CN112400863A - Clinical NK cell cryopreservation liquid and cryopreservation method - Google Patents
Clinical NK cell cryopreservation liquid and cryopreservation method Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
Abstract
The invention discloses a clinical NK cell cryopreservation solution and a cryopreservation method, and the clinical NK cell cryopreservation solution comprises the following components in percentage by volume: 3 to 10 percent of dimethyl sulfoxide, 2 to 4 percent of glycerol, 13 to 20 percent of dextran, 0.1 to 0.2 percent of polyvinylpyrrolidone, 5 to 10 percent of N-acetylcysteine and 60 to 70 percent of glucose solution; wherein the glucose solution is formed by compounding medical glucose and medical pure water; the mass concentration of glucose in the glucose solution is 5 wt%; the invention can be directly used for intravenous injection, has lower DMSO content, greatly reduces the toxicity to human bodies or frozen cells, and is safer; can better preserve the activity and killing capacity of cells and reduce the damage to the cells in the process of cryopreservation.
Description
Technical Field
The invention relates to the technical field of cell cryopreservation, in particular to clinical NK cell cryopreservation liquid and a cryopreservation method.
Background
Natural Killer (NK) cell is a kind of lymphocyte, and is derived from CD34+Hematopoietic progenitor cellsKey immunomodulatory functions are achieved by modulating Dendritic Cells (DCs). The NK cells have the functions of resisting virus invasion and killing tumors as immune cells, are the first line of defense of human bodies against tumors, and have good application prospects in adoptive immunotherapy of malignant tumors. However, the compound occupies a small proportion in peripheral blood, and occupies 5% -15% of lymphocytes, and the function of NK cells in a tumor patient body is deficient to different degrees, so that a good anti-tumor effect is difficult to generate. Therefore NK cells are one of the common classes of immune cell therapy. At present, NK cells are mainly obtained from peripheral mononuclear cells through in vitro induction and expansion culture. Has good effect in clinically treating tumors (such as leukemia, lymphoma and melanoma). However, effective storage of NK cells for long periods of time is always a problem.
The cryopreservation technology is to freeze and preserve the living cells at ultralow temperature (liquid nitrogen at-198 deg.C), so that the enzyme activity for controlling the cell growth and metabolism is inhibited, the cell metabolism speed is reduced, the internal biochemical reaction of the cells is slowed down, and the cell activity is maintained, and the cell preservation life is prolonged. Cell freezing methods are mainly divided into slow freezing and fast freezing according to the technical difference of cryopreservation and the way of cell cryopreservation. Slow freezing, also known as slow programmed freezing, refers to placing cells in a cryoprotectant and using a program freezer or a refrigerator with different temperature differences to achieve staged cooling. The principle of the technology is that the water in the extracellular solution is frozen to raise the concentration of the solution, the water in the cell permeates the cell membrane to seep outwards, the cell volume is contracted, the concentration of the intracellular fluid and the osmotic pressure are increased, and the freezing point is lowered. The rapid freezing method is, for example, the conventional vitrification freezing technique, in which a cryoprotectant with a high concentration is rapidly solidified in an ultra-low temperature environment to form an irregular vitrified solid. Normal molecules and ionic molecules of cells in a liquid state are preserved, the cell structure and intracellular components are protected, and low-cell-damage cryopreservation is realized. The high-concentration cryoprotectant added in the rapid freezing process has a large cytotoxic effect and can cause certain cell damage.
