CN108552156A - A kind of the induction versatile stem cell frozen stock solution and cryopreservation methods of serum-free - Google Patents
A kind of the induction versatile stem cell frozen stock solution and cryopreservation methods of serum-free Download PDFInfo
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- CN108552156A CN108552156A CN201711126234.0A CN201711126234A CN108552156A CN 108552156 A CN108552156 A CN 108552156A CN 201711126234 A CN201711126234 A CN 201711126234A CN 108552156 A CN108552156 A CN 108552156A
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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
Abstract
The present invention provides a kind of induction versatile stem cell frozen stock solution and its cryopreservation methods, wherein frozen stock solution is made of DMEM basal mediums, NEAA, KSR, DMSO and hydroxyethyl starch etc., frozen stock solution definite ingredients, no animal blood serum pollution, great application value.Cryopreservation methods are easy to operate, do not need multigelation and now with the current.The cell that frozen stock solution and cryopreservation methods using the present invention freeze has many advantages, such as that cell viability is good, and growth rate is fast.It is of great significance to promoting the research of induction versatile stem cell, establishing induction versatile stem cell library.
Description
Technical field
The present invention relates to stem cell biologies and regenerative medicine field, freeze more particularly to a kind of induction versatile stem cell
Liquid storage and induction versatile stem cell cryopreservation methods.
Background technology
The multipotential stem cell of induction refers to by the reprogramming of somatic cells of differentiation and the pluripotent stem cell that obtains, has self
The potential of update and Multidirectional Differentiation, it is in stem cell protein marker gene and protein expression level, teratoma formation, self-renewing
Many aspects such as potential and Multidirectional Differentiation ability are similar to embryonic stem cell.It induces versatile stem cell not by cell origin, exempt from
All various limitations such as epidemic disease repulsion, ethics, religion and law, it is great in terms of drug development, disease treatment and mechanism study
Application value is the hot spot of Present Global stem-cell research.
Since the acquisition of versatile stem cell is time and labor-intensive, and the cell phenotype of stem cell can be passed
The influence of generation number, therefore often it is related to freezing for stem cell in the course of the research.It can be significantly changed during freezing more
The thermodynamics of function stem cell, chemically and physically environment cause to damage to cell, freeze and improper result even in cell death.
Influence factor during freezing has：Cell concentration, freezing rate and frozen stock solution.It is carried out with higher cell concentration
It freezes, the survival rate higher of cell, it is to carry out freezing best results with the rate of 1 DEG C/min to freeze rate.It is most common at present
Stem cell cryopreserving liquid is+10% dimethyl sulfoxide (DMSO) (DMSO) of 90% fetal calf serum (FBS) or 50% stem cell complete medium
+ 40%FBS+10%DMSO.Although DMSO is stronger to the penetration power of cell, the freezing point of cell can be reduced, the shape of ice crystal is reduced
At during cooling protective effect can be reached to cell.But high-concentration dimethyl sulfoxide has toxicity, it can be with cell
The hydrophobic grouping of internal protein is had an effect, and protein denaturation is caused, and causes cellular damage or inactivation.And it existing freezes
Liquid causes to participate in non-personnel's substance in cells frozen storing liquid due to the use of the serum of animal sources, can cause to remain to versatile stem cell
Or pollution.
To obtain non-animal derived pollution, the big induction versatile stem cell of quantity, and versatile stem cell library is established, studied
Develop a kind of no animal foreign constituents, frozen stock solution and jelly smaller to cellular damage and that versatile stem cell can be preserved for a long time
Method is deposited, to inducing the research of versatile stem cell to be of great significance.
Invention content
To help to understand the present invention, some terms are defined below.Term defined herein has field of the present invention
The normally understood meaning of those of ordinary skill.
Unless otherwise stated, iPSCs herein is the English abbreviation for inducing versatile stem cell, and DMSO is that dimethyl is sub-
The English abbreviation of sulfone, KSR are the English abbreviation of serum replacement, and DMEM is the English abbreviation of basic culture medium.
Unless otherwise stated, versatile stem cell herein and cell refer both to induction versatile stem cell.
