CN107251893A - A kind of novel cell frozen stock solution - Google Patents
A kind of novel cell frozen stock solution Download PDFInfo
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- CN107251893A CN107251893A CN201710524490.9A CN201710524490A CN107251893A CN 107251893 A CN107251893 A CN 107251893A CN 201710524490 A CN201710524490 A CN 201710524490A CN 107251893 A CN107251893 A CN 107251893A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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Abstract
The invention provides a kind of novel cell frozen stock solution, including 80 90 parts of 5 15 parts of dimethyl sulfoxide (DMSO), 25 parts of HES, 5 10 parts of chitosan, 25 parts of glutathione, 14 parts of vitamin C, 0.3 4 parts of dextran, 0.5 2 parts of propane diols, 0.1 1 parts of N (benzyl) 2 (4 pyrimdinyl-amino) 4 thiazole carboxamides and basal medium.Novel cell frozen stock solution component of the present invention is rationally, nutritious, can also substantially reduce the damage to cell while the nutrition of rest cell is ensured, improve the survival rate of freeze-stored cell, and also keeps in terms of propagation good state.
Description
Technical field
The invention belongs to biological technology application, in particular it relates to a kind of novel cell frozen stock solution.
Background technology
Cell culture is a biological important technology with healthy Related Research Domain.With the rapid hair of life science
Exhibition, current cell culture turns into the important base of the disciplinary studies such as cell biology, molecular biology, science of heredity and immunology
Plinth.In order to further investigated cell vegetative activity rule, have the pathology of related disorders and the pharmacology of medicine（Toxicity）Mechanism, exploitation tool
There are different targetedly cell culture processes significant.
Cell cryopreservation technology is a very important means in cytobiology technology, is also important in cell culture
Technology.People are increasing to use histocyte culture multiplication technique in the research of cell biology and its related discipline,
There to be Research Significance or have the active somatic cell of application prospect, carry out preserving several months, several years, many decades even for a long time using low temperature
Centuries, once needing, can recover to the cell preserved, recover its complete morphosis and biological characteristics at any time
Property, tested for life science.
Novel cell frozen stock solution security of the present invention, stability are good, and survival rate is high after cell recovery, can be applicable
The culture of the various kinds of cell in Yu Renyuan and inhuman source.
The content of the invention
Goal of the invention：The invention provides a kind of novel cell frozen stock solution, for a variety of people sources and non-human archeocyte
Culture.
Technical scheme：The invention provides a kind of novel cell frozen stock solution, by weight part, including following components：Two
5-15 parts of methyl sulfoxide, 2-5 parts of HES, 5-10 parts of chitosan, 2-5 parts of glutathione, 1-4 parts of vitamin C, dextrose
0.3-4 parts of acid anhydride, 0.5-2 parts of propane diols, 0.1-1 parts of N- (benzyl) -2- (4- pyrimdinyl-aminos) -4- thiazole carboxamides and basis training
Support 80-90 parts of base.Novel cell frozen stock solution component of the present invention is rationally, nutritious, wherein, dextran is permeability
Liquid is protected, and N- (benzyl) -2- (4- pyrimdinyl-aminos) -4- thiazole carboxamides then suppress cellular activity and gene expression, dimension life
Plain C good in oxidation resistance, can also substantially reduce the damage to cell, raising freezes while the nutrition of rest cell is ensured
The survival rate of cell, and also keep in terms of propagation good state.
Further, above-mentioned novel cell frozen stock solution, the basal medium by weight part, including with the following group
Point：82-88 parts of glucose, 12-25 parts of sodium acid carbonate, 10-16 parts of Sodium Pyruvate, 5-9 parts of sericin, potassium chloride 35-45
Part, 6-10 parts of anhydrous magnesium sulfate, 70-78 parts of sodium chloride, 10-14 parts of AMSP, 0.3-0.5 parts of folic acid, inositol
0.8-1.2 parts, 0.3-0.6 parts of niacinamide, 25-35 parts of anhydrous calcium chloride, 0.2-0.4 parts of ferric nitrate, 6-8 parts of succinic acid, fourth two
Sour sodium 12-16 parts, 0.3-0.6 parts of D-VB5 calcium, 0.6-0.8 parts of choline tartrate, 0.1-0.3 parts of riboflavin, thiamine hydrochloride
0.2-0.4 parts, 0.3-0.5 parts of pyridoxine hydrochloride, 0.2-0.4 parts of NIPA, 1-2 parts of leukotrienes, monoethanolamine
0.6-0.8 parts, 0.3-0.8 parts of aromatic esters, phenol red sodium 1-1.3 parts and 100 parts of deionized water.Basal medium component is reasonable,
It is nutritious, nutriment during cell cryopreservation can be improved, it is ensured that the activity of freeze-stored cell.
