CN117397675A - Protective liquid for long-term preservation of cells and preparation method thereof - Google Patents
Protective liquid for long-term preservation of cells and preparation method thereof Download PDFInfo
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- CN117397675A CN117397675A CN202311343155.0A CN202311343155A CN117397675A CN 117397675 A CN117397675 A CN 117397675A CN 202311343155 A CN202311343155 A CN 202311343155A CN 117397675 A CN117397675 A CN 117397675A
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- Prior art keywords
- solution
- chloride
- long
- cells
- term preservation
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- 238000004321 preservation Methods 0.000 title claims abstract description 38
- 230000001681 protective effect Effects 0.000 title claims abstract description 32
- 230000007774 longterm Effects 0.000 title claims abstract description 28
- 239000007788 liquid Substances 0.000 title claims abstract description 13
- 238000002360 preparation method Methods 0.000 title abstract description 11
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 51
- 239000000243 solution Substances 0.000 claims abstract description 51
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims abstract description 30
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 30
- 239000007853 buffer solution Substances 0.000 claims abstract description 22
- 239000003755 preservative agent Substances 0.000 claims abstract description 22
- 230000002335 preservative effect Effects 0.000 claims abstract description 22
- 239000001103 potassium chloride Substances 0.000 claims abstract description 15
- 235000011164 potassium chloride Nutrition 0.000 claims abstract description 15
- 239000011780 sodium chloride Substances 0.000 claims abstract description 15
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims abstract description 13
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims abstract description 13
- 235000019799 monosodium phosphate Nutrition 0.000 claims abstract description 13
- 239000001509 sodium citrate Substances 0.000 claims abstract description 13
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims abstract description 13
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims abstract description 13
- 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 12
- 239000008103 glucose Substances 0.000 claims abstract description 12
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims abstract description 11
- 229930195725 Mannitol Natural products 0.000 claims abstract description 11
- 229920001213 Polysorbate 20 Polymers 0.000 claims abstract description 11
- 239000000594 mannitol Substances 0.000 claims abstract description 11
- 235000010355 mannitol Nutrition 0.000 claims abstract description 11
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims abstract description 11
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims abstract description 11
- 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 8
- 235000002639 sodium chloride Nutrition 0.000 claims abstract description 6
- 229910021642 ultra pure water Inorganic materials 0.000 claims abstract description 6
- 239000012498 ultrapure water Substances 0.000 claims abstract description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 30
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 12
- 239000004094 surface-active agent Substances 0.000 claims description 10
- 150000001720 carbohydrates Chemical class 0.000 claims description 9
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 229920002866 paraformaldehyde Polymers 0.000 claims description 7
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 150000005846 sugar alcohols Polymers 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 5
- 238000005303 weighing Methods 0.000 claims description 5
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 4
- 150000002016 disaccharides Chemical class 0.000 claims description 4
- 150000004676 glycans Chemical class 0.000 claims description 4
- 150000002772 monosaccharides Chemical class 0.000 claims description 4
- 239000008055 phosphate buffer solution Substances 0.000 claims description 4
- 229920001282 polysaccharide Polymers 0.000 claims description 4
- 239000005017 polysaccharide Substances 0.000 claims description 4
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 4
- 150000001298 alcohols Chemical class 0.000 claims description 3
- 150000001299 aldehydes Chemical class 0.000 claims description 3
- -1 alkyl glucosides Chemical class 0.000 claims description 3
- 239000003945 anionic surfactant Substances 0.000 claims description 3
- 150000001540 azides Chemical class 0.000 claims description 3
- 239000003093 cationic surfactant Substances 0.000 claims description 3
- 239000002736 nonionic surfactant Substances 0.000 claims description 3
- JNYAEWCLZODPBN-JGWLITMVSA-N (2r,3r,4s)-2-[(1r)-1,2-dihydroxyethyl]oxolane-3,4-diol Chemical compound OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O JNYAEWCLZODPBN-JGWLITMVSA-N 0.000 claims description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 2
- 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 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 claims description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 2
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims description 2
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 claims description 2
- 229920002307 Dextran Polymers 0.000 claims description 2
- 229920002527 Glycogen Polymers 0.000 claims description 2
- 239000007995 HEPES buffer Substances 0.000 claims description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 2
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 claims description 2
- 229920002472 Starch Polymers 0.000 claims description 2
