A NK cell freezing solution and its preparation method and application

Abstract

The present invention relates to a NK cell freezing solution and its preparation method and application. The present invention uses N-tris(hydroxymethyl)methylglycine and sodium glycinate as permeable cryoprotectants, and carboxymethyl chitosan, polyglutamic acid and sodium polyglutamate as non-permeable cryoprotectants, so that the freezing solution formed after the two are blended with the culture medium is applied to the freezing of NK cells. On the one hand, the permeable cryoprotectant can penetrate into the cell, balance the electrolyte concentration and combine with water molecules, so as to protect the cell from solute and mechanical damage, and on the other hand, the non-permeable cryoprotectant can also efficiently combine with the water in the system to reduce ice crystal formation, and at the same time, it also induces intracellular dehydration and forms a protection zone, so that mechanical damage can be reduced both inside and outside the cell, and the freezing solution is endowed with excellent freezing ability for NK cells through the synergistic effect of the two without adding DMSO and fetal bovine serum, and the survival rate of NK cells after recovery is high.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell cryopreservation, and in particular relates to a NK cell cryopreservation solution and a preparation method and application thereof. Background Art

[0002] NK cells, or natural killer cells, are an important type of immune cell in the body. They are not only related to anti-tumor, anti-viral infection and immune regulation, but also participate in the occurrence of hypersensitivity reactions and autoimmune diseases in some cases. Therefore, NK cells have been widely used in the field of cell therapy. In the field of cell therapy, cell cryopreservation is a very common, critical and important link. At present, many technologies still use the classic formula of dimethyl sulfoxide, fetal bovine serum and culture medium to mix and prepare them in a certain proportion for the cryopreservation of NK cells. This results in that although this type of cryopreservation solution can better ensure the survival rate of NK cells after recovery, dimethyl sulfoxide (DMSO) itself has certain toxic effects. Even if it is removed later, trace amounts of residues may still have adverse effects on the human body when used clinically. At the same time, fetal bovine serum, as an animal-derived component, also has a high risk of microbial contamination during actual production and use, and it is difficult to ensure quality.

[0003] Therefore, the current NK cell cryopreservation solution still has many disadvantages and urgently needs to be improved. Summary of the invention

[0004] In view of the problems existing in the prior art, the purpose of the present invention is to provide a NK cell freezing solution and a preparation method and application thereof. The present invention uses N-tri(hydroxymethyl)methylglycine and sodium glycine as permeable cryoprotectants, and uses carboxymethyl chitosan, polyglutamic acid and sodium polyglutamate as non-permeable cryoprotectants. When the freezing solution formed by mixing the two with a culture medium is applied to the freezing of NK cells, on the one hand, the permeable cryoprotectant can penetrate into the cells, balance the electrolyte concentration and bind to water molecules, thereby protecting the cells from solute and mechanical damage. On the other hand, the non-permeable cryoprotectant can also efficiently bind to water in the system to reduce ice crystal formation. At the same time, it also induces intracellular dehydration and forms a protection zone, so that mechanical damage can be reduced both inside and outside the cells. Without adding DMSO and fetal bovine serum, the synergistic effect of the two together endows the freezing solution with excellent freezing ability for NK cells, and the survival rate of NK cells after recovery is high.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] A NK cell freezing solution comprises a culture medium and N-tricine, sodium glycine, carboxymethyl chitosan, polyglutamic acid and sodium polyglutamate added to the culture medium.

[0007] As a preferred technical solution of the present invention, the culture medium is one of RPMI-1640 culture medium and DMEM culture medium.

[0008] As a preferred technical solution of the present invention, the concentration of N-tris(hydroxymethyl)methylglycine in the culture medium is 1-2 g / L.

[0009] As a preferred technical solution of the present invention, the concentration of sodium glycinate in the culture medium is 20-30 mg / L.

[0010] As a preferred technical solution of the present invention, the concentration of the carboxymethyl chitosan in the culture medium is 100-150 mg / L.

[0011] As a preferred technical solution of the present invention, the concentration of the polyglutamic acid in the culture medium is 250-300 mg / L; the molecular weight of the polyglutamic acid is 50,000-80,000.

