CN103255103B - Serum-free adipose tissue-derived mesenchymal stem cell culture medium - Google Patents
Serum-free adipose tissue-derived mesenchymal stem cell culture medium Download PDFInfo
- Publication number
- CN103255103B CN103255103B CN201310134502.9A CN201310134502A CN103255103B CN 103255103 B CN103255103 B CN 103255103B CN 201310134502 A CN201310134502 A CN 201310134502A CN 103255103 B CN103255103 B CN 103255103B
- Authority
- CN
- China
- Prior art keywords
- serum
- stem cell
- mesenchymal stem
- culture medium
- cell culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The invention relates to a serum-free adipose tissue-derived mesenchymal stem cell culture medium, which consists of a basic culture medium and added ingredients, wherein the basic culture medium is DMEM-LG, and the added ingredients and the content of each added ingredient are shown as follows: 5 to 20ng/mL of alkaline fiberblast growth factors, 5 to 20ng/mL of epidermal growth factors, 100U/mL of penicillin, 100 micrograms/mL of streptomycin, 50 to 200 micrograms/mL of heparin, 2 to 8mM of L-glutamine, 100 to 300 microM of 2-mercaptoethanol, 500 to 2000U/mL of leukaemia inhibitory factors and 0.5 to 2mM of sodium pyruvate. The serum-free adipose tissue-derived mesenchymal stem cell culture medium does not contain the serum, so that the inter-batch difference and the influence of the serum component on the cell culture can be avoided; the exogenous pollution of the serum and the toxicity of the serum on the cells can be avoided; and the ingredients are clear, so that the research of the psychological regulation mechanism of the cells can be facilitated.
Description
Technical field
The present invention relates to mescenchymal stem cell substratum, especially a kind of fat mesenchymal stem cell substratum without animal serum.
Background technology
Mescenchymal stem cell (Mesenchymal Stem Cells, MSCs) is the important member of stem cell line, derives from and grows early stage mesoderm and ectoderm.Mescenchymal stem cell finds at first in marrow, because of its there is multi-lineage potential, the feature such as hematopoiesis support and promotion stem cell are implanted, immunoregulation and self-replacation and day by day receive the concern of people.Mescenchymal stem cell is in vivo or under external specific inductive condition, the Various Tissues cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium can be divided into, still there is multi-lineage potential after continuous passage cultivation and freezen protective, can be used as the injuries of tissues and organs reparation that desirable seed cell causes for old and feeble and pathology.
At present the domestic and international amplification system for mescenchymal stem cell mainly basic medium add the foetal calf serum (FBS) of 5-10% concentration.Containing foreign protein matter in FBS, itself has the risk of Carried bacteria, virus, albumen communicable disease or Protein virus.In addition, there are some researches show that stem cell can engulf the albumen in substratum in culturing process, interior containing bovine serum albumin (7mg-30mg/10
8individual cell), can make to produce anti-bovine protein antibody in recipient's body, cause immune response, thus cause patient's especially failure in treatment after the treatment of repetition infusion mescenchymal stem cell.Therefore the unfavorable factor of FBS in the clinical large scale culturing of mescenchymal stem cell comes out gradually, now the substitute of existing a lot of scholar's research FBS.Current more existing company's Development of New Generation serum free mediums.But existing serum free medium or chemical composition uncertain, or expensive, or be difficult to ensure the consistence between substratum batch.
Summary of the invention
In view of this, be necessary for the problems referred to above, a kind of fat mesenchymal stem cell substratum of serum-free is provided.
To achieve these goals, the present invention adopts following technical scheme:
A fat mesenchymal stem cell substratum for serum-free, is made up of basic medium and interpolation composition; Wherein, basic medium is DMEM-LG, add composition and content as follows:
Basic Fibroblast Growth Factor: 5-20ng/mL;
Urogastron: 5-20ng/mL;
Penicillin: 100U/mL;
Streptomycin sulphate: 100 μ g/mL;
Heparin: 50-200 μ g/mL;
L-glutaminate: 2-8mM;
2 mercapto ethanol: 100-300 μM;
Leukaemia inhibitory factor: 500-2000U/mL;
Sodium.alpha.-ketopropionate: 0.5-2mM.
