CN114041456A

CN114041456A - Clinical-grade NK cell cryopreservation liquid and using method thereof

Info

Publication number: CN114041456A
Application number: CN202111497839.7A
Authority: CN
Inventors: 高雅丽; 周桃; 俞君英; 张颖; 焦璐琰
Original assignee: Nuwacell Ltd
Current assignee: Nuwacell Ltd
Priority date: 2021-12-09
Filing date: 2021-12-09
Publication date: 2022-02-15
Anticipated expiration: 2041-12-09
Also published as: CN114041456B

Abstract

The invention provides a clinical-grade NK cell cryopreservation liquid and a using method thereof, and belongs to the technical field of NK cell cryopreservation and recovery. The components contained in the clinical-grade NK cell frozen stock solution are all injection grade or clinical grade, and can be directly infused into a human body. In addition, the invention reduces the dosage of injection-grade albumin and DMSO, reduces the preparation cost so as to be suitable for mass production, and provides conditions for storage and clinical application of clinical-grade NK cells.

Description

Clinical-grade NK cell cryopreservation liquid and using method thereof

Technical Field

The invention relates to the technical field of NK cell cryopreservation and resuscitation, and particularly relates to a clinical-grade NK cell cryopreservation solution and a using method thereof.

Background

NK cells generally refer to natural killer cells (NK), are important immune cells of the body, are not only related to tumor resistance, virus infection resistance and immune regulation, but also participate in the occurrence of hypersensitivity and autoimmune diseases under certain conditions, and can recognize target cells and killing media. Common NK cells include NK cells derived from peripheral blood, NK cells derived from cord blood, NK cells derived from hPSC, and the like.

In recent years, NK cells have been used for cell therapy of tumor immunity and have a good effect in clinical treatment of tumors (such as leukemia, lymphoma and melanoma); however, the long-term efficient storage of NK cells is always a problem, both for third party cell banks and for clinical administration of delivery and application. The prior art mainly preserves the activity and function of NK cells by cryopreservation.

The prior NK cell frozen stock solution has various formulas. For example, chinese patent CN202010240894.7 discloses a NK cell cryopreservation solution, the cryopreservation solution formula comprises dextran, polylysine (CPLL) and basal medium, wherein CPLL is prepared from reagent grade succinic anhydride and epsilon-polylysine solution, the preparation process is complex, batch-to-batch differences are easy to occur, and CPLL and basal medium are reagent grade and cannot be used for clinical and direct infusion. Chinese patent CN201510727721.7 discloses a frozen stock solution of cultured NK cells, which contains tea polyphenols, grape seed extract, vitamin C and autologous serum, wherein the tea polyphenols, grape seed extract and vitamin C are not clinical grade components and the components of the extract are not clear, and can not be used for clinical and direct infusion. Chinese patent CN201910274568.5 discloses a cryopreservation method of NK cells, wherein the cryopreservation liquid is added with AB plasma and 5-glutenin-3-ol to improve the activity of the cells after cryopreservation recovery. The AB plasma has undefined components and different batches, and is not suitable for large-scale and standardized production and preparation; 5-glutenin-3-ol is a non-clinical grade component and cannot be used for clinical and direct infusion. In summary, the cryopreservation solution disclosed in the prior art can only be used for preservation of scientific research-grade products, but cannot be used for clinical injection.

Disclosure of Invention

In view of the above, the invention aims to provide a clinical-grade NK cell frozen stock solution, and the components of the clinical-grade NK cell frozen stock solution are injection-grade and clinical-grade medicines, and can be used for clinical injection.

The invention provides a clinical-grade NK cell frozen stock solution, which comprises a first frozen stock solution and a second frozen stock solution;

the first cryopreservation liquid comprises clinical grade basic cryopreservation liquid and an impermeable cryoprotectant; the mass concentration of the non-permeable cryoprotectant in the first frozen stock solution is less than or equal to 10 percent;

the second frozen stock solution comprises clinical grade basic frozen stock solution, an impermeable cryoprotectant and a permeable cryoprotectant; the mass concentration of the non-permeable cryoprotectant in the second frozen stock solution is less than or equal to 10 percent; the mass concentration of the permeable cryoprotectant in the second freezing solution is less than or equal to 20 percent;

the clinical grade basic freezing solution comprises dextran sodium chloride injection;

the non-osmotic cryoprotectant comprises injection grade albumin;

the osmotic cryoprotectant comprises injection grade DMSO.

