A kind of NKT cell freezing solution and the preservation method of NKT cell

Abstract

The present application relates to a kind of NKT cell freezing solution and the storage method of NKT cell freezing solution, the components of the NKT cell freezing solution include: dimethyl sulfoxide, human blood albumin, dextran, alginic polysaccharide, tremella polysaccharide and physiological saline.The NKT cell freezing solution involved in the present application is obtained by simultaneously adding dimethyl sulfoxide, human blood albumin, dextran, alginic polysaccharide and tremella polysaccharide in physiological saline, the freezing solution greatly reduces the injury suffered by cells in the process of freezing recovery, can well increase the survival rate of NKT cells, and cells after recovery also have good killing ability.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cell cryopreservation, and relates to an immune cell cryopreservation solution and a preservation method thereof, in particular to an NKT cell cryopreservation solution and a preservation method of NKT cells. BACKGROUND

[0002] NKT (natural killer T) cells are a special T cell subpopulation with both T cell receptor TCR and NK cell receptor on the cell surface. In recent years, great progress has been made in the research on the phenotypic characteristics, distribution and development, immunological effects, relationship with diseases, tumor treatment and autoimmune treatment of NKT cells. Due to the research on NKT cells in the treatment of tumors and immune diseases, the importance of cryopreservation of NKT cells after culture is determined. In the treatment aspect, the low cell activity caused by delayed culture can be avoided, in the economic aspect, the cost increase caused by delayed culture can be avoided, and in the research aspect, NKT cells at different periods can be cryopreserved to study the activity of NKT cells at different cryopreservation times.

[0003] At present, glycerol or dimethyl sulfoxide is often used as a protective agent for cell cryopreservation. These two substances can improve the permeability of the cell membrane to water, and slow freezing can make the water in the cell seep out of the cell, reducing the formation of ice crystals in the cell and reducing the damage to the cell caused by the formation of ice crystals. The resuscitated cells should be thawed quickly, which can ensure that the extracellular crystals are melted in a very short time, avoiding the damage to the cells caused by slow thawing and the formation of intracellular recrystallization. When cells are directly cryopreserved without protective agents, water in the cell and the external environment will form ice crystals, which can cause a series of changes in the cell, such as mechanical damage, increase of electrolyte concentration, change of osmotic pressure, dehydration, change of pH value, protein denaturation, etc., causing irreversible permanent damage to the cell, and even causing cell death.

[0004] The existing cryopreservation method has problems of low cell survival rate and low cell activity after resuscitation for NKT cells, and the clinical application of NKT cells needs a cryopreservation method that can better maintain the activity of NKT cells.

[0005] CN105211052A discloses a cryopreservation solution for cultured NKT cells and a preparation method thereof. The cryopreservation solution comprises lentinan, fucoidan, autologous serum and DMSO. Since autologous serum is used, there is no pollution of exogenous animal viruses and introduction of exogenous proteins, and the safety is high. The added lentinan and fucoidan can effectively maintain the activity of the cells and have no harm to the human body, and the state of the cells after cryopreservation and resuscitation is basically close to the state before cryopreservation, and the cryopreservation effect is good.

[0006] CN105211051A Cryopreservation solution of NKT cells after culture and preparation method thereof, the cryopreservation solution contains tea polyphenol, grape seed extract, vitamin C and autologous serum, wherein the tea polyphenol, grape seed extract and vitamin C added can effectively inhibit cell oxidation, maintain the activity of cells, and have no harm to human body, and the state of the cells after cryopreservation recovery is basically close to the state before cryopreservation, and the cryopreservation effect is good.

[0007] However, the cryopreservation solution for NKT cells in the prior art is still very limited, therefore, it is very meaningful to develop more cryopreservation solutions that can make the survival rate and killing activity of NKT cells after cryopreservation recovery excellent. SUMMARY

[0008] In view of the deficiencies of the prior art, the purpose of the present application is to provide an immune cell cryopreservation solution and a preservation method thereof, in particular to a NKT cell cryopreservation solution and a preservation method of NKT cells.

[0009] To achieve the purpose of the present application, the following technical solutions are adopted:

[0010] In a first aspect, the present application provides a NKT cell cryopreservation solution, the components of the NKT cell cryopreservation solution comprising: dimethyl sulfoxide, human blood albumin, dextran, algal polysaccharide, tremella polysaccharide and physiological saline.

