CN117598288A - Immune cell cryopreservation liquid and cryopreservation method - Google Patents
Immune cell cryopreservation liquid and cryopreservation method Download PDFInfo
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- CN117598288A CN117598288A CN202311632208.0A CN202311632208A CN117598288A CN 117598288 A CN117598288 A CN 117598288A CN 202311632208 A CN202311632208 A CN 202311632208A CN 117598288 A CN117598288 A CN 117598288A
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- 210000002865 immune cell Anatomy 0.000 title claims abstract description 156
- 238000005138 cryopreservation Methods 0.000 title claims abstract description 80
- 239000007788 liquid Substances 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 26
- 210000004027 cell Anatomy 0.000 claims abstract description 91
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 44
- 230000000087 stabilizing effect Effects 0.000 claims abstract description 31
- 239000006143 cell culture medium Substances 0.000 claims abstract description 22
- 150000001413 amino acids Chemical class 0.000 claims abstract description 21
- 230000001681 protective effect Effects 0.000 claims abstract description 21
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 14
- 239000004372 Polyvinyl alcohol Substances 0.000 claims abstract description 14
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 14
- 229920002451 polyvinyl alcohol Polymers 0.000 claims abstract description 14
- 108091006905 Human Serum Albumin Proteins 0.000 claims abstract description 11
- 102000008100 Human Serum Albumin Human genes 0.000 claims abstract description 11
- 102000008070 Interferon-gamma Human genes 0.000 claims abstract description 11
- 108010074328 Interferon-gamma Proteins 0.000 claims abstract description 11
- 229940044627 gamma-interferon Drugs 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims description 119
- 239000011550 stock solution Substances 0.000 claims description 73
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- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 40
- 238000007710 freezing Methods 0.000 claims description 40
- 230000008014 freezing Effects 0.000 claims description 40
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims description 29
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 28
- 239000001963 growth medium Substances 0.000 claims description 28
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- 108010024636 Glutathione Proteins 0.000 claims description 20
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- 101001002657 Homo sapiens Interleukin-2 Proteins 0.000 claims description 20
- 101001002709 Homo sapiens Interleukin-4 Proteins 0.000 claims description 20
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- 229930003427 Vitamin E Natural products 0.000 claims description 20
- 235000001014 amino acid Nutrition 0.000 claims description 20
- 229940024606 amino acid Drugs 0.000 claims description 20
- 238000001816 cooling Methods 0.000 claims description 20
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 claims description 20
- 102000055277 human IL2 Human genes 0.000 claims description 20
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- 238000004321 preservation Methods 0.000 claims description 20
- 229910052711 selenium Inorganic materials 0.000 claims description 20
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- 235000019154 vitamin C Nutrition 0.000 claims description 20
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- 229940046009 vitamin E Drugs 0.000 claims description 20
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- 229910052757 nitrogen Inorganic materials 0.000 claims description 14
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 10
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 10
- CYDQOEWLBCCFJZ-UHFFFAOYSA-N 4-(4-fluorophenyl)oxane-4-carboxylic acid Chemical compound C=1C=C(F)C=CC=1C1(C(=O)O)CCOCC1 CYDQOEWLBCCFJZ-UHFFFAOYSA-N 0.000 claims description 10
- UZOVYGYOLBIAJR-UHFFFAOYSA-N 4-isocyanato-4'-methyldiphenylmethane Chemical group C1=CC(C)=CC=C1CC1=CC=C(N=C=O)C=C1 UZOVYGYOLBIAJR-UHFFFAOYSA-N 0.000 claims description 10
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims description 10
- 229920002307 Dextran Polymers 0.000 claims description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 10
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 10
- 239000004471 Glycine Substances 0.000 claims description 10
- 229920001612 Hydroxyethyl starch Polymers 0.000 claims description 10
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 10
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 10
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 10
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 10
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 10
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 10
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 10
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 10
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims description 10
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 10
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 10
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 10
- 235000004279 alanine Nutrition 0.000 claims description 10
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 10
- 235000009582 asparagine Nutrition 0.000 claims description 10
- 229960001230 asparagine Drugs 0.000 claims description 10
- 235000003704 aspartic acid Nutrition 0.000 claims description 10
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 10
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 10
- 210000004369 blood Anatomy 0.000 claims description 10
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- 235000018417 cysteine Nutrition 0.000 claims description 10
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 10
- 239000008103 glucose Substances 0.000 claims description 10
- 235000013922 glutamic acid Nutrition 0.000 claims description 10
- 239000004220 glutamic acid Substances 0.000 claims description 10
