CN106834220A - A kind of serum-free cultured chondrocytes base and preparation method thereof - Google Patents
A kind of serum-free cultured chondrocytes base and preparation method thereof Download PDFInfo
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- C12N5/0602—Vertebrate cells
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Abstract
The invention belongs to cell culture medium technical field, and in particular to a kind of serum-free cultured chondrocytes base and preparation method thereof.Serum-free cultured chondrocytes base of the present invention include basal medium and additive, the composition of the additive with final concentration, including：The 32ng/mL of cell factor 24, the 10mg/mL of polylysine 7,25 μM of arachidonic acid, the μ g/mL of vitamin C 10 15, the μ g/mL of parthenolide 58,59 μM of butanediamine, the μ g/mL of insulin 79,36 μM of leukotrienes, the μ g/mL of flavones 14 18, the μ g/mL of transferrins 12 16, polyvinylpyrrolidone 20 26 μ g/mL, the μ g/mL of hyperglycemic factor 8 13 and the fine 32ng/mL of laminins 20.The serum-free cultured chondrocytes base that the present invention is provided is free of serum, and cost is relatively low, is conducive to scale application.
Description
Technical field
The invention belongs to cell culture medium technical field, and in particular to a kind of serum-free cultured chondrocytes base and its preparation
Method.
Background technology
Articular cartilage is mainly the vesselless tissue being made up of cartilage cell and extracellular matrix, relies primarily on the fortune in joint
Dynamic and extruding absorbs nutriment, so self-repairing capability is weaker after cartilage damage, the repairing transplant after its damage is always
Since be one of problem of medical field.In recent years, with the development of tissue engineering technique, Cartilage transplantation is in cartilage damage
Clinical treatment at home and abroad has application.Clinical research is limited to patient tissue materials, and also because cartilage cell is whole end
Noble cells, in-vitro multiplication is limited in one's ability and easily dedifferentes, so the culture and amplification to chondrocytes in vitro cell turn at present
The key of solution.
Chinese patent application CN104120105A discloses a kind of serum-free for cultivating older population cartilage cell and trains
Base, including basal medium and additive are supported, additive concentration composition is：233 μM of -287 μM of vitamin Cs, 3.7 μM of -8.9 μM of Asias
Oleic acid, 9 μM of -17 μM of cholesterol, 6nM-15nM dexamethasone, 43 μM of -58 μM of acetylcysteines, 18 μ g/mL-32 μ g/mL turn
Ferritin, 22nM-52nM sodium selenites, 13 μM of -24 μM of sodium pantothenates, 28 μM of -43 μM of biotins, 7 μ g/mL-18 μ g/mL pancreas islet
Element, 2ng/mL-9ng/mL EGFs, 2ng/mL-9ng/mL fibroblast growth factors, 2ng/mL-9ng/mL blood platelets
Derivative growth factor, 0.5%-2.5% human serum albumins.It is thin that Chinese patent application CN104480066A discloses a kind of cartilage
Born of the same parents' culture medium and cultured chondrocytes method, the cultured chondrocytes base include：Basal medium, concentration are the blood of 5-20%
Clearly, concentration is that the platelet derived growth factor and concentration of 1-20ng/ml are (1-5) * 10-5M/L beta -mercaptoethanols.
What the existing culture medium suitable for cartilage cell's in vitro culture had contains serum, because serum is by many sizes
Different biological molecules composition extremely complex mixture, it can provide growth factor required when cell is cultivated in vitro,
Hormone, associated proteins, and protective effect is provided；And mitogenic factor is also rich in serum itself, also beneficial to cell line and
Original cuiture.But the drawbacks of blood serum medium has certain, it is impossible to evade the alloplasm in animal blood serum, if using animal blood
, can there is human body alloplasm and repel and conflict in the cell that clear culture medium is cultivated.Although some culture mediums are free of serum,
Be there are problems that it is expensive.
The content of the invention
In view of the shortcomings of the prior art, it is an object of the invention to provide a kind of serum-free cultured chondrocytes base and its system
Preparation Method.The serum-free cultured chondrocytes base that the present invention is provided is free of serum, eliminates the pathogen contamination of animal origin, carries
Cartilage cell's amplification rate high, substantially reduces the time of cultured chondrocytes, the serum-free cartilage cell that the present invention is provided
The cost of culture medium is relatively low, is conducive to scale application.