The technical problem to be solved in the present invention is to provide it is a kind of it is easy to operate, without animal foreign constituents, cell survival rate more
High induction versatile stem cell frozen stock solution and cryopreservation methods.
It is an object of the present invention to provide a kind of frozen stock solutions of induction versatile stem cell.
Frozen stock solution of the present invention is made of A liquid and B liquid.The volume ratio of A liquid and B liquid is 1:1.
In the A liquid 100mL containing 0-3% beta -mercaptoethanols, 0-5% LIF ELISAs, 0-4% NEAA,
The KSR and 40-58% of the L-Glutamine of 0-4%, 0-5 μM of CHIR99021,0-5 μM of PD0325901,38-55%
DMEM。
Preferably, in the A liquid 100mL containing 0.5-2% beta -mercaptoethanols, 0.5-2% LIF ELISAs,
The L-Glutamine of NEAA, 0.3-3% of 0.3-3%, 1-4 μM of CHIR99021,1-4 μM of PD0325901,40-50%
The DMEM of KSR and 45-55%.
It is highly preferred that containing 0.8% beta -mercaptoethanol, 0.8% LIF ELISA, 1% in the A liquid 100mL
NEAA, 1% L-Glutamine, 2 μM of CHIR99021,1 μM of PD0325901,45% KSR and 51.4% DMEM.
The L-Glutamine of NEAA, 0-3.5% containing 6-12%KSR, 0-3.5%, 0.5- in the B liquid 100mL
1.5% beta -mercaptoethanol, 1-2% LIF ELISAs, 2-5 μM of CHIR99021,0.1-1 μM of PD0325901,7-15%
DMSO, 6-15% hydroxyethyl starch and 55-68% DMEM.
Preferably, the L-Glutamine of NEAA, 1-3% containing 8-11%KSR, 1-3% in the B liquid 100mL,
0.8-1.2% beta -mercaptoethanols, 1.5-1.8% LIF ELISAs, 2.5-3 μM of CHIR99021,0.5-0.8 μM
The hydroxyethyl starch of DMSO, 8-13% of PD0325901,8-13% and the DMEM of 60-65%.
It is highly preferred that containing 10%KSR, 2% NEAA, 2% L-Glutamine, 1% β-in the B liquid 100mL
Mercaptoethanol, 1.6% LIF ELISA, 3 μM of CHIR99021,0.6 μM of PD0325901,10% DMSO, 10%
Hydroxyethyl starch and 63.4% DMEM.
Another object of the present invention is to provide a kind of cryopreservation methods of induction versatile stem cell, and this method includes following step
Suddenly：A. appropriate A liquid is added after after the versatile stem cell of digestion being centrifuged.B. equivalent B liquid mixings are added.C. by stem cell and jelly
The mixed liquor of liquid storage, which is put in program temperature reduction box, to be freezed, and the cell suspension of freezing is transferred in liquid nitrogen after 24 hours.
Preferably, the cell suspension final concentration of 1 × 106-6×106A/mL.
The frozen stock solution definite ingredients of the present invention do not contain animal blood serum, avoid animal derived pollution.KSR is instead of dynamic
Object serum, no animal exogenous components, entire formula composition is clear, safer, for further grinding for versatile stem cell
Studying carefully will not interfere.Beta -mercaptoethanol and fibroblast growth factor in KSR and the DMSO in B liquid and ethoxy
Starch can increase the permeability of cell during freezing, and cell can be protected during freezing, and reduce cell
Freezing point, reduce the generation of ice crystal, to keep cell viability, prevent versatile stem cell from breaking up.
Compared with the prior art scheme, beneficial effects of the present invention：First, frozen stock solution of the invention does not contain animal blood
Clearly, stem cell is polluted without exogenous animal component.Second, The present invention reduces the dosage of DMSO, reduce high in frozen stock solution
Damages of the concentration DMSO to cell effectively prevent cell differentiation to keep cell activity.Third, frozen stock solution of the invention can
In 4 DEG C of preservations, multigelation and now with the current is not needed.4th, cryopreservation methods of the invention are easy to operate, save manpower.