[0011] The dimethyl sulfoxide in the NKT cell cryopreservation solution of the present application can penetrate into the cells through the cell membrane before the complete freezing of the cell freezing suspension, generate a certain molar concentration inside and outside the cells, reduce the concentration of electrolytes in the non-frozen solution inside and outside the cells, thereby protecting the cells from damage by high-concentration electrolytes; at the same time, the water in the cells will not excessively exosmosis, avoiding excessive dehydration and shrinkage of the cells, changing the permeability of the cell membrane, protecting the cell membrane and reducing the formation of ice crystals in the cells during the cryopreservation process.

[0012] The human blood albumin, dextran, algal polysaccharide and tremella polysaccharide in the NKT cell cryopreservation solution of the present application can potentially positively affect each other, and have a synergistic promotion effect on maintaining the survival rate and target cell killing ability of NKT cells after recovery. They can preferentially combine with water molecules in the solution, reduce the content of free water in the solution; lower the freezing point and reduce the formation of ice crystals; at the same time, due to their large molecular weight, the electrolyte concentration in the solution is reduced, thereby reducing the damage of solutes. Moreover, the amount of dimethyl sulfoxide can be reduced, thereby greatly reducing the toxicity of the cryopreservation solution to the cryopreserved cells and the human body.

[0013] Therefore, the NKT cell freezing solution relates to the present application, which is obtained by adding dimethyl sulfoxide, human blood albumin, dextran, algal polysaccharide and tremella polysaccharide in physiological saline at the same time, greatly reduces the damage of cells in the freezing and recovery process, can well increase the survival rate of NKT cells, and has good killing ability after cell recovery, and has wide application prospect in the biological treatment of NKT cells.

[0014] Preferably, the components of the NKT cell freezing solution include, in terms of mass percentage: dimethyl sulfoxide 5-10%, human blood albumin 0.1-2%, dextran 0.2-2%, algal polysaccharide 0.5-3%, tremella polysaccharide 0.5-3% and physiological saline.

[0015] When the components of the NKT cell freezing solution are combined in the above specific mass ratio, the combination has better effect, and has better technical effect in reducing the damage of cells in the freezing and recovery process, increasing the survival rate and killing ability of NKT cells.

[0016] The mass percentage of dimethyl sulfoxide in the NKT cell freezing solution can be selected as 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10% and the like.

[0017] The mass percentage of human blood albumin in the NKT cell freezing solution can be selected as 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 1.7%, 2% and the like.

[0018] The mass percentage of dextran in the NKT cell freezing solution can be selected as 0.2%, 0.3%, 0.5%, 0.7%, 1%, 1.2%, 1.4%, 1.5%, 1.6%, 1.8%, 2% and the like.

[0019] The mass percentage of algal polysaccharide in the NKT cell freezing solution can be selected as 0.5%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5%, 2.8%, 3% and the like.

[0020] The mass percentage of tremella polysaccharide in the NKT cell freezing solution can be selected as 0.5%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5%, 2.8%, 3% and the like.

[0021] Other specific point values in the above numerical range can be selected, which will not be described here.

[0022] Preferably, the components of the NKT cell cryopreservation solution further comprise vitamin B12.

[0023] The addition of coenzyme vitamin B12 in the NKT cell cryopreservation solution according to the present application helps to further improve the survival rate and the ability to kill target cells of the NKT cells after resuscitation.

[0024] Preferably, the concentration of vitamin B12 in the NKT cell cryopreservation solution is 5-50 ng / mL, such as 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, etc. Other specific point values within this numerical range can also be selected, which will not be repeated here.

[0025] In a second aspect, the present application provides a preparation method of the NKT cell cryopreservation solution according to the first aspect, which comprises uniformly dissolving dimethyl sulfoxide, human blood albumin, dextran, fucoidan, and tremella polysaccharide in physiological saline as a dissolving medium.

[0026] The preparation process of the NKT cell cryopreservation solution according to the present application is simple and easy to operate, and is very suitable for industrialized large-scale production.

[0027] In a third aspect, the present application provides the use of the NKT cell cryopreservation solution according to the first aspect in the preservation of NKT cells.