The technical scheme is that：
A kind of serum-free cultured chondrocytes base, including the addition of basal medium and addition in the basal medium
Agent, the composition of the additive with final concentration, including：Cell factor 24-32ng/mL, polylysine 7-10mg/mL, peanut
2-5 μM of tetraenoic acid, vitamin C 10-15 μ g/mL, parthenolide 5-8 μ g/mL, 5-9 μM of butanediamine, insulin 7-9 μ g/mL,
3-6 μM of leukotrienes, flavones 14-18 μ g/mL, transferrins 12-16 μ g/mL, polyvinylpyrrolidone 20-26 μ g/mL, pancreas blood high
Sugar element 8-13 μ g/mL and fine laminins 20-32ng/mL.
Further, the serum-free cultured chondrocytes base includes basal medium and addition in the basal medium
In additive, the composition of the additive with final concentration, including：Cell factor 28ng/mL, polylysine 9mg/mL, flower
Raw 3 μM of tetraenoic acid, the μ g/mL of vitamin C 12, the μ g/mL of parthenolide 6,7 μM of butanediamine, the μ g/mL of insulin 8,5 μM of leukotrienes,
The μ g/mL of flavones 16, the μ g/mL of transferrins 14, the μ g/mL of polyvinylpyrrolidone 22, the μ g/mL of hyperglycemic factor 11 and fibre are adhered egg
White 25ng/mL.
Further, the basal medium is RMPI 1640 or IMEM culture mediums.
Further, the cell factor by serum spread factor, TGF and EGF by weight
Than 8-10: 3-7: 1-3 composition.
Further, the cell factor by serum spread factor, TGF and EGF by weight
Than 9: 5: 2 compositions.
It is a further object of the present invention to provide the preparation method of the serum-free cultured chondrocytes base, including following step
Suddenly：
S1 in basal medium add cell factor, polylysine, arachidonic acid, vitamin C, parthenolide,
Butanediamine, insulin, leukotrienes, flavones, transferrins and fine laminins, stir 20-40 minutes, add polyvinylpyrrolidine
Ketone and hyperglycemic factor, continue to stir 18-25 minutes, obtain mixture；
S2 is the sodium hydroxide solution of 4-7% to mass fraction is added in step S1 gained mixtures, regulation culture medium
PH to 7.0-7.3, sterilising temp is 117-122 DEG C, and sterilization time is 18-23 minutes, is obtained final product.
Preferably, the step S1 is stirred 26 minutes.
Preferably, the step S1 continues to stir 22 minutes.
Preferably, the step S2 is to the sodium hydroxide solution that addition mass fraction in step S1 gained mixtures is 6%.
Preferably, the step S2 adjusts the pH to 7.1 of culture medium.
Preferably, the step S2 sterilising temps are 120 DEG C.
Preferably, the step S2 sterilization times are 20 minutes.
The serum-free cultured chondrocytes base that the present invention is provided, including basal medium and addition are in the basal medium
In additive, the additive of addition includes cell factor, polylysine, arachidonic acid, vitamin C and parthenolide
Deng.In the composition of various culture mediums of the invention, basal medium can provide the existence of cartilage cell and minimum physiology
Activity.In the present invention, cell factor is mainly as the supplement needed for maintaining the existence of cartilage cell's in vitro culture, propagation and differentiation
The factor.In the present invention, polylysine and fine laminins collective effect, can promote the adhesion of cartilage cell, and its adherent
Growth.In the present invention, arachidonic acid is the main component of cell membrane, decides the bioactivity of cell membrane.In the present invention
In, vitamin C can participate in cell metabolism as nutritional agents, and also act as oxidation resistant effect.In the present invention, butanediamine
Lipid needed for cell membrane synthesis can be provided and the water-soluble lipid needed for cell growth.In the present invention, insulin can
Promote the synthesis of RNA, protein and aliphatic acid, suppress Apoptosis.In the present invention, leukotrienes can provide cell membrane synthesis
Required lipid, and acted synergistically with other raw materials in the present invention, cell can be promoted to breed, improve cell yield.
