CN116473051A - Serum-free non-program cell frozen stock solution and preparation method and application thereof - Google Patents
Serum-free non-program cell frozen stock solution and preparation method and application thereof Download PDFInfo
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- 239000011550 stock solution Substances 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title abstract description 5
- 210000004027 cell Anatomy 0.000 claims abstract description 122
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 53
- 210000000170 cell membrane Anatomy 0.000 claims abstract description 49
- 239000000203 mixture Substances 0.000 claims abstract description 43
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 36
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 claims abstract description 36
- 239000003085 diluting agent Substances 0.000 claims abstract description 33
- 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 abstract description 22
- 229920001612 Hydroxyethyl starch Polymers 0.000 claims abstract description 22
- 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 abstract description 22
- 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 abstract description 22
- 229940050526 hydroxyethylstarch Drugs 0.000 claims abstract description 22
- PFTAWBLQPZVEMU-DZGCQCFKSA-N (+)-catechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-DZGCQCFKSA-N 0.000 claims abstract description 18
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims abstract description 18
- AFSDNFLWKVMVRB-UHFFFAOYSA-N Ellagic acid Chemical compound OC1=C(O)C(OC2=O)=C3C4=C2C=C(O)C(O)=C4OC(=O)C3=C1 AFSDNFLWKVMVRB-UHFFFAOYSA-N 0.000 claims abstract description 18
- ATJXMQHAMYVHRX-CPCISQLKSA-N Ellagic acid Natural products OC1=C(O)[C@H]2OC(=O)c3cc(O)c(O)c4OC(=O)C(=C1)[C@H]2c34 ATJXMQHAMYVHRX-CPCISQLKSA-N 0.000 claims abstract description 18
- 229920002079 Ellagic acid Polymers 0.000 claims abstract description 18
- 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 abstract description 18
- 239000007995 HEPES buffer Substances 0.000 claims abstract description 18
- ADRVNXBAWSRFAJ-UHFFFAOYSA-N catechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3ccc(O)c(O)c3 ADRVNXBAWSRFAJ-UHFFFAOYSA-N 0.000 claims abstract description 18
- 235000005487 catechin Nutrition 0.000 claims abstract description 18
- 229950001002 cianidanol Drugs 0.000 claims abstract description 18
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims abstract description 18
- 229960002852 ellagic acid Drugs 0.000 claims abstract description 18
- 235000004132 ellagic acid Nutrition 0.000 claims abstract description 18
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 claims abstract description 18
- IQPNAANSBPBGFQ-UHFFFAOYSA-N luteolin Chemical compound C=1C(O)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(O)C(O)=C1 IQPNAANSBPBGFQ-UHFFFAOYSA-N 0.000 claims abstract description 18
- LRDGATPGVJTWLJ-UHFFFAOYSA-N luteolin Natural products OC1=CC(O)=CC(C=2OC3=CC(O)=CC(O)=C3C(=O)C=2)=C1 LRDGATPGVJTWLJ-UHFFFAOYSA-N 0.000 claims abstract description 18
- 235000009498 luteolin Nutrition 0.000 claims abstract description 18
- FAARLWTXUUQFSN-UHFFFAOYSA-N methylellagic acid Natural products O1C(=O)C2=CC(O)=C(O)C3=C2C2=C1C(OC)=C(O)C=C2C(=O)O3 FAARLWTXUUQFSN-UHFFFAOYSA-N 0.000 claims abstract description 18
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 18
- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 18
- 239000011780 sodium chloride Substances 0.000 claims abstract description 18
- 229940054269 sodium pyruvate Drugs 0.000 claims abstract description 18
- PVLHOJXLNBFHDX-XHJPDDKBSA-N Panaxadiol Chemical compound C[C@]1([C@H]2CC[C@@]3([C@@H]2[C@H](O)C[C@H]2[C@]3(CC[C@H]3C(C)(C)[C@@H](O)CC[C@@]32C)C)C)CCCC(C)(C)O1 PVLHOJXLNBFHDX-XHJPDDKBSA-N 0.000 claims abstract description 13
- SYFJYASKXNAXKC-UHFFFAOYSA-N Panaxadiol Natural products CC1(C)CCCC(O1)C2CCC3(C)C2C(O)CC4C5(C)CCC(O)C(C)(C)C5CCC34C SYFJYASKXNAXKC-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000002577 cryoprotective agent Substances 0.000 claims abstract description 11
- 238000004321 preservation Methods 0.000 claims abstract description 11
- 210000004962 mammalian cell Anatomy 0.000 claims abstract description 7
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims abstract description 5
- 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 abstract description 5
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims abstract description 5
- 210000002865 immune cell Anatomy 0.000 claims abstract description 4
- 210000000130 stem cell Anatomy 0.000 claims abstract description 4
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- 239000000243 solution Substances 0.000 claims description 44
- 238000005138 cryopreservation Methods 0.000 claims description 40
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 26
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 26
- -1 0.1-0.5g/L Chemical compound 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 15
- 235000011164 potassium chloride Nutrition 0.000 claims description 13
- 239000001103 potassium chloride Substances 0.000 claims description 13
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 13
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 13
