CN107475185A - A kind of non-animal derived culture medium for being used to cultivate human mesenchymal stem cell - Google Patents
A kind of non-animal derived culture medium for being used to cultivate human mesenchymal stem cell Download PDFInfo
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Abstract
The present invention relates to a kind of non-animal derived culture medium for being used to cultivate human mesenchymal stem cell, including following ingredients：Rh-insulin：1~100ug/ml；Basic fibroblast growth factor：1~50ng/ml；Human transferrin：1~50ug/ml；Selenous acid：1~80ug/ml；Serine：1 ~ 300ug/ml Choline Chlorides：1~200ug/ml；Adenine：1~60ug/ml；Single acetyl amine：1~100ug/ml；Phosphatidyl ethanolamine：0.01~10mmol/ml；Progesterone:1~40 pg/ml；Triiodothyronine:1~50pg/ml；Human plasma：1~100ul/ml.The culture medium of the present invention is in the adipose-derived mescenchymal stem cell of culture people and human umbilical cord mesenchymal stem cells, achieve the culture efficiency for being significantly better than conventional medium, good stem cell properties are kept with cell, ability of cell proliferation, the characteristics of biology performance is stronger；Dependence of the stem cell to hyclone is reduced, hyclone is also mitigated to stem cell properties and the adverse effect of differentiation, thoroughly removes hyclone, avoid, because the bad caused viral pollution risk of animal blood serum quality be present, more conforming to clinical practice.
Description
Technical field
The present invention relates to technical field of stem cell culture, and in particular to a kind of nothing for being used to cultivate human mesenchymal stem cell is moved
Material resource culture medium.
Background technology
Mescenchymal stem cell【mesenchymal stem cells,MSC】It is the important member of stem cell line, derives from
The mesoderm and ectoderm of mesoderm growing early stage, belong to multipotential stem cell, MSC initially has found in marrow, because it has Multidirectional Differentiation
Potential, hematopoiesis support and promote stem cell implantation, immunoregulation and be increasingly subject to the concern of people the features such as self-replacation.Such as
Mescenchymal stem cell is in vivo or in vitro under specific inductive condition, can be divided into fat, bone, cartilage, muscle, tendon, ligament,
The Various Tissues cell such as nerve, liver, cardiac muscle, endothelium, still there is multi-lineage potential after continuous passage culture and freezen protective, can
It is used for injuries of tissues and organs reparation caused by aging and lesion as preferable seed cell.
The culture medium that traditional mescenchymal stem cell cultural method uses mostly contains animal blood serum, on the one hand causes into fibre
The pollution of cell is tieed up, influences Keratiocyte growth, accelerates keratinocyte differentiation；The egg that another aspect is failed to understand due to serum and composition
White presence, the basic research such as cell biology, toxicology, pathology are formed and disturbed；Simultaneously using the culture of addition serum
The artificial organ of base structure is transplanted in vivo, and the anti-bovine protein antibody of generation in recipient's body can be made to cause immune response, so as to
Cause patient's unsafe factor such as failure in treatment especially after infusion stem-cell therapy is repeated.
The current serum free medium of new generation for also thering are some companies to develop, such as Invitrogen exploitation MSC SFM
XenoFree serum free mediums, Canadian stemcell companies develop the training of MesenCultTM-XF Medium serum-frees
Support base, the Serum-free MSC that BDMOSAICTM Hmsc the SF Medium and CellGenix of the exploitation of BD companies are developed
Medium etc..But these culture mediums are expensive, it is required for carrying out cell culture using auxiliary product during MSC is cultivated
The coating processing of bottle.
The content of the invention
The purpose of the present invention is in view of the shortcomings of the prior art, there is provided a kind of multiplication capacity is strong, is suitable for clinical practice
The non-animal derived culture medium of mescenchymal stem cell.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of non-animal derived culture medium for being used to cultivate human mesenchymal stem cell, including following ingredients, with densimeter：
Rh-insulin：1~100ug/ml；
Basic fibroblast growth factor：1~50ng/ml；
Human transferrin：1~50ug/ml；
Selenous acid：1~80ug/ml；
Serine：1~300ug/ml；
Choline Chloride：1~200ug/ml；
Adenine：1~60ug/ml；
Single acetyl amine：1~100ug/ml；
Phosphatidyl ethanolamine：0.01~10mmol/ml；
Progesterone:1~40pg/ml
Triiodothyronine:1~50pg/ml；
Human plasma：1~100ul/ml.