In the present invention, flavones is mainly used as antioxidant, can eliminate infringement of the oxygen radical to cell, in addition it is possible to culture medium in
Other raw materials synergy, promote cartilage cell propagation.In the present invention, transferrins can adjust internal ferro element
, be transferred to for ferro element by the TfR of cell surface intracellular by metabolism；In addition, it can also be with other micro units
Element is combined, so as to adjust the growing multiplication and functional expression of cartilage cell.In the present invention, polyvinylpyrrolidone can absorb
Aldehydes matter, mitigates murder by poisoning of the aldehydes matter to cell.In the present invention, hyperglycemic factor participate in cartilage cell glycometabolism,
Lipid metabolism etc., can adjust the propagation and functional expression of cartilage cell.
Research finds that parthenolide and flavones are added in serum-free cultured chondrocytes base, and both compositions can be with
Acted synergistically between other compositions, cartilage cell's amplification rate is further improved.Serum-free cartilage cell training of the present invention
Support base collocation rationally, mutually coordinated between each composition, collective effect improves cartilage cell's amplification rate, substantially reduces cartilage
The time of cell culture.
Compared with prior art, the serum-free cultured chondrocytes base that the present invention is provided has the advantage that：
(1) the serum-free cultured chondrocytes base that the present invention is provided is free of serum, and the pathogen for eliminating animal origin is dirty
Dye.
(2) the present invention is to provide the serum-free cultured chondrocytes base that a kind of non-animal derived property composition, composition determine, can
So that the quality stability of product batches is effectively ensured, be conducive to the standardization of products.
(3) cost of the serum-free cultured chondrocytes base that the present invention is provided is relatively low, is conducive to scale application.
(4) the serum-free cultured chondrocytes base that the present invention is provided improves cartilage cell's amplification rate, substantially reduces
The time of cultured chondrocytes.
Specific embodiment
Below by way of the description of specific embodiment, the invention will be further described, but this is not to limit of the invention
System, those skilled in the art's basic thought of the invention, various modifications may be made or improves, but without departing from this
The basic thought of invention, within the scope of the present invention.
The culture mediums of RMPI 1640 are purchased from the vast Tyke biological gene technology Co., Ltd in Beijing in the present invention,
IMEM culture mediums are purchased from the vast Tyke biological gene technology Co., Ltd in Beijing, and it is public that hyperglycemic factor is purchased from Sigma
Department, polylysine is purchased from Zhengzhou Bai Nafo bioengineering limited company, and flavones is purchased from Harbin hundred and likes that science and technology is limited
Company, parthenolide is purchased from Sigma companies.
Embodiment 1, a kind of serum-free cultured chondrocytes base
The serum-free cultured chondrocytes base, including the addition of basal medium and addition in the basal medium
Agent, the composition of the additive with final concentration, including：Cell factor 24ng/mL, polylysine 7mg/mL, arachidonic acid 2
μM, the μ g/mL of vitamin C 10, the μ g/mL of parthenolide 5,5 μM of butanediamine, the μ g/mL of insulin 7,3 μM of leukotrienes, the μ g/ of flavones 14
ML, the μ g/mL of transferrins 12, polyvinylpyrrolidone 20 μ g/mL, the μ g/mL of hyperglycemic factor 8 and fine laminins 20ng/mL.
The basal medium is the culture mediums of RMPI 1640.
The cell factor is by serum spread factor, TGF and EGF by weight 8: 7: 3 groups
Into.
Preparation method：
S1 in basal medium add cell factor, polylysine, arachidonic acid, vitamin C, parthenolide,
Butanediamine, insulin, leukotrienes, flavones, transferrins and fine laminins, stir 20 minutes, add polyvinylpyrrolidone
And hyperglycemic factor, continue to stir 18 minutes, obtain mixture；
S2 adjusts the pH of culture medium to the sodium hydroxide solution that mass fraction is 4% is added in step S1 gained mixtures
To 7.0, sterilising temp is 117 DEG C, and sterilization time is 18 minutes, is obtained final product.
Embodiment 2, a kind of serum-free cultured chondrocytes base
The serum-free cultured chondrocytes base, including the addition of basal medium and addition in the basal medium
Agent, the composition of the additive with final concentration, including：Cell factor 32ng/mL, polylysine 10mg/mL, arachidonic acid
5 μM, the μ g/mL of vitamin C 15, the μ g/mL of parthenolide 8,9 μM of butanediamine, the μ g/mL of insulin 9,6 μM of leukotrienes, the μ of flavones 18
G/mL, the μ g/mL of transferrins 16, polyvinylpyrrolidone 26 μ g/mL, the μ g/mL of hyperglycemic factor 13 and fine laminins 32ng/
mL。
The basal medium is IMEM culture mediums.