[0028] In a fourth aspect, the present application provides a preservation method of NKT cells, which comprises mixing NKT cells to be preserved with the NKT cell cryopreservation solution according to the first aspect to obtain a cell suspension, placing the cell suspension in a sterile cryopreservation tube, and cryopreserving in liquid nitrogen.

[0029] Preferably, the concentration of the NKT cells in the cell suspension is (1×10 6 ) to (3×10 7 ) cells / mL, such as 1×10 6 cells / mL, 3×10 6 cells / mL, 5×10 6 cells / mL, 8×10 6 cells / mL, 1×10 7 cells / mL, 2×10 7 cells / mL, 3×10 7 cells / mL, etc. Other specific point values within this numerical range can also be selected, which will not be repeated here.

[0030] Preferably, the preservation method further comprises: mixing the NKT cells to be preserved with L-glutamine for 24-72 hours (for example, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 72 hours, etc.) before preservation.

[0031] The present application also creatively finds that adding L-glutamine to the culture medium in the last stage of NKT cell culture helps to improve the survival rate and target cell killing activity of the cryopreserved cells after recovery.

[0032] Preferably, the concentration of L-glutamine in the mixed culture system is 0.5-10 mM, for example, 0.5 mM, 1 mM, 3 mM, 5 mM, 6 mM, 7 mM, 8 mM, 10 mM, etc. Other specific point values in this range can also be selected, which will not be repeated here.

[0033] Compared with the prior art, the present application has the following beneficial effects:

[0034] The NKT cell cryopreservation solution involved in the present application is obtained by simultaneously adding dimethyl sulfoxide, human blood albumin, dextran, algal polysaccharide and tremella polysaccharide in physiological saline. The cryopreservation solution greatly reduces the damage to cells during the cryopreservation and recovery process, can well increase the survival rate of NKT cells, and has good killing ability after cell recovery, and has broad application prospects in the biological treatment of NKT cells.

[0035] Among them, human blood albumin, dextran, algal polysaccharide and tremella polysaccharide can potentially positively affect each other, and have a synergistic promotion effect on maintaining the survival rate and target cell killing ability of NKT cells after recovery. Moreover, it can reduce the amount of dimethyl sulfoxide, greatly reducing the toxicity of the cryopreservation solution to the cryopreserved cells and the human body.

[0036] Moreover, the present application also finds that adding coenzyme vitamin B12 to the cryopreservation solution helps to further improve the survival rate and the ability to kill target cells of NKT cells after recovery. DETAILED DESCRIPTION

[0037] In order to further illustrate the technical means adopted by the present application and its effects, the technical solutions of the present application will be further described below in combination with preferred embodiments of the present application, but the present application is not limited in the scope of the embodiments.

[0038] The source of the efficacy components involved in the following content is as follows (only the actual efficacy components are embodied in the examples and comparative examples, and other necessary auxiliary ingredient components existing in the commercially available raw materials are not described again) :

[0039] The human blood albumin is derived from the human blood albumin injection product of the Instituto Grifols company;

[0040] Dextran was derived from Dextran 40 in normal saline injection product with type number 1179708 from US Pharmacopeia Corporation;

[0041] Laminarin was derived from Laminarin product with type number 6138-23-4 from Sigma-Aldrich Corporation;

[0042] Tremella Polysaccharide was derived from Tremella Polysaccharide product with type number 9075-53-0 from Macklin Corporation;

[0043] Vitamin B12 was derived from Vitamin B12 product with type number 68-19-9 from Sigma-Aldrich Corporation;

[0044] Dimethyl Sulfoxide was derived from Dimethyl Sulfoxide product with type number 7-68-5 from Sigma-Aldrich Corporation;

[0045] L-Glutamine was derived from L-Glutamine product with type number 56-85-9 from Sigma-Aldrich Corporation;

[0046] Other raw materials can be purchased through commercial channels.

[0047] Preparation Example 1

[0048] Expansion of NKT cells:

[0049] Peripheral blood of volunteers was taken; mononuclear cells were isolated by Ficoll density gradient centrifugation; NKT cell culture medium (RPMI1640 medium containing 5% human serum, 100 μ / mL penicillin, 100 μg / mL streptomycin) was prepared. The obtained peripheral blood mononuclear cells were cultured in NKT cell culture medium, and a-GalCer (100 ng / mL) (NKT cell activator KRN7000) and IL-15 20 ng / mL were added to the culture medium on day 0 and day 7. NKT cell culture medium was added according to cell proliferation. L-Glutamine was added on day 13 with a final concentration of 5 mM. Cells were harvested after 24 h. It was detected that 11.1% were Vα24+Vβ11+ cells and 21.3% were CD3+CD56+ cells.

