CN103255103A - Serum-free adipose tissue-derived mesenchymal stem cell culture medium - Google Patents
Serum-free adipose tissue-derived mesenchymal stem cell culture medium Download PDFInfo
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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 the mescenchymal stem cell substratum, especially a kind of fat mesenchymal stem cell substratum that does not have animal serum.
Background technology
(Mesenchymal Stem Cells is the important member of stem cell family MSCs) to mescenchymal stem cell, derives to grow early stage mesoderm and ectoderm.Mescenchymal stem cell is found in marrow at first, because it has multidirectional differentiation potential, hematopoiesis support and promotes characteristics such as stem cell implantation, immunoregulation and self-replacation to be subjected to people's attention day by day.Mescenchymal stem cell is in vivo or under the external specific inductive condition, can be divided into multiple histocytes such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium, still have multidirectional differentiation potential after continuous passage cultivation and the freezing preservation, can be used as desirable seed cell and be used for injuries of tissues and organs reparation old and feeble and that pathology causes.
Domestic and international amplification system for mescenchymal stem cell mainly is the foetal calf serum (FBS) that basic medium adds 5-10% concentration at present.Contain foreign protein matter among the FBS, itself has the risk of carrying bacterium, virus, albumen communicable disease or Protein virus.In addition, there are some researches show that stem cell can engulf the albumen in the substratum in culturing process, contain bovine serum albumin (7mg-30mg/10
8Individual cell), can make to produce anti-bovine protein antibody in the recipient's body, cause immune response, thereby cause the patient especially to be lost efficacy in repetition infusion mescenchymal stem cell treatment back treatment.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 ingredients are uncertain, or expensive, or are difficult to guarantee the consistence between substratum batch.
Summary of the invention
In view of this, be necessary at 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 kind of fat mesenchymal stem cell substratum of serum-free is made up of basic medium and interpolation composition; Wherein, basic medium is DMEM-LG, and it is as follows to add composition and content thereof:
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 μ M;
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 does not contain serum, can avoid differences between batches and serum component to the influence of cell cultures, and cost is low; Avoid the exogenous pollution of serum and to cytotoxic effect; Definite ingredients is conducive to study the cells physiological regulation mechanism.
Description of drawings
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 the fat mesenchymal stem cell cultivated of common stem cell media the form picture.
Embodiment
In order better to understand the present invention, be described further below in conjunction with embodiment.
(Basic Fibroblast Growth Factor is to contain 155 amino acid whose mitogenetic cationic polypeptides bFGF) to Prostatropin, and molecular weight is 16～18.5KD.BFGF has biological action widely, and is influential to growth, differentiation and the function of various kinds of cell, plays a role in normal physiological and pathologic process, and its main biological action has: (1) is as angiogenesis factor; (2) promote wound healing and tissue repair; (3) promote tissue regeneration; (4) participate in neurotization etc.
BFGF has very strong heparin avidity, is the high-affinity district in its 114～123 amino acids, and then there is the low-affinity district at other position.The monoclonal antibody of anti-bFGF and receptors bind does not have influence to itself and heparin-binding capacity, and cancellation has hydroxyl terminal 42 amino acids, and heparin avidity namely disappears, and can lose part biological and learn active.
Heparin is a kind of mucopolysaccharide sulfuric ester of alternately being made up of D-glycosamine, 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 mainly is to be produced by mastocyte and basophilic granulocyte, and content is abundant in tissues such as lung, the heart, liver, muscle, under the physiological conditions in the blood plasma content very little.No matter still external in vivo, the anticoagulation of heparin is all very strong, so clinical widely-used as antithrombotics it.
(Epidermal Growth Factor EGF) is a kind of little peptide to Urogastron, is made up of 53 amino-acid residues, and EGF is a kind of multifunctional growth factor, and external in vivo all have strong short splitting action to multiple histocyte.
L-glutaminate plays very important effect when cell cultures.Behind the desamidizate, L-glutaminate can be used as the energy derive of culturing cell, the synthetic and nucleic acid metabolism of participation protein; When lacking glutamine, cell poor growth and death.L-glutaminate is very unstable in solution, should put-20 ℃ of stored frozen, in preceding adding substratum.Be added with 4 ℃ of refrigerator storage of liquid nutrient medium two week of L-glutaminate when above, should add the L-glutaminate of original amount again.