[0012] As a preferred technical solution of the present invention, the concentration of the sodium polyglutamate in the culture medium is 10-15 mg / L; the molecular weight of the sodium polyglutamate is 50-100 kDa.

[0013] A method for preparing a NK cell cryopreservation solution, the method comprising the following steps:

[0014] (1) Add N-tris(hydroxymethyl)methylglycine, sodium glycinate, carboxymethyl chitosan, polyglutamic acid and sodium polyglutamate to the culture medium under a sterile environment, stir and mix, and the preparation is completed.

[0015] As a preferred technical solution of the present invention, the stirring time in step (1) is 5-10 minutes.

[0016] An application of a NK cell freezing solution, wherein the NK cell freezing solution is applied to the freezing of NK cells.

[0017] A method for using a NK cell cryopreservation solution, the method comprising the following steps:

[0018] Step A: Add the NK cell freezing solution as described above to the NK cells to form a cell density of 10 6 -10 8 cells / mL of cell suspension;

[0019] Step B: The cell suspension is kept at 4°C for 10 minutes, then cooled to -12-15°C at a rate of 1°C / min, kept at the constant temperature for 1-2 hours, then cooled to -80°C at a rate of 15-20°C / min, kept at the constant temperature for 8-10 hours, and finally transferred to liquid nitrogen for storage;

[0020] Step C: When thawing, take it out from the liquid nitrogen, then heat it in a water bath at 37°C until it is completely melted, centrifuge it, and remove the supernatant.

[0021] As a preferred technical solution of the present invention, the storage period in step B is within 3 years.

[0022] As a preferred technical solution of the present invention, the centrifugal speed in step C is 1500-2000 r / min, and the centrifugal time is 5-8 min.

[0023] Beneficial effects of the present invention:

[0024] (1) The present invention uses N-tris(hydroxymethyl)methylglycine and sodium glycinate as permeable cryoprotectants, and carboxymethyl chitosan, polyglutamic acid and sodium polyglutamate as non-permeable cryoprotectants. When the two are mixed with a culture medium to form a cryopreservation solution, when applied to the cryopreservation of NK cells, on the one hand, the permeable cryoprotectant can penetrate into the cells, balance the electrolyte concentration and bind to water molecules, thereby protecting the cells from solute and mechanical damage. On the other hand, the non-permeable cryoprotectant can also efficiently bind to water in the system to reduce ice crystal formation. At the same time, it also induces intracellular dehydration and forms a protection zone, thereby reducing mechanical damage both inside and outside the cells. Without adding DMSO and fetal bovine serum, the synergistic effect of the two together endows the cryopreservation solution with excellent cryopreservation ability for NK cells, and the survival rate of NK cells after recovery is high.

[0025] (2) The present invention creatively uses N-tris(hydroxymethyl)methylglycine and sodium glycinate as permeable cryoprotectants, and carboxymethyl chitosan, polyglutamic acid and sodium polyglutamate as non-permeable cryoprotectants, so that the freezing solution formed by physically mixing the two with the culture medium has excellent freezing ability when actually used for freezing NK cells, and the survival rate of NK cells after recovery is high; N-tris(hydroxymethyl)methylglycine, sodium glycinate, polyglutamic acid and sodium polyglutamate are all derivatives of amino acids, and have the characteristics of high biosafety and good biocompatibility. Among them, N-tris(hydroxymethyl)methylglycine and polyglutamic acid contain a large number of hydrophilic groups, which can effectively exert their intracellular It can combine with water molecules outside, thereby destroying the hydrogen bond network between water molecules and inhibiting the growth of ice crystals. At the same time, polyglutamic acid can also induce NK cells to gradually dehydrate, further reducing the possible mechanical damage. A certain amount of sodium glycine and sodium polyglutamate can better balance the electrolyte concentration by regulating sodium ions, so that the solute damage to NK cells is reduced. In addition, carboxymethyl chitosan not only has good biosafety as a water-soluble derivative of chitosan, but is also an amphoteric electrolyte that can gather around NK cells to form a protective isolation zone, thereby effectively reducing the damage of ice recrystallization to NK cells during resuscitation and ensuring survival rate.