[0050] Preparation Example 2

[0051] Expansion of NKT cells:

[0052] Peripheral blood of a volunteer was taken; mononuclear cells were separated by Ficoll density gradient centrifugation; NKT cell culture medium (RPMI 1640 medium containing 5% human serum, 100 μ / mL penicillin, 100 μg / mL streptomycin) was prepared. The obtained peripheral blood mononuclear cells were cultured in the NKT cell culture medium, the difference between the operation mode of Preparation Example 1 being that L-glutamine was added at a final concentration of 5 mM on the 11th day. The cells were harvested after 72 h, and the total culture time was consistent. It was detected that 10.6% of them were Vα24+Vβ11+ cells, and 24.0% were CD3+CD56+ cells.

[0053] Preparation Example 3

[0054] Expansion of NKT cells:

[0055] Peripheral blood of a volunteer was taken; mononuclear cells were separated by Ficoll density gradient centrifugation; NKT cell culture medium (RPMI 1640 medium containing 5% human serum, 100 μ / mL penicillin, 100 μg / mL streptomycin) was prepared. The obtained peripheral blood mononuclear cells were cultured in the NKT cell culture medium, the difference between the operation mode of Preparation Example 1 being that L-glutamine was added at a final concentration of 5 mM on the 11th day. The cells were harvested after 72 h, and the total culture time was consistent. It was detected that 10.6% of them were Vα24+Vβ11+ cells, and 24.0% were CD3+CD56+ cells.

[0056] Example 1

[0057] An NKT cell freezing solution was prepared in this example, and the formula thereof was: dimethyl sulfoxide 5%, human blood albumin 0.8%, dextran 0.8%, alginate 1%, tremella polysaccharide 1%, vitamin B12 10 ng / mL, prepared with a total volume of 100 mL of normal saline.

[0058] Example 2

[0059] An NKT cell freezing solution was prepared in this example, and the formula thereof was: dimethyl sulfoxide 8%, human blood albumin 0.2%, dextran 0.3%, alginate 0.8%, tremella polysaccharide 0.5%, vitamin B12 20 ng / mL, prepared with a total volume of 100 mL of normal saline.

[0060] Example 3

[0061] An NKT cell freezing solution was prepared in this example, and the formula thereof was: dimethyl sulfoxide 5%, human blood albumin 0.8%, dextran 0.8%, alginate 1%, tremella polysaccharide 1%, prepared with a total volume of 100 mL of normal saline.

[0062] Comparative Example 1

[0063] The NKT cell cryopreservation solution of the present comparative example is prepared with the same composition as that of Example 1 except that it does not contain human blood albumin, and the mass of which is proportionally distributed to the mass of the three components of dextran, fucoidan and tremella polysaccharide, and the other components and contents remain unchanged.

[0064] Comparative Example 2

[0065] The NKT cell cryopreservation solution of the present comparative example is prepared with the same composition as that of Example 1 except that it does not contain dextran, and the mass of which is proportionally distributed to the mass of the three components of human blood albumin, fucoidan and tremella polysaccharide, and the other components and contents remain unchanged.

[0066] Comparative Example 3

[0067] The NKT cell cryopreservation solution of the present comparative example is prepared with the same composition as that of Example 1 except that it does not contain fucoidan, and the mass of which is proportionally distributed to the mass of the three components of human blood albumin, dextran and tremella polysaccharide, and the other components and contents remain unchanged.

[0068] Comparative Example 4

[0069] The NKT cell cryopreservation solution of the present comparative example is prepared with the same composition as that of Example 1 except that it does not contain tremella polysaccharide, and the mass of which is proportionally distributed to the mass of the three components of human blood albumin, dextran and fucoidan, and the other components and contents remain unchanged.

[0070] Comparative Example 5

[0071] The NKT cell cryopreservation solution of the present comparative example is prepared with the following composition: DMEM basic medium 70%, fetal bovine serum 20%, DMSO 10%.