Preferably, the dextran sodium chloride injection comprises dextran sodium chloride injection with a mass concentration of 10% or dextran sodium chloride injection with a mass concentration of 6%.

Preferably, the dextran sodium chloride injection is selected from one or more of dextran 40 sodium chloride injection, dextran 70 sodium chloride injection or dextran 20 sodium chloride injection.

Preferably, the mass concentration of the non-permeable cryoprotectant in the first frozen stock solution and the second frozen stock solution is independently 1-10%.

Preferably, the mass concentration of the osmotic cryoprotectant in the second frozen stock solution is 2 to 20 percent.

The invention also provides application of the clinical-grade NK cell cryopreservation liquid in the scheme in NK cell cryopreservation and/or iNK cell cryopreservation.

The invention also provides a use method of the clinical NK cell frozen stock solution, which comprises the following steps:

and mixing the NK cells and/or iNK cells to be cryopreserved, the first cryopreserving liquid and the second cryopreserving liquid, and then carrying out sectional cooling, wherein the cryopreserving is carried out after the sectional cooling.

Preferably, the mixing of the NK cells and/or iNK cells to be cryopreserved, the first cryopreservation solution and the second cryopreservation solution is that the NK cells and/or iNK cells to be cryopreserved are mixed with the first cryopreservation solution and then are mixed with the second cryopreservation solution.

Preferably, the final freezing density of the NK cells and/or iNK cells to be frozen is 1-8 x 10⁷/mL。

Preferably, the volume ratio of the first frozen stock solution to the second frozen stock solution is (1-2): (1-2).

The invention provides a clinical-grade NK cell frozen stock solution, which comprises a first frozen stock solution and a second frozen stock solution; the first cryopreservation liquid comprises clinical grade basic cryopreservation liquid and an impermeable cryoprotectant; the mass concentration of the non-permeable cryoprotectant in the first frozen stock solution is less than or equal to 10 percent; the second frozen stock solution comprises clinical grade basic frozen stock solution, an impermeable cryoprotectant and a permeable cryoprotectant; the mass concentration of the non-permeable cryoprotectant in the second frozen stock solution is less than or equal to 10 percent; the mass concentration of the permeable cryoprotectant in the second freezing solution is less than or equal to 20 percent; the clinical grade basic freezing solution comprises dextran sodium chloride injection; the non-osmotic cryoprotectant comprises injection grade albumin; the osmotic cryoprotectant comprises injection grade DMSO. The components contained in the clinical-grade NK cell frozen stock solution are all injection grade or clinical grade, and can be directly infused into a human body. In addition, the invention reduces the dosage of injection-grade albumin and DMSO, reduces the preparation cost so as to be suitable for mass production, and provides conditions for storage and clinical application of clinical-grade NK cells. The cells frozen by the clinical-grade NK cell frozen stock solution can be directly infused into a human body, so that the clinical application of NK cell products is greatly promoted, the safety and the stability of clinical application of NK cell preparations can be improved, the pollution risk caused by operations such as centrifugal resuspension after cell resuscitation and the like can be avoided, the damage of the cells and the quality difference before infusion caused by the damage of the cells can be avoided, and the threshold of clinical application of the cell products can be reduced. The vicinity of the clinical infusion point does not need to be equipped with sterile operating facilities and experienced operators.

Secondly, the clinical-grade NK cell cryopreservation protective solution can maintain high survival rate and high recovery rate of the recovered NK cells and maintain the function of the NK cells in cracking tumor cells. The cell preparation prepared by the frozen stock solution has high stability after recovery, can maintain the cell survival rate and the recovery rate for a long time (8 hours) higher than 90 percent under the condition of high cell density and different temperatures, and ensures the stability of the cell preparation in clinical application.