Cryoprotectants are required for both rapid and slow freezing. Generally, cryoprotectants are classified as osmotic and non-osmotic. They all reduce the freezing point and electrolyte concentration of the solution and reduce the formation of ice crystals. Osmotic cryoprotectants are generally small molecule substances, mainly composed of dimethyl sulfoxide (DMSO), Glycerol (GLY), Methanol (METH), ethylene Glycol (GE), Propylene Glycol (PG), Dimethylformamide (DMF), Dimethylacetamide (DMA), and the like. The time for its penetration (equilibration) into the cell varies depending on the type of freezing material and cryoprotectant. Osmotic protectants are often used to enhance membrane fluidity and partially dehydrate cells, resulting in freezing point depression, reducing the number and size of ice crystal formations within cells. However, osmotic cryoprotectants have toxic effects on cells themselves, and specific applications require technical optimization for specific preserved subjects. The non-osmotic cryoprotectant is also called as an extramembranous cryoprotectant, such as Fructose (Fructose), Sucrose (SUC), trehalose (trehalose, TRE) honey, and some high molecular compounds, such as polyvinylpyrrolidone (PVP), Dextran (Dextran), Yolk (Yolk), albumin (alum), and the like. Such substances cannot cross the cell membrane and function to protect the cell by maintaining the stability of the cell membrane. The non-permeable cryoprotectant can increase the extracellular osmotic pressure, rapidly exude the water in the cells to the outside of the cells in the freezing process, reduce the water in the cells, thereby reducing the formation of ice crystals in the cells and protecting the cells from mechanical damage caused by the formation of the ice crystals. In addition, during the rewarming of frozen cells, due to the high osmotic concentration of the non-osmotic protective agent formed in the extracellular fluid, the frozen preservation of the cells can be effectively prevented from being damaged by swelling caused by the rapid entry of water into the cells.
The existing cell freezing protective agent generally adopts an osmotic protective agent (DMSO, glycerol, fetal calf serum) or an osmotic protective agent combined with a non-osmotic protective agent (DMSO, hydroxyethyl starch, fetal calf serum), and generally the cryoprotectant cannot leave away from the fetal calf serum. In clinical treatment, autologous serum of a patient is generally used to replace fetal bovine serum, or some antioxidants (such as VC) are added to increase the antioxidant capacity of cell membranes, so as to achieve the purpose of protecting cells. However, the DMSO is harmful to human bodies and cells when the dosage is large, and only frozen and revived cells can be washed if DMSO is removed, so that the operation is complex, and a large amount of cells are lost in the operation process. However, human autologous serum has limited sources and cannot meet the requirements of mass production, and if the human autologous serum is replaced by fetal calf serum, animal-derived components are introduced, so that the risk of virus infection is increased.
Disclosure of Invention
The invention provides a clinical NK cell cryopreservation solution and a cryopreservation method which have high clinical safety and can well maintain the activity and killing capability of cryopreserved cells according to the problems in the prior art.
The technical scheme adopted by the invention is as follows:
a clinical NK cell frozen stock solution comprises the following components in percentage by volume:
3 to 10 percent of dimethyl sulfoxide, 2 to 4 percent of glycerol, 13 to 20 percent of dextran, 0.1 to 0.2 percent of polyvinylpyrrolidone, 5 to 10 percent of N-acetylcysteine and 60 to 70 percent of glucose solution; wherein the glucose solution is formed by compounding medical glucose and medical pure water; the mass concentration of glucose in the glucose solution was 5 wt%.
Further, the dextran is dextran 40 sodium chloride injection.
Further, the polyvinylpyrrolidone is medical grade polyvinylpyrrolidone K15.
Further, the paint comprises the following components in percentage by volume:
5% of dimethyl sulfoxide, 3% of glycerol, 15% of dextran, 0.1% of polyvinylpyrrolidone, 8% of N-acetylcysteine and 68.9% of glucose solution.
A clinical NK cell cryopreservation method of NK cell cryopreservation liquid comprises the following steps:
step 1: fully and uniformly mixing glycerol, dextran, polyvinylpyrrolidone, N-acetylcysteine, glucose and part of water to prepare solution A;
step 2: adding dimethyl sulfoxide and the rest water to prepare solution B;
and step 3: keeping the solution A and the solution B obtained in the steps 1 and 2 away from light and at the temperature of 2-8 ℃ for 30 min;
and 4, step 4: mixing solution A and solution B obtained in step 3, adding NK cells with cell density of 1 × 106cells/ml～8×107cells/ml；
And 5: and (4) standing the cell suspension obtained in the step (4) for 8min at the temperature of 4-6 ℃ in a dark place, then cooling, and transferring to liquid nitrogen for storage after cooling.