[0072] Test Example 1

[0073] (1) Cryopreservation of NKT cells: The cell samples prepared in Preparation Examples 1-3 were mixed with the NKT cell cryopreservation solution prepared in Example 1, and the NKT cells were resuspended at a density of 10 7 cells / mL, and then 1 mL / branch was aliquoted into cell cryopreservation tubes, and the cell cryopreservation tubes were placed in a cryopreservation box containing isopropanol and placed at -80°C. After 18 hours, the cell cryopreservation tubes were transferred to a liquid nitrogen tank for cryopreservation for 3 months.

[0074] (2) NKT cell recovery: Take the cell cryopreservation tube out of liquid nitrogen and put it into a 37℃ water bath for about 1 minute. Take the cells out and put them into 0.9% sodium chloride injection at a volume ratio of 1:10, mix gently, centrifuge, and aspirate the supernatant to wash away the lymphocyte cryopreservation solution. Resuspend the cells with 1 mL of NKT cell culture medium (RPMI1640 culture medium, 5% human serum, 100 u / ml penicillin, 100 lg / ml streptomycin), and put them into 20 mL of 37℃ preheated culture medium, and place them in a 37℃, 5% CO2 incubator for culture recovery.

[0075] (3) Measurement of cell survival rate: After recovery, the cells were placed in a 37℃, 5% CO2 incubator for 20 hours, then stained with fluorescent antibodies, and the survival rates of whole cells, Vα24+Vβ11+ cells and CD3+CD56+ cells before freezing and after freezing for 3 months were detected by flow cytometry, and the results are shown in Table 1.

[0076] Table 1

[0077]

[0078] As can be seen from the data results in Table 1, the cryopreservation solution involved in the present application can greatly reduce the damage to NKT cells during the freezing and recovery process, and can well increase the survival rate of NKT cells, with a survival rate of more than 85%; and the addition of L-glutamine to the culture medium in the last stage of NKT cell culture helps to further improve the survival rate of the recovered frozen cells.

[0079] (4) Measurement of target cell killing activity of cells: After recovery, the cells were placed in a 37℃, 5% CO2 incubator for 20 hours, then the cell density was adjusted to 1×10 6 / mL, 5×10 5 / mL, 1×10 5 / mL. The K562 cell density was adjusted to 1×10 5 / mL. NKT cells (effector) and K562 cells (target) with an effector-to-target ratio of 10:1 were added to a 96-well round-bottom plate at 50 μL / well, with 3 replicate wells. At the same time, target cell wells, effector cell wells and blank (only NKT cell culture medium) were set as controls. After mixing the cells, they were placed in a 37℃, 5% CO2 incubator for culture, and at 4, 6 and 10 hours of culture, Cell Counting Kit-8 (CCK-8) reagent kit was used for detection. The OD value at 450 nm wavelength was detected by a microplate reader. The target cell killing rate (%) = [1-(effector and target cell well OD value-effector cell well OD value) / target cell OD value]×100%. The results are shown in Table 2.

[0080] Table 2

[0081] Target cell killing rate (%) 4h 6h 12h Preparation Example 1 63.0 77.5 85.9 Preparation Example 2 64.1 74.6 83.5 Preparation Example 3 61.0 68.4 78.3

[0082] From the data results of Table 2, it can be seen that the freezing solution involved in the present application can greatly reduce the damage to NKT cells during the freezing and recovery process, can well maintain the target cell killing ability of NKT cells, and the target cell killing rate of 12h is more than 78%; and adding L-glutamine to the culture medium in the last stage of NKT cell culture helps to further improve the target cell killing rate after the recovery of frozen cells.

[0083] Test Example 2

[0084] (1) Freezing of NKT cells: The cell sample prepared in Preparation Example 1 was mixed with the NKT cell freezing solution prepared in Examples 1-3 and Comparative Examples 1-5, respectively, and the NKT cells were resuspended at a density of 10 7 cells / mL, and then 1 mL / branch was aliquoted into a cell freezing tube, and the cell freezing tube was placed in a freezing box with isopropanol and placed at -80°C. After 18 hours, the cell freezing tube was transferred to a liquid nitrogen tank for 3 months.