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 13
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 13
- 239000010413 mother solution Substances 0.000 claims description 6
- 241000208340 Araliaceae Species 0.000 claims description 5
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 claims description 5
- 235000003140 Panax quinquefolius Nutrition 0.000 claims description 5
- 150000002009 diols Chemical class 0.000 claims description 5
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- 238000001914 filtration Methods 0.000 claims description 2
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- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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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
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the field of cell culture and preservation, and particularly discloses a serum-free non-program cell frozen stock solution, a preparation method and application thereof, wherein the serum-free non-program cell frozen stock solution comprises the following components: 40-70v/v% of basic diluent, 2.5-10v/v% of dimethyl sulfoxide, 20-45w/v% of cell membrane external cryoprotectant, 2.5-10w/v% of cell membrane protectant composition: the base diluent contains the following components: sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium bicarbonate, D-glucose, HEPES, and sodium pyruvate; the cell membrane external cryoprotectant contains trehalose and hydroxyethyl starch; the cell membrane protectant composition comprises: luteolin, catechin, panaxadiol, ellagic acid, and combinations thereof. The frozen stock solution does not contain any serum and protein components, has definite components, is convenient to use, has high resurrection rate of frozen stock cells and wide application range, and can be applied to long-term preservation of various tumor cells, stem cells, immune cells and most other mammalian cells.
Description
Technical Field
The invention belongs to the field of cell culture and preservation, and particularly discloses a serum-free non-program cell frozen stock solution, and a preparation method and application thereof.
Background
Cell culture technology is the most basic and important experimental technology in the field of life sciences. In the cell culture process, various biological characteristics of cells are gradually changed along with the increase of passage times, so that timely preservation of original cell seeds is very important. The preservation of cells is usually carried out by freezing the cells in a low temperature environment to temporarily remove the cells from growth, preserving their cellular properties, and resuscitating the cells when necessary. In addition, cells also need to be stored in frozen form during purchase and transport. Therefore, it is necessary to perform the actual work of freezing and storing cells in time and conveniently.
The existing cell cryopreservation solution generally consists of DMSO, serum and a cell culture medium. The frozen stock solution is generally a ready-to-use frozen stock solution, needs to be prepared and used at present, has large batch difference, short holding period and unstable preservation effect. In addition, the existing freezing solution has complex freezing procedures, needs to be subjected to programmed cooling, and utilizes a programmed cooling box formed by isopropanol or special materials, so that the whole freezing process is long in time consumption and multiple in steps, the freezing operation is complex, the treatment capacity is small, the freezing cost is high, and the requirement of quick freezing of large-scale cells cannot be met.
Along with the development of the medical scientific research fields such as gene editing, cell therapy and the like, the requirement of in vitro cell culture is higher and higher, on one hand, the serum-free cell culture medium and the frozen stock solution can avoid the introduction of animal source components and allergic sources, and the serum components are unknown to have unpredictable influence on the growth and differentiation of cells. In cell therapy, the presence of foreign proteins, particularly unknown proteins, can cause unwanted, even unknown adverse effects, severely affecting the therapeutic outcome. Therefore, the medical research field is extremely important for the research and development of serum-free cell culture media and frozen stock solutions. In addition, due to the fact that the components of the serum are variable, the stability among the frozen stock solutions is poor, and due to the fact that proteins in the serum exist, the quality guarantee period of the cell frozen stock solution is short, and the preservation condition is harsh.
Disclosure of Invention
In order to solve the problems, the invention discloses a serum-free non-program cell cryopreservation liquid, which can remove instability caused by exogenous serum and rejection caused by exogenous protein, can also efficiently maintain cell activity and function, protect cell membrane structure and functional integrity, resist oxidative damage, is suitable for non-program cryopreservation, and can be conveniently and rapidly widely applied to cryopreservation of various mammalian cells.