- 235000004554 glutamine Nutrition 0.000 claims description 10
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 10
- 229940050526 hydroxyethylstarch Drugs 0.000 claims description 10
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 claims description 10
- 235000013930 proline Nutrition 0.000 claims description 10
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- 235000002374 tyrosine Nutrition 0.000 claims description 10
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 10
- 229960003180 glutathione Drugs 0.000 claims description 9
- 230000000694 effects Effects 0.000 abstract description 10
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- 210000000170 cell membrane Anatomy 0.000 abstract description 3
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 3
- 238000011084 recovery Methods 0.000 description 9
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- 238000009098 adjuvant therapy Methods 0.000 description 1
- 230000007416 antiviral immune response Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 235000019996 baijiu Nutrition 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
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- 150000007949 saponins Chemical class 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
Classifications
-
- 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
-
- 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
-
- 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
-
- 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
- A01N1/0278—Physical preservation processes
-
- 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
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Biophysics (AREA)
- Physiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of cell cryopreservation, in particular to immune cell cryopreservation liquid, which comprises the following components: 0.01 to 0.03mg/m of polyvinyl alcohol, 0.03 to 0.07mg/m of dimethyl sulfoxide, 0.01 to 0.03mg/m of polyethylene glycol, 13 to 18mg/m of human serum albumin, 0.03 to 0.07mg/m of gamma-interferon, 0.1 to 0.3mg/m of cell balancing solution, 2 to 10mg/m of cell stabilizing solution, 10 to 16mg/m of amino acid, 5 to 10mg/m of protective solution and cell culture medium. The immune cell cryopreservation liquid and the cryopreservation method can avoid the introduction of harmful components by adopting serum-free and DMSO-free cryopreservation liquid, so that the immune cell cryopreservation liquid is safer and more reliable in clinical application, and the permeability of cell membranes can be effectively changed by adopting polyvinyl alcohol, dimethyl sulfoxide and polyethylene glycol, so that free water in cells is discharged out of the cells, the damage to the cells caused by ice crystals formed in the cells in the cryopreservation process is reduced, and the potential risk of damaging the activity of the cells is reduced by adopting safer components.
Description
Technical Field
The invention relates to the technical field of cell cryopreservation, in particular to immune cell cryopreservation liquid and a cryopreservation method.
Background
The biological immune cell treatment can induce an autologous antiviral immune response by in vitro culture, proliferation and activation and return to the body, and once activated, the human antiviral immunity can continuously generate antiviral substances to kill viruses. T cells with the function of killing tumor cells are activated and are mostly changed into memory cells to be stored in lymphoid tissues in vivo, so that long-term protection is provided for thoroughly eliminating tumor cells and preventing and treating metastasis and recurrence. With the rapid development of immunocytobiology and immune molecular biology, somatic cell immunotherapy has become one of the important means for adjuvant therapy after radiotherapy and chemotherapy of tumor patients, and has good effects of promoting the reconstruction of the immune system of patients, eliminating residual focus and purifying bone marrow. The immune cells are cultured and frozen in advance, and are recovered and returned immediately when needed, so that the method is quite beneficial to the treatment of patients. However, the cell cryopreservation process can significantly change the thermodynamic, chemical and physical environment of the cells with the concomitant risk of biological damage. In order to minimize the damage of cells during the freezing and recovery processes, it is necessary to add a freezing protecting agent before freezing and remove the cells after dissolution. The most commonly used frozen stock solution at present is dimethyl sulfoxide, and the material has small molecular weight and high solubility, is easy to penetrate cells, can lower the freezing point, and reduces the chance of forming ice crystals in the cells, thereby reducing the damage of the ice crystals to the cells. However, high-concentration DMSO is cytotoxic, and other liquid components such as fetal bovine serum and cell culture medium must be added to reduce the concentration of DMSO and reduce the damage to cells.
Through retrieval, the Chinese patent publication No. CN106665559B discloses an immune cell cryopreservation solution and application thereof, wherein the immune cell cryopreservation solution comprises: 2-10% by volume DMSO;2-15 vol.% HSA; 20-88% by volume of plasma, optionally adding baijiu saponin R; and the balance of culture medium. The frozen stock solution can effectively store immune cells, and has long storage time, strong recovery capability after cell recovery and high cell recovery rate.
At present, the immune cell frozen stock solution on the market is mostly prepared from fetal bovine serum, high-proportion DMSO, autologous plasma of patients and non-clinical application-level reagents, and the animal serum has the potential danger of introducing animal pathogenic bacteria pollution, and the DMSO has certain toxicity, so that the immune cell survival rate is lower when freezing immune cells. In view of this, we propose an immune cell cryopreservation solution and cryopreservation method.