Further, the cell density in the step 4 is 8 × 107cells/ml。
Further, the NK cells in step 4 are treated cells, and the treatment process is as follows: centrifuging the NK cell suspension, and then removing the supernatant to obtain the required NK cells.
Furthermore, the temperature reduction procedure in the step 5 can be carried out by placing a programmed temperature reduction box at-80 ℃ or by using a programmed temperature reduction instrument.
Further, the volume ratio of water in the step 1 to the step 2 is 1:1
The invention has the beneficial effects that:
(1) the invention has definite components, does not contain animal-derived components, and does not have the risk of introducing animal-derived antigens;
(2) the content of the used components is below the concentration of respective clinical adverse reactions, and the composition can be directly used for intravenous injection when in use;
(3) the DMSO content in the invention is lower, the toxicity of the DMSO content to human bodies or frozen cells is greatly reduced, and the DMSO content is safer;
(4) the invention can better maintain the activity and killing capability of the cryopreserved cells and reduce the damage to the cells in the cryopreservation process.
(5) The osmotic protective agent and the non-osmotic protective agent are used together, so that when the cell is frozen, cells can be protected from both inside and outside, the mechanical damage of ice crystals of the cells is reduced, and the mechanical damage to the cells in the recovery process can be protected;
(6) in the preparation method, firstly, glycerol, dextran, polyvinylpyrrolidone, N-acetylcysteine, glucose and part of water are uniformly mixed, and then DMSO and the rest water are mixed; finally, mixing the two solutions; the preparation method can avoid that DMSO and other components are added at the same time to form precipitates which influence the freezing preservation effect;
(7) the frozen stock solution of the invention is added with a certain amount of N-acetylcysteine, which can effectively prevent excessive reactive oxygen free radicals in cells from damaging the integrity of cytoplasmic membranes and DNA. In addition, when the revived cells have oxidative stress reaction, the N-acetylcysteine can be used as an antioxidant to provide reduced glutathione so as to achieve the effect of protecting the cells. The N-acetylcysteine also has strong bactericidal activity, and can obviously destroy the mycoderm of common clinical pathogenic bacteria, including pseudomonas, staphylococcus aureus, enterococcus, enterobacter and the like; is more favorable for clinical use.
Drawings
FIG. 1 is a graph showing the cell killing rate of the frozen stock solutions obtained in the examples of the present invention and the comparative examples.
Detailed Description
The invention is further described with reference to the following figures and specific embodiments.
A clinical NK cell frozen stock solution comprises the following components in percentage by volume:
3 to 10 percent of dimethyl sulfoxide, 2 to 4 percent of glycerol, 13 to 20 percent of dextran, 0.1 to 0.2 percent of polyvinylpyrrolidone, 5 to 10 percent of N-acetylcysteine and 60 to 70 percent of glucose solution; wherein the glucose solution is formed by compounding medical glucose and medical pure water; the mass concentration of glucose in the glucose solution was 5 wt%.
The components are preferably:
5% of dimethyl sulfoxide, 3% of glycerol, 15% of dextran, 0.1% of polyvinylpyrrolidone, 8% of N-acetylcysteine and 68.9% of glucose solution.
The dextran is dextran 40 sodium chloride injection. The polyvinylpyrrolidone is medical grade polyvinylpyrrolidone K15.
A clinical NK cell cryopreservation method of NK cell cryopreservation liquid comprises the following steps:
step 1: fully and uniformly mixing glycerol, dextran, polyvinylpyrrolidone, N-acetylcysteine, glucose and part of water to prepare solution A;
step 2: adding dimethyl sulfoxide and the rest water to prepare solution B;
and step 3: keeping the solution A and the solution B obtained in the steps 1 and 2 away from light and at the temperature of 2-8 ℃ for 30 min;
and 4, step 4: mixing solution A and solution B obtained in step 3, adding NK cells with cell density of 1 × 106cells/ml～8×107cells/ml; the cell density is preferably 8X 107cells/ml。
And 5: standing the cell suspension obtained in the step (4) for 8min at 4-6 ℃ in a dark place, and transferring the cell suspension to liquid nitrogen for preservation after cooling and cooling are completed; the temperature reduction procedure can be carried out by placing a program temperature reduction box at-80 ℃ or by adopting a program temperature reduction instrument.