2 mercapto ethanol (being called beta-mercaptoethanol again) is a kind of organic compound, and its chemical formula is HOCH
2CH
2SH, it has ethylene glycol (HOCH concurrently
2CH
2OH) and dithioglycol (HSCH
2CH
2SH) functional group is generally used for the reduction of disulfide linkage, the protection disulfide linkage, thus make not oxidized fall of protein and inactivation.
(Leukemia Inhibitory Factor LIF) has the effect of propagation, differentiation and the phenotype of regulating cell to leukaemia inhibitory factor; Sodium.alpha.-ketopropionate (molecular formula CH
3COCOONa, molecular weight 110.04g/mol) can be used as the alternative carbon source in the cell cultures.
Embodiment 1
A kind of fat mesenchymal stem cell substratum of serum-free is made up of basic medium and interpolation composition; Wherein, basic medium is DMEM-LG, and it is as follows to add composition and content thereof:
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 μ M;
Leukaemia inhibitory factor: 1000U/mL;
Sodium.alpha.-ketopropionate: 1mM.
The effect of the fat mesenchymal stem cell culture medium culturing fat mesenchymal stem cell of use serum-free of the present invention is as follows.
1. the frozen pipe of fat mesenchymal stem cell separating obtained through conventional collagenase I digestion method and that liquid nitrogen is preserved is taken out from liquid nitrogen, drop into immediately in 37 ℃ of water-baths, slightly shake, liquid all melts back (about 1-1.5 minute), takes out a little alcohol of spray and is put in the Bechtop.
2. above-mentioned fat mesenchymal stem cell after 37 ℃ thaw is drawn onto in the centrifuge tube of the 15mL that the 10mL substratum is housed, with substratum frozen pipe is washed one time, the cell that is bonded on the wall is all washed, 1000 left the heart 5 minutes.
3. supernatant liquor is outwelled, added the above-mentioned substratum that 1mL prepares respectively, cell suspension is got up; Be drawn onto in the 10cm culture dish that the 10mL substratum is housed and all around shake gently, the cell in the culture dish is evenly distributed.
4. mark cell category and date, kinds of culture medium people etc., be put into CO
2Cultivate in the incubator, change substratum behind the cell attachment.
5. when the cell fraction of coverage in the culture dish reaches 80%-90%, adopt the full-automatic cell calculating instrument Countess of Invitrogen company to carry out cell counting.
In basic medium DMEM-LG, add 10% foetal calf serum (volume fraction), in contrast the culture medium culturing fat mesenchymal stem cell.
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, as to use control medium to cultivate fat mesenchymal stem cell, 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 that is 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 fibrous cellular form of one-tenth of fusiformis.
The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.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 (7)
1. the fat mesenchymal stem cell substratum of a serum-free is made up of basic medium and interpolation composition, and it is characterized in that: basic medium is DMEM-LG, and it is as follows to add composition and content thereof:
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 μ M;
Leukaemia inhibitory factor: 500-2000U/mL;
Sodium.alpha.-ketopropionate: 0.5-2mM.
2. the fat mesenchymal stem cell substratum of serum-free according to claim 1, it is characterized in that: described Basic Fibroblast Growth Factor is 15ng/mL; Urogastron is 15ng/mL.
3. the fat mesenchymal stem cell substratum of serum-free according to claim 1, it is characterized in that: described heparin is 100 μ g/mL.
4. the fat mesenchymal stem cell substratum of serum-free according to claim 1, it is characterized in that: described L-glutaminate is 5mM.
5. the fat mesenchymal stem cell substratum of serum-free according to claim 1, it is characterized in that: described 2 mercapto ethanol is 0.1mM.
6. the fat mesenchymal stem cell substratum of serum-free according to claim 1, it is characterized in that: described leukaemia inhibitory factor is 1000U/mL.
7. the fat mesenchymal stem cell substratum of serum-free according to claim 1, it is characterized in that: described Sodium.alpha.-ketopropionate is 1mM.
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