The cell factor is by serum spread factor, TGF and EGF by weight 10: 3: 1 groups
Into.
Preparation method：
S1 in basal medium add cell factor, polylysine, arachidonic acid, vitamin C, parthenolide,
Butanediamine, insulin, leukotrienes, flavones, transferrins and fine laminins, stir 40 minutes, add polyvinylpyrrolidone
And hyperglycemic factor, continue to stir 25 minutes, obtain mixture；
S2 adjusts the pH of culture medium to the sodium hydroxide solution that mass fraction is 7% is added in step S1 gained mixtures
To 7.3, sterilising temp is 122 DEG C, and sterilization time is 23 minutes, is obtained final product.
Embodiment 3, a kind of serum-free cultured chondrocytes base
The serum-free cultured chondrocytes base, including the addition of basal medium and addition in the basal medium
Agent, the composition of the additive with final concentration, including：Cell factor 28ng/mL, polylysine 9mg/mL, arachidonic acid 3
μM, the μ g/mL of vitamin C 12, the μ g/mL of parthenolide 6,7 μM of butanediamine, the μ g/mL of insulin 8,5 μM of leukotrienes, the μ g/ of flavones 16
ML, the μ g/mL of transferrins 14, polyvinylpyrrolidone 22 μ g/mL, the μ g/mL of hyperglycemic factor 11 and fine laminins 25ng/
mL。
The basal medium is IMEM culture mediums.
The cell factor is by serum spread factor, TGF and EGF by weight 9: 5: 2 groups
Into.
Preparation method：
S1 in basal medium add cell factor, polylysine, arachidonic acid, vitamin C, parthenolide,
Butanediamine, insulin, leukotrienes, flavones, transferrins and fine laminins, stir 26 minutes, add polyvinylpyrrolidone
And hyperglycemic factor, continue to stir 22 minutes, obtain mixture；
S2 adjusts the pH of culture medium to the sodium hydroxide solution that mass fraction is 6% is added in step S1 gained mixtures
To 7.1, sterilising temp is 120 DEG C, and sterilization time is 20 minutes, is obtained final product.
Comparative example 1, a kind of serum-free cultured chondrocytes base
The serum-free cultured chondrocytes base, including the addition of basal medium and addition in the basal medium
Agent, the composition of the additive with final concentration, including：Cell factor 28ng/mL, polylysine 9mg/mL, arachidonic acid 3
μM, the μ g/mL of vitamin C 12, the μ g/mL of capsule of weeping forsythia aglycon 6,7 μM of butanediamine, the μ g/mL of insulin 8,5 μM of leukotrienes, the μ g/ of flavones 16
ML, the μ g/mL of transferrins 14, polyvinylpyrrolidone 22 μ g/mL, the μ g/mL of hyperglycemic factor 11 and fine laminins 25ng/
mL。
The basal medium is IMEM culture mediums.
The cell factor is by serum spread factor, TGF and EGF by weight 9: 5: 2 groups
Into.
Preparation method is similar to Example 3.
Difference with embodiment 3 is that parthenolide is replaced with into capsule of weeping forsythia aglycon.
Comparative example 2, a kind of serum-free cultured chondrocytes base
The serum-free cultured chondrocytes base, including the addition of basal medium and addition in the basal medium
Agent, the composition of the additive with final concentration, including：Cell factor 28ng/mL, polylysine 9mg/mL, arachidonic acid 3
μM, the μ g/mL of vitamin C 12, the μ g/mL of parthenolide 6,7 μM of butanediamine, the μ g/mL of insulin 8,5 μM of leukotrienes, the μ of isoflavones 16
G/mL, the μ g/mL of transferrins 14, polyvinylpyrrolidone 22 μ g/mL, the μ g/mL of hyperglycemic factor 11 and fine laminins 25ng/
mL。
The basal medium is IMEM culture mediums.
The cell factor is by serum spread factor, TGF and EGF by weight 9: 5: 2 groups
Into.
Preparation method is similar to Example 3.
Difference with embodiment 3 is that flavones is replaced with into isoflavones.