[0085] (2) Recovery of NKT cells: The cell freezing tube was taken out from the liquid nitrogen and put into a 37°C water bath for about 1 minute. The cells were taken out and placed in 0.9% sodium chloride injection at a volume ratio of 1:10, mixed gently and centrifuged, and the supernatant was aspirated to wash away the lymphocyte freezing solution. The cells were resuspended with 1 mL of NKT cell culture medium (RPMI1640 culture medium, 5% human serum, 100u / ml penicillin, 100lg / ml streptomycin), and placed in 37°C preheated 20mL medium, and cultured in a 37°C, 5% CO2 incubator for recovery.

[0086] (3) Determination of cell survival rate: The recovered cells were placed in a 37°C, 5% CO2 incubator for 20 hours, then stained with fluorescent antibody, and the survival rates of whole cells, Vα24+Vβ11+ cells and CD3+CD56+ cells before freezing and after freezing for 3 months were detected by flow cytometry. The results are shown in Table 3.

[0087] Table 3

[0088]

[0089]

[0090] The results of the data in Table 3 show that the four components of human blood albumin, dextran, algal polysaccharide and tremella polysaccharide in the NKT cell freezing solution can potentially positively affect each other, and have a synergistic promotion effect on maintaining the survival rate of the recovered NKT cells; and the addition of coenzyme vitamin B12 in the NKT cell freezing solution helps to further improve the survival rate of the recovered NKT cells.

[0091] (4) Measurement of target cell killing activity of the cells: the recovered cells were cultured in a 37℃, 5% CO2 incubator for 20 hours, and then the cell density was adjusted to 1×10 6 / mL, 5×10 5 / mL, 1×10 5 / mL. The K562 cell density was adjusted to 1×10 5 / mL. NKT cells (effector) and K562 cells (target) with an effector-to-target ratio of 10:1 were added to a 96-well round-bottom plate at 50 μL / well, with three replicate wells. At the same time, target cell wells, effector cell wells and blank (only NKT cell medium) were set as controls. After mixing the cells, they were placed in a 37℃, 5% CO2 incubator for culture, and the Cell Counting Kit-8 (CCK-8) kit was used for detection at 4, 6 and 10 hours of culture, respectively. The OD450nm wavelength OD value was detected by a microplate reader. The target cell killing rate (%) = [1-(effector and target cell well OD value-effector cell well OD value) / target cell OD value]×100%. The results are shown in Table 4.

[0092] Table 4

[0093]

[0094]

[0095] The results of the data in Table 4 show that the four components of human blood albumin, dextran, algal polysaccharide and tremella polysaccharide in the NKT cell freezing solution can potentially positively affect each other, and have a synergistic promotion effect on maintaining the target cell killing ability of the recovered NKT cells; and the addition of coenzyme vitamin B12 in the NKT cell freezing solution helps to further improve the ability of the recovered NKT cells to kill target cells.

[0096] The applicant declares that the NKT cell freezing solution and the NKT cell preservation method of the present application are illustrated by the above examples, but the present application is not limited to the above examples, i.e. it does not mean that the present application must rely on the above examples to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. fall within the protection scope and disclosure scope of the present application.

[0097] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details of the above-described embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

[0098] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not describe various possible combinations again.

Claims

1. A method for preserving NKT cells, characterized by, The preservation method comprises the following steps: mixing the NKT cells to be preserved with L-glutamine for 24-72 hours before preservation, mixing with an NKT cell freezing solution to obtain a cell suspension, placing the cell suspension in a sterile freezing tube, and freezing in liquid nitrogen; The components of the NKT cell freezing solution comprise dimethyl sulfoxide 5-10%, human blood albumin 0.1-2%, dextran 0.2-2%, algal polysaccharide 0.5-3%, tremella polysaccharide 0.5-3%, vitamin B12 and physiological saline; The concentration of vitamin B12 in the NKT cell freezing solution is 5-50 ng / mL.

2. The method of claim 1, wherein the NKT cells are preserved at a concentration of 1 x 106 cells / mL to 1 x 108 cells / mL. The concentration of the NKT cells in the cell suspension is (1 x 10 6 )-(3 x 10 7 ) cells / mL.

3. The method of claim 1, wherein the NKT cells are preserved at a concentration of about 1 x 106 cells / mL to about 1 x 108 cells / mL. The concentration of L-glutamine in the mixed culture system is 0.5-10 mM.

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