In addition, the NK cell frozen stock solution provided by the invention reduces the use concentration of DMSO (dimethyl sulfoxide), and can avoid or reduce adverse reactions of a patient caused by infusion of a large amount of DMSO. DMSO is infused into a patient in a large amount in a short time, which is easy to cause adverse reactions, and has main systemic side reactions: headache, nausea, dizziness, and in patients who are secondarily sensitive to DMSO, respiratory disturbance and mild cardiotoxicity, hypertension or bradycardia occur, so that lowering the DMSO content in the clinically injected cells can reduce adverse reactions caused by cell infusion. Meanwhile, DMSO has certain toxic and side effects on cells at normal temperature, and the reduction of the content of DMSO is beneficial to the maintenance of the cell survival rate and functions after recovery. In addition, the common frozen stock solution has the problems of great cell loss, cell viability, proliferation capacity and differentiation capacity reduction caused by the operation of centrifugally removing DMSO after cells need to be recovered, the content of DMSO in the frozen stock solution is reduced, the centrifugation step can be omitted, the operation can be simplified, the subsequent cell differentiation and proliferation are facilitated, and the problems of pollution and the like in the production process of cell products are reduced.

Thirdly, the clinical-grade NK cell freezing solution has high freezing cell density, and the freezing final density of the NK cells to be frozen can reach 8-10 multiplied by 10⁷and/mL. The clinical single infusion amount of NK cells is large, so that the cryopreserved high-density cell preparation has a plurality of benefits for clinical application, the storage and the transportation are convenient, the cryopreserved volume is reduced, the resuscitation time is shortened, the cell injury and the pollution risk can be avoided, and the infusion frequency of a tested body is reduced.

Finally, the NK cell cryopreservation liquid provided by the invention can be used for cryopreservation of not only primary NK cells but also iNK cells. iNK cells induced by iPSC have obvious clinical application advantages compared with primary NK cells, and the NK cell frozen stock solution provided by the invention can promote clinical application of the primary NK cells and iNK cells.

Drawings

FIG. 1 is a flow chart of the cryopreservation and resuscitation of NK cells in the present invention;

FIG. 2 recovery rate and recovery rate of frozen stock solution after cryopreservation of NK cells;

FIG. 3 morphology of NK cells after 2 days of resuscitation;

FIG. 4 survival rate and recovery rate of NK cells after cryopreservation recovery under different proportions of albumin;

FIG. 5 survival rate and recovery rate of NK cells after cryopreservation recovery under different cryopreservation densities of the cells;

FIG. 6 survival and recovery of NK cells after cryopreservation recovery at different DMSO concentrations;

FIG. 7 CD56+/CD 3-expression of NK cryopreserved cells and fresh cells;

FIG. 8 expression of surface marker receptors of NK cryopreserved cells and fresh cells;

FIG. 9 cell viability after recovery of NK cells (preservation in frozen stock solution for a certain time after recovery);

FIG. 10 recovery rate after recovery of NK cells (preservation in frozen stock solution for a certain time after recovery);

FIG. 11 tumor cell lysis capacity after NK cell recovery (after 8 hours of preservation in frozen stock solution after recovery);

FIG. 12 shows that NK cryopreservation solution has cryopreservation protection effect on primary NK cells;

FIG. 13 lytic capacity of NK cryopreserved cells and fresh cells on different tumor cells;

FIG. 14NK cryopreserved cells can significantly inhibit the proliferation of tumor cells in K562 mouse tumor model.

Detailed Description

the first cryopreservation liquid comprises clinical grade basic cryopreservation liquid and an impermeable cryoprotectant; the mass concentration of the non-permeable cryoprotectant in the first frozen stock solution is less than or equal to 10 percent; the second frozen stock solution comprises clinical grade basic frozen stock solution, an impermeable cryoprotectant and a permeable cryoprotectant; the mass concentration of the non-permeable cryoprotectant in the second frozen stock solution is less than or equal to 10 percent; the mass concentration of the permeable cryoprotectant in the second freezing solution is less than or equal to 20 percent;

the non-osmotic cryoprotectant comprises injection grade albumin;

the osmotic cryoprotectant comprises injection grade DMSO.