Further, above-mentioned novel cell frozen stock solution, the basal medium also includes 3-8 parts of amino acid, the ammonia
Base acid is by weight part, composed of the following components：5-10 parts of L- R-genes, 3-8 parts, L- ammonia of L- hydrochloric acid cystines
Sour 3-6 parts, 1-5 parts of glycine, 4-6 parts of L- histidine monohydrochlorides, 8-20 parts of ILE, 7-18 parts of L-Leu, L- hydrochloric acid
10-20 parts of lysine, 1-6 parts of METHIONINE, 2-8 parts of L-phenylalanine, 5-15 parts of L-threonine, 1-3 parts of L-Trp, L-
8-12 parts of -9 parts of tyrosinase 15 and Valine.
Further, above-mentioned novel cell frozen stock solution, the basal medium also includes 2-5 parts of confactors, described
Confactor is by weight part, composed of the following components：2-10 parts of recombination human basic fibroblast growth factor, interleukin
1-4 parts of 2 3-8 parts, 3-6 parts of IL-4,1-5 parts of interleukin-11 4,3-6 parts of IFN-γ and neuroleukin.
Further, above-mentioned novel cell frozen stock solution, the basal medium also includes volume ratio 10-30% tire ox
Serum.Can be as needed, further increase hyclone, improve the survival rate of freeze-stored cell, keep cytoactive.
Further, above-mentioned novel cell frozen stock solution, the basal medium by weight part, in addition to 0.006
Part penicillin and 0.01 part of streptomysin.Freeze-stored cell can be effectively prevented to be contaminated.
Further, above-mentioned novel cell frozen stock solution, the penicillin is selected from 10000U/ml, and the streptomysin is selected from
10000μg/ml.Penicillin and streptomysin activity are good, and effect is good.
Beneficial effect：Novel cell frozen stock solution of the present invention, its component is rationally, nutritious, is ensureing that dormancy is thin
The damage to cell can also be substantially reduced while the nutrition of born of the same parents, the survival rate of freeze-stored cell is improved, and in terms of propagation
Also good state is kept.
Brief description of the drawings
Fig. 1 is the measure figure of embodiment 1-4 cell survival rates.
Embodiment
Below will be by several specific embodiments, the present invention is furture elucidated, these embodiments simply to illustrate that problem,
It is not a kind of limitation.
Embodiment 1
A kind of novel cell frozen stock solution, by weight part, including following components：5 parts of dimethyl sulfoxide (DMSO), 2 parts of HES,
5 parts of chitosan, 2 parts of glutathione, 1 part of vitamin C, 0.3 part of dextran, 0.5 part of propane diols, (4- is phonetic by N- (benzyl) -2-
Pyridine amino) 80 parts of 0.1 part of -4- thiazole carboxamides and basal medium.
Wherein, the basal medium by weight part, including following components：82 parts of glucose, sodium acid carbonate 12
Part, 10 parts of Sodium Pyruvate, 5 parts of sericin, 35 parts of potassium chloride, 6 parts of anhydrous magnesium sulfate, 70 parts of sodium chloride, anhydrous phosphoric acid dihydro
10 parts of sodium, 0.3 part of folic acid, 0.8 part of inositol, 0.3 part of niacinamide, 25 parts of anhydrous calcium chloride, 0.2 part of ferric nitrate, 6 parts of succinic acid,
12 parts of sodium succinate, 0.3 part of D-VB5 calcium, 0.6 part of choline tartrate, 0.1 part of riboflavin, 0.2 part of thiamine hydrochloride, hydrochloric acid pyrrole
Tremble pungent 0.3 part, 0.2 part of NIPA, 1 part of leukotrienes, 0.6 part of monoethanolamine, 0.3 part of aromatic esters, phenol red sodium 1
100 parts of part and deionized water.
In addition, the basal medium also includes 3 parts of amino acid, the amino acid by weight part, by following components group
Into：5 parts of L- R-genes, 3 parts of L- hydrochloric acid cystine, 3 parts of Serine, 1 part of glycine, 4 parts of L- histidine monohydrochlorides,
8 parts of ILE, 7 parts of L-Leu, 10 parts of LYS, 1 part of METHIONINE, 2 parts of L-phenylalanine, L- Soviet Unions ammonia
8 parts of 5 parts of acid, 1 part of L-Trp, 5 parts of TYR and Valine.