Preferably, a kind of non-animal derived culture medium for being used to cultivate human mesenchymal stem cell of the present invention,
Including following ingredients：
Rh-insulin：1~50ug/ml；
Basic fibroblast growth factor：1~20ng/ml；
Human transferrin：1~20ug/ml；
Selenous acid：1~30ug/ml；
Serine：1~150ug/ml；
Choline Chloride：1~100ug/ml；
Adenine：1~30ug/ml；
Single acetyl amine：1~20ug/ml；
Phosphatidyl ethanolamine：0.05~1mmol/ml；
Progesterone:1~10pg/ml
Triiodothyronine:1~20pg/ml；
Human plasma：1~50ul/ml.
More electedly, a kind of non-animal derived culture medium for being used to cultivate human mesenchymal stem cell of the present invention,
Including following ingredients：
Rh-insulin：5ug/ml；
Basic fibroblast growth factor：4ng/ml；
Human transferrin：5ug/ml；
Selenous acid：6.84ug/ml；
Serine：105ug/ml；
Choline Chloride：89.3ug/ml；
Adenine：12.2ug/ml；
Single acetyl amine：14ug/ml；
Phosphatidyl ethanolamine：0.1mmol/ml；
Progesterone:3.145pg/ml
Triiodothyronine:13.46pg/ml；
Human plasma：15ul/ml.
Preferably, described a kind of non-animal derived culture medium for being used to cultivate human mesenchymal stem cell, in addition to base
Basal culture medium, the basal medium are DMEM in high glucose culture medium.
A kind of described non-animal derived culture medium for being used to cultivate human mesenchymal stem cell is done in people's fat mesenchymal
Application in cell and human umbilical cord mesenchymal stem cells culture.
Beneficial effects of the present invention are：
1st, for culture medium of the present invention on the basis of universal DMEM culture mediums, the kind of addition more than ten promotes the material of cell growth, drop
Low dependence of the stem cell to hyclone, hyclone is also mitigated to stem cell properties and the adverse effect of differentiation, thoroughly
Hyclone is removed, more conforms to clinical practice.
2nd, contrast universal mescenchymal stem cell culture medium, above-mentioned culture medium have cell keep good stem cell properties,
The characteristics of ability of cell proliferation is stronger；
3rd, compared with certain external well-known low blood serum medium, cultivate and fill between most-often used people's adipose-derived stem cells/people's umbilical cord
Matter stem cell, in terms of cellular morphology and multiplication capacity, all show advantage；
4th, culture medium production cost of the present invention can control same type culture medium price 1/10 or so abroad；
5th, single acetyl amine, Choline Chloride are added in the medium, contribute to the division of new cell to synthesize.In the activity of cell
In, cell can synthesize phosphatidyl ethanolamine by single acetyl amine kinases by substrate of single acetyl amine, be turned choline by choline kinase
Phosphocholine is turned to, while single acetyl amine is also the part of glycosyl-phosphatidyl inositol, glycosyl-phosphatidyl inositol is cell table
The necessary material that face albumen sticks；Triiodothyronine, RNA polymerases I and II synthesis are stimulated, increases the conjunction of protein
Into；Serine, the biosynthesis of purine and pyrimidine is participated in, is several amino acids（Glycine, cysteine etc.）Before folic acid
Body.
Brief description of the drawings：
Accompanying drawing 1 is culture medium Fibroblast cell-culture experimental cell analysis of accounts of the present invention；
Accompanying drawing 2 is culture medium Fibroblast cell-culture experimental cell morphologic observation figure of the present invention（x40）；
Accompanying drawing 3 is culture medium Fibroblast cell-culture experimental cell morphologic observation figure of the present invention（x100）.
Embodiment：
The present invention is described in detail with reference to embodiment, but they are not the further limitations to the present invention.
Embodiment one
A kind of non-animal derived culture medium for being used to cultivate human mesenchymal stem cell of the present invention, including following ingredients,
With densimeter：
Rh-insulin：5ug/ml；
Basic fibroblast growth factor：4ng/ml；
Human transferrin：5ug/ml；
Selenous acid：6.84ug/ml；
Serine：105ug/ml；
Choline Chloride：89.3ug/ml；
Adenine：12.2ug/ml；
Single acetyl amine：14ug/ml；
Phosphatidyl ethanolamine：0.1mmol/ml；
Progesterone:3.145pg/ml
Triiodothyronine:13.46pg/ml；
Human plasma：15ul/ml.