Example 1
Preparing clinical NK cell frozen stock solution according to the following steps:
step 1: weighing the following components in percentage by volume: fully and uniformly mixing 3% of glycerol, 15% of dextran, 0.1% of polyvinylpyrrolidone, 8% of N-acetylcysteine, a glucose solution and a half of water to obtain a solution A;
step 2: and mixing the DMSO and the residual water fully and uniformly to obtain solution B.
And step 3: and (3) keeping the solution A and the solution B obtained in the steps 1 and 2 away from light, and storing at 2 ℃ for 30 min.
And 4, step 4: mixing the solution A and the solution B obtained in step 3, adding NK cells, and gently pumping until the cell suspension density is 8 × 107cells/ml; the NK cell processing procedure was as follows: the NK cell suspension was centrifuged at 300g for 8min and the supernatant was discarded.
And 5: and (4) standing the cell suspension obtained in the step (4) for 8min at the temperature of 4 ℃ in a dark place, then cooling, and transferring to liquid nitrogen for storage after cooling.
Transferring to a program cooling box, placing at-80 deg.C, cooling according to a specific program, and transferring to liquid nitrogen for long-term storage after cooling.
Example 2
Preparing clinical NK cell frozen stock solution according to the following steps:
step 1: weighing the following components in percentage by volume: fully and uniformly mixing 3% of glycerol, 15% of dextran, 0.1% of polyvinylpyrrolidone, 8% of N-acetylcysteine, a glucose solution and a half of water to obtain a solution A;
step 2: and mixing the DMSO and the residual water fully and uniformly to obtain solution B.
And step 3: and (3) keeping the solution A and the solution B obtained in the steps 1 and 2 away from light, and storing at 8 ℃ for 30 min.
And 4, step 4: mixing the solution A and the solution B obtained in step 3, adding NK cells, and gently pumping until the cell suspension density is 1 × 107cells/ml; the NK cell processing procedure was as follows: the NK cell suspension was centrifuged at 300g for 8min and the supernatant was discarded.
And 5: and (4) standing the cell suspension obtained in the step (4) for 8min at 6 ℃ in the dark, then cooling, and transferring to liquid nitrogen for storage after cooling.
Transferring to a program cooling box, placing at-80 deg.C, cooling according to a specific program, and transferring to liquid nitrogen for long-term storage after cooling.
Example 3
Preparing clinical NK cell frozen stock solution according to the following steps:
step 1: weighing the following components in percentage by volume: fully and uniformly mixing 3% of glycerol, 15% of dextran, 0.1% of polyvinylpyrrolidone, 8% of N-acetylcysteine, a glucose solution and a half of water to obtain a solution A;
step 2: and mixing the DMSO and the residual water fully and uniformly to obtain solution B.
And step 3: and (3) keeping the solution A and the solution B obtained in the steps 1 and 2 away from light, and storing at 6 ℃ for 30 min.
And 4, step 4: mixing the solution A and the solution B obtained in step 3, adding NK cells, and gently pumping until the cell suspension density is 8 × 107cells/ml; the NK cell processing procedure was as follows: the NK cell suspension was centrifuged at 300g for 8min and the supernatant was discarded.
And 5: and (4) standing the cell suspension obtained in the step (4) for 8min at the temperature of 4 ℃ in a dark place, then cooling, and transferring to liquid nitrogen for storage after cooling.
Transferring to a program cooling box, placing at-80 deg.C, cooling according to a specific program, and transferring to liquid nitrogen for long-term storage after cooling.
Comparative example
Step 1: weighing the following components according to the volume ratio and uniformly mixing: 10% of DMSO, 20% of FBS and 70% of basal medium, and fully and uniformly mixing to obtain a frozen stock solution.
Step 2: after the preparation is finished, putting the prepared preservative in a refrigerator for freezing and preserving for 30min in a dark place; the refrigerator temperature was 2 ℃.
And step 3: after freezing, adding the frozen stock solution into the treated NK cells, and gently pumping until the cell suspension density is 8 multiplied by 107cells/ml; the NK cell processing procedure was as follows: the NK cell suspension was centrifuged at 300g for 8min and the supernatant was discarded.
And 4, step 4: and (4) uniformly blowing the frozen stock solution obtained in the step (3), keeping out of the sun, standing in a refrigerator for 8min, and setting the temperature of the refrigerator to be 4 ℃.
Transferring the mixture into a program cooling box for cooling, cooling according to a specific program, and transferring the mixture into liquid nitrogen for long-term storage after cooling.
The survival rate and the killing ability against K562 of NK cells in comparative example and examples 1, 2, 3 were tested after a certain period of time, and the results are shown in tables 1 and 2.
The method for detecting the survival rate of the NK cells before and after cryopreservation comprises the following steps:
staining the NK cells before cryopreservation and after recovery, and then counting the cells by using a cell counter to calculate the cell survival rate. The results are shown in Table 1.
Detection of killing capacity of NK cells before and after cryopreservation
And (3) detecting the killing activity of the cultured NK cells before and after cryopreservation by adopting a 7AAD/CFSE labeling method. Taking K562 cells as target cells, respectively detecting the killing activity of NK cells (effector cells) before and after cryopreservation (effective-target ratio of 20:1, 3 parallel control wells at 37 deg.C and 5% CO concentration)2Flow detection and analysis are carried out after 3h in the incubator), and the results are shown in the table2。
TABLE 1 comparison of cell viability (%)
TABLE 2 comparison of cell killing Rate (%) (20:1)
As can be seen from Table 1, the cell viability rates of the groups were decreased from the initial data, and the cell viability rates of the experimental groups were higher than those of the control group per time period. Over time, the data of 12 months show that the survival rate of the cells frozen by the freezing medium is still about 94 percent after recovery, while the survival rate of the control group is obviously inferior. Compared with the freezing preservation protective agent of the comparative example, the NK cells revived by the freezing preservation solution of the invention obviously has higher survival rate.
As can be seen from Table 2, in the killing capacity of each group of NK cells to K562 cells (effective target ratio of 20:1), the killing capacity of NK cells revived by the frozen stock solution of the invention is not obviously different from that before freezing. And under the same effective target ratio, the killing capability of the control group is reduced by about one time compared with that before freezing storage. The frozen stock solution has small influence on the killing activity of NK cells after recovery. Compared with the frozen stock solution of the comparative example, the NK cells recovered by the frozen stock solution of the invention have higher killing capability.
The invention takes DMSO and glycerol as osmotic protective agents and dextran 40 and polyvinylpyrrolidone as non-osmotic protective agents. The permeability protective agent can enhance the fluidity of cell membranes and partially dehydrate cells, so that the freezing point is reduced, the formation amount and size of ice crystals in the cells are reduced, and the combined use of glycerol and DMSO can reduce the DMSO content to a certain extent without influencing the freezing effect. Because DMSO has high cytotoxicity at normal temperature and high toxicity to human bodies. The non-permeable protective agent can effectively improve the extracellular osmotic pressure and reduce the water in the cells, thereby reducing the formation of ice crystals in the cells and protecting the cells from mechanical damage caused by the formation of the ice crystals. In addition, during cell recovery, due to the high osmotic concentration of the non-osmotic protective agent formed in extracellular fluid, the cell cryopreservation can be effectively prevented from being damaged by swelling caused by rapid water entering into cells. The osmotic protective agent and the non-osmotic protective agent are used together, so that when the cell is frozen, cells can be protected from both inside and outside, the mechanical damage of ice crystals of the cells is reduced, and the mechanical damage to the cells in the recovery process can also be protected. The frozen stock solution is added with a certain amount of N-acetylcysteine, so that excessive active oxygen free radicals in cells can be effectively prevented, and the integrity of plasma membranes and DNA of the cells is damaged. In addition, when the revived cells have oxidative stress reaction, the N-acetylcysteine can be used as an antioxidant to provide reduced glutathione so as to achieve the effect of protecting the cells. Meanwhile, the N-acetylcysteine has strong bactericidal activity, and can obviously destroy the mycoderm of common clinical pathogenic bacteria, including pseudomonas, staphylococcus aureus, enterococcus, enterobacter and the like. The glucose solution can provide nutrients for energy consumption caused by external changes in the processes of freezing and reviving cells, has simple components, convenient and fast operation and high clinical safety, can well maintain the activity and killing capacity of the frozen cells, and reduces the damage to the cells in the freezing process.
Claims (9)
1. The clinical NK cell freezing solution is characterized by comprising the following components in percentage by volume:
3 to 10 percent of dimethyl sulfoxide, 2 to 4 percent of glycerol, 13 to 20 percent of dextran, 0.1 to 0.2 percent of polyvinylpyrrolidone, 5 to 10 percent of N-acetylcysteine and 60 to 70 percent of glucose solution; wherein the glucose solution is formed by compounding medical glucose and medical pure water; the mass concentration of glucose in the glucose solution was 5 wt%.
2. The clinical NK cell cryopreservation solution according to claim 1, wherein the dextran is dextran 40 sodium chloride injection.
3. The clinical NK cell cryopreservation solution according to claim 1, wherein the polyvinylpyrrolidone is medical grade polyvinylpyrrolidone K15.
4. The clinical NK cell cryopreservation solution according to claim 1, comprising the following components by volume percent:
5% of dimethyl sulfoxide, 3% of glycerol, 15% of dextran, 0.1% of polyvinylpyrrolidone, 8% of N-acetylcysteine and 68.9% of glucose solution.
5. The clinical NK cell cryopreservation method using the clinical NK cell cryopreservation solution according to claims 1 to 4, comprising the following steps:
step 1: fully and uniformly mixing glycerol, dextran, polyvinylpyrrolidone, N-acetylcysteine, glucose and part of water to prepare solution A;
step 2: adding dimethyl sulfoxide and the rest water to prepare solution B;
and step 3: keeping the solution A and the solution B obtained in the steps 1 and 2 away from light and at the temperature of 2-8 ℃ for 30 min;
and 4, step 4: mixing solution A and solution B obtained in step 3, adding NK cells with cell density of 1 × 106cells/ml～8×107cells/ml；
And 5: and (4) standing the cell suspension obtained in the step (4) for 8min at the temperature of 4-6 ℃ in a dark place, then cooling, and transferring to liquid nitrogen for storage after cooling.
6. The method for cryopreserving a clinical NK cell cryopreserving solution according to claim 5, wherein the cell density in the step 4 is 8 x 107cells/ml。
7. The method according to claim 5, wherein the NK cells in step 4 are processed cells by the following steps: centrifuging the NK cell suspension, and then removing the supernatant to obtain the required NK cells.
8. The method for cryopreserving the clinical NK cell cryopreserving liquid according to claim 5, wherein the cooling process in the step 5 can be performed by using a programmed cooling box at-80 ℃ or using a programmed cooling instrument.
9. The method for cryopreserving the clinical NK cell cryopreserving liquid according to claim 5, wherein the volume ratio of water in the step 1 to the step 2 is 1: 1.
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| CN114041456A (en) * | 2021-12-09 | 2022-02-15 | 安徽中盛溯源生物科技有限公司 | Clinical-grade NK cell cryopreservation liquid and using method thereof |
| CN114766471A (en) * | 2022-05-31 | 2022-07-22 | 宁波建顺生物科技有限公司 | Cryopreservation liquid and cryopreservation method for long-term storage of stem cells and products thereof |
| CN115005199A (en) * | 2022-07-14 | 2022-09-06 | 北京鼎成肽源生物技术有限公司 | Cryopreservation solution and cryopreservation method for natural killer cells and application of cryopreservation solution and cryopreservation method |
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