Description of the drawings
The specific implementation mode for illustrating the present invention for clarity, provides the following drawings, to describe the one of the present invention
A little embodiments.
Fig. 1 show versatile stem cell recover 1 day after cellular morphology figure.
Fig. 2 shows the cell that the frozen stock solution of the cell that frozen stock solution and cryopreservation methods of the present invention freeze and routine freezes
The growing state of recovery rear clone.
Fig. 3 shows the cell that the frozen stock solution of the cell that frozen stock solution and cryopreservation methods of the present invention freeze and routine freezes
The growth curve measured after recovery.
Fig. 4 shows alkaline phosphatase detection figure after versatile stem cell recovery.
Fig. 5 indicates versatile stem cell identified by immunofluorescence figure.
Specific implementation mode
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with the drawings and specific embodiments pair
The present invention is described further.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1, the preparation for inducing versatile stem cell frozen stock solution
Frozen stock solution is made of A liquid and B liquid.The volume ratio of A liquid and B liquid is 1:1.
Contain following components in per 100mL frozen stock solution A liquid：
Contain following components in per 100mL frozen stock solution B liquid：
Preparation method:A liquid and B liquid, 4 DEG C of preservations will be respectively obtained after each ingredient mixing.
The application of embodiment 2, frozen stock solution in freezing induction versatile stem cell
Cryopreservation methods：
After the induction versatile stem cell digestion in exponential phase, supernatant is removed in 800rpm/min centrifugations after five minutes,
It is resuspended with the A liquid of the appropriate present invention, after carrying out cell count, the B liquid of equivalent is added, it is 1 × 10 to make cell concentration6-6×
106A/mL, then the amount of 1mL/ pipes be sub-packed in the cell cryopreservation tube of 1.8mL, be put in program temperature reduction box be built in -80 DEG C into
Line program cools down, and is transferred in liquid nitrogen and is preserved after 24 hours.
Freeze product test：
1, morphologic observation after versatile stem cell recovery
Liquid is changed after cell recovery after freezing daily, morphologic observation is carried out under the microscope, takes pictures after clone to be formed.It is multiple
Versatile stem cell form after Soviet Union is as shown in Figure 1, it is seen that versatile stem cell clones rounded, edge clear, clones edge
The cell of no differentiation climbs out of.Illustrate that the frozen stock solution of the present invention can effective protection cell activity during freezing.Prevent cell point
Change.
2, the growing state comparison after versatile stem cell recovery
It is thin with generation induction multifunctional dry that the frozen stock solution of the present invention and conventional cryopreservation liquid are frozen into a batch under the same conditions
Born of the same parents, recovery cell after half a year, and the metamorphosis to form clone is observed, the results are shown in Figure 2.By Fig. 2 results as it can be seen that the present invention
The multi-functional cell recovery that freezes of frozen stock solution after, the speed of clonal growth will be faster than the cell that conventional cryopreservation liquid freezes, and gram
Grand sharpness of border.Wherein conventional cryopreservation liquid is to contain 10% DMSO and 90%FBS.
3, the growth curve after versatile stem cell recovery measures
Same a batch of cell recovery that the frozen stock solution of the present invention and conventional cryopreservation liquid are frozen, with 1 × 10 after counting4/
The density 1mL of mL is laid in one hole of 6 orifice plates, and the cell that each frozen stock solution freezes spreads 35 holes.Later every taking the digestion of 3 holes for 24 hours
It counts, its average is taken, until cell starts death by plateau.Cell growth curve is as shown in figure 3, can from figure
Go out, exponential phase was entered by 3 days demurrages after the cell recovery that frozen stock solution of the invention freezes, was reached at the 7th day
To plateau, and cell density can reach 1 × 106/mL.The cell that the frozen stock solution of the present invention that compares freezes, uses conventional jelly
Demurrage is longer after the cell recovery that liquid storage freezes, to the 9th talent enter plateau, and final cell density will be less than 1 ×
106/ mL illustrates that the cell viability of frozen stock solution freeze-stored cell of the present invention is better than the cell that conventional cryopreservation liquid freezes.
