cell culture scheme in Example 3. The cell density was adjusted to 5E+05 / mL, 1E+06 / mL, 1E+07 / mL, and 1E+08 / mL when the cells were harvested, and they were frozen according to the traditional freezing program (using CoolCell cell program cooling box to freeze the cells) and the rapid freezing program, respectively.

[0171] The results are shown in Table 8, which compares the viability, tumor cell killing ratio and IFN-γ secretion level of CAR-T cells frozen at different densities by two freezing schemes. The revived cells were inoculated in a medium containing 10% FBS and continued to be cultured for 24 hours, and the cell survival rate after culture was detected.

[0172] The results are as follows Figure 6 As shown, the horizontal axis represents the different concentrations of frozen cells, and the vertical axis represents the 24-hour viability of cells recovered by the two freezing schemes. The results show that the cell viability of the rapid freezing scheme is better than that of the programmed freezing at different frozen cell concentrations, especially when frozen at 5E+05 / mL and 1E+06 / mL densities. The cells frozen by the non-programmed freezing scheme protected by the present invention have better killing and factor secretion capabilities after recovery. The recovered CAR-T was co-cultured with the tumor cell line expressing the CD19 antigen for 24 hours to detect the tumor killing ability of the CAR-T cells, and the culture supernatant was collected at the same time, and the IFN-γ secretion level in the supernatant was detected by ELISA.

[0173] The results are as follows Figure 7 As shown, Figure 7 A The horizontal axis represents the different concentrations of cryopreserved cells, and the vertical axis represents the tumor cell killing ratio of the two cryopreservation schemes. Figure 7 B The horizontal axis represents the different concentrations of cryopreserved cells, and the vertical axis represents the IFN-γ secretion of CAR-T cells in the two cryopreservation schemes. The paired T test was used to analyze the results. The results showed that the rapid cryopreservation scheme significantly improved the tumor killing function and IFN-γ secretion level of CAR-T cells at different cryopreservation cell concentrations. ( Figure 7 A, Paired t test P = 0.0188; Figure 7 B, Paired ttest P=0.0392).

[0174] Table 8 Comparison of the viability, killing and IFN-γ secretion levels of CAR-T cells frozen at different densities under two cryopreservation schemes

[0175]

[0176] Example 7 Rapid freezing scheme is applicable to different types of cryopreservation protection solutions and is superior to programmed freezing

[0177] Cryopreservation medium:

[0178] Compound electrolyte injection 31.25% (v / v)

[0179] Glucose and Sodium Chloride Injection 15.63% (v / v)

[0180] Human albumin injection 20.00% (v / v)

[0181] Dextran 40 Glucose Injection 16.67% (v / v)

[0182] DMSO 7.5%(v / v)

[0183] Glucose injection 8.95% (v / v).

[0184] According to the culture schemes in Examples 2 and 3, PBMC and CAR-T cells with different targets were prepared. After collecting the cells, the cells were resuspended with the above-mentioned cryopreservation protection medium, and the cells were cryopreserved and revived according to the traditional cryopreservation program (using CoolCell cell program cooling box to cryopreserve cells) and the rapid cryopreservation program, respectively. After resuscitation, the cells were tested for viability, and the cells containing cryopreservation protection agents were placed at room temperature for at least 5 hours, during which the cell viability was monitored, and the two cryopreservation programs were compared to maintain the viability and stability of different immune cells. The revived cells were inoculated in a culture medium containing 10% FBS and continued to be cultured for 24 hours, and the cell survival rate after culture was detected.

[0185] The results are shown in Table 9 and Figure 8 As shown, the viability and stability of CAR-T cells after CAR-T cells are revived by freezing the CAR-T cells using different freezing methods in the cryopreservation protection solution of the present invention are shown. Figure 8 A The horizontal axis represents the different test times after cell recovery, and the vertical axis represents the cell viability of the two freezing schemes. Figure 8 B The horizontal axis represents different freezing schemes, and the vertical axis represents the 24-hour cell recovery rate of the two freezing schemes ( Figure 8 B, Paired ttest P = 0.0032). The results showed that the cell viability stability of rapid frozen cells was better than that of programmed frozen cells after 24 hours of cryopreservation. CAR-T cells were co-cultured with tumor cell lines expressing target antigens for 24 hours to detect the tumor killing ability of CAR-T cells. The culture supernatant was collected and the IFN-γ secretion level in the supernatant was detected by ELISA.

[0186] The results are shown in Table 10 and Fig. 9 As shown in Table 10, the target cell killing ability and IFN-γ secretion level of CAR-T cells frozen by cryopreservation protection solution using different freezing schemes are shown. Fig. 9The horizontal axis represents different cryopreservation schemes, and the vertical axis represents the IFN-γ secretion of CAR-T cells in the two cryopreservation schemes. The paired T test was used to analyze the results. The results showed that the rapid cryopreservation scheme maintained a more stable CAR-T cell tumor killing function under different types of cryopreservation fluid conditions, and the IFN-γ secretion level was significantly better than that of the programmed cryopreservation ( Fig. 9 , Paired t test P=0.0105).

[0187] Table 9 Viability and stability of CAR-T cells cryopreserved by cryoprotectant program and rapid cryopreservation

[0188]

[0189]

[0190] Table 10 Target cell killing ability and IFN-γ secretion level of CAR-T cells frozen in different cryopreservation protection solutions

[0191]

[0192] Example 8 Rapid freezing solution is applicable to different freezing packaging methods and is superior to programmed freezing

[0193] PBMC and CAR-T cells with different targets were prepared according to the culture schemes in Examples 2 and 3, and the cells were cryopreserved using three different types and materials of packaging materials, namely PP cryopreservation tubes, glass vials, and EVA cryopreservation bags. The revived cells were inoculated in a culture medium containing 10% FBS and cultured for 24 hours, and the cell survival rate after culture was detected.

[0194] The results are shown in Table 11 and Fig.10 As shown in the results, Table 11 shows the viability of CAR-T cells frozen in different cryopreservation materials and packaging. Fig.10 A The horizontal axis represents different cryopreservation packaging materials, and the vertical axis represents the 24-hour viability of cells recovered under the two cryopreservation schemes. Fig.10 B The horizontal axis represents two freezing schemes, and the vertical axis represents the 24-hour viability of frozen cells recovered by 3 different types and materials of packaging for a total of 15 batches. The paired T test was used to analyze the results. The results showed that the cell viability after recovery of the rapid freezing scheme was better than that of the programmed freezing scheme (using Thermo's programmable cooling instrument model 7451TF to freeze cells and using the CoolCell cell program cooling box to freeze cells) ( Fig.10 B, Paired t test P = 0.0059).

[0195] Table 11 Viability of CAR-T cells cryopreserved in different cryopreservation materials and packaging

[0196]

[0197]

[0198] Example 9 Rapid freezing scheme is applicable to different freezing specifications and is superior to programmed freezing

[0199] CAR-T cells were prepared according to the culture scheme in Example 3. After the cells were collected, they were cryopreserved using two cryopreservation schemes in different volumes of 1 mL, 5 mL, 25 mL, 50 mL and 70 mL, respectively. After recovery, the cells were tested for viability. At the same time, the cells containing cryopreservation protectants were placed at room temperature for at least 5 hours, during which the cell viability was monitored, and the two cryopreservation schemes were compared to maintain the viability and stability of different immune cells. After recovery, the cells were inoculated in a culture medium containing 10% FBS and cultured for 24 hours, and the cell survival rate after culture was detected.

[0200] The results are shown in Table 12 and Fig.11 As shown in the results, Table 12 shows the viability and stability of CAR-T cells frozen in different freezing specifications. Fig.11 A The horizontal axis represents the different test times after cell recovery, and the vertical axis represents the cell viability of the two freezing schemes. Fig.11 B The horizontal axis represents different freezing specifications, and the vertical axis represents the 24-hour cell viability of the two freezing schemes. Paired T test was used for data analysis, and the results showed that the stability and cell viability of the rapid freezing scheme were better than those of programmed freezing (using Thermo's programmable cooling instrument model 7451TF to freeze cells and using CoolCell cell program cooling box to freeze cells) ( Fig.11 B, Paired ttest P=0.0027).

[0201] Table 12 Viability and stability of CAR-T cells frozen in different cryopreservation specifications

[0202]

[0203]

[0204] Example 10 Rapid freezing scheme improves CAR-T cell tolerance to freezing solution

[0205] CAR-T cells were prepared according to the culture scheme in Example 3. After the cells were collected, they were resuspended in the freezing solution for 10 min, 30 min, 60 min and 120 min, and then cryopreserved by programmed freezing and rapid freezing. After recovery, the cells were inoculated in a culture medium containing 10% FBS and cultured for 24 hours, and the cell survival rate after culture was detected.