As a kind of preferred implementation of the fat mesenchymal stem cell substratum of serum-free of the present invention, described Basic Fibroblast Growth Factor is 15ng/mL; Urogastron is 15ng/mL.
As a kind of preferred implementation of the fat mesenchymal stem cell substratum of serum-free of the present invention, described heparin is 100 μ g/mL.
As a kind of preferred implementation of the fat mesenchymal stem cell substratum of serum-free of the present invention, described L-glutaminate is 5mM.
As a kind of preferred implementation of the fat mesenchymal stem cell substratum of serum-free of the present invention, described 2 mercapto ethanol is 0.1mM.
As a kind of preferred implementation of the fat mesenchymal stem cell substratum of serum-free of the present invention, described leukaemia inhibitory factor is 1000U/mL.
As a kind of preferred implementation of the fat mesenchymal stem cell substratum of serum-free of the present invention, described Sodium.alpha.-ketopropionate is 1mM.
The fat mesenchymal stem cell substratum of serum-free of the present invention, not containing serum, differences between batches and serum component can be avoided the impact of cell cultures, and cost is low; Avoid serum exogenous pollution and to cytotoxic effect; Definite ingredients, is conducive to the Physiological Mechanism in Regulation studying cell.
Accompanying drawing explanation
The form picture of the fat mesenchymal stem cell that Fig. 1 different dry cell culture medium is cultivated; Figure 1A is the form picture of the fat mesenchymal stem cell of the fat mesenchymal stem cell culture medium culturing of serum-free of the present invention; Figure 1B be common stem cell media cultivate fat mesenchymal stem cell form picture.
Embodiment
In order to better understand the present invention, be described further below in conjunction with embodiment.
Prostatropin (Basic Fibroblast Growth Factor, bFGF) is that molecular weight is 16 ~ 18.5KD containing 155 amino acid whose mitogenetic cationic polypeptides.BFGF has biological action widely, and have impact to the growth of various kinds of cell, differentiation and function, play a role in normal physiological and pathologic process, its principal biological effect has: (1) is as angiogenesis factor; (2) wound healing and tissue repair is promoted; (3) tissue regeneration is promoted; (4) neurotization etc. is participated in.
BFGF has very strong heparin avidity, is high-affinity district in its 114 ~ 123 amino acids, Ze You low-affinity district, other position.The monoclonal antibody of anti-bFGF and receptors bind is on itself and heparin-binding capacity without impact, and cancel and have hydroxyl terminal 42 amino acids, namely heparin avidity disappear, and can lose partial biological.
Heparin is a kind of mucopolysaccharide sulfuric ester be alternately made up of GLUCOSAMINE, L-iduronic acid, N-acetyl-glucosamine and glucuronic acid, and molecular weight is from 5 to 30kDa, and wherein sulfate radical accounts for 40%.Heparin is mainly produced by mastocyte and basophilic granulocyte, and rich content in the tissues such as lung, the heart, liver, muscle, under physiological conditions, in blood plasma, content is very micro-.No matter in vivo or external, the anticoagulation of heparin is all very strong, therefore clinical it is widely used as antithrombotics.
Urogastron (Epidermal Growth Factor, EGF) is a kind of little peptide, is made up of 53 amino-acid residues, and EGF is a kind of multi-functional somatomedin, and external in vivo all have strong short splitting action to Various Tissues cell.
L-glutaminate plays very important effect when cell cultures.After desamidizate, L-glutaminate can be used as the energy derive of culturing cell, the synthesis participating in protein and nucleic acid metabolism; When lacking glutamine, Growth of Cells is bad and dead.L-glutaminate is very unstable in the solution, should put-20 DEG C of stored frozen, with front adding in substratum.When being added with liquid nutrient medium 4 DEG C of refrigerator storage more than two weeks of L-glutaminate, the L-glutaminate of original amount should be rejoined.