[0026] (3) The NK cell cryopreservation solution prepared by the present invention does not contain components with certain toxic effects such as dimethyl sulfoxide, fetal bovine serum, and glycerol, and has very good biological safety. DETAILED DESCRIPTION

[0027] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation methods, structures, features and effects of the present invention are described in detail below in combination with the embodiments.

[0028] The RPMI-1640 culture medium in all embodiments and comparative examples of the present invention is purchased directly from the market, purchased from Wuhan Pronosai Life Science Technology Co., Ltd.; carboxymethyl chitosan is purchased directly from the market, purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a carboxylation degree of ≥80%; the source of NK cells is human natural killer NK cells produced by Qingqi (Shanghai) Biotechnology Development Co., Ltd., with a cargo code of BFN60808601.

[0029] Example 1

[0030] Take the culture medium with a volume of 100 mL as an example:

[0031] A NK cell freezing solution comprises a culture medium and N-tricine, sodium glycine, carboxymethyl chitosan, polyglutamic acid and sodium polyglutamate added to the culture medium.

[0032] The culture medium is RPMI-1640 culture medium.

[0033] The concentration of the N-tricine in the culture medium is 1 g / L.

[0034] The concentration of sodium glycinate in the culture medium is 20 mg / L.

[0035] The concentration of the carboxymethyl chitosan in the culture medium is 100 mg / L.

[0036] The concentration of the polyglutamic acid in the culture medium is 250 mg / L; the molecular weight of the polyglutamic acid is 50,000.

[0037] The concentration of the sodium polyglutamate in the culture medium is 10 mg / L; the molecular weight of the sodium polyglutamate is 50 kDa.

[0038] A method for preparing a NK cell cryopreservation solution, the method comprising the following steps:

[0039] (1) Add N-tris(hydroxymethyl)methylglycine, sodium glycinate, carboxymethyl chitosan, polyglutamic acid and sodium polyglutamate to the culture medium under a sterile environment, stir and mix, and the preparation is completed.

[0040] The stirring time in step (1) is 5 min.

[0041] An application of a NK cell freezing solution, wherein the NK cell freezing solution is applied to the freezing of NK cells.

[0042] A method for using a NK cell cryopreservation solution, the method comprising the following steps:

[0043] Step A: Add the NK cell freezing solution as described above to the NK cells to form a cell density of 10 6 cells / mL of cell suspension;

[0044] Step B: the cell suspension was kept at 4°C for 10 min, then cooled to -12°C at a rate of 1°C / min, kept at the constant temperature for 1 h, then cooled to -80°C at a rate of 15°C / min, kept at the constant temperature for 8 h, and finally transferred to liquid nitrogen for storage;

[0045] Step C: When thawing, take it out from the liquid nitrogen, then heat it in a water bath at 37°C until it is completely melted, centrifuge it, and remove the supernatant.

[0046] The storage period in step B is 90 days.

[0047] The centrifugal speed in step C is 1500 r / min, and the centrifugal time is 5 min.

[0048] Example 2

[0049] Take the culture medium with a volume of 100 mL as an example:

[0050] A NK cell freezing solution comprises a culture medium and N-tricine, sodium glycine, carboxymethyl chitosan, polyglutamic acid and sodium polyglutamate added to the culture medium.

[0051] The culture medium is RPMI-1640 culture medium.

[0052] The concentration of N-tricine in the culture medium is 2 g / L.

[0053] The concentration of sodium glycinate in the culture medium is 30 mg / L.

[0054] The concentration of the carboxymethyl chitosan in the culture medium is 150 mg / L.

[0055] The concentration of the polyglutamic acid in the culture medium is 300 mg / L; the molecular weight of the polyglutamic acid is 80,000.

[0056] The concentration of the sodium polyglutamate in the culture medium is 15 mg / L; the molecular weight of the sodium polyglutamate is 100 kDa.

[0057] A method for preparing a NK cell cryopreservation solution, the method comprising the following steps:

[0058] (1) Add N-tris(hydroxymethyl)methylglycine, sodium glycinate, carboxymethyl chitosan, polyglutamic acid and sodium polyglutamate to the culture medium under a sterile environment, stir and mix, and the preparation is completed.