Again, the basal medium also includes 2 parts of confactors, the confactor by weight part, by with the following group
It is grouped into：2 parts of recombination human basic fibroblast growth factor, 3 parts of interleukin-22,3 parts of IL-4,41 parts of interleukin-11,
1 part of 3 parts of IFN-γ and neuroleukin.
Further, the basal medium also includes the hyclone of volume ratio 10%.In addition, the basal medium
By weight part, in addition to 0.006 part of penicillin and 0.01 part of streptomysin.Also, the penicillin is selected from 10000U/
Ml, the streptomysin is selected from 10000 μ g/ml.
By above-mentioned cells frozen storing liquid, applied to freezing for humanized's liver cancer cells A549.
Embodiment 2
A kind of novel cell frozen stock solution, by weight part, including following components：15 parts of dimethyl sulfoxide (DMSO), HES 5
Part, 10 parts of chitosan, 5 parts of glutathione, 4 parts of vitamin C, 4 parts of dextran, 2 parts of propane diols, (4- is phonetic by N- (benzyl) -2-
Pyridine amino) 90 parts of 1 part of -4- thiazole carboxamides and basal medium.
Wherein, the basal medium by weight part, including following components：88 parts of glucose, sodium acid carbonate 25
Part, 16 parts of Sodium Pyruvate, 9 parts of sericin, 45 parts of potassium chloride, 10 parts of anhydrous magnesium sulfate, 78 parts of sodium chloride, anhydrous phosphoric acid two
14 parts of hydrogen sodium, 0.5 part of folic acid, 1.2 parts of inositol, 0.6 part of niacinamide, 35 parts of anhydrous calcium chloride, 0.4 part of ferric nitrate, succinic acid 8
Part, 16 parts of sodium succinate, 0.6 part of D-VB5 calcium, 0.8 part of choline tartrate, 0.3 part of riboflavin, 0.4 part of thiamine hydrochloride, hydrochloric acid
0.5 part of Benadon, 0.4 part of NIPA, 2 parts of leukotrienes, 0.8 part of monoethanolamine, 0.8 part of aromatic esters, phenol red sodium
1.3 parts and 100 parts of deionized water.
In addition, the basal medium also includes 8 parts of amino acid, the amino acid by weight part, by following components group
Into：10 parts of L- R-genes, 8 parts of L- hydrochloric acid cystine, 6 parts of Serine, 5 parts of glycine, 6 parts of L- histidine monohydrochlorides,
20 parts of ILE, 18 parts of L-Leu, 20 parts of LYS, 6 parts of METHIONINE, 8 parts of L-phenylalanine, L- Soviet Unions
12 parts of 15 parts of propylhomoserin, 3 parts of L-Trp, 9 parts of TYR and Valine.
Again, the basal medium also includes 5 parts of confactors, the confactor by weight part, by with the following group
It is grouped into：8 parts of recombination human basic fibroblast growth factor, 8 parts of interleukin-22,4 parts of IL-4,45 parts of interleukin-11,
4 parts of 6 parts of IFN-γ and neuroleukin.
Further, the basal medium also includes the hyclone of volume ratio 30%.In addition, the basal medium
By weight part, in addition to 0.006 part of penicillin and 0.01 part of streptomysin.Also, the penicillin is selected from 10000U/
Ml, the streptomysin is selected from 10000 μ g/ml.
By above-mentioned cells frozen storing liquid, the melanoma cells B16's originated applied to mouse freezes.
Embodiment 3
A kind of novel cell frozen stock solution, by weight part, including following components：10 parts of dimethyl sulfoxide (DMSO), HES 3
Part, 6 parts of chitosan, 3 parts of glutathione, Catergen part, 3 parts of dextran, 1 part of propane diols, (4- is phonetic by N- (benzyl) -2-
Pyridine amino) 85 parts of 0.8 part of -4- thiazole carboxamides and basal medium.
Wherein, the basal medium by weight part, including following components：86 parts of glucose, sodium acid carbonate 18
Part, 12 parts of Sodium Pyruvate, 6 parts of sericin, 40 parts of potassium chloride, 8 parts of anhydrous magnesium sulfate, 75 parts of sodium chloride, anhydrous phosphoric acid dihydro
12 parts of sodium, 0.4 part of folic acid, 1 part of inositol, 0.5 part of niacinamide, 30 parts of anhydrous calcium chloride, 0.3 part of ferric nitrate, 7 parts of succinic acid, fourth
14 parts of diacid sodium, 0.5 part of D-VB5 calcium, 0.7 part of choline tartrate, 0.2 part of riboflavin, 0.3 part of thiamine hydrochloride, hydrochloric acid pyrrole are trembled
Pungent 0.4 part, 0.3 part of NIPA, 1.6 parts of leukotrienes, 0.7 part of monoethanolamine, 0.5 part of aromatic esters, phenol red sodium
1.2 parts and 100 parts of deionized water.