Disclosure of Invention
The invention aims to provide immune cell cryopreservation liquid and cryopreservation method, which are used for solving the problems in the background technology.
In order to achieve the above purpose, the present invention provides the following technical solutions:
an immune cell cryopreservation solution comprises the following components: 0.01 to 0.03mg/m of polyvinyl alcohol, 0.03 to 0.07mg/m of dimethyl sulfoxide, 0.01 to 0.03mg/m of polyethylene glycol, 13 to 18mg/m of human serum albumin, 0.03 to 0.07mg/m of gamma-interferon, 0.1 to 0.3mg/m of cell balancing solution, 2 to 10mg/m of cell stabilizing solution, 10 to 16mg/m of amino acid, 5 to 10mg/m of protective solution and cell culture medium.
Preferably, the amino acid comprises one or a combination of more of glycine, alanine, proline, tyrosine, serine, cysteine, asparagine, glutamine, aspartic acid and glutamic acid.
Preferably, the cell stabilizing solution comprises vitamin C, vitamin E, insulin-transferrin-selenium, recombinant human interleukin 2, recombinant human interleukin 4 and reduced glutathione.
Preferably, the content of each component in the cell stabilizing solution in the immune cell frozen stock solution is 20-300 mug/ml of vitamin C, 50-350 mug/ml of vitamin E, 15-55 mug/ml of insulin-transferrin-selenium, 50-350U/ml of recombinant human interleukin 2, 30-400U/ml of recombinant human interleukin 4 and 0.5-2 mg/ml of reduced glutathione.
Preferably, the cell culture medium is IMDM medium, RPMI-1640 medium or McCoy5A medium.
Preferably, the cell balancing solution is one of a grignard solution or a sodium lactate ringer's solution.
Preferably, the protective liquid is one or more of trehalose, dextran, glucose and hydroxyethyl starch.
A cryopreservation method of immune cell cryopreservation liquid comprises the following steps:
s1, extracting immune cells by using whole blood;
s2, adding the extracted immune cells into the prepared immune cell frozen stock solution according to a proportion, and placing the immune cell frozen stock solution into an environment of 4-8 ℃ for preservation for 2-3 hours for precooling;
s3, cooling the frozen stock solution filled with the immune cells to the temperature of minus 80 ℃ at the speed of 3-5 ℃/h after the pre-cooling step is finished, and transferring the frozen stock solution into liquid nitrogen for preservation after the frozen stock solution is frozen at the temperature of minus 80 ℃ for a period of time, namely, freezing the immune cells.
Preferably, 1.5-2.5 ml of immune cell frozen stock solution is added into every 1000-1500 ten thousand IU of immune cells in the step S2.
Preferably, the immune cell cryopreservation solution in the step S3 is frozen at 80 ℃ below zero for 2-3 hours.
Compared with the prior art, the invention has the beneficial effects that:
the immune cell cryopreservation liquid and the cryopreservation method can avoid the introduction of harmful components by adopting serum-free and DMSO-free cryopreservation liquid, so that the immune cell cryopreservation liquid is safer and more reliable in clinical application, the permeability of cell membranes can be effectively changed by adopting polyvinyl alcohol, dimethyl sulfoxide and polyethylene glycol, free water in cells is discharged out of the cells, the damage to the cells caused by ice crystals formed in the cells in the cryopreservation process is reduced, the cryopreservation recovery rate of immune cells is improved, the safer components are adopted, the potential risk of damaging the activity of the cells is reduced, and the immune cell cryopreservation liquid does not contain the components such as antibodies, complements and the like, has less impurity proteins, and reduces the influence on the activity of the immune cells.
Drawings
FIG. 1 is a schematic diagram of a refrigeration process according to the present invention;
FIG. 2 is a graphical representation of cell activity comparison after 14 days of resuscitation in conventional procedures of the present invention;
FIG. 3 is a graphical representation of cell activity comparison after 14 days of resuscitation in liquid nitrogen cryopreservation in accordance with the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
As shown in fig. 1-3, an immune cell cryopreservation solution comprises the following components: 0.01mg/m of polyvinyl alcohol, 0.03mg/m of dimethyl sulfoxide, 0.01mg/m of polyethylene glycol, 13mg/m of human albumin, 0.03mg/m of gamma-interferon, 0.1mg/m of cell balancing solution, 2mg/m of cell stabilizing solution, 10mg/m of amino acid, 5mg/m of protective solution and cell culture medium.
The amino acid comprises one or more of glycine, alanine, proline, tyrosine, serine, cysteine, asparagine, glutamine, aspartic acid and glutamic acid.
The cell stabilizing solution contains vitamin C, vitamin E, insulin-transferrin-selenium, recombinant human interleukin 2, recombinant human interleukin 4 and reduced glutathione.