In the present invention, the dextran sodium chloride injection preferably includes dextran 40 sodium chloride injection, dextran 70 sodium chloride injection or dextran 20 sodium chloride injection.

In the invention, the dextran sodium chloride injection preferably comprises dextran sodium chloride injection with the mass concentration of 10% or dextran sodium chloride injection with the mass concentration of 6%. In the invention, the mass concentration of sodium chloride in the dextran sodium chloride injection is preferably 0.9%. In the present invention, the mass concentration of the impermeable cryoprotectant in the first frozen stock solution and the second frozen stock solution is independently preferably 1% to 10%, and more preferably 3% to 8%.

In the present invention, the mass concentration of the osmotic cryoprotectant in the second cryopreservation solution is preferably 2% to 20%, more preferably 3% to 16%, even more preferably 4% to 10%, and most preferably 5% to 8%.

The source of the injection-grade albumin is not particularly limited in the invention, and commercially available clinical albumin can be used.

The source of the injection-grade DMSO is not particularly limited in the invention, and the injection-grade DMSO sold in the market can be used.

In the invention, the dextran sodium chloride injection is from conventional market. The dextran sodium chloride injection is not limited to the above, and other dextran sodium chloride injections which can be used in clinical grade can be included. Wherein the dextran can be selected from dextran 70, dextran 40 and dextran 20, i.e. the dextran sodium chloride injection can be selected from dextran 70, dextran 40 and dextran 20 sodium chloride injection. Further, the dextran sodium chloride injection may be selected from 10% or 6% dextran 70 or dextran 40 or dextran 20 sodium chloride injection. Such as: 6% dextran 20 sodium chloride injection: 500ml of dextran 20 30g and sodium chloride 4.5 g; 6% dextran 40 sodium chloride injection: 500ml, 30g of dextran 40 and 4.5g of sodium chloride and the like.

In the invention, the dextran sodium chloride injection contains dextran which belongs to macromolecular saccharides and has a freezing protection effect on cells, and the dextran partially replaces the freezing protection function of DMSO (dimethyl sulfoxide), so that the concentration of the DMSO can be greatly reduced. In addition, the dextran has the functions of protecting and promoting the survival of NK cells, and the great reduction of the concentration of the DMSO can reduce the damage to the cryopreserved cells, so that the clinical-grade cell cryopreservation solution can improve the stability of the cryopreserved cells.

The invention has no special limitation on the source and preparation method of the NK cells and iNK cells, and in the specific implementation process of the invention, the NK cells are preferably derived from peripheral blood or umbilical cord blood, and can be isolated, cultured and amplified by adopting the conventional technical means in the field; said iNK is preferably obtained by hPSC (pluripotent stem cell) differentiation, and the method of differentiation is preferably the method disclosed in Chinese patent CN202010153358.3 for differentiating human pluripotent stem cells into natural killer cells.

In the present invention, the NK cells and/or iNK cells to be cryopreserved, the first cryopreservation solution and the second cryopreservation solution are mixed in the order of mixing the NK cells and/or iNK cells to be cryopreserved with the first cryopreservation solution and then mixing with the second cryopreservation solution. The mixing sequence can improve the survival rate and the recovery rate of the cells after the cryopreservation recovery

In the present invention, the second cryopreservation solution is preferably added dropwise to a mixed system of the NK cells and/or iNK cells to be cryopreserved and the first cryopreservation solution.

In the invention, the freezing final density of the NK cells and/or iNK cells to be frozen is preferably 1-8 x 10⁷A volume of 5 to 8X 10 is more preferable⁷/mL。

In the present invention, the volume ratio of the first frozen stock solution to the second frozen stock solution is preferably (1-2): (1-2), more preferably 1: 1.

the sectional cooling procedure is not particularly limited, and the conventional freezing technology in the field can be adopted; the instrument adopted by the sectional cooling is preferably a program cooling instrument.