The traditional cell freezing solution is composed of a cell culture medium, serum and DMSO, and the formula can play a role in protecting cells in the cell freezing process. Through multiparty groping and experiments, the serum-free non-program cell frozen stock solution is developed, an ideal protection effect can be achieved in the cell freezing process, and because animal serum is not needed, unpredictable influence of unknown serum components on growth and differentiation of cells is avoided, and the possibility of virus pollution is avoided; in addition, the cost is greatly reduced because a cell culture medium and animal serum are not required, and the toxicity to cell growth is reduced because DMSO with lower concentration is used.
The invention comprises the following technical scheme:
a serum-free non-procedural cell cryopreservation solution comprising the following components: 40-70v/v% of basic diluent, 2.5-10v/v% of dimethyl sulfoxide, 20-45w/v% of external cell membrane cryoprotectant and 2.5-10w/v% of cell membrane protectant composition;
the base diluent contains the following components: sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium bicarbonate, D-glucose, HEPES, and sodium pyruvate;
the cell membrane external cryoprotectant contains trehalose and hydroxyethyl starch;
the cell membrane protectant composition is; luteolin, catechin, panaxadiol, ellagic acid, and combinations thereof.
The cell freezing solution contains a plurality of salt ions, so that the balance of key ions inside and outside cells and osmotic pressure can be effectively maintained, and the pH environment is maintained stable; the cell freezing protective agent contains a plurality of cell freezing protective agents, so that the freezing injury-preventing capability of cells is improved, and the good state of the cells before and after freezing is ensured; DMSO is used as one of main antifreeze components and is used as a classical osmotic antifreeze protective agent, the DMSO can quickly penetrate cell membranes to enter cells, the freezing point is reduced, the freezing process is delayed, meanwhile, the ion concentration in the cells is improved, the formation of ice crystals in the cells is reduced, the damage degree of the cells is reduced, and the resurrection rate of the cells is ensured; trehalose and hydroxyethyl starch are used as impermeable cryoprotectants; the trehalose is a typical stress metabolite, can form a unique protective film on the surface of cells in severe environments such as low temperature, high osmotic pressure and the like, and effectively protects the biological molecular structure from being damaged; the hydroxyethyl starch is a macromolecular substance, cannot penetrate cells, reduces solute damage by diluting extracellular electrolyte concentration, preferentially combines water molecules in a solution before ice crystal formation, reduces extracellular free water content, and reduces ice crystal formation. When cells are stressed by adversity, a large amount of superoxide radicals are generated, so that membrane lipids undergo peroxidation, and Malondialdehyde (MDA) is generated. Excessive accumulation of malondialdehyde causes cross-linked polymerization of living macromolecules such as proteins and nucleic acids, and changes in the structure and function of cell membranes. Thus, membrane lipid peroxidation is an important marker for cell membrane damage. Superoxide dismutase (SOD) is an important component member of an antioxidant enzyme system in a biological system, is widely distributed in various organisms, can catalyze superoxide anion free radical disproportionation to generate oxygen and hydrogen peroxide, and plays a vital role in organism oxidation and antioxidant balance. The cell membrane protectant composition of luteolin, catechin, panaxadiol, ellagic acid can protect cell membrane structure and functional stability during freezing process by enhancing SOD activity, inhibiting Lipid Peroxide (LPO) production, and resisting lipid peroxidation induced by oxygen free radical.
Further, the serum-free non-program cell cryopreservation solution comprises the following components: 5-10g/L of sodium chloride, 0.1-0.8g/L of potassium chloride, 0.01-0.1g/L of monopotassium phosphate, 0.02-0.1g/L of disodium hydrogen phosphate, 0.1-0.5g/L, D-glucose 0.2-2g/L, HEPES 1.0.0-5.0 g/L of sodium bicarbonate and 0.05-0.2g/L of sodium pyruvate.
Preferably, the serum-free non-program cell freezing solution comprises the following components in percentage by weight: 8g/L of sodium chloride, 0.4g/L of potassium chloride, 0.06g/L of monopotassium phosphate, 0.048g/L of disodium hydrogen phosphate, 0.35g/L, D-glucose 1g/L, HEPES 3.57.57 g/L of sodium bicarbonate and 0.11g/L of sodium pyruvate.
Further, the serum-free non-procedure cell frozen stock solution contains 37.8-113.4g/L of trehalose and 10-40g/L of hydroxyethyl starch.
Preferably, the serum-free non-procedural cell freezing solution comprises 75.6g/L trehalose and 30g/L hydroxyethyl starch.