2 mercapto ethanol (being also called beta-mercaptoethanol) is a kind of organic compound, and its chemical formula is HOCH
2cH
2sH, it has ethylene glycol (HOCH concurrently
2cH
2and dithioglycol (HSCH OH)
2cH
2sH) functional group, is generally used for the reduction of disulfide linkage, protection disulfide linkage, thus make protein not oxidized fall and inactivation.
Leukaemia inhibitory factor (Leukemia Inhibitory Factor, LIF) has the effect regulating the propagation of cell, differentiation and phenotype; Sodium.alpha.-ketopropionate (molecular formula CH
3cOCOONa, molecular weight 110.04g/mol) can as the alternative carbon source in cell cultures.
Embodiment 1
A fat mesenchymal stem cell substratum for serum-free, is made up of basic medium and interpolation composition; Wherein, basic medium is DMEM-LG, add composition and content as follows:
Basic Fibroblast Growth Factor: 15ng/mL;
Urogastron: 15ng/mL;
Penicillin: 100U/mL;
Streptomycin sulphate: 100 μ g/mL;
Heparin: 100 μ g/mL;
L-glutaminate: 5mM;
2 mercapto ethanol: 100 μMs;
Leukaemia inhibitory factor: 1000U/mL;
Sodium.alpha.-ketopropionate: 1mM.
Use the effect of the fat mesenchymal stem cell culture medium culturing fat mesenchymal stem cell of serum-free of the present invention as follows.
1. take out from liquid nitrogen through the separating obtained and fat mesenchymal stem cell cryopreservation tube of Liquid nitrogen storage of conventional collagenase I digestion method, drop into immediately in 37 DEG C of water-baths, gentle agitation, after liquid all melts (about 1-1.5 minute), takes out a little alcohol of spray and is put in Bechtop.
2. above-mentioned thaw through 37 DEG C after fat mesenchymal stem cell be drawn onto in the centrifuge tube of the 15mL that 10mL substratum is housed, then with substratum, cryopreservation tube is washed one time, the cell be bonded on wall is all washed, and 1000 leave the heart 5 minutes.
3. supernatant liquor is outwelled, add the above-mentioned substratum that 1mL prepares respectively, cell suspension is got up; Be drawn onto in the 10cm culture dish that 10mL substratum is housed and all around shake gently, the cell in culture dish is uniformly distributed.
4. mark cell category and date, kinds of culture medium people etc., be put into CO
2cultivate in incubator, after cell attachment, change substratum.
5., when the cell fraction of coverage in culture dish reaches 80%-90%, adopt the full-automatic cell calculating instrument Countess of Invitrogen company to carry out cell counting.
The foetal calf serum (volume fraction) of 10% is added, culture medium culturing fat mesenchymal stem cell in contrast in basic medium DMEM-LG.