The content of each component in the cell stabilizing solution in the immune cell frozen stock solution is 20-300 mug/ml vitamin C, 50-350 mug/ml vitamin E, 15-55 mug/ml insulin-transferrin-selenium, 50-350U/ml recombinant human interleukin 2, 30-400U/ml recombinant human interleukin 4 and 0.5-2 mg/ml reducing glutathione.
The cell culture medium is IMDM culture medium, RPMI-1640 culture medium or McCoy5A culture medium.
The cell balancing solution is one of Grignard solution or sodium lactate ringer's solution.
The protective liquid is one or more of trehalose, dextran, glucose and hydroxyethyl starch.
A cryopreservation method of immune cell cryopreservation liquid comprises the following steps:
s1, extracting immune cells by using whole blood;
s2, adding the extracted immune cells into the prepared immune cell frozen stock solution according to a proportion, and placing the immune cell frozen stock solution into an environment of 4-8 ℃ for preservation for 2-3 hours for precooling;
s3, cooling the frozen stock solution filled with the immune cells to the temperature of minus 80 ℃ at the speed of 3-5 ℃/h after the pre-cooling step is finished, and transferring the frozen stock solution into liquid nitrogen for preservation after the frozen stock solution is frozen at the temperature of minus 80 ℃ for a period of time, namely, freezing the immune cells.
In the step S2, 1.5-2.5 ml of immune cell frozen stock solution is added into every 1000-1500 ten thousand IU of immune cells.
In the step S3, the freezing time of the immune cell freezing solution at 80 ℃ below zero is 2-3 hours.
Example two
As shown in fig. 1-3, an immune cell cryopreservation solution comprises the following components: 0.03mg/m of polyvinyl alcohol, 0.07mg/m of dimethyl sulfoxide, 0.03mg/m of polyethylene glycol, 18mg/m of human albumin, 0.07mg/m of gamma-interferon, 0.3mg/m of cell balancing solution, 10mg/m of cell stabilizing solution, 16mg/m of amino acid, 10mg/m of protective solution and cell culture medium.
The amino acid comprises one or more of glycine, alanine, proline, tyrosine, serine, cysteine, asparagine, glutamine, aspartic acid and glutamic acid.
The cell stabilizing solution contains vitamin C, vitamin E, insulin-transferrin-selenium, recombinant human interleukin 2, recombinant human interleukin 4 and reduced glutathione.
The content of each component in the cell stabilizing solution in the immune cell frozen stock solution is 20-300 mug/ml vitamin C, 50-350 mug/ml vitamin E, 15-55 mug/ml insulin-transferrin-selenium, 50-350U/ml recombinant human interleukin 2, 30-400U/ml recombinant human interleukin 4 and 0.5-2 mg/ml reducing glutathione.
The cell culture medium is IMDM culture medium, RPMI-1640 culture medium or McCoy5A culture medium.
The cell balancing solution is one of Grignard solution or sodium lactate ringer's solution.
The protective liquid is one or more of trehalose, dextran, glucose and hydroxyethyl starch.
A cryopreservation method of immune cell cryopreservation liquid comprises the following steps:
s1, extracting immune cells by using whole blood;
s2, adding the extracted immune cells into the prepared immune cell frozen stock solution according to a proportion, and placing the immune cell frozen stock solution into an environment of 4-8 ℃ for preservation for 2-3 hours for precooling;
s3, cooling the frozen stock solution filled with the immune cells to the temperature of minus 80 ℃ at the speed of 3-5 ℃/h after the pre-cooling step is finished, and transferring the frozen stock solution into liquid nitrogen for preservation after the frozen stock solution is frozen at the temperature of minus 80 ℃ for a period of time, namely, freezing the immune cells.
In the step S2, 1.5-2.5 ml of immune cell frozen stock solution is added into every 1000-1500 ten thousand IU of immune cells.
In the step S3, the freezing time of the immune cell freezing solution at 80 ℃ below zero is 2-3 hours.
Example III
As shown in fig. 1-3, an immune cell cryopreservation solution comprises the following components: 0.03mg/m of polyvinyl alcohol, 0.07mg/m of dimethyl sulfoxide, 0.01mg/m of polyethylene glycol, 18mg/m of human albumin, 0.03mg/m of gamma-interferon, 0.3mg/m of cell balancing solution, 5mg/m of cell stabilizing solution, 13mg/m of amino acid, 7mg/m of protective solution and cell culture medium.
The amino acid comprises one or more of glycine, alanine, proline, tyrosine, serine, cysteine, asparagine, glutamine, aspartic acid and glutamic acid.
The cell stabilizing solution contains vitamin C, vitamin E, insulin-transferrin-selenium, recombinant human interleukin 2, recombinant human interleukin 4 and reduced glutathione.
The content of each component in the cell stabilizing solution in the immune cell frozen stock solution is 20-300 mug/ml vitamin C, 50-350 mug/ml vitamin E, 15-55 mug/ml insulin-transferrin-selenium, 50-350U/ml recombinant human interleukin 2, 30-400U/ml recombinant human interleukin 4 and 0.5-2 mg/ml reducing glutathione.
The cell culture medium is IMDM culture medium, RPMI-1640 culture medium or McCoy5A culture medium.
The cell balancing solution is one of Grignard solution or sodium lactate ringer's solution.
The protective liquid is one or more of trehalose, dextran, glucose and hydroxyethyl starch.
A cryopreservation method of immune cell cryopreservation liquid comprises the following steps:
s1, extracting immune cells by using whole blood;
s2, adding the extracted immune cells into the prepared immune cell frozen stock solution according to a proportion, and placing the immune cell frozen stock solution into an environment of 4-8 ℃ for preservation for 2-3 hours for precooling;
s3, cooling the frozen stock solution filled with the immune cells to the temperature of minus 80 ℃ at the speed of 3-5 ℃/h after the pre-cooling step is finished, and transferring the frozen stock solution into liquid nitrogen for preservation after the frozen stock solution is frozen at the temperature of minus 80 ℃ for a period of time, namely, freezing the immune cells.
In the step S2, 1.5-2.5 ml of immune cell frozen stock solution is added into every 1000-1500 ten thousand IU of immune cells.
In the step S3, the freezing time of the immune cell freezing solution at 80 ℃ below zero is 2-3 hours.
Example IV
As shown in fig. 1-3, an immune cell cryopreservation solution comprises the following components: 0.02mg/m of polyvinyl alcohol, 0.04mg/m of dimethyl sulfoxide, 0.03mg/m of polyethylene glycol, 15mg/m of human albumin, 0.06mg/m of gamma-interferon, 0.1mg/m of cell balancing solution, 10mg/m of cell stabilizing solution, 16mg/m of amino acid, 9mg/m of protective solution and cell culture medium.
The amino acid comprises one or more of glycine, alanine, proline, tyrosine, serine, cysteine, asparagine, glutamine, aspartic acid and glutamic acid.
The cell stabilizing solution contains vitamin C, vitamin E, insulin-transferrin-selenium, recombinant human interleukin 2, recombinant human interleukin 4 and reduced glutathione.
The content of each component in the cell stabilizing solution in the immune cell frozen stock solution is 20-300 mug/ml vitamin C, 50-350 mug/ml vitamin E, 15-55 mug/ml insulin-transferrin-selenium, 50-350U/ml recombinant human interleukin 2, 30-400U/ml recombinant human interleukin 4 and 0.5-2 mg/ml reducing glutathione.
The cell culture medium is IMDM culture medium, RPMI-1640 culture medium or McCoy5A culture medium.
The cell balancing solution is one of Grignard solution or sodium lactate ringer's solution.
The protective liquid is one or more of trehalose, dextran, glucose and hydroxyethyl starch.
A cryopreservation method of immune cell cryopreservation liquid comprises the following steps:
s1, extracting immune cells by using whole blood;
s2, adding the extracted immune cells into the prepared immune cell frozen stock solution according to a proportion, and placing the immune cell frozen stock solution into an environment of 4-8 ℃ for preservation for 2-3 hours for precooling;
s3, cooling the frozen stock solution filled with the immune cells to the temperature of minus 80 ℃ at the speed of 3-5 ℃/h after the pre-cooling step is finished, and transferring the frozen stock solution into liquid nitrogen for preservation after the frozen stock solution is frozen at the temperature of minus 80 ℃ for a period of time, namely, freezing the immune cells.
In the step S2, 1.5-2.5 ml of immune cell frozen stock solution is added into every 1000-1500 ten thousand IU of immune cells.
In the step S3, the freezing time of the immune cell freezing solution at 80 ℃ below zero is 2-3 hours.
Example five
As shown in fig. 1-3, an immune cell cryopreservation solution comprises the following components: 0.01mg/m of polyvinyl alcohol, 0.07mg/m of dimethyl sulfoxide, 0.03mg/m of polyethylene glycol, 15mg/m of human albumin, 0.04mg/m of gamma-interferon, 0.2mg/m of cell balancing solution, 7mg/m of cell stabilizing solution, 11mg/m of amino acid, 6mg/m of protective solution and cell culture medium.