4, alkaline phosphatase detection is carried out after versatile stem cell recovery
The versatile stem cell recovery that the frozen stock solution of the present invention is frozen waits for that clone grows to 100 μm or so progress alkali of diameter
Acid phosphatase detects.Enough alkaline phosphatase staining liquid is added after first fixing cell with paraformaldehyde, in 37 DEG C of insulating boxs
It is protected from light and is incubated 0.5-2h.It is washed with PBS, is eventually adding appropriate PBS and observes coloration result under the microscope and take pictures, alkaline phosphatase
The results are shown in Figure 4 for enzyme dyeing.From fig. 4, it can be seen that clone's sharpness of border, and all contaminated for bluish violet, illustrate frozen stock solution guarantor
Cell viability has been protected, cell differentiation is effectively prevented.
5, identified by immunofluorescence is carried out after versatile stem cell recovery
The versatile stem cell recovery that the frozen stock solution of the present invention is frozen waits for that clone grows to 100 μm or so of diameter and exempted from
Epidemic disease Fluorescence Identification.It carries out penetrating after fixed cell, then fluorescence primary antibody and fluorescence secondary antibody is separately added into, after anti-fluorescence quenching is added
Image is acquired under fluorescence microscope, the results are shown in Figure 5.As shown in Figure 4, the clonal expression label base of multipotential cell
Cause illustrates that during freezing, cell does not break up, and has the potential of Multidirectional Differentiation.
Conclusion：The stem cell frozen with induction versatile stem cell frozen stock solution and the cryopreservation methods of the present invention, after recovery
Stem cell can be proliferated rapidly, with marker gene that is good active and expressing versatile stem cell.
In conclusion the frozen stock solution and cryopreservation methods of the present invention can be such that induction versatile stem cell is protected for a long time under liquid nitrogen
It deposits, and cell activity can be kept, prevent cell differentiation, to promoting the research of induction versatile stem cell, establishing induction multifunctional dry
Cell bank is of great significance.
It should be noted that above-described embodiment is merely preferred embodiments of the present invention, do not limit in any form
The system present invention, the technical solution that all equivalent substitution or changes according to the technique and scheme of the present invention obtain are encompassed by the present invention's
In protection domain.
Claims (7)
1. a kind of induction versatile stem cell frozen stock solution of serum-free, which is characterized in that the frozen stock solution is made of A liquid and B liquid.
2. the induction versatile stem cell frozen stock solution of serum-free according to claim 1, which is characterized in that the A liquid
In 100mL containing 0-3% beta -mercaptoethanols, 0-5% LIF ELISAs, 0-4% NEAA, 0-4% L-Glutamine,
The DMEM of the KSR and 40-58% of 0-5 μM of CHIR99021,0-5 μM of PD0325901,38-55%；Contain in the B liquid 100mL
There are L-Glutamine, 0.5-1.5% beta -mercaptoethanols, the 1-2% leukaemia of NEAA, 0-3.5% of 6-12%KSR, 0-3.5%
Inhibiting factor, 2-5 μM of CHIR99021,0.1-1 μM of PD0325901,7-15% DMSO, 6-15% hydroxyethyl starch and
The DMEM of 55-68%.
3. the induction versatile stem cell frozen stock solution of serum-free according to claim 1 or 2, which is characterized in that the A liquid
In 100mL containing 0.5-2% beta -mercaptoethanols, 0.5-2% LIF ELISAs, 0.3-3% NEAA, 0.3-3% L-
Glutamine, 1-4 μM of CHIR99021,1-4 μM of PD0325901,40-50% KSR and 45-55% DMEM；The B liquid
The L-Glutamine of NEAA, 1-3% containing 8-11%KSR, 1-3%, 0.8-1.2% beta -mercaptoethanols, 1.5- in 100mL
DMSO, 8-13% of 1.8% LIF ELISA, 2.5-3 μM of CHIR99021,0.5-0.8 μM of PD0325901,8-13%
Hydroxyethyl starch and 60-65% DMEM.