Comparative example 3, a kind of serum-free cultured chondrocytes base
The serum-free cultured chondrocytes base, including the addition of basal medium and addition in the basal medium
Agent, the composition of the additive with final concentration, including：Cell factor 28ng/mL, polylysine 9mg/mL, arachidonic acid 3
μM, the μ g/mL of vitamin C 12, the μ g/mL of parthenolide 6,7 μM of butanediamine, the μ g/mL of insulin 8,5 μM of leukotrienes, the μ g/ of flavones 16
ML, the μ g/mL of transferrins 14, polyvinylpyrrolidone 22 μ g/mL, the μ g/mL of hyperglycemic factor 11 and fine laminins 25ng/
mL。
The basal medium is IMEM culture mediums.
The cell factor is by serum spread factor, TGF and EGF by weight 1: 1: 1 group
Into.
Preparation method is similar to Example 3.
Difference with embodiment 3 is that the cell factor is by serum spread factor, TGF and epidermal growth
The factor is by weight 1: 1: 1 composition.
Test example one, serum-free cultured chondrocytes base of the present invention are to chondrocyte growth culture effect
1st, subjects：Embodiment of the present invention 1-3 gained serum-free cultured chondrocytes base bases, and comparative example 1-3 institutes
Obtain serum-free cultured chondrocytes base
2nd, test method：
The cartilaginous tissue chopping that will be obtained, is then first digested 40 minutes at 37 DEG C with 0.25% trypsase, then with 0.1%
II Collagenase Type digests overnight.1200r/min be centrifuged 5 minutes reclaim cell, respectively obtained by embodiment of the present invention 1-3 without blood
It is resuspended in clear cultured chondrocytes base, and comparative example 1-3 gained serum-free cultured chondrocytes bases.Grow in the medium
Cell is with 5000 cell/cm2Density bed board.All experiments use T75 blake bottles.
Cell reach 50% to 80% converge when passed on.The cell for growing in the medium is rinsed with PBS, is harvested
Cell, counts inoculation of laying equal stress on.At the end of passage every time, determine cell yield and calculate population doubling.Result such as table 1 and table
Shown in 2.
Table 1：Different culture media measures cell yield and compares
Can be drawn by table 1, in the passage of all detections, be grown in embodiment of the present invention 1-3 gained serum-free cartilages
Cell yield in cell culture medium, hence it is evident that higher than be grown in comparative example 1-3 gained serum-free cultured chondrocytes base in it is thin
Born of the same parents' yield.
Table 2：Different culture media measures growth index and compares
Can be drawn by table 2, in the passage of all detections, be grown in embodiment of the present invention 1-3 gained serum-free cartilages
The growth index of cell in cell culture medium, hence it is evident that more than be grown in comparative example 1-3 gained serum-free cultured chondrocytes base in
The growth index of cell.
Claims (10)
1. a kind of serum-free cultured chondrocytes base, it is characterised in that including basal medium and addition in the basis culture
Additive in base, the composition of the additive with final concentration, including：Cell factor 24-32ng/mL, polylysine 7-
10mg/mL, 2-5 μM of arachidonic acid, vitamin C 10-15 μ g/mL, parthenolide 5-8 μ g/mL, 5-9 μM of butanediamine, pancreas islet
Plain 7-9 μ g/mL, 3-6 μM of leukotrienes, flavones 14-18 μ g/mL, transferrins 12-16 μ g/mL, polyvinylpyrrolidone 20-26 μ
G/mL, hyperglycemic factor 8-13 μ g/mL and fine laminins 20-32ng/mL.
2. serum-free cultured chondrocytes base as claimed in claim 1, it is characterised in that exist including basal medium and addition
Additive in the basal medium, the composition of the additive with final concentration, including：Cell factor 28ng/mL, gathers and relies
Propylhomoserin 9mg/mL, 3 μM of arachidonic acid, the μ g/mL of vitamin C 12, the μ g/mL of parthenolide 6,7 μM of butanediamine, the μ g/ of insulin 8
ML, 5 μM of leukotrienes, the μ g/mL of flavones 16, the μ g/mL of transferrins 14, polyvinylpyrrolidone 22 μ g/mL, the μ of hyperglycemic factor 11
G/mL and fine laminins 25ng/mL.
3. serum-free cultured chondrocytes base as claimed in claim 1 or 2, it is characterised in that the basal medium is
RMPI 1640 or IMEM culture mediums.