The amino acid comprises one or more of glycine, alanine, proline, tyrosine, serine, cysteine, asparagine, glutamine, aspartic acid and glutamic acid.
The cell stabilizing solution contains vitamin C, vitamin E, insulin-transferrin-selenium, recombinant human interleukin 2, recombinant human interleukin 4 and reduced glutathione.
The content of each component in the cell stabilizing solution in the immune cell frozen stock solution is 20-300 mug/ml vitamin C, 50-350 mug/ml vitamin E, 15-55 mug/ml insulin-transferrin-selenium, 50-350U/ml recombinant human interleukin 2, 30-400U/ml recombinant human interleukin 4 and 0.5-2 mg/ml reducing glutathione.
The cell culture medium is IMDM culture medium, RPMI-1640 culture medium or McCoy5A culture medium.
The cell balancing solution is one of Grignard solution or sodium lactate ringer's solution.
The protective liquid is one or more of trehalose, dextran, glucose and hydroxyethyl starch.
A cryopreservation method of immune cell cryopreservation liquid comprises the following steps:
s1, extracting immune cells by using whole blood;
s2, adding the extracted immune cells into the prepared immune cell frozen stock solution according to a proportion, and placing the immune cell frozen stock solution into an environment of 4-8 ℃ for preservation for 2-3 hours for precooling;
s3, cooling the frozen stock solution filled with the immune cells to the temperature of minus 80 ℃ at the speed of 3-5 ℃/h after the pre-cooling step is finished, and transferring the frozen stock solution into liquid nitrogen for preservation after the frozen stock solution is frozen at the temperature of minus 80 ℃ for a period of time, namely, freezing the immune cells.
In the step S2, 1.5-2.5 ml of immune cell frozen stock solution is added into every 1000-1500 ten thousand IU of immune cells.
In the step S3, the freezing time of the immune cell freezing solution at 80 ℃ below zero is 2-3 hours.
Example six
As shown in fig. 1-3, an immune cell cryopreservation solution comprises the following components: 0.02mg/m of polyvinyl alcohol, 0.07mg/m of dimethyl sulfoxide, 0.01mg/m of polyethylene glycol, 18mg/m of human albumin, 0.03mg/m of gamma-interferon, 0.1mg/m of cell balancing solution, 2mg/m of cell stabilizing solution, 10mg/m of amino acid, 5mg/m of protective solution and cell culture medium.
The amino acid comprises one or more of glycine, alanine, proline, tyrosine, serine, cysteine, asparagine, glutamine, aspartic acid and glutamic acid.
The cell stabilizing solution contains vitamin C, vitamin E, insulin-transferrin-selenium, recombinant human interleukin 2, recombinant human interleukin 4 and reduced glutathione.
The content of each component in the cell stabilizing solution in the immune cell frozen stock solution is 20-300 mug/ml vitamin C, 50-350 mug/ml vitamin E, 15-55 mug/ml insulin-transferrin-selenium, 50-350U/ml recombinant human interleukin 2, 30-400U/ml recombinant human interleukin 4 and 0.5-2 mg/ml reducing glutathione.
The cell culture medium is IMDM culture medium, RPMI-1640 culture medium or McCoy5A culture medium.
The cell balancing solution is one of Grignard solution or sodium lactate ringer's solution.
The protective liquid is one or more of trehalose, dextran, glucose and hydroxyethyl starch.
A cryopreservation method of immune cell cryopreservation liquid comprises the following steps:
s1, extracting immune cells by using whole blood;
s2, adding the extracted immune cells into the prepared immune cell frozen stock solution according to a proportion, and placing the immune cell frozen stock solution into an environment of 4-8 ℃ for preservation for 2-3 hours for precooling;
s3, cooling the frozen stock solution filled with the immune cells to the temperature of minus 80 ℃ at the speed of 3-5 ℃/h after the pre-cooling step is finished, and transferring the frozen stock solution into liquid nitrogen for preservation after the frozen stock solution is frozen at the temperature of minus 80 ℃ for a period of time, namely, freezing the immune cells.
In the step S2, 1.5-2.5 ml of immune cell frozen stock solution is added into every 1000-1500 ten thousand IU of immune cells.
In the step S3, the freezing time of the immune cell freezing solution at 80 ℃ below zero is 2-3 hours.
Example seven
As shown in fig. 1-3, an immune cell cryopreservation solution comprises the following components: 0.01mg/m of polyvinyl alcohol, 0.03mg/m of dimethyl sulfoxide, 0.03mg/m of polyethylene glycol, 18mg/m of human albumin, 0.07mg/m of gamma-interferon, 0.3mg/m of cell balancing solution, 8mg/m of cell stabilizing solution, 15mg/m of amino acid, 9mg/m of protective solution and cell culture medium.