In the present invention, after the cryopreservation, it is preferable to further comprise thawing the cryopreserved cells; the thawing temperature is 36-39 ℃, and more preferably 37 ℃; the thawing time is 1-2 min, and the ice crystals in the cell suspension are taken out when the ice crystals are about to completely disappear through visual observation; the thawing mode is preferably water bath thawing.

In the present invention, the cryopreservation method is preferably performed in liquid nitrogen, and the frozen stock can be stored for a long time.

In the present invention, the revived cells can be used directly for injection.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

Reagents used in the following examples: 1. human serum albumin, Chengdu Rong drug industry, 20%, 10g/50 ml/bottle; 2. shijiazhuang four drugs, dextran 40 sodium chloride injection, 500ml, 30g dextran 40 and 4.5g sodium chloride; 3. Cryosure-DMSO (dimethyl sulfoxide) cryopreservation solution 70 ml/bottle, OriGenbiochemical company in the United states; 4. 500ml of dextran 20 and sodium chloride injection of Shandongdi pharmaceutical industry Co., Ltd, 30g of dextran 20 and 4.5g of sodium chloride; 5. 500ml of Zhejiang Tianrui dextran 70 sodium chloride injection: 30g dextran 70 and 4.5g sodium chloride.

Example 1NK cells cryopreservation and Resuscitation

The specific freezing operation process is shown in figure 1.

1. Preparing clinical NK cell frozen stock solution:

1) first frozen stock solution (frozen stock solution A)

Adding injection-grade albumin into 6% dextran 40 sodium chloride injection, slightly inverting and mixing, wherein the mass concentration of the injection-grade albumin in the frozen solution A is 3g/100mL, and storing in a refrigerator at 4 ℃ for later use.

2) Second frozen stock solution (frozen stock solution B)

Adding injection-grade albumin into 6% dextran 40 sodium chloride injection, slightly reversing the mixture from top to bottom, uniformly mixing the mixture, adding injection-grade DMSO, wherein the mass concentration of the injection-grade albumin in the frozen B solution is 3g/100mL, the mass concentration of the injection-grade DMSO in the frozen B solution is 6g/100mL, and storing the mixture in a refrigerator at 4 ℃ for later use.

2. NK cell cryopreservation

1) Treatment group a + B: collecting NK cells, washing the NK cells with a conventional salt solution and centrifuging; discarding supernatant, resuspending the cells with a certain volume of frozen solution A, mixing, counting cells, and adjusting cell density to 10 according to counting result⁸Per mL; slowly dripping the frozen B solution with the same volume, uniformly mixing, and subpackaging the cell suspension.

2) Group AB: mixing the frozen solution A and the frozen solution B in advance according to the volume ratio of 1:1 under the aseptic condition, subsequently adding NK cells (the final cell density is consistent with that in the A + B treatment group), uniformly mixing, and subpackaging the cell suspension.

3. Programmed cooling

And (3) putting the frozen and stored subpackage tubes into a program cooling instrument for sectional cooling, and immediately transferring the frozen and stored cells into liquid nitrogen after the program cooling is finished, so that the frozen and stored cells can be stored for a long time. If the programmed cooling conditions are as follows: programmed cooling-program (4-0 deg.C, -10 deg.C/min, holingat 0 deg.C, 15min, 0-8 deg.C, -1 deg.C/min, -8 deg.C-45 deg.C, -50 deg.C/min, -45 deg.C-12 deg.C, +15 deg.C/min, -12 deg.C-100 deg.C, -1 deg.C/min).

4. NK cell resuscitation

Preheating a water bath to 37 ℃, transferring the cells stored in the liquid nitrogen to cells by using a dry ice box, taking out the freezing tube, quickly placing the freezing tube in the water bath at 37 ℃, slightly shaking the freezing tube within 1-2 min, and taking out the freezing tube when the ice crystals in the cell suspension are about to completely disappear by visual observation.

