Cell cryopreservation liquid, preparation method, application and cell cryopreservation method

Abstract

The invention provides a cell cryopreservation solution, a preparation method, application and a cell cryopreservation method, and belongs to the technical field of cell cryopreservation. According to the invention, CS10 is mixed with dextran 40 glucose injection, compound electrolyte injection and human serum albumin injection to prepare the cell freezing medium provided by the invention. The cell cryopreservation liquid can effectively reduce cell damage, improve cell activity and maintain the killing effect of cells, solves the problem that the cell survival rate or killing effect is obviously reduced after resuscitated cells are stored in an existing cell cryopreservation liquid, and compared with CS10 and a current common cell cryopreservation liquid, the cell cryopreservation liquid has a better protection effect, and the cell cryopreservation liquid is suitable for being used in the field of cell cryopreservation. The viability of the cells within 3 days after the cells are recovered can be recovered to a state similar to that before the cells are cryopreserved, and the CAR-NK cells which are frozen and recovered by using the cell cryopreservation liquid still have good killing activity.

Description

Technical Field

[0001] The present invention relates to the technical field of cell cryopreservation, and in particular to a cell cryopreservation solution, a preparation method, an application and a method for cryopreserving cells. Background Art

[0002] NK is an important cell type in the human immune system. They have the ability to recognize and kill cancer cells and infected cells without prior activation, so they are regarded as the first line of defense against cancer. CAR-NK is obtained by transcribing the artificially synthesized CAR structure onto NK cells through genetic engineering technology. This technology is equivalent to equipping NK cells with a "GPS" device, so that while retaining the original function of NK cells, it can accurately lock the target on specific antigen proteins, thereby killing cancer cells more effectively. As important immune cells, NK and CAR-NK cells play a key role in tumor immunotherapy. However, the cryopreservation of NK and CAR-NK cells still faces some technical challenges.

[0003] First, how to minimize or completely avoid the use of toxic substances such as DMSO during the cryopreservation of NK and CR-NK cells is an important issue. Researchers are exploring DMSO-free cryopreservation methods, such as using substitutes such as sucrose and glycerol, to improve the activity and function of NK and CR-NK cells after thawing. These alternative cryopreservation agents can effectively reduce cell damage and improve cell survival rate.

[0004] Secondly, how to maintain the activity and killing ability of NK and CR-NK cells after freezing is also a key issue. Studies have found that reasonable freezing conditions and thawing methods can protect the function of NK cells to the greatest extent. For example, the use of slow freezing and rapid thawing methods can effectively reduce cell damage and improve cell activity. In addition, adding some protective agents such as bovine serum albumin, lactose, etc. can also increase the survival rate of NK and CAR-NK cells during freezing and thawing.

[0005] At present, the decrease in viability and killing ability of NK and CAR-NK cells after cryopreservation is a technical difficulty that needs to be solved urgently. By developing new cryopreservation media, optimizing the preparation and cryopreservation process, and performing personalized treatment on cells from different sources, the performance of CAR-NK cells after cryopreservation can be effectively improved, laying the foundation for the clinical application of CAR-NK cell therapy. Therefore, a cryopreservation fluid with good cryopreservation effect is urgently needed. Summary of the invention

[0006] The purpose of the present invention is to provide a cell freezing solution, a preparation method, an application and a method for freezing cells. The cell freezing solution of the present invention can effectively reduce cell damage, improve cell activity, and maintain the cell killing effect, thereby solving the problem that the cell survival rate or killing effect of the existing cell freezing solution significantly decreases after the cells are stored and revived, and laying a good foundation for the clinical application of cell therapy.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The invention provides a cell freezing solution, which is composed of the following raw materials in percentage by volume: 50-75% of cell cryoprotectant solution CS10, 10-20% of dextran 40 glucose injection, 10-50% of compound electrolyte injection, and 5-30% of human albumin injection, and the total content of each raw material is 100%.

[0008] The present invention also provides a method for preparing a cell freezing solution, comprising the following steps: uniformly mixing a cell cryoprotectant solution CS10, a dextran 40 glucose injection, a compound electrolyte injection, and a human albumin injection in a certain proportion to obtain a cell freezing solution.

[0009] The present invention also provides a cell freezing solution or the use of the cell freezing solution prepared by the preparation method of the cell freezing solution in cell freezing.

[0010] Preferably, the cells include NK cells and CAR-NK cells.

[0011] The present invention also provides a method for freezing cells using a cell freezing solution, comprising the following steps: The cells are resuspended in a cell freezing solution to obtain a cell suspension, which is then placed in a freezing bag and cryopreserved using a programmed cooling device.

[0012] Preferably, the freezing program of the programmed cooling instrument is as follows: Step1: Wait at 4.0℃; Step2: 1.0℃ / mS to -10℃; Step3: 25.0℃ / mC to -50℃; Step4: 2℃ / mC to -40℃; Step5: 1.0℃ / mC to -45℃; Step6: 10.0℃ / mC to -90℃; Step7: End.

[0013] Preferably, the concentration of the cell suspension is 1×10 7 / mL~1×10 8 Pieces / mL.

[0014] The present invention also provides a method for freezing cells using a cell freezing solution, characterized in that it comprises the following steps: Resuspend the cells in cell freezing solution to obtain cell suspension, put the cell suspension into a cryopreservation tube, cover the lid, place the cryopreservation tube containing the cell suspension into the hole of the programmed cooling box, ensure that the tube is fully inserted, place the programmed cooling box in a -80°C refrigerator overnight, and transfer it to a liquid nitrogen tank for long-term storage the next day.

[0015] The beneficial effects of the present invention compared with the prior art are: (1) The present invention mixes CS10 with dextran 40 glucose injection, compound electrolyte injection, and human albumin injection to prepare the cell cryopreservation solution of the present invention. The cell cryopreservation solution of the present invention can effectively reduce cell damage, improve cell activity, and maintain the cell killing effect, solving the problem that the cell survival rate or killing effect is significantly reduced after the existing cell cryopreservation solution is used to store and resuscitate cells, and lays a good foundation for the clinical application of cell therapy. Compared with CS10 and the currently commonly used cell cryopreservation protection solution, the cell cryopreservation solution of the present invention has a better protection effect. The cell viability can be restored to a state similar to that before cell cryopreservation within 3 days after cell resuscitation, and the CAR-NK cells frozen and revived using the cell cryopreservation solution of the present invention still have good killing activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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 will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0017] Figure 1 is the viability of NK cells after cryopreservation and resuscitation in Experimental Example 1 of the present invention; Figure 2 is the viability of CAR-NK cells after cryopreservation and resuscitation in Experimental Example 2 of the present invention; Figure 3 This is the evaluation of the killing ability of CAR-NK cells after cryopreservation and resuscitation in Experimental Example 3 of the present invention, E:T=2:1. DETAILED DESCRIPTION

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0020] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0021] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0022] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0023] Example 1 Embodiment 1 of the present invention provides a method for preparing a cell freezing solution, which specifically comprises the following steps: Take cell cryoprotectant CS10 (US Biolife brand CryoStor clinical-grade ready-to-use cryopreservation solution), dextran 40 glucose injection (national medicine standard number: H51020230), compound electrolyte injection (national medicine standard number: H20000475), and human albumin injection, mix the above solutions in a volume ratio of 5:1:3:1 to obtain cell cryopreservation solution (QM cryopreservation solution).

[0024] Test Example 1 Experimental Example 1 of the present invention adopts a programmed cooling box freezing method, uses isopropanol as a freezing medium, and detects the freezing effect of the cell freezing solution (QM freezing solution) prepared in Example 1 on NK cells. The specific steps are as follows: (1) Take the same batch of NK cells with the same viability and resuspend the cells in cell cryopreservation solution (QM cryopreservation solution), 10% DMSO + autologous plasma, CS10, commercial cryopreservation solution (control-1), and commercial cryopreservation solution (control-2) to obtain a concentration of 1×10 8 / mL cell suspension, put the cell suspension into the cryopreservation tube, cover the lid, put the cryopreservation tube containing the cell suspension into the hole of the program cooling box, ensure that the tube is fully inserted, place the program cooling box in a -80℃ refrigerator overnight, and transfer it to a liquid nitrogen tank for storage the next day.

[0025] (2) The viability of NK cells after cryopreservation and resuscitation is directly related to the killing and proliferation ability of NK cells after resuscitation. A high viability after resuscitation means that there are more NK cells with killing and proliferation ability, and the effect of killing tumor cells is more obvious, and the clinical treatment effect is more obvious. Therefore, the present invention uses the viability after cryopreservation and resuscitation as an important observation indicator, revives the cells frozen in step (1), and detects the cell survival rate. The results are shown in Tables 1 and Figure 1 shown.

[0026] The steps for resuscitation are as follows: First, heat the water bath to 37°C. Then, immerse the cell cryopreservation tube or cryopreservation bag in the water bath and observe the cryopreservation solution gradually melting until it is completely melted. Finally, take out the cryopreservation tube or bag and spray the surface with 75% alcohol for disinfection. Transfer to the biosafety cabinet for the next step.

[0027] Table 1 Evaluation of viability of NK cells after cryopreservation and resuscitation

[0028] From Table 1 and Figure 1 It can be seen that the viability of the cell cryopreservation solution (QM cryopreservation solution) reached 93% immediately after recovery, with the smallest decrease among the five cryopreservation solutions. 24 hours after recovery, theoretically, the viability of NK cells drops to the lowest point, and usually the viability drops to 50-60%. However, the cell cryopreservation solution (QM cryopreservation solution) can protect NK cells very well, and the viability of NK cells is as high as 80% at this time. The viability of subsequent cultured NK cells will gradually increase, and the viability will return to 83% after 48 hours of recovery, and to 90% after 72 hours of recovery. Within 3 days, it returned to a state similar to that before NK cells were frozen.

[0029] 10% DMSO + autologous plasma, this formula is the most common laboratory self-prepared cryopreservation solution formula, generally including 10% DMSO, 10-20% autologous plasma and 70-80% culture medium. This formula is simple to prepare and has the lowest cost, but the effect of freezing NK cells is the worst. Immediately after recovery, the viability reached 87%, and the decline was not obvious, but 24 hours after recovery, the viability dropped to 49%. The viability of subsequent cultured NK cells did not recover significantly, with a viability of 50% after 48 hours of recovery and 56% after 72 hours of recovery.

[0030] CS10 is the most widely used commercial cryopreservation solution for cryopreservation of immune cell products. It is widely used for cryopreservation of CAR-T, NK, and CAR-NK cell products. From the experimental comparison results of the present invention, CS10 can better protect the activity of NK cells after cryopreservation and resuscitation. Immediately after resuscitation, the viability rate reached 90%, and 24 hours after resuscitation, the viability rate dropped to 61%. After 48 hours, the viability rate was 68%, and after 72 hours, the viability rate was 80%.

[0031] Commercial cryopreservation solution control-1 (batch number: MED2403008) and control-2 (batch number: 20240312) are two domestically produced commercial cryopreservation solutions. Both are claimed to be used for the cryopreservation of CAR-T, NK, and CAR-NK cell products. However, from the experimental results, the ability of these two products to protect the activity of NK cells is not as good as CS10, and the gap is more obvious than that of cell cryopreservation solution (QM cryopreservation solution).

[0032] Test Example 2 Experimental Example 2 of the present invention adopted a programmed cooling box freezing method, using isopropanol as the freezing medium, and detected the freezing effect of the cell freezing solution (QM freezing solution) prepared in Example 1 on CAR-NK cells. The specific steps are as follows: (1) Take the same batch of CAR-NK cells with the same viability and resuspend the cells in cell cryopreservation solution (QM cryopreservation solution), 10% DMSO + autologous plasma, CS10, commercial cryopreservation solution (control-1), and commercial cryopreservation solution (control-2) to obtain a concentration of 1×10 8 / mL cell suspension, put the cell suspension into the cryopreservation tube, cover the lid, put the cryopreservation tube containing the cell suspension into the hole of the program cooling box, ensure that the tube is fully inserted, place the program cooling box in a -80℃ refrigerator overnight, and transfer it to a liquid nitrogen tank for storage the next day.

[0033] (2) The viability of CAR-NK cells after cryopreservation and resuscitation is directly related to the killing and proliferation ability of CAR-NK cells after resuscitation. A high viability after resuscitation means that there are more CAR-NK cells with killing and proliferation ability, and the effect of killing tumor cells is more obvious, and the clinical treatment effect is more obvious. Therefore, the present invention uses the viability after cryopreservation and resuscitation as an important observation indicator, revives the cells frozen in step (1), and detects the cell survival rate. The results are shown in Table 2 and Figure 2 shown.

[0034] Table 2 Evaluation of viability of CAR-NK cells after cryopreservation and resuscitation

[0035] From Table 2 and Figure 2 It can be seen that the viability of the cell cryopreservation solution (QM cryopreservation solution) reached 90% immediately after recovery, with the smallest decrease among the five cryopreservation solutions. 24 hours after recovery, the viability of CAR-NK cells was as high as 80%. The viability was 82% after 48 hours of recovery and 89% after 72 hours of recovery. CAR-NK cells resuscitated with the cell cryopreservation solution (QM cryopreservation solution) quickly returned to their pre-freezing state within 3 days. The cell cryopreservation solution (QM cryopreservation solution) can well protect the activity of CAR-NK cells and ensure that the killing effect is the best among the five cryopreservation solutions.

[0036] 10% DMSO + autologous plasma, although this formula is simple to prepare and has the lowest cost, it has the worst protection for the activity of CAR-NK cells. The viability rate continued to decline after resuscitation, with the 24-hour viability rate dropping to 45%, the 48-hour viability rate to 33%, and the 72-hour viability rate to 2%. In other words, 3 days after resuscitation, the CAR-NK cells in this group died completely, and the cryopreservation solution did not play a role in protecting the activity of CAR-NK cells.

[0037] Although CS10 is the most recognized cryopreservation product, the viability of CAR-NK cells was only 57% after 24 hours of recovery, which is much lower than the data of cell cryopreservation solution during the same period. The viability was 63% after 48 hours and 78% after 72 hours, indicating that the viability of CAR-NK cells has recovered.

[0038] Commercial cryopreservation fluids Control-1 and Control-2 are two domestically produced commercial cryopreservation fluids. From the experimental results, the two products are weaker than CS10 in protecting the activity of CAR-NK cells. Control-2 is stronger than Control-1. From the results of the post-recovery viability, Control-2 is close to the cryopreservation effect of CS10, but there is a significant gap with the cell cryopreservation fluid (QM cryopreservation fluid).

[0039] Test Example 3 Experimental Example 3 of the present invention adopts the cryopreservation method of step (1) of Example 2, and detects the effect of the cell cryopreservation solution prepared in Example 1 (QM cryopreservation solution) on the killing ability of CAR-NK cells after cryopreservation and resuscitation. The specific steps are as follows: The CAR-NK cells were cryopreserved in step (1) of Example 2 and revived, and then mixed with the target cells MM.1S-GFP at an effector-target ratio of 2:1. The killing effect on the target cells at different time points was detected by flow cytometry. The results are as follows: Figure 3 shown.

[0040] Figure 3 It shows that at 0h, it can be seen that the target ratio of NK and CAR-NK cells after cryopreservation and resuscitation in a total of 6 groups, including the control NK cells, is about 2:1. At 24hrs, except for the CAR-NK cells in the cell cryopreservation solution (QM cryopreservation solution) group, which still has more CAR-NK cells than target cells, the proportion of CAR-NK cells in the other groups has decreased significantly and is far lower than the target cells. This means that the number of CAR-NK cells has decreased and cannot effectively kill target cells. At 48hrs, as shown by the arrow, the proportion of CAR-NK cells in the cell cryopreservation solution (QM cryopreservation solution) group has further increased, and the proportion of target cells is very low, which means that the CAR-NK cells revived by the cell cryopreservation solution (QM cryopreservation solution) have good killing activity against target cells, and almost completely clear the target cells. The proportion of CAR-NK cells in the CS10 group has gradually exceeded the target cells. The CAR-NK cells in the other groups are still far lower than the target cells. This means that the killing activity of CAR-NK cells cannot be well protected in the other cryopreservation solution groups.

[0041] 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. A cell freezing solution, characterized in that: The invention is composed of the following raw materials in the following volume percentages: 50-75% of cell cryoprotectant solution CS10, 10-20% of dextran 40 glucose injection, 10-50% of compound electrolyte injection, and 5-30% of human albumin injection, and the total content of each raw material is 100%.

2. A method for preparing the cell freezing solution according to claim 1, characterized in that: The method comprises the following steps: uniformly mixing cell cryoprotective solution CS10, dextran 40 glucose injection, compound electrolyte injection and human albumin injection according to a certain proportion to obtain cell cryopreservation solution.

3. Use of the cell freezing solution according to claim 1 or the cell freezing solution prepared by the preparation method of the cell freezing solution according to claim 2 in cell freezing.

4. The use of the cell freezing solution in cell freezing according to claim 3, characterized in that: The cells include NK cells and CAR-NK cells.

5. A method for freezing cells using the cell freezing solution according to claim 1, characterized in that: The steps include: The cells are resuspended in a cell freezing solution to obtain a cell suspension, which is then placed in a freezing bag and cryopreserved using a programmed cooling device.

6. The method for freezing cells using the cell freezing solution according to claim 5, characterized in that: The freezing procedure of the program cooling instrument is as follows: Step1: Wait at 4.0℃; Step2: 1.0℃ / mC to -10℃; Step3: 25.0℃ / mC to -50℃; Step4: 2℃ / mC to -40℃; Step5: 1.0℃ / mC to -45℃; Step6: 10.0℃ / mC to -90℃; Step7: End.

7. The method for freezing cells using the cell freezing solution according to claim 5, characterized in that: The concentration of the cell suspension was 1×10 7 / mL~1×10 8 Pieces / mL.

8. A method for freezing cells using the cell freezing solution of claim 1, characterized in that: The steps include: Resuspend the cells in cell freezing solution to obtain cell suspension, put the cell suspension into a cryopreservation tube, cover the lid, place the cryopreservation tube containing the cell suspension into the hole of the programmed cooling box, ensure that the tube is fully inserted, place the programmed cooling box in a -80°C refrigerator overnight, and transfer it to a liquid nitrogen tank for long-term storage the next day.

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