The amino acid comprises one or more of glycine, alanine, proline, tyrosine, serine, cysteine, asparagine, glutamine, aspartic acid and glutamic acid.
The cell stabilizing solution contains vitamin C, vitamin E, insulin-transferrin-selenium, recombinant human interleukin 2, recombinant human interleukin 4 and reduced glutathione.
The content of each component in the cell stabilizing solution in the immune cell frozen stock solution is 20-300 mug/ml vitamin C, 50-350 mug/ml vitamin E, 15-55 mug/ml insulin-transferrin-selenium, 50-350U/ml recombinant human interleukin 2, 30-400U/ml recombinant human interleukin 4 and 0.5-2 mg/ml reducing glutathione.
The cell culture medium is IMDM culture medium, RPMI-1640 culture medium or McCoy5A culture medium.
The cell balancing solution is one of Grignard solution or sodium lactate ringer's solution.
The protective liquid is one or more of trehalose, dextran, glucose and hydroxyethyl starch.
A cryopreservation method of immune cell cryopreservation liquid comprises the following steps:
s1, extracting immune cells by using whole blood;
s2, adding the extracted immune cells into the prepared immune cell frozen stock solution according to a proportion, and placing the immune cell frozen stock solution into an environment of 4-8 ℃ for preservation for 2-3 hours for precooling;
s3, cooling the frozen stock solution filled with the immune cells to the temperature of minus 80 ℃ at the speed of 3-5 ℃/h after the pre-cooling step is finished, and transferring the frozen stock solution into liquid nitrogen for preservation after the frozen stock solution is frozen at the temperature of minus 80 ℃ for a period of time, namely, freezing the immune cells.
In the step S2, 1.5-2.5 ml of immune cell frozen stock solution is added into every 1000-1500 ten thousand IU of immune cells.
In the step S3, the freezing time of the immune cell freezing solution at 80 ℃ below zero is 2-3 hours.
Example eight
As shown in fig. 1-3, an immune cell cryopreservation solution comprises the following components: 0.01mg/m of polyvinyl alcohol, 0.02mg/m of dimethyl sulfoxide, 0.06mg/m of polyethylene glycol, 14mg/m of human albumin, 0.06mg/m of gamma-interferon, 0.2mg/m of cell balancing solution, 8mg/m of cell stabilizing solution, 15mg/m of amino acid, 7mg/m of protective solution and cell culture medium.
The amino acid comprises one or more of glycine, alanine, proline, tyrosine, serine, cysteine, asparagine, glutamine, aspartic acid and glutamic acid.
The cell stabilizing solution contains vitamin C, vitamin E, insulin-transferrin-selenium, recombinant human interleukin 2, recombinant human interleukin 4 and reduced glutathione.
The content of each component in the cell stabilizing solution in the immune cell frozen stock solution is 20-300 mug/ml vitamin C, 50-350 mug/ml vitamin E, 15-55 mug/ml insulin-transferrin-selenium, 50-350U/ml recombinant human interleukin 2, 30-400U/ml recombinant human interleukin 4 and 0.5-2 mg/ml reducing glutathione.
The cell culture medium is IMDM culture medium, RPMI-1640 culture medium or McCoy5A culture medium.
The cell balancing solution is one of Grignard solution or sodium lactate ringer's solution.
The protective liquid is one or more of trehalose, dextran, glucose and hydroxyethyl starch.
A cryopreservation method of immune cell cryopreservation liquid comprises the following steps:
s1, extracting immune cells by using whole blood;
s2, adding the extracted immune cells into the prepared immune cell frozen stock solution according to a proportion, and placing the immune cell frozen stock solution into an environment of 4-8 ℃ for preservation for 2-3 hours for precooling;
s3, cooling the frozen stock solution filled with the immune cells to the temperature of minus 80 ℃ at the speed of 3-5 ℃/h after the pre-cooling step is finished, and transferring the frozen stock solution into liquid nitrogen for preservation after the frozen stock solution is frozen at the temperature of minus 80 ℃ for a period of time, namely, freezing the immune cells.
In the step S2, 1.5-2.5 ml of immune cell frozen stock solution is added into every 1000-1500 ten thousand IU of immune cells.
In the step S3, the freezing time of the immune cell freezing solution at 80 ℃ below zero is 2-3 hours.