After recovery, the survival rate and recovery rate of the cells (not washed) were directly tested, and the results are shown in FIG. 2, AB indicates group AB (solution A and solution B were mixed at a ratio of 1:1 before the cells were added), and A + B indicates group A + B (cells were resuspended in solution A first, and then solution B was added dropwise).

Experiments show that the survival rate and recovery rate of iNK cells (prepared by referring to iNK preparation method in CN 202010153358.3) are high and can reach more than 90% no matter what application mode. And the cells are treated by using a mode of firstly freezing the solution A and uniformly mixing the cells and then dripping the solution B, and the survival rate and the recovery rate of iNK cells are higher after freezing recovery, so that the method is a better freezing mode.

In other embodiments, the NK cells are cryopreserved and recovered by using dextran 70 or dextran 40 or dextran 20 sodium chloride injection with 10% or 6% according to the experimental processes and steps, wherein the mass concentration of injection-grade albumin is 4-6 g/100mL or the mass concentration of injection-grade DMSO in cryopreserved B liquid is 10-16 g/100mL, and it is found that the survival rate and recovery rate of iNK cells can reach more than 90% no matter what method is used, the difference from the above results is not great, and the method of uniformly mixing the cells by using the cryopreserved A liquid and then dropping the cryopreserved B liquid is more preferable.

Example 2: NK cells show typical aggregation growth form after 2 days of resuscitation

After thawing the cryopreserved NK cells in example 1, the cells were thawed at 1X 10⁶After the cells are inoculated into a cell culture plate for suspension culture for 2 days, the cells form a typical NK cell clustering phenomenon, the clustering phenomenon indicates that the state and the activity of the cells are higher, and indicates that the cryopreservation protection effect of a cryopreservation solution on the NK cells is better, so that the cells can quickly recover the cell activity and the amplification capacity after recovery, and the result is shown in figure 3.

Example 3: influence of albumin concentration on survival rate and recovery rate of cryopreserved NK cells after resuscitation.

Freezing and storing NK cells, testing whether the addition and concentration of albumin can influence the survival rate and recovery rate of the recovered NK cells, and testing the concentration of albumin in a frozen storage solution to be 0, 1%, 3%, 5%, 8% and 10%, wherein the survival rate and recovery rate of the recovered cells are the cell survival rate/total cell number; the cell recovery rate is the number of viable cells after resuscitation/the number of viable cells before resuscitation, and the results are shown in fig. 4.

It can be seen that the albumin contained in the frozen stock solution can obviously improve the survival rate and the recovery rate of the cells after the frozen stock recovery. The concentration of albumin in the frozen stock solution is preferably 3% to 8%. Compared with the prior art, the albumin concentration is obviously reduced while the high recovery rate and the high survival rate of cells are maintained, and the cost of the frozen stock solution can be reduced. For large-scale production and clinical application, the total application amount of albumin can be greatly saved, so that the large-scale production cost is reduced.

Example 4: influence of cell cryopreservation density on survival rate and recovery rate of cryopreserved NK cells after recovery

Setting the cell density at 2X 10 for freezing NK⁶/mL、5×10⁶/mL、1×10⁷/mL、2×10⁷/mL、5×10⁷/mL、8×10⁷/mL、10×10⁷Performing cryopreservation test, and detecting the survival rate and recovery rate of the recovered NK cells, wherein the cell survival rate is the number of viable cells/the total number of the cells; cell recovery rate-number of viable cells after resuscitation/number of viable cells before resuscitation, and the results are shown in fig. 5.

Therefore, when the NK cells are frozen, the freezing density of the cells can reach 10 multiplied by 10⁷A volume of 1 to 8X 10⁷The best effect is 5-8 multiplied by 10 per mL⁷The viability and recovery rate of the/mL cell are better. Compared with the prior art, the density of the frozen cells can be obviously improved, and the clinical use is facilitated.

Example 5: influence of DMSO on survival and recovery rate after NK cell resuscitation.

When the cell freezing density is 5-8 multiplied by 10⁷In the case of/mL, the final concentration of DMSO in the frozen stock is tested to be 0%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 20%, and the survival rate and recovery rate of the cells after recovery, wherein the cell survival rate is the number of viable cells/the total number of cells; the cell recovery rate is the number of viable cells after resuscitation/the number of viable cells before resuscitation, and the results are shown in fig. 6.