[0059] The stirring time in step (1) is 10 min.

[0060] An application of a NK cell freezing solution, wherein the NK cell freezing solution is applied to the freezing of NK cells.

[0061] A method for using a NK cell cryopreservation solution, the method comprising the following steps:

[0062] Step A: Add the NK cell freezing solution as described above to the NK cells to form a cell density of 10 8 cells / mL of cell suspension;

[0063] Step B: the cell suspension was kept at 4°C for 10 min, then cooled to -15°C at a rate of 1°C / min, kept at the constant temperature for 2 h, then cooled to -80°C at a rate of 20°C / min, kept at the constant temperature for 10 h, and finally transferred to liquid nitrogen for storage;

[0064] Step C: When thawing, take it out from the liquid nitrogen, then heat it in a water bath at 37°C until it is completely melted, centrifuge it, and remove the supernatant.

[0065] The storage period in step B is 90 days.

[0066] The centrifugal speed in step C is 2000 r / min, and the centrifugal time is 8 min.

[0067] Example 3

[0068] Take the culture medium with a volume of 100 mL as an example:

[0069] A NK cell freezing solution comprises a culture medium and N-tricine, sodium glycine, carboxymethyl chitosan, polyglutamic acid and sodium polyglutamate added to the culture medium.

[0070] The culture medium is RPMI-1640 culture medium.

[0071] The concentration of N-tricine in the culture medium is 1.5 g / L.

[0072] The concentration of sodium glycinate in the culture medium is 25 mg / L.

[0073] The concentration of the carboxymethyl chitosan in the culture medium is 130 mg / L.

[0074] The concentration of the polyglutamic acid in the culture medium is 270 mg / L; the molecular weight of the polyglutamic acid is 60,000.

[0075] The concentration of sodium polyglutamate in the culture medium is 13 mg / L; the molecular weight of sodium polyglutamate is 75 kDa.

[0076] A method for preparing a NK cell cryopreservation solution, the method comprising the following steps:

[0077] (1) Add N-tris(hydroxymethyl)methylglycine, sodium glycinate, carboxymethyl chitosan, polyglutamic acid and sodium polyglutamate to the culture medium under a sterile environment, stir and mix, and the preparation is completed.

[0078] The stirring time in step (1) is 8 minutes.

[0079] An application of a NK cell freezing solution, wherein the NK cell freezing solution is applied to the freezing of NK cells.

[0080] A method for using a NK cell cryopreservation solution, the method comprising the following steps:

[0081] Step A: Add the NK cell freezing solution as described above to the NK cells to form a cell density of 10 7 cells / mL of cell suspension;

[0082] Step B: the cell suspension was kept at 4°C for 10 min, then cooled to -14°C at a rate of 1°C / min, kept at the constant temperature for 1.5 h, then cooled to -80°C at a rate of 18°C / min, kept at the constant temperature for 9 h, and finally transferred to liquid nitrogen for storage;

[0083] Step C: When thawing, take it out from the liquid nitrogen, then heat it in a water bath at 37°C until it is completely melted, centrifuge it, and remove the supernatant.

[0084] The storage period in step B is 90 days.

[0085] The centrifugal speed in step C is 1800 r / min, and the centrifugal time is 7 min.

[0086] Comparative Example 1

[0087] Based on Example 1, N-tricine was replaced by an equal weight of sodium glycinate, and the rest remained unchanged.

[0088] Comparative Example 2

[0089] Based on Example 1, sodium glycinate was replaced with an equal weight of N-tris(hydroxymethyl)methylglycine, and the rest remained unchanged.

[0090] Comparative Example 3

[0091] Based on Example 1, carboxymethyl chitosan was replaced with polyglutamic acid of equal weight, and the rest remained unchanged.

[0092] Comparative Example 4

[0093] On the basis of Example 1, carboxymethyl chitosan was replaced by sodium polyglutamate of equal weight, and the rest remained unchanged.

[0094] Comparative Example 5

[0095] Based on Example 1, polyglutamic acid was replaced with an equal weight of carboxymethyl chitosan, and the rest remained unchanged.