Embodiment two：
A kind of non-animal derived culture medium for being used to cultivate human mesenchymal stem cell of the present invention, including following ingredients,
With densimeter：
Rh-insulin：1ug/ml；
Basic fibroblast growth factor：20ng/ml；
Human transferrin：50ug/ml；
Selenous acid：1ug/ml；
Serine：150ug/ml；
Choline Chloride：1ug/ml；
Adenine：60ug/ml；
Single acetyl amine：20ug/ml；
Phosphatidyl ethanolamine：0.05mmol/ml；
Progesterone:1pg/ml
Triiodothyronine:1pg/ml；
Human plasma：10ul/ml.
Embodiment three：
A kind of non-animal derived culture medium for being used to cultivate human mesenchymal stem cell of the present invention, including following ingredients,
With densimeter：
Rh-insulin：50ug/ml；
Basic fibroblast growth factor：50ng/ml；
Human transferrin：1ug/ml；
Selenous acid：80ug/ml；
Serine：300ug/ml；
Choline Chloride：100ug/ml；
Adenine：1ug/ml；
Single acetyl amine：1ug/ml；
Phosphatidyl ethanolamine：10mmol/ml；
Progesterone:10pg/ml
Triiodothyronine:50pg/ml；
Human plasma：100ul/ml.
Example IV：
A kind of non-animal derived culture medium for being used to cultivate human mesenchymal stem cell of the present invention, including following ingredients,
With densimeter：
Rh-insulin：100ug/ml；
Basic fibroblast growth factor：1ng/ml；
Human transferrin：20ug/ml；
Selenous acid：30ug/ml；
Serine：1ug/ml；
Choline Chloride：200ug/ml；
Adenine：30ug/ml；
Single acetyl amine：100ug/ml；
Phosphatidyl ethanolamine：0.01mmol/ml；
Progesterone:40pg/ml
Triiodothyronine:20pg/ml；
Human plasma：50ul/ml.
Example IV：Non-animal derived culture medium Fibroblast cell-culture experiment
1. the culture medium based on DMEM, carrying out 9 groups of difference additive addition culture experiments altogether, and carry out cytomorphology sight
Examine and analyzed with cell count.
As shown in Figure 1, after the distinctive composition for promoting to grow is added in DMEM basal mediums, 2%FCS (hyclone)
（Culture medium 2 ~ 5）Addition be greatly facilitated the propagation of human fibroblasts, far superior to add 10%FCS conventional medium
（Culture medium 1）；And with 1%（10ul/ml）Human plasma（Culture medium 9）With culture medium 8（Culture medium of the present invention）Replace 2%FCS, cell
Propagation efficiency be more significantly increased, culture medium of the present invention（Culture medium 8）Culture effect is best.
Cellular morphology compares：A ~ I in Fig. 2 ~ 3 corresponds to the cellular morphology of culture medium 1 ~ 9 respectively, culture medium 1 (figure A)
Conventional medium, fibroblast cell space is big, into ribbon strand；Additive+2%FCS groups（Scheme E）Cell body is small, in short shuttle
Type；Culture medium of the present invention（Scheme H）Group, cell body is minimum, and in star cerioid, cell karyoplasmic ratio is maximum, has stronger increasing
Grow ability.
Claims (5)
1. a kind of non-animal derived culture medium for being used to cultivate human mesenchymal stem cell, it is characterised in that including following ingredients：
Rh-insulin：1~100ug/ml；
Basic fibroblast growth factor： 1~50ng/ml；
Human transferrin：1~50ug/ml；
Selenous acid：1~80ug/ml；
Serine：1~300ug/ml；
Choline Chloride：1~200ug/ml；
Adenine：1~60ug/ml；
Single acetyl amine：1~100ug/ml；
Phosphatidyl ethanolamine：0.01~10mmol/ml；
Progesterone:1~40 pg/ml；
Triiodothyronine:1~50pg/ml；
Human plasma：1~100ul/ml.