As shown in fig. 2, after 14 days of cryopreservation and recovery under a conventional program cooling and freezing environment (namely, preset for 20min at 4 ℃, preset for 30min at-20 ℃, overnight cryopreservation at-80 ℃ and direct storage in liquid nitrogen), the cell activity of the immune cell cryopreservation solution is far higher than that of the common cell cryopreservation solution after cell recovery compared with that of the two common cell cryopreservation solutions;
as shown in fig. 3, after 14 days of resuscitating in a liquid nitrogen freezing environment (i.e., directly placed at-80 ℃ for freezing and directly placed at liquid nitrogen for storage after overnight freezing), the cell activity of the immune cell freezing solution is far higher than that of the two common cell freezing solutions after resuscitating, compared with that of the two common cell freezing solutions;
therefore, as can be seen from fig. 2 and fig. 3, the immune cell cryopreservation solution has lower damage to cell irradiation, and compared with the common cell cryopreservation solution, the immune cell cryopreservation solution improves the cryopreservation recovery survival rate of immune cells.
The serum-free and DMSO-free cryopreservation liquid is adopted in the invention, so that the introduction of harmful ingredients can be avoided, the clinical application is safer and more reliable, the permeability of cell membranes can be effectively changed by adopting polyvinyl alcohol, dimethyl sulfoxide and polyethylene glycol, free water in cells is discharged out of the cells, the damage of ice crystals formed in the cells to the cells in the cryopreservation process is reduced, the cryopreservation recovery rate of immune cells is improved, safer components are adopted, the potential danger of damaging the activity of the cells is reduced, and the immune cell cryopreservation liquid has no antibody, complement and other components, less mixed proteins and less influence on the activity of the immune cells.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the above-described embodiments, and that the above-described embodiments and descriptions are only preferred embodiments of the present invention, and are not intended to limit the invention, and that various changes and modifications may be made therein without departing from the spirit and scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. An immune cell cryopreservation solution is characterized by comprising the following components: 0.01 to 0.03mg/m of polyvinyl alcohol, 0.03 to 0.07mg/m of dimethyl sulfoxide, 0.01 to 0.03mg/m of polyethylene glycol, 13 to 18mg/m of human serum albumin, 0.03 to 0.07mg/m of gamma-interferon, 0.1 to 0.3mg/m of cell balancing solution, 2 to 10mg/m of cell stabilizing solution, 10 to 16mg/m of amino acid, 5 to 10mg/m of protective solution and cell culture medium.
2. The immune cell cryopreservation solution according to claim 1, wherein: the amino acid comprises one or more of glycine, alanine, proline, tyrosine, serine, cysteine, asparagine, glutamine, aspartic acid and glutamic acid.
3. The immune cell cryopreservation solution according to claim 1, wherein: the cell stabilizing solution comprises vitamin C, vitamin E, insulin-transferrin-selenium, recombinant human interleukin 2, recombinant human interleukin 4 and reduced glutathione.
4. The immunocyte cryopreservation solution of claim 3, wherein: the content of each component in the cell stabilizing solution in the immune cell frozen stock solution is 20-300 mug/ml vitamin C, 50-350 mug/ml vitamin E, 15-55 mug/ml insulin-transferrin-selenium, 50-350U/ml recombinant human interleukin 2, 30-400U/ml recombinant human interleukin 4 and 0.5-2 mg/ml reducing glutathione.
5. The immune cell cryopreservation solution according to claim 1, wherein: the cell culture medium is IMDM culture medium, RPMI-1640 culture medium or McCoy5A culture medium.
6. The immune cell cryopreservation solution according to claim 1, wherein: the cell balancing liquid is one of Grignard liquid or sodium lactate ringer's liquid.
7. The immune cell cryopreservation solution according to claim 1, wherein: the protective liquid is one or a combination of more of trehalose, dextran, glucose and hydroxyethyl starch.
8. A method of cryopreserving an immune cell cryopreservation solution according to claim 1, comprising the steps of:
s1, extracting immune cells by using whole blood;
s2, adding the extracted immune cells into the prepared immune cell frozen stock solution according to a proportion, and placing the immune cell frozen stock solution into an environment of 4-8 ℃ for preservation for 2-3 hours for precooling;
s3, cooling the frozen stock solution filled with the immune cells to the temperature of minus 80 ℃ at the speed of 3-5 ℃/h after the pre-cooling step is finished, and transferring the frozen stock solution into liquid nitrogen for preservation after the frozen stock solution is frozen at the temperature of minus 80 ℃ for a period of time, namely, freezing the immune cells.
9. The method for cryopreserving an immune cell cryopreservation solution according to claim 8, wherein: in the step S2, 1.5-2.5 ml of immune cell frozen stock solution is added into every 1000-1500 ten thousand IU of immune cells.
10. The method for cryopreserving an immune cell cryopreservation solution according to claim 8, wherein: the freezing time of the immune cell freezing solution in the step S3 at the temperature of 80 ℃ below zero is 2-3 hours.
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