4. serum-free cultured chondrocytes base as claimed in claim 1 or 2, it is characterised in that the cell factor is by serum
Spreading factor, TGF and EGF are by weight 8-10: 3-7: 1-3 composition.
5. serum-free cultured chondrocytes base as claimed in claim 4, it is characterised in that the cell factor is sprawled by serum
The factor, TGF and EGF are by weight 9: 5: 2 compositions.
6. the preparation method of the serum-free cultured chondrocytes base as described in claim 1-5 is any, it is characterised in that including with
Lower step：
S1 is to addition cell factor, polylysine, arachidonic acid, vitamin C, parthenolide, fourth two in basal medium
Amine, insulin, leukotrienes, flavones, transferrins and fine laminins, stir 20-40 minute, add polyvinylpyrrolidone with
Hyperglycemic factor, continues to stir 18-25 minutes, obtains mixture；
S2 to step S1 gained mixture in add mass fraction for 4-7% sodium hydroxide solution, adjust culture medium pH to
7.0-7.3, sterilising temp is 117-122 DEG C, and sterilization time is 18-23 minutes, is obtained final product.
7. the preparation method of serum-free cultured chondrocytes base as claimed in claim 6, it is characterised in that the step S2 to
The sodium hydroxide solution that mass fraction is 6% is added in step S1 gained mixtures.
8. the preparation method of serum-free cultured chondrocytes base as claimed in claim 6, it is characterised in that the step S2 is adjusted
Save the pH to 7.1 of culture medium.
9. the preparation method of serum-free cultured chondrocytes base as claimed in claim 6, it is characterised in that the step S2 goes out
Bacterium temperature is 120 DEG C.
10. the preparation method of serum-free cultured chondrocytes base as claimed in claim 6, it is characterised in that the step S2
Sterilization time is 20 minutes.
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| CN201611210407.2A CN106834220A (en) | 2016-12-24 | 2016-12-24 | A kind of serum-free cultured chondrocytes base and preparation method thereof |
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| CN201611210407.2A CN106834220A (en) | 2016-12-24 | 2016-12-24 | A kind of serum-free cultured chondrocytes base and preparation method thereof |
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Cited By (3)
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| CN107267462A (en) * | 2017-08-07 | 2017-10-20 | 广州润虹医药科技股份有限公司 | The serum free medium that a kind of induced multi-potent stem cell is quickly produced |
| CN110438069A (en) * | 2019-08-22 | 2019-11-12 | 石学高 | A kind of phillygenol for promoting human adipose mesenchymal stem cells at the purposes of cartilage differentiation in vitro |
| CN113025572A (en) * | 2021-04-09 | 2021-06-25 | 太东(镇江)生物科技有限公司 | Amplification culture medium and application thereof in NK cell culture |
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| CN104630138A (en) * | 2013-11-06 | 2015-05-20 | 陕西瑞盛生物科技有限公司 | Serum-free cartilage cell culture solution |
| CN105200008A (en) * | 2015-10-23 | 2015-12-30 | 新乡医学院 | Stem cell culture medium |
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| CN104630138A (en) * | 2013-11-06 | 2015-05-20 | 陕西瑞盛生物科技有限公司 | Serum-free cartilage cell culture solution |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107267462A (en) * | 2017-08-07 | 2017-10-20 | 广州润虹医药科技股份有限公司 | The serum free medium that a kind of induced multi-potent stem cell is quickly produced |
| CN107267462B (en) * | 2017-08-07 | 2020-02-21 | 广州润虹医药科技股份有限公司 | Serum-free culture medium for inducing pluripotent stem cells to rapidly generate |
| CN110438069A (en) * | 2019-08-22 | 2019-11-12 | 石学高 | A kind of phillygenol for promoting human adipose mesenchymal stem cells at the purposes of cartilage differentiation in vitro |
| CN110438069B (en) * | 2019-08-22 | 2022-11-22 | 深圳泽医细胞治疗集团有限公司 | Application of forsythiaside in promoting chondrogenic differentiation of human adipose mesenchymal stem cells in vitro |
| CN113025572A (en) * | 2021-04-09 | 2021-06-25 | 太东(镇江)生物科技有限公司 | Amplification culture medium and application thereof in NK cell culture |
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