[0206] The results are shown in Table 13 and Fig.12The results are shown in Table 13. The viability of cells frozen for different freezing solution contact times and inoculated in a medium containing 10% FBS for 24 hours after thawing. Fig.12 A The horizontal axis represents the resuspension time of cells in the freezing solution, and the vertical axis represents the 24-hour viability of cells recovered under the two freezing solutions. Fig.12 B The horizontal axis represents two freezing schemes, and the vertical axis represents the summary of cells resuspended in freezing solution for different time periods and then frozen, and the cell viability after 24 hours of recovery. The results showed that the cell survival rate of programmed freezing (using CoolCell cell programmed cooling box to freeze cells) decreased when the cell resuspended time in freezing solution was extended to 60min and 120min. Although there was no statistical difference in cell viability, the cell viability of the rapid freezing method was more stable, which is beneficial to reduce the impact of long-term contact with freezing solution on cell freezing effect in industrial mass production and complex cell freezing procedures.

[0207] Table 13 Viability of cells frozen at different cryopreservation solution contact times

[0208]

[0209] Example 11 Rapid freezing solution can keep cells in -80 ultra-low temperature freezer for a long time

[0210] CAR-T cells were prepared according to the culture scheme in Example 3, and the cells were frozen by programmed freezing (using Thermo's programmable cooling instrument model 7451TF to freeze the cells) and rapid freezing. The programmed frozen cells were cooled to -80°C and immediately stored in liquid nitrogen. The rapidly frozen cells continued to be stored in an environment of -80°C and transferred to liquid nitrogen overnight. After 3 months of storage, the cells were revived and the cell viability was tested. At the same time, the cells containing the cryoprotectant were placed at room temperature for at least 5 hours, during which the cell viability was monitored, and the two freezing and storage schemes were compared to maintain cell viability and stability.

[0211] The results are shown in Table 14 and Fig.13 The results show that Table 14 shows the cell viability and stability under different freezing and storage schemes. Fig.13 A The horizontal axis represents the different test times after cell recovery, and the vertical axis represents the cell viability of the two freezing schemes. Fig.13 B The horizontal axis represents the two freezing schemes, and the vertical axis represents the viability of cells after recovery. The unpaired T test was used for data analysis. The results showed that the cells frozen by the rapid freezing scheme were stored in a -80℃ refrigerator and liquid nitrogen for at least 3 months. The viability and stability of cells after recovery were still better than those stored in liquid nitrogen by programmed freezing ( Fig.13 B, Pairedt test P = 0.0465).

[0212] Table 14 Cell viability and stability under different freezing and storage schemes

[0213]

[0214] Example 12 Rapid freezing scheme The frozen cells were thawed and revived in a 37°C water bath and at room temperature of 15-37°C

[0215] CAR-T cells were prepared according to the culture scheme in Example 3, and the cells were frozen by programmed freezing (using CoolCell cell programmed cooling box to freeze cells) and rapid freezing. When resuscitating cells, traditional 37°C water bath thawing and resuscitation were used, and the rapidly frozen cells were thawed and resuscitated at room temperature. The cells were inoculated in a medium containing 10% FBS and cultured for 24 hours, and the cell survival rate after culture was detected.

[0216] The results are shown in Table 15 and Fig.14 The results are shown in Table 15. The cell viability of the rapid freezing solution was obtained by water bath and room temperature recovery. The cells were inoculated in a medium containing 10% FBS and cultured for 24 hours. Fig.14 The horizontal axis represents different freezing and recovery schemes, and the vertical axis represents the cell survival rate after 24 hours of cell recovery and culture. The results show that the survival rate of cells frozen by the rapid freezing scheme is better than that of cells frozen by programmed freezing, and room temperature recovery has less effect on cell survival rate. This is conducive to simplifying the recovery method and reducing dependence on recovery equipment.

[0217] Table 15 Cell viability in water bath and room temperature thawing scheme

[0218]

[0219] Example 13 Rapid Cryopreservation and Programmed Cryopreservation Schemes for Cryopreservation of Stem Cells

[0220] To further test the feasibility of the rapid freezing scheme for stem cell cryopreservation, mesenchymal stem cells were passaged to the 5th to 9th generation, and after trypsin digestion, the cell concentration was adjusted to 5E+05 / ml~1.5E+07 / ml and resuspended in freezing solution. A total of 10 batches of cells were cryopreserved according to the two schemes of rapid freezing and programmed freezing (using CoolCell cell programmed cooling box to freeze cells). After recovery, the cell viability was tested, the cells were inoculated into culture flasks, cultured in mesenchymal stem cell culture medium for 3 days, and counted after trypsin digestion to calculate the cell proliferation multiples (proliferation multiples = total number of cells in culture for 3 days / total number of starting cells).