2. a kind of non-animal derived culture medium for being used to cultivate human mesenchymal stem cell according to claim 1, it is special
Sign is, including following ingredients：
Rh-insulin：1~50ug/ml；
Basic fibroblast growth factor： 1~20ng/ml；
Human transferrin：1~20ug/ml；
Selenous acid：1~30ug/ml；
Serine：1~150ug/ml；
Choline Chloride：1~100ug/ml；
Adenine：1~30ug/ml；
Single acetyl amine：1~20ug/ml；
Phosphatidyl ethanolamine：0.05~1mmol/ml；
Progesterone:1~10pg/ml；
Triiodothyronine:1~20pg/ml；
Human plasma：1~50ul/ml.
3. a kind of non-animal derived culture medium for being used to cultivate human mesenchymal stem cell according to claim 1, it is special
Sign is, including following ingredients：
Rh-insulin：5ug/ml；
Basic fibroblast growth factor： 4ng/ml；
Human transferrin：5ug/ml；
Selenous acid：6.84ug/ml；
Serine：105ug/ml；
Choline Chloride：89.3ug/ml；
Adenine：12.2ug/ml；
Single acetyl amine：14ug/ml；
Phosphatidyl ethanolamine：0.1mmol/ml；
Progesterone:3.145pg/ml；
Triiodothyronine:13.46pg/ml；
Human plasma：15ul/ml.
A kind of 4. non-animal derived composition culture for being used to cultivate human mesenchymal stem cell according to any one of claim 1 ~ 3
Base, it is characterised in that also including basal medium, the basal medium is DMEM in high glucose culture medium.
A kind of 5. non-animal derived composition culture for being used to cultivate human mesenchymal stem cell according to any one of claim 1 ~ 3
Application of the base in human adipose mesenchymal stem cells and human umbilical cord mesenchymal stem cells culture.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110042079A (en) * | 2019-04-16 | 2019-07-23 | 深圳大学 | It is a kind of for cultivating the culture medium of mescenchymal stem cell |
| CN111440764A (en) * | 2020-02-21 | 2020-07-24 | 广东国科细胞科技有限公司 | Serum-free culture medium of mesenchymal stem cells and clinical-grade large-scale culture method of mesenchymal stem cells |
| WO2021227573A1 (en) * | 2020-05-14 | 2021-11-18 | 梦芊科技知识产权有限公司 | Xeno-free culture medium and method for expansion of mesenchymal stem cells by means of using same |
| CN115369080A (en) * | 2022-08-08 | 2022-11-22 | 北京大学人民医院 | Clinical-grade autologous urinary stem cell culture medium and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1871343A (en) * | 2003-11-04 | 2006-11-29 | 株式会社宝玛斯特 | Method and system for preparing stem cells from fat tissue |
| CN102433301A (en) * | 2011-12-02 | 2012-05-02 | 上海安集协康生物技术有限公司 | Method for extracting and amplifying monoclonal mesenchymal stem cells and culture solution for same |
| WO2015169762A1 (en) * | 2014-05-06 | 2015-11-12 | F. Hoffmann-La Roche Ag | Method for differentiation of pluripotent stem cells into cardiomyocytes |
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2016
- 2016-06-07 CN CN201610398256.1A patent/CN107475185A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1871343A (en) * | 2003-11-04 | 2006-11-29 | 株式会社宝玛斯特 | Method and system for preparing stem cells from fat tissue |
| CN102433301A (en) * | 2011-12-02 | 2012-05-02 | 上海安集协康生物技术有限公司 | Method for extracting and amplifying monoclonal mesenchymal stem cells and culture solution for same |
| WO2015169762A1 (en) * | 2014-05-06 | 2015-11-12 | F. Hoffmann-La Roche Ag | Method for differentiation of pluripotent stem cells into cardiomyocytes |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110042079A (en) * | 2019-04-16 | 2019-07-23 | 深圳大学 | It is a kind of for cultivating the culture medium of mescenchymal stem cell |
| CN111440764A (en) * | 2020-02-21 | 2020-07-24 | 广东国科细胞科技有限公司 | Serum-free culture medium of mesenchymal stem cells and clinical-grade large-scale culture method of mesenchymal stem cells |
| WO2021227573A1 (en) * | 2020-05-14 | 2021-11-18 | 梦芊科技知识产权有限公司 | Xeno-free culture medium and method for expansion of mesenchymal stem cells by means of using same |
| CN115369080A (en) * | 2022-08-08 | 2022-11-22 | 北京大学人民医院 | Clinical-grade autologous urinary stem cell culture medium and application thereof |
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