Therefore, when the cryopreservation density of NK cells is 5-8 multiplied by 10⁷at/mL, the final concentration of DMSO can be reduced to 2-8% without affecting the survival rate and recovery rate of the cells after recovery. Compared with the prior art, the final concentration of DMSO is remarkably reduced, and clinical use is facilitated.

Example 6: after the NK cells are recovered, the cell survival rate and the recovery rate are higher, the purity can be still kept higher, and the expression of NK cell surface marker receptors is maintained.

The cryopreservation liquid and the corresponding program of the invention are used for cryopreservation of NK cells, and the cells still have higher cell survival rate and recovery rate after recovery. Further, identification and detection of specific markers (CD3 and CD56) are carried out on the recovered NK cells by a flow cytometry method; meanwhile, the expression of NK cell surface marker receptors is detected by a flow cytometry method (NKG2A and KIR are NK cell surface inhibitory receptors, and NKG2D and Nkp30 are NK cell surface activating receptors). Compared with fresh NK cells, the cryopreserved recovered NK cells in the invention still have higher purity (CD56+ CD3- > 95%) and maintain the expression of cell surface marker receptors, and the results are shown in FIG. 7 and FIG. 8.

Therefore, compared with the fresh cells which are not frozen, the NK cells frozen by using the freezing solution and the freezing method have higher purity after cell recovery, and the expression of important surface marker receptors of the cells is maintained.

Example 7: after the NK cells are recovered, the stability of the cell preparation can be maintained in a frozen stock solution.

Because the NK cells frozen by the frozen stock solution can be directly used for clinical infusion, the preparation stability of the cell preparation after recovery is tested. After recovering NK cells, respectively standing the recovered NK cells in the temperature environments of 37 ℃, room temperature (25-28 ℃) and 4 ℃ for a certain time, and taking the cells at different time points (0h, 2h, 4h, 6h and 8h) to detect the survival rate and the recovery rate of the cells, wherein the cell survival rate is the number of the living cells/the total number of the cells; the cell recovery rate is the number of viable cells after resuscitation/the number of viable cells before resuscitation, and the results are shown in fig. 9 and fig. 10. NK cells incubated in the frozen stock for 8h were compared to K562 tumor cells at an effective target ratio of 3: 1 co-culture for 24h, and testing the capability of killing tumor cells in vitro, wherein the result shows that the NK cells stored in the frozen stock solution can maintain the tumor cell lysis function, and the result is shown in figure 11.

As can be seen, after the NK cells are recovered, the stability of the NK cell preparation can be maintained for at least 8 hours, the NK cell preparation can adapt to different temperature environments, the cell survival rate can be maintained to be more than 90%, the cell recovery rate is more than 90%, and the function of the NK cells in tumor cell lysis can be maintained.

Example 8: the NK frozen stock solution has the freezing preservation protection effect on primary NK cells (PBNK cells derived from peripheral blood and CBNK cells derived from umbilical cord blood).

Extracting mononuclear cells from peripheral blood or umbilical cord blood, enriching by using CD56 antibody magnetic beads to obtain cells with the CD56 positive rate of more than 95%, activating and amplifying in a CD16 antibody and cytokine mode, or co-culturing and activating and amplifying by using trophoblasts expressing stimulating factors, amplifying for two weeks to obtain PBNK cells derived from the peripheral blood and CBNK cells derived from the umbilical cord blood, freezing the PBNK and the CBNK cells by using the frozen stock solution, detecting the cell viability and the recovery rate after recovery, wherein the cell viability is the viable cell number/the total cell number; the cell recovery rate is the number of viable cells after resuscitation/the number of viable cells before resuscitation, and the results are shown in fig. 12. Recovered PBNK and CBNK cells were compared to K562 tumor cells at an effective target ratio of 3: 1 co-culture for 24h, testing the capability of killing tumor cells in vitro, and showing that the expanded PBNK and CBNK cells can be preserved in the frozen stock solution of the invention and maintain the tumor cell lysis function, and the result is shown in figure 12.