- 235000021355 Stearic acid Nutrition 0.000 claims description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 2
- 229930006000 Sucrose Natural products 0.000 claims description 2
- 239000007983 Tris buffer Substances 0.000 claims description 2
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 claims description 2
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 claims description 2
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- 239000001913 cellulose Substances 0.000 claims description 2
- 229920002678 cellulose Polymers 0.000 claims description 2
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 claims description 2
- GVGUFUZHNYFZLC-UHFFFAOYSA-N dodecyl benzenesulfonate;sodium Chemical compound [Na].CCCCCCCCCCCCOS(=O)(=O)C1=CC=CC=C1 GVGUFUZHNYFZLC-UHFFFAOYSA-N 0.000 claims description 2
- 229930182830 galactose Natural products 0.000 claims description 2
- 229930182478 glucoside Natural products 0.000 claims description 2
- 235000011187 glycerol Nutrition 0.000 claims description 2
- 229940096919 glycogen Drugs 0.000 claims description 2
- 239000008101 lactose Substances 0.000 claims description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 2
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 claims description 2
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 claims description 2
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 claims description 2
- 229920000136 polysorbate Polymers 0.000 claims description 2
- 229950008882 polysorbate Drugs 0.000 claims description 2
- 150000003856 quaternary ammonium compounds Chemical class 0.000 claims description 2
- 229940080264 sodium dodecylbenzenesulfonate Drugs 0.000 claims description 2
- 239000000600 sorbitol Substances 0.000 claims description 2
- 235000010356 sorbitol Nutrition 0.000 claims description 2
- 239000008107 starch Substances 0.000 claims description 2
- 235000019698 starch Nutrition 0.000 claims description 2
- 239000008117 stearic acid Substances 0.000 claims description 2
- 239000005720 sucrose Substances 0.000 claims description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 2
- 239000000811 xylitol Substances 0.000 claims description 2
- 235000010447 xylitol Nutrition 0.000 claims description 2
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 claims description 2
- 229960002675 xylitol Drugs 0.000 claims description 2
- 239000011592 zinc chloride Substances 0.000 claims description 2
- 235000005074 zinc chloride Nutrition 0.000 claims description 2
- 235000014113 dietary fatty acids Nutrition 0.000 claims 2
- 239000000194 fatty acid Substances 0.000 claims 2
- 229930195729 fatty acid Natural products 0.000 claims 2
- 239000000872 buffer Substances 0.000 claims 1
- 235000014633 carbohydrates Nutrition 0.000 claims 1
- 150000004665 fatty acids Chemical class 0.000 claims 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 72
- 108090000623 proteins and genes Proteins 0.000 abstract description 6
- 102000004169 proteins and genes Human genes 0.000 abstract description 6
- 210000000170 cell membrane Anatomy 0.000 abstract description 4
- 230000001086 cytosolic effect Effects 0.000 abstract description 4
- 239000000306 component Substances 0.000 abstract 1
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 9
- 239000012530 fluid Substances 0.000 description 7
- 238000002073 fluorescence micrograph Methods 0.000 description 7
- 238000003908 quality control method Methods 0.000 description 6
- 239000003761 preservation solution Substances 0.000 description 5
- 238000005057 refrigeration Methods 0.000 description 5
- 238000001514 detection method Methods 0.000 description 4
- 238000003745 diagnosis Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 239000012124 Opti-MEM Substances 0.000 description 3
- 230000002421 anti-septic effect Effects 0.000 description 3
- 210000005266 circulating tumour cell Anatomy 0.000 description 3
- 210000000130 stem cell Anatomy 0.000 description 3
- 230000008901 benefit Effects 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 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 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 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 description 1
- 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 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000008151 electrolyte solution Substances 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 125000005456 glyceride group Chemical group 0.000 description 1
- 230000034659 glycolysis Effects 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 description 1
- 229960001008 heparin sodium Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000005406 washing Methods 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
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
-
- 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
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 discloses a protective solution for long-term preservation of cells, and relates to the technical field of biological cells. The invention also provides a preparation method of the protective solution, which is used for preserving cells for a long time, wherein according to the weight percentage of each component, sodium citrate, citric acid, glucose, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride, mannitol and a preservative BiocareGPL are weighed by an analytical balance, dissolved in 1L of ultrapure water, tween 20 is removed into a buffer solution by a liquid transfer device, the solution is uniformly mixed, and the pH is adjusted to 6.8-7.5,2-8 ℃ for preservation. The invention is used for long-term preservation of cells at 2-8 ℃ for 12 months, and ensures that cell membranes and cytoplasmic target proteins are not degraded while maintaining cell morphology.