4. the induction versatile stem cell frozen stock solution of serum-free according to claim 3, which is characterized in that the A liquid
In 100mL containing 0.8% beta -mercaptoethanol, 0.8% LIF ELISA, 1% NEAA, 1% L-Glutamine, 2 μM
CHIR99021,1 μM of PD0325901,45% KSR and 51.4% DMEM；Contain 10%KSR, 2% in the B liquid 100mL
NEAA, 2% L-Glutamine, 1% beta -mercaptoethanol, 1.6% LIF ELISA, 3 μM of CHIR99021,0.6 μM
PD0325901,10% DMSO, 10% hydroxyethyl starch and 63.4% DMEM.
5. the induction versatile stem cell frozen stock solution of serum-free according to claim 3, which is characterized in that the frozen stock solution
The volume ratio of middle A liquid and B liquid is 1：1.
6. a kind of induction versatile stem cell cryopreservation methods, this approach includes the following steps：
A. the frozen stock solution A liquid will be added after postdigestive induction versatile stem cell centrifugation, it is thin obtains induction multifunctional dry
Born of the same parents' suspension；
B. the frozen stock solution B liquid mixings of equivalent are added, obtain the mixed liquor of induction versatile stem cell and frozen stock solution；
C. the mixed liquor is freezed, then the mixed liquor after freezing is preserved in liquid nitrogen.
7. according to the method described in claim 6, it is characterized in that, wherein inducing versatile stem cell in the mixed liquor
A concentration of 1 × 106-6×106A/mL.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111808799A (en) * | 2020-08-28 | 2020-10-23 | 广州同康生物科技有限公司 | Multifunctional stem cell culture method and culture medium used by same |
| CN115299434A (en) * | 2022-08-09 | 2022-11-08 | 广州明迅生物科技有限责任公司 | Cell cryopreservation liquid, cell cryopreservation and recovery method and application thereof |
| CN115363016A (en) * | 2022-08-09 | 2022-11-22 | 广州明迅生物科技有限责任公司 | Cell storage liquid and application thereof |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102550542A (en) * | 2011-08-09 | 2012-07-11 | 臻景生物技术（上海）有限公司 | Establishment for serum-free freezing medium and adipose-derived stem cell library of adipose-derived stem cells |
| CN102907416A (en) * | 2012-08-01 | 2013-02-06 | 臻景生物技术（上海）有限公司 | Tissue cryopreservation liquid capable of maintaining cell activity |
| CN103958669A (en) * | 2011-11-25 | 2014-07-30 | 住友化学株式会社 | Method of cryopreservation of tissue derived from pluripotent stem cells |
| CN104928251A (en) * | 2014-03-21 | 2015-09-23 | 中国科学院动物研究所 | Culture system for inducing pluripotent stem cells |
| CN105087472A (en) * | 2015-05-20 | 2015-11-25 | 广州赛莱拉干细胞科技股份有限公司 | Freezing medium for induced pluripotent stem cells, application of freezing medium, and freezing method of induced pluripotent stem cells |
| CN105238742A (en) * | 2015-11-19 | 2016-01-13 | 湖南师范大学 | Inducing method for danio-rerio inductivity multi-potential stem cells and inductive culture medium and iPS culture medium used for the same |
| CN105432597A (en) * | 2014-08-29 | 2016-03-30 | 马燕琳 | Kit and method for cryopreserving induced pluripotent stem cells |
| CN106561632A (en) * | 2016-11-01 | 2017-04-19 | 浙江译美生物科技有限公司 | Freezing preservation solution used for adipose-derived stem cells as well as preparation method for freezing preservation solution and culture medium |
| CN106900695A (en) * | 2017-04-01 | 2017-06-30 | 北京焕生汇生物技术研究院 | A kind of mankind's inductive pluripotent stem cells are special to freeze reagent and cryopreservation methods |