[0221] The results are shown in Table 16 and Fig.15 Table 16 shows the survival rate of mesenchymal stem cells after thawing by different cryopreservation methods and the proliferation multiples after 3 days of culture. Fig.15A The horizontal axis represents the two cryopreservation schemes, and the vertical axis represents the cell recovery rate. The paired T test was used to compare and analyze the cell viability. The results showed that the cell viability of stem cells recovered by rapid cryopreservation was significantly better than that of programmed cryopreservation ( Fig.15 A,Paired t test P=0.0424). Fig.15 B The horizontal axis represents the two freezing schemes, and the vertical axis represents the proliferation times of the revived cells after 3 days of culture. The results show that the proliferation times of cells frozen rapidly are better than those of programmed freezing.

[0222] In summary, the rapid freezing process is more suitable for cryopreservation of mesenchymal stem cells than the traditional programmed freezing scheme.

[0223] Table 16 Viability and proliferation of mesenchymal stem cells after thawing by different cryopreservation methods

[0224]

[0225] Example 14 Rapid freezing and programmed freezing protocols for freezing non-immune cells

[0226] Two non-immune cells, 293-T cells and MRC-5 fibroblasts, were digested with trypsin and resuspended in cryoprotectant. They were cryopreserved according to two schemes: rapid cryopreservation and programmed cryopreservation (using CoolCell cell programmed cooling box to cryopreserve cells). After cell recovery, they were inoculated in FBS-containing culture medium and cultured for 24 hours and 48 hours. The cells were digested with trypsin and counted. The cell viability and number after cell recovery of the two cryopreservation schemes were compared.

[0227] The results are shown in Table 17 and Fig.16 As shown in Table 17, the viability and cell number of non-immune cells after cryopreservation and recovery are shown in Table 17. Fig.16 The horizontal axis represents the two freezing schemes, and the vertical axis represents the cell viability and cell number. The paired T test was used to analyze the results and showed that the cell viability and number after rapid freezing of 293-T cells were significantly better than those after programmed freezing ( Fig.16 A, Paired t test P=0.0071, Fig.16 B, Paired t test P = 0.0037). Two cryopreservation schemes were used to cryopreserve MRC-5 fibroblasts. The programmed cryopreservation scheme after cell recovery was superior to rapid cryopreservation in maintaining cell viability and cell number. The results suggest that the traditional programmed cryopreservation scheme still has advantages for non-immune cells and non-stem cells.

[0228] Table 17 Viability and cell number of non-immune cells after cryopreservation and thawing

[0229]

[0230] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Cell cryopreservation method, It is characterized in that The cells to be frozen are mixed with a cryoprotectant, pre-frozen, and then frozen to obtain the frozen cells; The pre-freezing temperature is -86°C to -70°C, and the time is 1 hour to 1 month; The pre-cryostorage and the cryopreservation do not include a gradient cooling step or a programmed cooling step.

2. The cryopreservation method according to claim 1, It is characterized in that The pre-freezing time is 1 hour to 24 hours or 7 days or 1 month.

3. The cryopreservation method according to claim 1 or 2, It is characterized in that The cryoprotectant comprises: compound electrolyte, human serum albumin and DMSO.

4. The cryopreservation method according to any one of claims 1 to 3, It is characterized in that The cryoprotectant also includes glucose and dextran 40.

5. The cryopreservation method according to claim 4, It is characterized in that The composite electrolyte, the human serum albumin, the glucose and the dextran 40 are all added in the form of injection.

6. The cryopreservation method according to any one of claims 1 to 5, It is characterized in that The cryoprotectant comprises:

7. The cryopreservation method according to any one of claims 1 to 6, It is characterized in that The pre-freezing carrier includes: one or more of a PP cryotube, a glass vial, and / or an EVA cryobag.

8. The cryopreservation method according to any one of claims 1 to 7, It is characterized in that The cells include: immune cells and / or stem cells.

9. The cryopreservation method according to any one of claims 1 to 8, It is characterized in that The immune cells include: one or more of PBMC cells, DC cells, αβT cells, γδT cells, NK cells, CAR-T cells and / or CAR-NK cells.

10. The cryopreservation method according to any one of claims 1 to 9, It is characterized in that The cell density is: 5×10 5 / mL~1×10 8 / mL.

11. Cell recovery method, It is characterized in that The frozen cells obtained by the freezing method according to any one of claims 1 to 10 are thawed to obtain thawed cells.

12. The resuscitation method according to claim 11, It is characterized in that The resuscitation temperature is: 15°C ~ 37°C.

13. Cell culture method, It is characterized in that The method comprises the cryopreservation method according to any one of claims 1 to 10 and / or the resuscitation method according to claim 11 or 12.

14. The culture method according to claim 13, It is characterized in that The cells obtained by the resuscitation method are cultured; the culture time is 1 hour to 72 hours.

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