[0096] Comparative Example 6

[0097] Based on Example 1, polyglutamic acid was replaced with sodium polyglutamate of equal weight, and the rest remained unchanged.

[0098] Comparative Example 7

[0099] On the basis of Example 1, sodium polyglutamate was replaced with carboxymethyl chitosan of equal weight, and the rest remained unchanged.

[0100] Comparative Example 8

[0101] Based on Example 1, sodium polyglutamate was replaced with polyglutamic acid of equal weight, and the rest remained unchanged.

[0102] Test Example 1

[0103] Survival test:

[0104] Take 10 mL of the completely thawed cell suspension in step C of Example 1-3 and Comparative Example 1-8, then add the same volume of trypan blue dye (the mass fraction of trypan blue is 0.2%, and the solvent is PBS buffer with a pH of 7.4), dye for 5 minutes, then take 20 μL of each and add it to the cell counting plate, and finally use Countstar BioLab automatic cell counter to count the living cells, and the survival rate can be calculated. Repeat the test 3 times and take the average.

[0105] Table 1. Survival test results

[0106]

[0107]

[0108] By comparing Examples 1-3, Comparative Examples 1-8 and Test Example 1, it can be seen that:

[0109] The difference between Comparative Example 1 and Example 1 is that N-tris(hydroxymethyl)methylglycine is replaced by an equal weight of sodium glycinate.

[0110] The difference between Comparative Example 2 and Example 1 is that sodium glycinate is replaced by an equal weight of N-tris(hydroxymethyl)methylglycine.

[0111] The difference between Comparative Example 3 and Example 1 is that carboxymethyl chitosan is replaced by polyglutamic acid of equal weight.

[0112] The difference between Comparative Example 4 and Example 1 is that carboxymethyl chitosan is replaced by an equal weight of sodium polyglutamate.

[0113] The difference between Comparative Example 5 and Example 1 is that polyglutamic acid is replaced by an equal weight of carboxymethyl chitosan.

[0114] The difference between Comparative Example 6 and Example 1 is that polyglutamic acid is replaced by sodium polyglutamate of equal weight.

[0115] The difference between Comparative Example 7 and Example 1 is that sodium polyglutamate is replaced by an equal weight of carboxymethyl chitosan.

[0116] The difference between Comparative Example 8 and Example 1 is that sodium polyglutamate is replaced by polyglutamic acid of equal weight.

[0117] From the comparison between Examples 1-3, Comparative Examples 1-8 and Test Example 1, it can be seen that the NK cell cryopreservation solution provided by the present invention has excellent cryopreservation ability when applied to the cryopreservation of NK cells, and the survival rate of NK cells after recovery is high.

[0118] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A NK cell freezing solution, characterized in that: The NK cell freezing solution comprises a culture medium and N-tris(hydroxymethyl)methylglycine, sodium glycine, carboxymethyl chitosan, polyglutamic acid and sodium polyglutamate added to the culture medium; The concentration of N-tris(hydroxymethyl)methylglycine in the culture medium is 1-2 g / L; The concentration of sodium glycinate in the culture medium is 20-30 mg / L; The concentration of the carboxymethyl chitosan in the culture medium is 100-150 mg / L; The concentration of the polyglutamic acid in the culture medium is 250-300 mg / L; the molecular weight of the polyglutamic acid is 50000-80000; The concentration of sodium polyglutamate in the culture medium is 10-15 mg / L; the molecular weight of sodium polyglutamate is 50-100 kDa; The culture medium is RPMI-1640 culture medium.

2. A method for preparing the NK cell cryopreservation solution according to claim 1, characterized in that: The preparation method comprises the following steps: (1) Add N-tris(hydroxymethyl)methylglycine, sodium glycinate, carboxymethyl chitosan, polyglutamic acid and sodium polyglutamate to the culture medium under a sterile environment, stir and mix, and the preparation is completed.

3. The method for preparing a NK cell cryopreservation solution according to claim 2, characterized in that: The stirring time in step (1) is 5-10 min.

4. A use of the NK cell freezing solution as claimed in claim 1, characterized in that: The NK cell freezing solution is used for freezing NK cells.

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