| CN106942203A (en) * | 2017-05-23 | 2017-07-14 | 广州润虹医药科技有限公司 | A kind of IPS cell-preservation liquids and preparation method thereof |
| CN107002042A (en) * | 2014-10-15 | 2017-08-01 | 瑞泽恩制药公司 | Method and composition for producing or maintaining pluripotent cell |
| CN107027743A (en) * | 2017-06-14 | 2017-08-11 | 深圳市泰华细胞工程有限公司 | Cells frozen storing liquid and cell freezing method |
-
2017
- 2017-11-13 CN CN201711126234.0A patent/CN108552156A/en active Pending
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102550542A (en) * | 2011-08-09 | 2012-07-11 | 臻景生物技术（上海）有限公司 | Establishment for serum-free freezing medium and adipose-derived stem cell library of adipose-derived stem cells |
| CN103958669A (en) * | 2011-11-25 | 2014-07-30 | 住友化学株式会社 | Method of cryopreservation of tissue derived from pluripotent stem cells |
| CN102907416A (en) * | 2012-08-01 | 2013-02-06 | 臻景生物技术（上海）有限公司 | Tissue cryopreservation liquid capable of maintaining cell activity |
| CN104928251A (en) * | 2014-03-21 | 2015-09-23 | 中国科学院动物研究所 | Culture system for inducing pluripotent stem cells |
| CN105432597A (en) * | 2014-08-29 | 2016-03-30 | 马燕琳 | Kit and method for cryopreserving induced pluripotent stem cells |
| CN107002042A (en) * | 2014-10-15 | 2017-08-01 | 瑞泽恩制药公司 | Method and composition for producing or maintaining pluripotent cell |
| CN105087472A (en) * | 2015-05-20 | 2015-11-25 | 广州赛莱拉干细胞科技股份有限公司 | Freezing medium for induced pluripotent stem cells, application of freezing medium, and freezing method of induced pluripotent stem cells |
| CN105238742A (en) * | 2015-11-19 | 2016-01-13 | 湖南师范大学 | Inducing method for danio-rerio inductivity multi-potential stem cells and inductive culture medium and iPS culture medium used for the same |
| CN106561632A (en) * | 2016-11-01 | 2017-04-19 | 浙江译美生物科技有限公司 | Freezing preservation solution used for adipose-derived stem cells as well as preparation method for freezing preservation solution and culture medium |
| CN106900695A (en) * | 2017-04-01 | 2017-06-30 | 北京焕生汇生物技术研究院 | A kind of mankind's inductive pluripotent stem cells are special to freeze reagent and cryopreservation methods |
| CN106942203A (en) * | 2017-05-23 | 2017-07-14 | 广州润虹医药科技有限公司 | A kind of IPS cell-preservation liquids and preparation method thereof |
| CN107027743A (en) * | 2017-06-14 | 2017-08-11 | 深圳市泰华细胞工程有限公司 | Cells frozen storing liquid and cell freezing method |
Non-Patent Citations (3)
| Title |
|---|
| 刘鸿燕 等: "人多能干细胞的冷冻与保存", 《中国组织工程研究》 * |
| 成德 等: "RHO相关蛋白激酶抑制剂Y-27632对猪诱导多能干细胞冻存和传代效率的影响", 《农药生物技术学报》 * |
| 陈露萍等: "不同冻存液对人诱导性多能干细胞冻存与复苏效果的影响", 《中国临床神经科学》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111808799A (en) * | 2020-08-28 | 2020-10-23 | 广州同康生物科技有限公司 | Multifunctional stem cell culture method and culture medium used by same |
| CN111808799B (en) * | 2020-08-28 | 2021-07-13 | 河南昆仑精准医疗科技有限公司 | Multifunctional stem cell culture method and culture medium used by same |
| CN115299434A (en) * | 2022-08-09 | 2022-11-08 | 广州明迅生物科技有限责任公司 | Cell cryopreservation liquid, cell cryopreservation and recovery method and application thereof |
| CN115363016A (en) * | 2022-08-09 | 2022-11-22 | 广州明迅生物科技有限责任公司 | Cell storage liquid and application thereof |
| CN115299434B (en) * | 2022-08-09 | 2023-11-21 | 广州明迅生物科技有限责任公司 | Cell cryopreservation liquid, cell cryopreservation and resuscitation method and application thereof |
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