In addition, the basal medium also includes 5 parts of amino acid, the amino acid by weight part, by following components group
Into：8 parts of L- R-genes, 6 parts of L- hydrochloric acid cystine, 5 parts of Serine, 3 parts of glycine, 5 parts of L- histidine monohydrochlorides,
12 parts of ILE, 10 parts of L-Leu, 15 parts of LYS, 3 parts of METHIONINE, 4 parts of L-phenylalanine, L- Soviet Unions
9 parts of 9 parts of propylhomoserin, 2 parts of L-Trp, 6 parts of TYR and Valine.
Again, the basal medium also includes 3 parts of confactors, the confactor by weight part, by with the following group
It is grouped into：6 parts of recombination human basic fibroblast growth factor, 5 parts of interleukin-22,4 parts of IL-4,45 parts of interleukin-11,
3 parts of 5 parts of IFN-γ and neuroleukin.
Further, the basal medium also includes the hyclone of volume ratio 15%.In addition, the basal medium
By weight part, in addition to 0.006 part of penicillin and 0.01 part of streptomysin.Also, the penicillin is selected from 10000U/
Ml, the streptomysin is selected from 10000 μ g/ml.
By above-mentioned cells frozen storing liquid, applied to freezing for MK cells Marc145.
Embodiment 4
A kind of novel cell frozen stock solution, by weight part, including following components：15 parts of dimethyl sulfoxide (DMSO), HES 2
Part, 6 parts of chitosan, 3 parts of glutathione, 1 part of vitamin C, 4 parts of dextran, 1 part of propane diols, (4- is phonetic by N- (benzyl) -2-
Pyridine amino) 90 parts of 0.8 part of -4- thiazole carboxamides and basal medium.
Wherein, the basal medium by weight part, including following components：82 parts of glucose, sodium acid carbonate 25
Part, 12 parts of Sodium Pyruvate, 6 parts of sericin, 45 parts of potassium chloride, 6 parts of anhydrous magnesium sulfate, 72 parts of sodium chloride, anhydrous phosphoric acid dihydro
14 parts of sodium, 0.3 part of folic acid, 1 part of inositol, 0.3 part of niacinamide, 35 parts of anhydrous calcium chloride, 0.3 part of ferric nitrate, 6 parts of succinic acid, fourth
12 parts of diacid sodium, 0.6 part of D-VB5 calcium, 0.7 part of choline tartrate, 0.2 part of riboflavin, 0.3 part of thiamine hydrochloride, hydrochloric acid pyrrole are trembled
Pungent 0.4 part, 0.4 part of NIPA, 2 parts of leukotrienes, 0.6 part of monoethanolamine, 0.3 part of aromatic esters, phenol red sodium 1.3
100 parts of part and deionized water.
In addition, the basal medium also includes 4 parts of amino acid, the amino acid by weight part, by following components group
Into：5 parts of L- R-genes, 8 parts of L- hydrochloric acid cystine, 6 parts of Serine, 3 parts of glycine, 5 parts of L- histidine monohydrochlorides,
12 parts of ILE, 18 parts of L-Leu, 10 parts of LYS, 5 parts of METHIONINE, 4 parts of L-phenylalanine, L- Soviet Unions
8 parts of 10 parts of propylhomoserin, 3 parts of L-Trp, 5 parts of TYR and Valine.
Again, the basal medium also includes 3 parts of confactors, the confactor by weight part, by with the following group
It is grouped into：2 parts of recombination human basic fibroblast growth factor, 8 parts of interleukin-22,3 parts of IL-4,44 parts of interleukin-11,
2 parts of 5 parts of IFN-γ and neuroleukin.
Further, the basal medium also includes the hyclone of volume ratio 30%.In addition, the basal medium
By weight part, in addition to 0.006 part of penicillin and 0.01 part of streptomysin.Also, the penicillin is selected from 10000U/
Ml, the streptomysin is selected from 10000 μ g/ml.
By above-mentioned cells frozen storing liquid, the immortalization stellate cells HSC-T6 applied to rat freezes.