Description
Technical Field
The invention belongs to the technical field of biological cells, and particularly relates to a protective solution for long-term preservation of cells and a preparation method thereof.
Background
Cells are one of the main sample sources for in vitro diagnosis, and the proportion of cells as detection objects in diagnosis is increasing, for example, CTCs or TCTs are all the diagnosis objects of cells. However, the problem that does not have standard quality control product to control the quality of the whole process at present, can't judge the accuracy of the detection result and can't trace the source and cause the reason that some problems appear.
The cell is used as a biological product, and the current preservation method is as follows: long-term preservation: storing at low temperature (-80deg.C) or deep temperature (-196 deg.C); short-term preservation: whereas for fresh cell products, the short time (usually not more than 24 hours) of normal temperature or cold storage results in a limited range of applications.
The preparation method of the quality control product for detecting the Chinese patent CN116499848ACTC in the prior art comprises the following steps: disclosed is a CTC detection quality control product which has the advantage of being capable of long-term transportation and storage. The method is obtained by mixing human T lymphocyte leukemia cells and tumor cells, and then performing primary freezing, secondary freezing and drying treatment.
The prior art Chinese patent CN116569917A discloses a cell, a cell preservation solution, a preservation method and application, wherein a solution rich in glycolysis inhibitor is added into a culture solution for incubation and digestion, and then the cell is collected by centrifugation; preparing cell suspension from the solution rich in trehalose, incubating, centrifuging, washing by using PBS buffer solution, and collecting cells; adding the collected cells into cell preservation solution, and preserving at low temperature of 2-8deg.C or room temperature of 18-30deg.C. The method protects living cells for no more than 1 week.
The prior art Chinese patent CN116584479A discloses a cell refrigeration protective liquid and a preparation method and application thereof, and the formula comprises a compound electrolyte solution, a red blood cell preservation liquid, heparin sodium, human serum albumin, human platelet lysate and glucose. When the cell refrigeration protective solution is prepared, the cell refrigeration protective solution is uniformly mixed and prepared under the sterile condition at room temperature. When the cell refrigeration protective solution is used for refrigerating the stem cell preparation, the activity of the stem cells is still more than 85% after 96 hours of refrigeration, and the growth and proliferation capacity and differentiation potential of the stem cells can be maintained. The method protects living cells for no more than 1 week.
Disclosure of Invention
The invention aims to solve the defects that in the prior art, the preservation time of living cells is short, the quality control of the whole process is carried out without a standard quality control product, the accuracy of a detection result cannot be judged, and the source cannot be traced. The protective solution is used for preserving cells for a long time at 2-8 ℃ for 12 months, and can ensure that cell membranes and cytoplasmic target proteins are not degraded while maintaining cell morphology.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a protective solution for long-term preservation of cells is designed, wherein the protective solution comprises a buffer solution, a saccharide substance, a chloride, an alcohol substance, a surfactant and a preservative.
Further, the buffer solution is one or a mixture solution of more than one of phosphate buffer solution, TRIS buffer solution, HEPES buffer solution and citric acid buffer solution.
Further, the buffer solution is a mixed solution of phosphate buffer solution and citric acid buffer solution, and comprises sodium citrate, citric acid, sodium dihydrogen phosphate and disodium hydrogen phosphate, wherein the weight percentages of the components are 0.001% -1% of sodium citrate, 0.00001% -0.1% of citric acid, 0.00001% -0.1% of sodium dihydrogen phosphate and 0.001% -1% of disodium hydrogen phosphate.
Further, the sugar is 0.1-1% of monosaccharide, disaccharide or polysaccharide substance, wherein the monosaccharide comprises glucose, galactose and ribose, the disaccharide comprises sucrose, maltose, cellobiose and lactose, and the polysaccharide comprises dextran, starch, glycogen and cellulose.