The NK cryopreservation solution can be simultaneously used for cryopreservation of primary NK cells and iNK cells.

Example 9: the frozen NK cells can crack various tumor cell strains, and have similar function with the fresh NK cells. Meanwhile, the recovered NK cells are directly injected into a K562 mouse tumor model by tail vein injection, and the proliferation of tumor cells in a mouse body can be obviously inhibited.

Cryopreserved NK cells were recovered immediately after resuscitating at an effective target ratio of 3: 1 co-culturing for 24H, and testing the capability of killing tumor cells in vitro, wherein MOML13, NB4 and Kasumi-1 are human acute myelocytic leukemia cell strains, K562 and KU812 are human chronic myelocytic leukemia cell strains, Raji is a human lymphoma cell strain, A549 and H460 are human lung cancer cell strains, Huh7 is a human hepatoma cell strain, SKOV-3 is a human ovarian cancer cell strain, and SKMEL-2 is a human melanoma cell strain. The results show that the cryopreserved NK cells can maintain the lysis function of different tumor cells, and the results are shown in FIG. 13.

And (3) injecting the cryopreserved NK cells into a K562 mouse tumor model in tail vein immediately after recovery, injecting the cryopreserved recovered NK cells one day after inoculating the tumor cells, and detecting tumor cell signals in the mouse body by using a living body imaging instrument on the 21 st day after inoculating the tumor cells. The results show that cryopreserved NK cells can inhibit proliferation of K562 tumor cells in tumor model mice, and the results are shown in fig. 14.

After the cryopreserved cells are recovered, the cryopreserved cells can directly kill various tumor cells cultured in vitro to different degrees, and can also be directly used for injection treatment of tumor-bearing mice, which shows that the NK cryopreservation solution disclosed by the invention has small damage to the NK cells and can maintain the tumor cell lysis function of the NK cells in vitro and in vivo. Compared with the prior art, the adverse effect of the cryopreservation recovery process on the activity and the function of the NK cells is reduced.

Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Claims

1. A clinical-grade NK cell cryopreservation solution comprises a first cryopreservation solution and a second cryopreservation solution;

the non-osmotic cryoprotectant comprises injection grade albumin;

the osmotic cryoprotectant comprises injection grade DMSO.

2. The clinical-grade NK cell cryopreservation solution of claim 1, wherein the dextran sodium chloride injection comprises dextran sodium chloride injection with a mass concentration of 10% or dextran sodium chloride injection with a mass concentration of 6%.

3. The clinical-grade NK cell cryopreservation solution of claim 1 or 2, wherein the dextran sodium chloride injection is selected from one or more of dextran 40 sodium chloride injection, dextran 70 sodium chloride injection or dextran 20 sodium chloride injection.

4. The clinical-grade NK cell cryopreservation solution of claim 1, wherein the mass concentration of the non-permeable cryoprotectant in the first and second cryopreservation solutions is independently 1% to 10%.

5. The clinical-grade NK cell cryopreservation solution of claim 1, wherein the mass concentration of the osmotic cryoprotectant in the second cryopreservation solution is 2% to 20%.

6. The use of the clinical grade NK cell cryopreservation solution of any one of claims 1 to 5 in NK cell cryopreservation and/or iNK cell cryopreservation.

7. The method of using the clinical grade NK cell frozen stock solution of any one of claims 1 to 5, comprising the steps of:

8. The use of claim 7, wherein the NK cells and/or iNK cells to be cryopreserved, the first cryopreservation solution and the second cryopreservation solution are mixed together after the NK cells and/or iNK cells to be cryopreserved are mixed with the first cryopreservation solution and then mixed with the second cryopreservation solution.

9. The use method according to claim 7, wherein the final cryopreservation density of the NK cells and/or iNK cells to be cryopreserved is 1-8 x 10⁷/mL。

10. The use method according to claim 7, wherein the volume ratio of the first frozen stock solution to the second frozen stock solution is (1-2): (1-2).