The cell that embodiment 1-4 is frozen take respectively freeze after 3 months, 6 months, 9 months, 12 months, 18 months and
The freeze-stored cell of 24 months is recovered, and cell survival rate is measured, and cell survival rate is more than 85%.
Described above is only several embodiments of invention, it is noted that for those skilled in the art
For, on the premise of inventive principle is not departed from, some improvement can also be made, these improvement also should be regarded as the protection of the present invention
Scope.
Claims (7)
1. a kind of novel cell frozen stock solution, it is characterised in that：By weight part, including following components：Dimethyl sulfoxide (DMSO) 5-15
Part, 2-5 parts of HES, 5-10 parts of chitosan, 2-5 parts of glutathione, 1-4 parts of vitamin C, 0.3-4 parts of dextran, third
80-90 parts of 0.5-2 parts of glycol, 0.1-1 parts of N- (benzyl) -2- (4- pyrimdinyl-aminos) -4- thiazole carboxamides and basal medium.
2. novel cell frozen stock solution according to claim 1, it is characterised in that：The basal medium is with components by weight percent
Meter, including following components：82-88 parts of glucose, 12-25 parts of sodium acid carbonate, 10-16 parts of Sodium Pyruvate, 5-9 parts of sericin,
35-45 parts of potassium chloride, 6-10 parts of anhydrous magnesium sulfate, 70-78 parts of sodium chloride, 10-14 parts of AMSP, folic acid 0.3-
0.5 part, 0.8-1.2 parts of inositol, 0.3-0.6 parts of niacinamide, 25-35 parts of anhydrous calcium chloride, 0.2-0.4 parts of ferric nitrate, succinic acid
6-8 parts, 12-16 parts of sodium succinate, 0.3-0.6 parts of D-VB5 calcium, 0.6-0.8 parts of choline tartrate, 0.1-0.3 parts of riboflavin,
0.2-0.4 parts of thiamine hydrochloride, 0.3-0.5 parts of pyridoxine hydrochloride, 0.2-0.4 parts of NIPA, 1-2 parts of leukotrienes,
0.6-0.8 parts of monoethanolamine, 0.3-0.8 parts of aromatic esters, phenol red sodium 1-1.3 parts and 100 parts of deionized water.
3. novel cell frozen stock solution according to claim 2, it is characterised in that：The basal medium also includes 3-8 parts
Amino acid, the amino acid is by weight part, composed of the following components：5-10 parts of L- R-genes, L- hydrochloric acid cystines
3-8 parts, 3-6 parts of Serine, 1-5 parts of glycine, 4-6 parts of L- histidine monohydrochlorides, 8-20 parts of ILE, L-Leu
7-18 parts, 10-20 parts of LYS, 1-6 parts of METHIONINE, 2-8 parts of L-phenylalanine, 5-15 parts of L-threonine, L-
8-12 parts of 1-3 parts of tryptophan, 5-9 parts of TYR and Valine.
4. the novel cell frozen stock solution according to Claims 2 or 3, it is characterised in that：The basal medium also includes 2-5
Part confactor, the confactor is by weight part, composed of the following components：Recombination human basic fibroblast growth because
Sub- 2-10 parts, 3-8 parts of interleukin-22,3-6 parts of IL-4,1-5 parts of interleukin-11 4,3-6 parts of IFN-γ and neuroleukin
1-4 parts.
5. novel cell frozen stock solution according to claim 4, it is characterised in that：The basal medium also includes volume ratio
10-30% hyclone.
6. novel cell frozen stock solution according to claim 5, it is characterised in that：The basal medium is with components by weight percent
Meter, in addition to 0.006 part of penicillin and 0.01 part of streptomysin.
7. novel cell frozen stock solution according to claim 6, it is characterised in that：The penicillin is selected from 10000U/ml, institute
State streptomysin and be selected from 10000 μ g/ml.
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| WO2019085271A1 (en) * | 2017-10-30 | 2019-05-09 | 华南农业大学 | Glyphosate resistant seed soaking agent for no-tillage dry direct-seeding rice |
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| CN114451401A (en) * | 2022-01-21 | 2022-05-10 | 深圳市茵冠生物科技有限公司 | Cell cryopreservation solution and preparation method and application thereof |
| CN115443985A (en) * | 2022-08-18 | 2022-12-09 | 宁波希诺赛生物科技有限公司 | Stem cell cryopreservation liquid and cryopreservation method and recovery method thereof |
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| CN117264876A (en) * | 2023-11-21 | 2023-12-22 | 妙顺（上海）生物科技有限公司 | Method for preparing multi-donor liver cells |
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