Further, the chloride is one or more of magnesium chloride, calcium chloride, sodium chloride, potassium chloride, copper chloride or zinc chloride.
Further, the chloride is mixed by sodium chloride and potassium chloride, and the weight percentage of each component is 0.5% -5% of sodium chloride and 0.001% -1% of potassium chloride.
Further, the alcohol substance is monohydric alcohol, dihydric alcohol or polyhydric alcohol with the weight percentage of 0.001% -1%, the monohydric alcohol comprises ethanol, the dihydric alcohol comprises ethylene glycol, and the polyhydric alcohol comprises glycerol, mannitol, xylitol and sorbitol.
Further, the surfactant is 0.05-2% by weight of anionic surfactant, cationic surfactant or nonionic surfactant, wherein the anionic surfactant comprises stearic acid and sodium dodecyl benzene sulfonate, the cationic surfactant comprises quaternary ammonium compound, and the nonionic surfactant comprises alkyl glucoside (APG), fatty glyceride, fatty sorbitan, polysorbate and tween 20.
Further, the preservative is 0.5-5% by weight of alcohols, aldehydes or azides, wherein the alcohols comprise methanol and ethanol, the aldehydes comprise formaldehyde and paraformaldehyde, and the azides comprise sodium azide and BiocareGPL.
In order to solve the technical problems, the invention also provides a preparation method of the protective liquid, which is used for preparing the protective liquid for long-term preservation of cells, and specifically comprises the following steps:
step 1), weighing sodium citrate, citric acid, glucose, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride, mannitol and a preservative biocaseGPL according to the weight percentage of each component by using an analytical balance, and dissolving in 1L of ultrapure water;
step 2), taking Tween 20 into a buffer solution by a liquid transfer device according to the weight percentage, uniformly mixing the solution, and storing the solution in an environment of 2-8 ℃;
step 3), adjusting the pH value to 6.8-7.5,2-8 ℃ and preserving.
Compared with the prior art, the protective solution for long-term preservation of cells and the preparation method thereof have the beneficial effects that: the preservation solution disclosed by the invention is simple in preparation method, controllable in cost and free from losing the structure of proteins on the cell surface and in cytoplasm while ensuring the cell morphology by using the preservative Biocare GPL and the polyalcohol in a mixed manner. The cell is used for long-term preservation of cells at the temperature of 2-8 ℃ for 12 months, and the cell membrane and cytoplasmic target protein are not degraded while the cell morphology is maintained.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 is a flow chart of the method for preparing the protection liquid.
FIG. 2 is a fluorescence image of 293 cells expressing EGFP of example 2 stored in a preservative fluid for 0 month at 2-8℃according to the present invention;
FIG. 3 is a fluorescence image of 293 cells expressing EGFP of example 2 stored in a preservative fluid for 1 month at 2-8℃according to the present invention;
FIG. 4 is a fluorescence image of 293 cells expressing EGFP of example 2 stored in a preservative fluid for 2 months at 2-8deg.C;
FIG. 5 is a fluorescence image of 293 cells expressing EGFP of example 2 stored in a preservative fluid for 3 months at 2-8deg.C;
FIG. 6 is a fluorescence image of 293 cells expressing EGFP of example 2 stored in a preservative fluid for 6 months at 2-8℃according to the present invention;
FIG. 7 is a fluorescence image of 293 cells expressing EGFP of example 2 stored in a preservative fluid for 10 months at 2-8deg.C;
FIG. 8 is a fluorescence image of 293 cells expressing EGFP of example 2 stored in a preservative fluid for 12 months at 2-8deg.C;
FIG. 9 is a graph of cell fluorescence for 0 days in paraformaldehyde solution for cell preservation in example 2 according to the present invention;
FIG. 10 is a graph of cell fluorescence for 3 days in paraformaldehyde solution for cell preservation in example 2 in accordance with the present invention;
FIG. 11 is a graph of cell fluorescence for 7 days in paraformaldehyde solution with respect to cell preservation in example 2.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention; it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments, and that all other embodiments obtained by persons of ordinary skill in the art without making creative efforts based on the embodiments in the present invention are within the protection scope of the present invention.
The structural features of the present invention will now be described in detail with reference to the accompanying drawings.
Example 1
A protective solution for long-term preservation of cells comprises buffer solution, saccharide, chloride, alcohol, surfactant and antiseptic.
Wherein the buffer solution is a mixed solution of 0.001% of sodium citrate, 0.00001% of citric acid, 0.00001% of sodium dihydrogen phosphate and 0.001% of disodium hydrogen phosphate, the saccharide is 0.1% of glucose, the chloride is a mixture of 0.5% of sodium chloride and 0.001% of potassium chloride, the alcohol is 0.001% of mannitol, the surfactant is 0.05% of Tween 20, and the preservative is 0.5% of biocareGPL.
Referring to fig. 1, a method for preparing a protective solution for long-term preservation of cells, specifically comprises the following steps:
step 1), weighing sodium citrate, citric acid, glucose, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride, mannitol and a preservative biocaseGPL according to the weight percentage of each component by using an analytical balance, and dissolving in 1L of ultrapure water;
step 2), taking Tween 20 into a buffer solution by a liquid transfer device according to the weight percentage, uniformly mixing the solution, and storing the solution in an environment of 2-8 ℃;
step 3), adjusting the pH value to 7.0-7.2,2-8 ℃ and preserving.
Example 2
A protective solution for long-term preservation of cells comprises buffer solution, saccharide, chloride, alcohol, surfactant and antiseptic.
Wherein the buffer solution is a mixed solution of 0.0015% of sodium citrate, 0.0002% of citric acid, 0.0094% of sodium dihydrogen phosphate and 0.114% of disodium hydrogen phosphate, the saccharide is 0.10793% of glucose, the chloride is a mixture of 1% of sodium chloride and 0.02% of potassium chloride, the alcohol is 0.01457% of mannitol, the surfactant is 0.1% of Tween 20, and the preservative is 1% of biocareGPL.
Referring to fig. 1, a method for preparing a protective solution for long-term preservation of cells, specifically comprises the following steps:
step 1), weighing sodium citrate, citric acid, glucose, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride, mannitol and a preservative biocaseGPL according to the weight percentage of each component by using an analytical balance, and dissolving in 1L of ultrapure water;
step 2), taking Tween 20 into a buffer solution by a liquid transfer device according to the weight percentage, uniformly mixing the solution, and storing the solution in an environment of 2-8 ℃;
step 3), adjusting the pH value to 7.0-7.2,2-8 ℃ and preserving.
Cell culture and transfection of EGFP plasmid: transfection reagents and 293 cells were prepared. Using Opti-MEM TM The Lipo3000 reagent was diluted in the medium and thoroughly mixed. To Opti-MEM TM EGFP plasmid solution (1 ug) was added. Plus is put into TM Regent (4 ul) was added to opti-MEM TM And uniformly mixing, and standing for 15 minutes. The cell culture medium was replaced with fresh 600ul serum-free medium, and liposomes were added and mixed slowly. After 2-4 hours (2 hours) 800ul of 20% serum medium was added. After 24 hours of transfection, the transfection condition of the cells is observed under a microscope, and the transfection efficiency can reach more than 95 percent.
Referring to FIGS. 2-8, preservation of cells: the transfected cells were digested, stored in a cell preservation solution, and placed in an environment of 2-8deg.C, and the cells were observed for 0 month, 1 month, 2 months, 3 months, 6 months, 10 months, and 12 months, respectively.
Referring to fig. 9-11, paraformaldehyde preserved cells: the transfected cells were digested, stored in paraformaldehyde, and placed in an environment at 2-8deg.C for 0 day, 3 days, and 7 days, respectively, to observe cells.
Example 3
A protective solution for long-term preservation of cells comprises buffer solution, saccharide, chloride, alcohol, surfactant and antiseptic.
Wherein the buffer solution is a mixed solution of 1% sodium citrate, 0.1% citric acid, 0.1% sodium dihydrogen phosphate and 1% disodium hydrogen phosphate, the saccharide is 1% glucose, the chloride is a mixture of 5% sodium chloride and 1% potassium chloride, the alcohol is 1% mannitol, the surfactant is 2% Tween 20, and the preservative is 5% biocareGPL.
Referring to fig. 1, a method for preparing a protective solution for long-term preservation of cells, specifically comprises the following steps:
step 1), weighing sodium citrate, citric acid, glucose, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride, mannitol and a preservative biocaseGPL according to the weight percentage of each component by using an analytical balance, and dissolving in 1L of ultrapure water;
step 2), taking Tween 20 into a buffer solution by a liquid transfer device according to the weight percentage, uniformly mixing the solution, and storing the solution in an environment of 2-8 ℃;
step 3), adjusting the pH value to 7.0-7.2,2-8 ℃ and preserving.
The preservation solution for preserving cells for a long time has the advantages of simple preparation method, controllable cost, and mixed use of the preservative BiocareGPL and the polyalcohol, ensures the cell morphology, does not lose the structure of proteins on the cell surface and in cytoplasm, is used for preserving cells for a long time under the condition of 2-8 ℃ for 12 months, and ensures the cell membrane and the cytoplasmic target protein not to degrade while maintaining the cell morphology. The long-term preservation of the cells is realized at the temperature of 2-8 ℃ for a long time, and a feasible quality control product preservation scheme is provided for diagnosis taking the cells as objects, such as CTC, TCT and the like.
The foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A protective solution for long-term preservation of cells, the protective solution comprising a buffer, a carbohydrate, a chloride, an alcohol, a surfactant, and a preservative.
2. The protective solution for long-term preservation of cells according to claim 1, wherein the buffer solution is one or more of phosphate buffer solution, TRIS buffer solution, HEPES buffer solution, and citric acid buffer solution.
3. The protective solution for long-term preservation of cells according to claim 2, wherein the buffer solution is a mixed solution of phosphate buffer solution and citric acid buffer solution, and comprises sodium citrate, citric acid, sodium dihydrogen phosphate and disodium hydrogen phosphate, wherein the weight percentages of the components are 0.001% -1% of sodium citrate, 0.00001% -0.1% of citric acid, 0.00001% -0.1% of sodium dihydrogen phosphate and 0.001% -1% of disodium hydrogen phosphate.
4. The protective solution for long-term preservation of cells according to claim 1, wherein the saccharide is 0.1-1% by weight of monosaccharide, disaccharide or polysaccharide substance, wherein the monosaccharide comprises glucose, galactose, ribose, the disaccharide comprises sucrose, maltose, cellobiose, lactose, and the polysaccharide comprises dextran, starch, glycogen, cellulose.
5. The protective solution for long-term preservation of cells according to claim 1, wherein the chloride is one or more of magnesium chloride, calcium chloride, sodium chloride, potassium chloride, copper chloride and zinc chloride.
6. The protective solution for long-term preservation of cells according to claim 5, wherein the chloride is a mixed chloride of sodium chloride and potassium chloride, and the weight percentages of the components are 0.5% -5% sodium chloride and 0.001% -1% potassium chloride.
7. The protective solution for long-term preservation of cells according to claim 1, wherein the alcohol is 0.001% -1% by weight of monohydric alcohol, dihydric alcohol or polyhydric alcohol, the monohydric alcohol comprises ethanol, the dihydric alcohol comprises ethylene glycol, and the polyhydric alcohol comprises glycerol, mannitol, xylitol, sorbitol.
8. The protective solution for long-term preservation of cells according to claim 1, wherein the surfactant is 0.05-2% by weight of an anionic surfactant including stearic acid, sodium dodecylbenzenesulfonate, a cationic surfactant including a quaternary ammonium compound, or a nonionic surfactant including alkyl glucosides (APG), fatty acid glycerides, fatty acid sorbitan, polysorbate, tween 20.
9. The protective solution for long term preservation of cells according to claim 1, wherein the preservative is 0.5-5% by weight of alcohols including methanol, ethanol, aldehydes including formaldehyde, paraformaldehyde, or azides including sodium azide, biocareGPL.
10. A method for preparing a protective solution for long-term preservation of cells according to any one of claims 1 to 7, comprising the steps of:
step 1), weighing sodium citrate, citric acid, glucose, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride, mannitol and a preservative biocaseGPL according to the weight percentage of each component by using an analytical balance, and dissolving in 1L of ultrapure water;
step 2), taking Tween 20 into a buffer solution by a liquid transfer device according to the weight percentage, uniformly mixing the solution, and storing the solution in an environment of 2-8 ℃;
step 3), adjusting the pH value to 6.8-7.5,2-8 ℃ and preserving.
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