Further, the serum-free non-program cell freezing solution comprises the following components: luteolin 5-20 mu mol/L, catechin 10-40 mu mol/L, ginseng diol 5-20 mu g/mL, ellagic acid 20-60 mu g/mL.
Preferably, the serum-free non-procedure cell cryopreservation solution comprises the following components: luteolin 10. Mu. Mol/L, catechin 20. Mu. Mol/L, panaxadiol 10. Mu.g/mL, ellagic acid 40. Mu.g/mL.
Further, the serum-free non-program cell freezing solution comprises the following components: 60v/v% of the basic diluent, 5v/v% of dimethyl sulfoxide, 30w/v% of the extracellular cryoprotectant, and 5w/v% of the cell membrane protectant composition.
Further, the pH of the basic diluent is 7.2.
Further, the preparation method of the serum-free non-program cell frozen stock solution comprises the following steps:
1) Preparing a basic diluent, sequentially adding sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium bicarbonate, D-glucose and sodium pyruvate, and adjusting the pH to 7.0-7.5 by using HEPES buffer;
2) Preparing trehalose mother solution, hydroxyethyl starch mother solution and cell membrane protectant composition mother solution;
3) Mixing the basic diluent, dimethyl sulfoxide, trehalose, hydroxyethyl starch and cell membrane protectant composition uniformly according to a certain proportion;
4) And (3) filtering and sterilizing the solution obtained in the step (3) by using a filter to obtain the serum-free non-program cell frozen stock solution.
Further, the serum-free non-procedural cell cryopreservation solution is applied to preservation of mammalian cells, including but not limited to tumor cells, stem cells and immune cells.
Further, the use of a serum-free non-procedural cell cryopreservation solution as described above for preservation of mammalian cells, including one of 293T cells, NK92 cells and RBMSC cells.
Compared with the prior art, the invention has the following beneficial effects:
(1) The cell frozen stock solution provided by the invention has no any exogenous serum component, avoids the introduction of animal-derived components and allergic sources, avoids unpredictable influence of unknown serum components on the growth and differentiation of cells, and avoids the possibility of virus pollution.
(2) The cell freezing solution adopts an innovative cell membrane protective agent combination, namely, the combination of four substances including luteolin, catechin, ginseng diol, ellagic acid and the like, and the composition can be found to protect the stability of cell membrane structure and function and resist freezing injury in the cell freezing process.
(3) The cell suspension frozen by the cell frozen stock solution can be directly placed in a refrigerator with the temperature of-80 ℃ or below, does not need to adopt program cooling equipment, does not need complicated frozen stock steps, and saves a great deal of time and energy.
(4) The cell freezing solution adopts lower DMSO content, so that on one hand, DMSO can be ensured to quickly penetrate cell membranes into cells, the freezing point is lowered, the freezing process is delayed, and the formation of ice crystals in the cells is reduced, thereby reducing cell damage; on the other hand, the potential toxic effect on cells can be greatly reduced.
(5) The cell freezing solution provided by the invention does not contain cell culture medium, serum and protein components, so that the cost is greatly reduced, and the freezing solution has long shelf life.
Drawings
FIG. 1 is a photograph of rat bone marrow mesenchymal stem cells (RBMSCs) taken prior to cryopreservation;
FIG. 2 is a photograph of cells 48h after recovery of rat bone marrow mesenchymal stem cells (RBMSC) (example 2);
FIG. 3 is a photograph of natural killer cells (NK 92) pre-cryopreserved cells of a human malignant non-Hodgkin's lymphoma patient;
FIG. 4 is a photograph of natural killer cells (NK 92) cells of a human malignant non-Hodgkin's lymphoma patient 48h after resuscitation (example 2);
FIG. 5 is a photograph of human embryonic kidney cells (HEK-293T) taken prior to cryopreservation;
FIG. 6 is a photograph of 48h of cells after recovery of human embryonic kidney cells (HEK-293T) (example 2);
FIG. 7 is a summary of the cell proliferation after resuscitation of tables 2, 4, and 6.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments 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.
The reagents or instruments used in the examples of the present invention were not manufacturer-identified and were conventional reagent products commercially available.
Example 1
The composition of the cell cryopreservation solution is as follows: 50v/v% of the base diluent, 25w/v% of trehalose, 20w/v% of hydroxyethyl starch, 2.5v/v% of dimethyl sulfoxide and 2.5w/v% of the cell membrane protective agent composition.
The base diluent contains the following components: 8g/L of sodium chloride, 0.4g/L of potassium chloride, 0.06g/L of monopotassium phosphate, 0.048g/L of disodium hydrogen phosphate, 0.35g/L, D-glucose 1g/L, HEPES 3.57.57 g/L of sodium bicarbonate and 0.11g/L of sodium pyruvate;
the cell membrane protective agent composition comprises the following components: luteolin 10. Mu. Mol/L, catechin 20. Mu. Mol/L, panaxadiol 10. Mu.g/mL, ellagic acid 40. Mu.g/mL.
Example 2
The composition of the cell cryopreservation solution is as follows: 60v/v% of a basic diluent, 20w/v% of trehalose, 10w/v% of hydroxyethyl starch, 5v/v% of dimethyl sulfoxide and 5w/v% of a cell membrane protective agent composition.
The base diluent contains the following components: 8g/L of sodium chloride, 0.4g/L of potassium chloride, 0.06g/L of monopotassium phosphate, 0.048g/L of disodium hydrogen phosphate, 0.35g/L, D-glucose 1g/L, HEPES 3.57.57 g/L of sodium bicarbonate and 0.11g/L of sodium pyruvate;
the cell membrane protective agent composition comprises the following components: luteolin 10. Mu. Mol/L, catechin 20. Mu. Mol/L, panaxadiol 10. Mu.g/mL, ellagic acid 40. Mu.g/mL.
Example 3
The composition of the cell cryopreservation solution is as follows: 40v/v% of the base diluent, 25w/v% of trehalose, 15w/v% of hydroxyethyl starch, 10v/v% of dimethyl sulfoxide and 10w/v% of the cell membrane protective agent composition.
The base diluent contains the following components: 8g/L of sodium chloride, 0.4g/L of potassium chloride, 0.06g/L of monopotassium phosphate, 0.048g/L of disodium hydrogen phosphate, 0.35g/L, D-glucose 1g/L, HEPES 3.57.57 g/L of sodium bicarbonate and 0.11g/L of sodium pyruvate;
the cell membrane protective agent composition comprises the following components: luteolin 10. Mu. Mol/L, catechin 20. Mu. Mol/L, panaxadiol 10. Mu.g/mL, ellagic acid 40. Mu.g/mL.
Example 4
The composition of the cell cryopreservation solution is as follows: 70v/v% of base dilution, 15w/v% of trehalose, 5w/v% of hydroxyethyl starch, 5v/v% of dimethyl sulfoxide and 5w/v% of cell membrane protective agent composition.
The base diluent contains the following components: 8g/L of sodium chloride, 0.4g/L of potassium chloride, 0.06g/L of monopotassium phosphate, 0.048g/L of disodium hydrogen phosphate, 0.35g/L, D-glucose 1g/L, HEPES 3.57.57 g/L of sodium bicarbonate and 0.11g/L of sodium pyruvate;
the cell membrane protective agent composition comprises the following components: luteolin 10. Mu. Mol/L, catechin 20. Mu. Mol/L, panaxadiol 10. Mu.g/mL, ellagic acid 40. Mu.g/mL.
Comparative example 1
The composition of the cell cryopreservation solution is as follows: 65v/v% of basic diluent, 20w/v% of trehalose, 10w/v% of hydroxyethyl starch and 5v/v% of dimethyl sulfoxide.
The base diluent contains the following components: 8g/L of sodium chloride, 0.4g/L of potassium chloride, 0.06g/L of monopotassium phosphate, 0.048g/L of disodium hydrogen phosphate, 0.35g/L, D-glucose 1g/L, HEPES 3.57.57 g/L of sodium bicarbonate and 0.11g/L of sodium pyruvate;
in comparison with example 2, the present comparative example 1 does not contain the cell membrane protectant composition, and the other components are the same.
Comparative example 2
The composition of the cell cryopreservation solution is as follows: 60v/v% of a basic diluent, 20w/v% of trehalose, 10w/v% of hydroxyethyl starch, 5v/v% of dimethyl sulfoxide and 5w/v% of a cell membrane protective agent composition.
The base diluent contains the following components: 8g/L of sodium chloride, 0.4g/L of potassium chloride, 0.06g/L of monopotassium phosphate, 0.048g/L of disodium hydrogen phosphate, 0.35g/L, D-glucose 1g/L, HEPES 3.57.57 g/L of sodium bicarbonate and 0.11g/L of sodium pyruvate;
the cell membrane protective agent composition comprises the following components: catechin 20. Mu. Mol/L, panaxadiol 10. Mu.g/mL, ellagic acid 40. Mu.g/mL.
This control does not contain luteolin, otherwise it is the same as in example 2.
Comparative example 3
The composition of the cell cryopreservation solution is as follows: 60v/v% of a basic diluent, 20w/v% of trehalose, 10w/v% of hydroxyethyl starch, 5v/v% of dimethyl sulfoxide and 5w/v% of a cell membrane protective agent composition.
The base diluent contains the following components: 8g/L of sodium chloride, 0.4g/L of potassium chloride, 0.06g/L of monopotassium phosphate, 0.048g/L of disodium hydrogen phosphate, 0.35g/L, D-glucose 1g/L, HEPES 3.57.57 g/L of sodium bicarbonate and 0.11g/L of sodium pyruvate;
the cell membrane protective agent composition comprises the following components: luteolin 10. Mu. Mol/L, panaxadiol 10. Mu.g/mL, ellagic acid 40. Mu.g/mL.
The comparative example does not contain catechin, and the procedure of example 2 is otherwise repeated.
Comparative example 4
The composition of the cell cryopreservation solution is as follows: 60v/v% of a basic diluent, 20w/v% of trehalose, 10w/v% of hydroxyethyl starch, 5v/v% of dimethyl sulfoxide and 5w/v% of a cell membrane protective agent composition.
The base diluent contains the following components: 8g/L of sodium chloride, 0.4g/L of potassium chloride, 0.06g/L of monopotassium phosphate, 0.048g/L of disodium hydrogen phosphate, 0.35g/L, D-glucose 1g/L, HEPES 3.57.57 g/L of sodium bicarbonate and 0.11g/L of sodium pyruvate;
the cell membrane protective agent composition comprises the following components: luteolin 10. Mu. Mol/L, catechin 20. Mu. Mol/L, ellagic acid 40. Mu.g/mL.
This comparative example does not contain panaxadiol, and is otherwise identical to example 2.
Comparative example 5
The composition of the cell cryopreservation solution is as follows: 60v/v% of a basic diluent, 20w/v% of trehalose, 10w/v% of hydroxyethyl starch, 5v/v% of dimethyl sulfoxide and 5w/v% of a cell membrane protective agent composition.
The base diluent contains the following components: 8g/L of sodium chloride, 0.4g/L of potassium chloride, 0.06g/L of monopotassium phosphate, 0.048g/L of disodium hydrogen phosphate, 0.35g/L, D-glucose 1g/L, HEPES 3.57.57 g/L of sodium bicarbonate and 0.11g/L of sodium pyruvate;
the cell membrane protective agent composition comprises the following components: luteolin 10. Mu. Mol/L, catechin 20. Mu. Mol/L, and panaxadiol 10. Mu.g/mL.
The comparative example does not contain ellagic acid, and is otherwise identical to example 2.
Comparative example 6
Serum-containing commercial control
The composition of the cell cryopreservation solution is as follows: 60v/v% of basal medium, 30v/v% of fetal calf serum and 10v/v% of dimethyl sulfoxide.
The basal medium is a DMEM high-sugar medium.
Test case
Cell lysates of each group were prepared according to the above examples 1 to 4 and the control protocol. The 293T cells, NK92 cells and RBMSC cells are recovered and expanded, and then the prepared cell cryopreservation solution of each group is used for cryopreserving the three cells. Cell counting and photographing are respectively carried out before freezing, cell viability is detected after recovery, cell photographing is carried out after recovery for 48 hours, the cells of the example 2 with the highest viability are inoculated into a 12-hole plate after recovery, continuous culture is carried out for 6 days, cell counting is carried out every day, and cell proliferation is detected.
1. Rat bone marrow mesenchymal stem cells (RBMSC)
1. Cell count 1.2E+6 before cryopreservation;
2. cell counts after resuscitation are shown in table 1;
TABLE 1 rat bone marrow mesenchymal Stem cell count after resuscitation
| Cell count | Cell viability | |
| Example 1 | 1.08E+6 | 90.0％ |
| Example 2 | 1.18E+6 | 98.3％ |
| Example 3 | 1.13E+6 | 94.2％ |
| Example 4 | 1.15E+6 | 95.8％ |
| Comparative example 1 | 8.21E+5 | 68.4％ |
| Comparative example 2 | 9.78E+5 | 81.5％ |
| Comparative example 3 | 9.92E+5 | 82.6％ |
| Comparative example 4 | 9.87E+5 | 82.2％ |
| Comparative example 5 | 9.97E+5 | 83.1％ |
| Comparative example 6 | 1.07E+6 | 89.2％ |
3. Photographing the cells before freezing, wherein the figure 1 is shown;
4. cells were photographed 48h after resuscitation (example 2), see FIG. 2;
5. the proliferation of cells after resuscitation (example 2) was seeded with 2 x 10≡4 cells, see Table 2;
TABLE 2 proliferation of cells after resuscitation (example 2)
| Units: 10≡4 | 1d | 2d | 3d | 4d | 5d | 6d |
| RBMSC cells | 2.66 | 3.78 | 6.26 | 9.86 | 17.8 | 33.4 |
From the above results, the serum-free non-program cell cryopreservation solution provided by the invention can keep the recovery activity rate of rat bone marrow mesenchymal stem cells at more than 90% after cryopreservation and can keep the normal growth and proliferation capacity of cells after recovery, wherein the effect of example 2 is the best.
2. Natural killer cells (NK 92) of human malignant non-Hodgkin lymphoma patients
1. Cell count 1.2E+6 before cryopreservation;
2. cell count after resuscitation, see table 3;
TABLE 3 cell count after resuscitation
| Cell count | Cell viability | |
| Example 1 | 9.65E+5 | 80.4％ |
| Example 2 | 1.12E+6 | 93.3％ |
| Example 3 | 1.09E+6 | 90.8％ |
| Example 4 | 1.10E+6 | 91.7％ |
| Comparative example 1 | 7.52E+5 | 62.6％ |
| Comparative example 2 | 8.47E+5 | 70.6％ |
| Comparative example 3 | 8.25E+5 | 68.7％ |
| Comparative example 4 | 8.34E+5 | 69.5％ |
| Comparative example 5 | 8.51E+5 | 70.9％ |
| Comparative example 6 | 9.82E+5 | 81.8％ |
3. Photographing the cells before freezing, wherein the figure 3 is shown;
4. cells were photographed 48h after resuscitation (example 2), see FIG. 4;
5. the proliferation of cells after resuscitation (example 2) was seeded with 1 x 10≡5 cells, see Table 4;
TABLE 4 cell proliferation after resuscitation (example 2)
| Units: 10≡5 | 1d | 2d | 3d | 4d | 5d | 6d |
| NK92 cells | 1.0 | 1.42 | 2.01 | 2.69 | 5.57 | 10.3 |
From the results, the serum-free non-program cell freezing solution provided by the invention can keep the recovery activity rate of the human malignant non-Hodgkin lymphoma patient natural killer cells at more than 90% after freezing, and can keep the normal growth and proliferation capacity of the cells after recovery, wherein the effect of the embodiment 2 is the best.
3. Human embryonic kidney cells (HEK-293T)
1. Cell count 1.2E+6 before cryopreservation;
2. cell count after resuscitation, results are shown in table 5;
TABLE 5 cell count after resuscitation
| Cell count | Cell viability | |
| Example 1 | 1.07E+6 | 89.2％ |
| Example 2 | 1.18E+6 | 98.3％ |
| Example 3 | 1.13E+6 | 94.2％ |
| Example 4 | 1.16E+6 | 96.7％ |
| Comparative example 1 | 8.78E+5 | 73.1％ |
| Comparative example 2 | 9.57E+5 | 79.7％ |
| Comparative example 3 | 9.62E+5 | 80.0％ |
| Comparative example 4 | 9.47E+5 | 78.9％ |
| Comparative example 5 | 9.35E+5 | 78.1％ |
| Comparative example 6 | 1.11E+6 | 92.5％ |
3. Photographing the cells before freezing, and referring to figure 5;
4. cells were photographed 48h after resuscitation (example 2), see FIG. 6;
5. the proliferation of cells after resuscitation (example 2) was seeded with 2 x 10≡4 cells, see Table 6;
TABLE 6 proliferation of cells after resuscitation (example 2)
| Units: 10≡4 | 1d | 2d | 3d | 4d | 5d | 6d |
| 293T cells | 2.2 | 4.76 | 13.7 | 42.6 | 91.8 | 196 |
From the above results, the serum-free non-procedure cell cryopreservation solution provided by the invention can keep the recovery activity rate of human embryonic kidney cells at more than 90% after cryopreservation and can keep the normal growth and proliferation capacity of cells after recovery, wherein the best effect of example 2 is obtained.
In summary, as can be seen from the cell count conditions after resuscitating in tables 1, 3 and 5, when four cell membrane protectants luteolin, catechin, ginseng diol and ellagic acid are all present, the cryopreservation recovery rate of rat mesenchymal stem cells, natural killer cells of patients suffering from malignant non-hodgkin lymphoma and human embryo kidney cells reaches the highest, and when one cell is absent, the recovery rate is greatly reduced, which proves that the complementary and synergistic effects exist among the cell membrane protectants luteolin, catechin, ginseng diol and ellagic acid, and the recovery rate of the cells is greatly influenced.
From the above results, the invention provides a serum-free non-procedure cell cryopreservation solution, which comprises a basic diluent and a cryoprotectant outside the cell membrane, and is particularly added with a cell membrane protectant composition, wherein the cell membrane protectant composition can enhance SOD activity, inhibit the generation of Lipid Peroxide (LPO), resist lipid peroxidation caused by free radicals, protect the cell membrane structure and function stability during freezing process, improve the cell membrane permeability, and promote the permeation type protectant inside the cell membrane to rapidly penetrate the cell membrane into the cell; the cryopreservation liquid does not contain any serum or protein component, has definite components, is convenient to use, has high resurrection rate of the cryopreserved cells and wide application range, and can be applied to long-term preservation of various tumor cells, stem cells, immune cells and most mammalian cells.
The foregoing is a description of only a limited number of preferred embodiments of the invention, which are described in greater detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Claims (10)
1. A serum-free non-procedural cell cryopreservation solution, comprising the following components: 40-70v/v% of basic diluent, 2.5-10v/v% of dimethyl sulfoxide, 20-45w/v% of external cell membrane cryoprotectant and 2.5-10w/v% of cell membrane protectant composition;
the base diluent contains the following components: sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium bicarbonate, D-glucose, HEPES, and sodium pyruvate;
the cell membrane external cryoprotectant contains trehalose and hydroxyethyl starch;
the cell membrane protectant composition is; luteolin, catechin, panaxadiol, ellagic acid, and combinations thereof.
2. The serum-free non-procedural cell cryopreservation solution of claim 1 wherein the base diluent comprises the following components: 5-10g/L of sodium chloride, 0.1-0.8g/L of potassium chloride, 0.01-0.1g/L of monopotassium phosphate, 0.02-0.1g/L of disodium hydrogen phosphate, 0.1-0.5g/L, D-glucose 0.2-2g/L, HEPES 1.0.0-5.0 g/L of sodium bicarbonate and 0.05-0.2g/L of sodium pyruvate.
3. The serum-free non-procedural cell cryopreservation solution of claim 1 wherein the base diluent comprises the following components: 8g/L of sodium chloride, 0.4g/L of potassium chloride, 0.06g/L of monopotassium phosphate, 0.048g/L of disodium hydrogen phosphate, 0.35g/L, D-glucose 1g/L, HEPES 3.57.57 g/L of sodium bicarbonate and 0.11g/L of sodium pyruvate.
4. The serum-free non-procedural cell cryopreservation solution of claim 1 wherein the extracellular cryoprotectant comprises trehalose 37.8-113.4g/L and hydroxyethyl starch 10-40g/L.
5. The serum-free non-procedural cell cryopreservation solution of claim 4 wherein the extracellular cryoprotectant comprises 75.6g/L trehalose and 30g/L hydroxyethyl starch.
6. The serum-free non-procedural cell cryopreservation solution of claim 1 wherein the cell membrane protectant composition comprises the following components: luteolin 5-20 mu mol/L, catechin 10-40 mu mol/L, ginseng diol 5-20 mu g/mL, ellagic acid 20-60 mu g/mL.
7. The serum-free non-procedural cell cryopreservation solution of claim 1 wherein the pH of the base diluent is 7.2.
8. The method for preparing serum-free non-procedural cell cryopreservation solution according to any one of claims 1 to 7, comprising the steps of:
1) Preparing a basic diluent, sequentially adding sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium bicarbonate, D-glucose and sodium pyruvate, and adjusting the pH to 7.0-7.5 by using HEPES buffer;
2) Preparing trehalose mother solution, hydroxyethyl starch mother solution and cell membrane protectant composition mother solution;
3) Mixing the basic diluent, dimethyl sulfoxide, trehalose, hydroxyethyl starch and cell membrane protectant composition uniformly according to a certain proportion;
4) And (3) filtering and sterilizing the solution obtained in the step (3) by using a filter to obtain the serum-free non-program cell frozen stock solution.
9. Use of the serum-free non-procedural cell cryopreservation solution of any one of claims 1-7 for the preservation of mammalian cells including, but not limited to, tumor cells, stem cells, immune cells.
10. The use of claim 9, wherein the mammalian cells comprise one of 293T cells, NK92 cells, and RBMSC cells.
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