Use the fat mesenchymal stem cell culture medium culturing fat mesenchymal stem cell of serum-free of the present invention, cell density is 2 × 10
7individual/mL, use the fat mesenchymal stem cell that control medium is cultivated, cell density is 2 × 10
6individual/mL.Serum free medium provided by the invention contains the glutamine of high density, can significantly improve the speed of growth of fat mesenchymal stem cell, effectively improves the growth conditions of fat mesenchymal stem cell, keeps the biological characteristics of fat mesenchymal stem cell constant.As shown in Figure 1, the fat mesenchymal stem cell be incubated in the fat mesenchymal stem cell substratum of serum-free of the present invention keeps good adherent characteristic, and fat mesenchymal stem cell all presents the one-tenth threadiness cellular form of fusiformis.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (1)
1. a fat mesenchymal stem cell substratum for serum-free, by basic medium and add composition form, it is characterized in that: basic medium is DMEM-LG, add composition and content as follows:
Basic Fibroblast Growth Factor: 15ng/mL;
Urogastron: 15ng/mL;
Penicillin: 100U/mL;
Streptomycin sulphate: 100 μ g/mL;
Heparin: 100 μ g/mL;
L-glutaminate: 5mM;
2 mercapto ethanol: 100 μMs;
Leukaemia inhibitory factor: 1000U/mL;
Sodium.alpha.-ketopropionate: 1mM.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310134502.9A CN103255103B (en) | 2013-04-17 | 2013-04-17 | Serum-free adipose tissue-derived mesenchymal stem cell culture medium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310134502.9A CN103255103B (en) | 2013-04-17 | 2013-04-17 | Serum-free adipose tissue-derived mesenchymal stem cell culture medium |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103255103A CN103255103A (en) | 2013-08-21 |
| CN103255103B true CN103255103B (en) | 2015-01-14 |
Family
ID=48959320
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310134502.9A Active CN103255103B (en) | 2013-04-17 | 2013-04-17 | Serum-free adipose tissue-derived mesenchymal stem cell culture medium |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103255103B (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103555661B (en) * | 2013-10-25 | 2015-07-29 | 中国科学院动物研究所 | A kind of serum-free, multipotential stem cell cultural method without feeder layer |
| CN103740641A (en) * | 2013-12-26 | 2014-04-23 | 广州爱菲科生物科技有限公司 | Inducing culture medium and inducing method thereof for inducing human adipose-derived mesenchymal stem cell to be differentiated into islet-like cell |
| CN105112360A (en) * | 2015-07-17 | 2015-12-02 | 深圳爱生再生医学科技有限公司 | Mass culture method of umbilical cord mesenchymal stem cells |
| CN108410813A (en) * | 2016-03-06 | 2018-08-17 | 李倩 | A kind of culture medium for cultivating human adipose-derived stem cell |
| US10513689B2 (en) | 2016-04-29 | 2019-12-24 | Hope Biosciences, Llc | Culture media for multipotent stem cells |
| CN106434549A (en) * | 2016-12-20 | 2017-02-22 | 江西宜信堂医疗科技有限公司 | Serum-free stem cell culture medium and preparation method thereof |
| CN106754675B (en) * | 2016-12-21 | 2019-11-22 | 广东科玮生物技术股份有限公司 | A kind of fat stem cell serum free medium and its preparation method and application |
| CN106635979A (en) * | 2017-01-16 | 2017-05-10 | 广东万海细胞生物科技有限公司 | Serum-free culture medium for adipose tissue-derived stromal cells |
| CN106906179A (en) * | 2017-04-28 | 2017-06-30 | 四川农业大学 | A kind of Seedling height versatile stem cell culture medium |
| CN111150746A (en) * | 2020-01-06 | 2020-05-15 | 湖南护宫福生物科技有限公司 | Stem cell repair liquid for treating gynecological inflammation by vaginal administration |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974486A (en) * | 2010-11-25 | 2011-02-16 | 王泰华 | Method for extracting mesenchymal stem cells from trace human fatty tissues and massively culturing |
| WO2011159359A2 (en) * | 2010-06-17 | 2011-12-22 | Stemrd, Inc. | Serum-free chemically defined cell culture medium |
| 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 |
| CN102827807A (en) * | 2012-09-19 | 2012-12-19 | 北京京蒙高科干细胞技术有限公司 | Serum-free culture medium for mesenchymal stem cells |
-
2013
- 2013-04-17 CN CN201310134502.9A patent/CN103255103B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011159359A2 (en) * | 2010-06-17 | 2011-12-22 | Stemrd, Inc. | Serum-free chemically defined cell culture medium |
| CN101974486A (en) * | 2010-11-25 | 2011-02-16 | 王泰华 | Method for extracting mesenchymal stem cells from trace human fatty tissues and massively culturing |
| 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 |
| CN102827807A (en) * | 2012-09-19 | 2012-12-19 | 北京京蒙高科干细胞技术有限公司 | Serum-free culture medium for mesenchymal stem cells |
Non-Patent Citations (1)
| Title |
|---|
| 大鼠皮下脂肪分离间充质干细胞的体外培养及鉴定(英文);秦宇红等;《中国组织工程研究与临床康复》;20070819;第11卷(第33期);第6701-6705页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103255103A (en) | 2013-08-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103255103B (en) | Serum-free adipose tissue-derived mesenchymal stem cell culture medium | |
| CN108251359B (en) | Mesenchymal stem cell serum-free medium and culture method | |
| Ji et al. | The differentiation of MSCs into functional hepatocyte-like cells in a liver biomatrix scaffold and their transplantation into liver-fibrotic mice | |
| CN103555665B (en) | A kind of serum-free medium for cultivating mescenchymal stem cell | |
| Chen et al. | Adipose-derived stem cells modified genetically in vivo promote reconstruction of bone defects | |
| EP2377542A2 (en) | Pharmaceutical composition for bone-disease treatment or countering inflammation, comprising cartilage stem cells as an active principle | |
| Yeasmin et al. | Stem cells derived from tooth periodontal ligament enhance functional angiogenesis by endothelial cells | |
| CN103070161A (en) | Cryopreservation liquid and cryopreservation method for adipose tissue-derived mesenchymal stem cells ( ADSC) | |
| CN104726406A (en) | Method for inducing dental pulp mesenchymal stem cells to be differentiated into nerve cells | |
| CN105534848A (en) | Cosmetic or pharmaceutical composition and application thereof | |
| US20050059152A1 (en) | In vitro culture of mesenchymal stem cells (MSC) and a process for the preparation thereof for therapeutic use | |
| Hutton et al. | Hypoxia inhibits de novo vascular assembly of adipose-derived stromal/stem cell populations, but promotes growth of preformed vessels | |
| CN109706115B (en) | Construction method of mouse bone marrow mesenchymal stem cell line | |
| de Paula et al. | Human serum is a suitable supplement for the osteogenic differentiation of human adipose-derived stem cells seeded on poly-3-hydroxibutyrate-co-3-hydroxyvalerate scaffolds | |
| CN105663168A (en) | Cell preparation for repairing ovarian functions | |
| CN108795855A (en) | A kind of serum free medium of mescenchymal stem cell | |
| Lei et al. | Biological character of human adipose-derived adult stem cells and influence of donor age on cell replication in culture | |
| CN105112362A (en) | Serum-free medium for placenta-derived mesenchymal stem cells and preparation method thereof | |
| CN110257328A (en) | A kind of mesenchymal stem cell serum-free culture medium | |
| CN111956668B (en) | Skin regeneration and repair cell composition and preparation method thereof | |
| Wang et al. | Muscular tubes of urethra engineered from adipose-derived stem cells and polyglycolic acid mesh in a bioreactor | |
| CN102965337A (en) | Method for separating and extracting human subcutaneous adipose-derived mesenchymal stem cells and special culture medium for extraction | |
| CN106434542A (en) | Method for enhancing proliferation and post-transplantation survival ability of adipose derived stem cells | |
| CN102146359A (en) | Method for extracting original mesenchymal stem cells from placenta and serum-free amplification | |
| CN105456293A (en) | Stem cell-based medicinal product for treating diabetes and preparing method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: SHE ZHIYONG CHEN JIANXING FANG HAIQING Effective date: 20140428 Owner name: FENG WENFENG Free format text: FORMER OWNER: GUANGZHOU ETHICAL BIOTECHNOLOGY CO., LTD. Effective date: 20140428 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 510070 GUANGZHOU, GUANGDONG PROVINCE TO: 510000 GUANGZHOU, GUANGDONG PROVINCE |
|
| TA01 | Transfer of patent application right |
Effective date of registration: 20140428 Address after: 510000 Guangdong city of Guangzhou province Haizhuqu District Xiaogang Road No. 74 room 503 Applicant after: Feng Wenfeng Applicant after: She Zhiyong Applicant after: Chen Jianxing Applicant after: Fang Haiqing Address before: Guangzhou City, Guangdong province 510070 martyrs Road No. 76 Kiu building 13 Building G room Applicant before: Guangzhou Ethical Biotech Co., Ltd. |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |