CN102433302A - Serum-free culture medium for mesenchymal stem cells - Google Patents
Serum-free culture medium for mesenchymal stem cells Download PDFInfo
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- CN102433302A CN102433302A CN2011104205399A CN201110420539A CN102433302A CN 102433302 A CN102433302 A CN 102433302A CN 2011104205399 A CN2011104205399 A CN 2011104205399A CN 201110420539 A CN201110420539 A CN 201110420539A CN 102433302 A CN102433302 A CN 102433302A
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Abstract
The invention discloses a serum-free culture medium for mesenchymal stem cells. The serum-free culture medium for the mesenchymal stem cells comprises the following ingredients: fibronectin at the final concentration of 25mu g/ml, basic fibroblast growth factors at the final concentration of 10ng/ml, human epidermal growth factors at the final concentration of 15ng/ml, recombinant human insulin at the final concentration of 1mg/ml, human transferrin at the final concentration of 0.55mg/ml, human blood albumin in a volume ratio of 5 percent, sodium selenite at the final concentration of 0.67mu g/ml, L-carnitine at the final concentration of 5mM and resveratrol at the final concentration of 30mu M. When the mesenchymal stem cells are cultured by the serum-free culture medium for the mesenchymal stem cells, animal-derived serum is not contained, so infection risks can be controlled; the L-carnitine and the resveratrol which are added into the serum-free culture medium for the mesenchymal stem cells can effectively improve the growth state of the mesenchymal stem cells; and the growth speed of the mesenchymal stem cells is remarkably improved, and the biological characteristics of the mesenchymal stem cells are kept unchanged.
Description
Technical field
The present invention relates to the stem cell media technical field, be specifically related to a kind of mesenchymal stem cell serum-free culture medium.
Background technology
Mescenchymal stem cell (Mesenchymal Stem Cells; MSCs) be the important member of stem cell family; Derive from and grow early stage mesoderm and ectoderm, have multidirectional differentiation potential, hematopoiesis support and promote characteristics such as stem cell implantation, immunoregulation and self-replacation and receive people's attention day by day.Mescenchymal stem cell extensively is present in the multiple tissue of whole body to be imprinted at cultured and amplified in vitro, and can be divided into myocardial cell, neurocyte and adipocyte etc. under given conditions.Along with the further investigation of Chinese scholars, can from multiple tissue, isolate mescenchymal stem cell at present to mesenchymal stem cell biological characteristic and function.Mescenchymal stem cell has effects such as hematopoiesis support and adjusting immunity, has broad clinical application prospect at aspects such as hematopoietic reconstitution, tissue repair, immunotherapies.
The substratum that existing mescenchymal stem cell cultural method uses mostly contains animal serum, causes fibroblastic pollution on the one hand, influences Keratiocyte growth, the differentiation of accelerator angle cell plastid; Owing to serum and the not clear proteic existence of composition, fundamental researchs such as pair cell biology, toxicology, pathology form to be disturbed on the other hand; Adopt the artificial organ of the substratum structure that adds serum to carry out transplanting in the body simultaneously, possibly cause immunoreation or introduce unsafe factor (as: virus or other pathogenic agent etc.), so need the serum free medium of exploitation specific chemical components.
Because therefore the deficiency of serum free medium composition aspect is utilizing these culture medium culturing cells during the course, the speed of growth is slow, occurs the aged characteristic easily, thereby influences a large amount of amplifications of cell.Mescenchymal stem cell pair cell quantity when being used for clinical treatment has very big requirement, but also must obtain enough cells at short notice to satisfy the needs of treatment.
Summary of the invention
The objective of the invention is deficiency, the transplanting mesenchymal stem cell serum-free culture medium is provided, can improve the mescenchymal stem cell ground speed of growth, improve rate of amplification, guarantee mesenchymal stem cell biological characteristic simultaneously to prior art.
For achieving the above object, the technical scheme that the present invention takes is: a kind of mesenchymal stem cell serum-free culture medium is provided, it is characterized in that, comprise following ingredients: in final concentration,
Fibronectin: 25 μ g/ml;
Prostatropin: 10ng/ml;
Human epidermal growth factor: 15ng/ml;
Recombinant human insulin: 1mg/ml;
HTrf: 0.55mg/ml;
RHSA: volume ratio is 5%;
Sodium Selenite: 0.67 μ g/ml;
Left-handed Carnitine: 5mM;
Trans-resveratrol: 30 μ M.
Said mesenchymal stem cell serum-free culture medium is characterized in that: also comprise basic medium, said basic medium is the DMEM/F12 substratum.
DMEM/F12 (the Dulbecco's Modified Eagle's Medium/ Ham's Nutrient Mixture F1) substratum that the present invention adopts adopts DMEM and F12 to mix than 1:1 by liquor capacity; The DMEM/F12 substratum is provided by Gibco company, and article No. is 11320-033.
The left-handed Carnitine (L-camitine) that the present invention adopts is provided the accurate word h2005107 of traditional Chinese medicines by Zhuhai Yibang Pharmaceutical Co., Ltd.; Left-handed Carnitine is a crude substance in the body that needs in the Mammals energy metabolism, and its major function is to promote lipid metabolism; When anoxic, ischemic, acyl-CoA piles up, and Intramitochondrial long-chain acyl vitamin BT is also piled up, and free vitamin BT reduces because of mass consumption; Anoxic, ischemic cause ATP (adenosine-triphosphate) level to descend, and cytolemma raises with the ubcellular membrane permeability, and the acyl-CoA of accumulation can cause the membrane structure change, film phase disintegration and cause necrocytosis; In addition, be main with sugared anaerobic glycolysis during anoxic, accumulations such as lipid acid cause oxypathy, ion imbalances, aqtocytolysis is dead; The free vitamin BT of q.s can make the acyl-CoA of accumulation get in the plastosome, reduces its inhibition to acenine nucleotide translocase, makes oxidative phosphorylation smooth.
The trans-resveratrol (Resveratrol) that the present invention adopts is a polyphenolic compound, and being mainly derived from polygonaceae plant giant knotweed Polygonum cuspidatum Sieb. et Zucc. rhizome extract trans-resveratrol is a kind of natural inhibitor; But blood viscosity lowering suppresses thrombocyte and condenses and vasorelaxation, keeps unobstructed blood; But the generation of preventing cancer and development; Has atherosclerosis and coronary heart disease, ischemic heart disease, the preventive and therapeutic effect of hyperlipidemia.The effect that suppresses tumour also has estrogen-like effects, can be used for treating diseases such as mammary cancer.
Adopt mesenchymal stem cell serum-free culture medium culturing mesenchymal stem cells provided by the invention, have following beneficial effect:
1, owing to do not contain the serum of animal-origin, therefore can the control infection risk;
2, the left-handed Carnitine and the trans-resveratrol that add in the mesenchymal stem cell serum-free culture medium can effectively improve the growth of mesenchymal stem cells state;
3, growth of mesenchymal stem cells speed significantly improves;
4 and guaranteed that mesenchymal stem cell biological learns characteristic and remain unchanged;
Can umbilical cord mesenchymal stem cells or fat mesenchymal stem cell number be increased about 1000 times 10 day time.The biological characteristics of the mescenchymal stem cell after the amplification does not change.
Embodiment
Below in conjunction with embodiment the present invention is carried out detailed description, but they not to further restriction of the present invention.
Mesenchymal stem cell serum-free culture medium provided by the invention comprises following ingredients: in final concentration,
Fibronectin: 25 μ g/ml;
Prostatropin: 10ng/ml;
Human epidermal growth factor: 15ng/ml;
Recombinant human insulin: 1mg/ml;
HTrf: 0.55mg/ml;
RHSA: volume ratio is 5%;
Sodium Selenite: 0.67 μ g/ml;
Left-handed Carnitine: 5mM;
Trans-resveratrol: 30 μ M.
Above-mentioned mesenchymal stem cell serum-free culture medium also comprises basic medium, and basic medium is the DMEM/F12 substratum.
Embodiment one: the fat mesenchymal stem cell cultivation of in substratum, going down to posterity;
Substratum 1: mesenchymal stem cell serum-free culture medium provided by the invention;
Substratum 2:L-DMEM+10%FBS (Fetal Bovine Serum);
Substratum 3:STEMPRO MSC SFM serum free medium.
Above-mentioned L-DMEM is provided by Gibco company, and article No. is 11885-084.
Above-mentioned STEMPRO MSC SFM serum free medium is provided by Gibco company, and article No. is A10332-01.
Operation steps:
1.1 with fat mesenchymal stem cell according to 10000 cells/cm
2Density plant in 3 T75 Tissue Culture Flasks, add substratum 1 respectively, substratum 2, substratum 3 behind substratum and the abundant mixing of fat mesenchymal stem cell suspension, is put into 37 ℃, CO with 3 Tissue Culture Flasks
2Concentration is to cultivate in 5% the cell culture incubator, and after this per 3 days according to 10000 cells/cm
2Density carry out passage;
1.2 the 10th day of cell cultures, with the cell harvesting in the Tissue Culture Flask; Trysinization 2 min with 0.25% blow and beat mescenchymal stem cell get off afterwards and stop digesting with each corresponding substratum;
1.3 the mescenchymal stem cell that step 1.2 is obtained is with the centrifugal 5min of the speed of 200g; Collect mescenchymal stem cell and with the phosphoric acid salt buffer memory solution of 10ml resuspended after; With the centrifugal 5min of the speed of 200g, the mesenchymal cell that obtains is with the phosphoric acid salt buffer memory solution mixing of 10ml again;
1.4 the cell that step 1.3 is obtained carries out cell counting with the full-automatic cell calculating instrument Countess of Invitrogen company.Survey 3 times for every group, results averaged is as described in Table 1.
Table 1: fat mesenchymal stem cell goes down to posterity in substratum 1, substratum 2, substratum 3 and cultivates after 10 days the cell count of fat mesenchymal stem cell and motility rate detected result
| ? | Substratum 1 | Substratum 2 | Substratum 3 |
| Cell count | 8.2×10 7 | 9.4×10 6 | 4.2×10 7 |
| Motility rate | 97.6% | 88.3% | 91.3% |
Find out that from table 1 mesenchymal stem cell serum-free culture medium provided by the invention can better be kept the activity of fat mesenchymal stem cell with respect to other two kinds of substratum, makes fat mesenchymal stem cell keep the better speed of growth.
Embodiment two: the umbilical cord mesenchymal stem cells cultivation of in substratum, going down to posterity;
Substratum 1: mesenchymal stem cell serum-free culture medium provided by the invention;
Substratum 2:L-DMEM+10%FBS (Fetal Bovine Serum);
Substratum 3:STEMPRO MSC SFM serum free medium.
Above-mentioned L-DMEM is provided by Gibco company, and article No. is 11885-084.
Above-mentioned STEMPRO MSC SFM serum free medium is provided by Gibco company, and article No. is A10332-01.
Operation steps:
2.1. with umbilical cord mesenchymal stem cells according to 10000 cells/cm
2Density plant in 3 T75 Tissue Culture Flasks, add substratum 1 respectively, substratum 2, substratum 3 behind substratum and the abundant mixing of umbilical cord mesenchymal stem cells suspension, is put into 37 ℃, CO with 3 Tissue Culture Flasks
2Concentration is to carry out cell cultures in 5% the cell culture incubator, and after this per 3 days according to 10000 cells/cm
2Density carry out passage;
2.2 the 10th day of cell cultures, with the cell harvesting in the Tissue Culture Flask; Trysinization 2 min with 0.25% blow and beat mescenchymal stem cell get off afterwards and stop digesting with each corresponding substratum;
2.3 the mescenchymal stem cell that step 2.2 is obtained with the centrifugal 5min of the speed of 200g, collect mescenchymal stem cell with the phosphoric acid salt buffer memory solution of 10ml resuspended after, again with the centrifugal 5min of the speed of 200g, the cell that obtains is with the phosphoric acid salt buffer memory solution mixing of 10ml;
2.4 the cell that step 2.3 obtains carries out cell counting with the full-automatic cell calculating instrument Countess of Invitrogen company.Survey 3 times for every group, results averaged is as described in Table 2.
Table 2: umbilical cord mesenchymal stem cells goes down to posterity in substratum 1, substratum 2, substratum 3 and cultivates after 10 days the cell count of umbilical cord mesenchymal stem cells and motility rate detected result
| ? | Substratum 1 | Substratum 2 | Substratum 3 |
| Cell count | 7.3×10 7 | 7.1×10 6 | 5.2×10 7 |
| Motility rate | 96.4% | 85.7% | 94.3% |
Find out that from table 2 mesenchymal stem cell serum-free culture medium provided by the invention can better be kept the activity of umbilical cord mesenchymal stem cells with respect to other two kinds of substratum, makes umbilical cord mesenchymal stem cells keep the better speed of growth.
Embodiment three:Umbilical cord mesenchymal stem cells and the biological characteristics of fat mesenchymal stem cell after cultivating go down to posterity in substratum;
Substratum: mesenchymal stem cell serum-free culture medium provided by the invention.
Operation steps:
3.1 with umbilical cord mesenchymal stem cells and fat mesenchymal stem cell according to 10000 cells/cm
2Density plant respectively in 2 T175 Tissue Culture Flasks, add the cultivation of going down to posterity in the mesenchymal stem cell serum-free culture medium provided by the invention, each stem cell is gone down to posterity respectively 5 times and 10 times;
3.2 with the cell harvesting in the Tissue Culture Flask, trysinization 2 min with 0.25% blow and beat mescenchymal stem cell get off afterwards and stop digesting with substratum;
3.3 umbilical cord mesenchymal stem cells that step 3.2 is obtained and fat mesenchymal stem cell are with the centrifugal 5min of the speed of 200g; Collect umbilical cord mesenchymal stem cells and fat mesenchymal stem cell with the phosphoric acid salt buffer memory solution of 10ml resuspended after; Again with the centrifugal 5min of the speed of 200g, umbilical cord mesenchymal stem cells that obtains and fat mesenchymal stem cell go down to posterity respectively after 5 times and 10 times stem cell totally four groups divide respectively and install in 13 streaming pipes; Add following traget antibody in the corresponding streaming pipe respectively: (1) mouse anti human IgG2a-PE; (2) mouse anti human CD11b-PE; (3) mouse anti human IgG1-PE; (4) mouse anti human CD19-PE; (5) mouse anti human HLA-DR-PE; (6) mouse anti human IgG1-PE; (7) mouse anti human CD73-PE; (8) mouse anti human IgG1-FITC; (9) mouse anti human CD90-FITC; (10) mouse anti human CD34-FITC; (11) mouse anti human CD105-FITC; (12) mouse anti human IgG2a-FITC; (13) mouse anti human CD45;
3.4 the cell that mark is handled well uses the FACSAria drain cell appearance of BD company to analyze; Analytical results is as described in Table 3.
Table 3: go down to posterity through mesenchymal stem cell serum-free culture medium provided by the invention and to cultivate umbilical cord mesenchymal stem cells and the biological characteristics parameter variation statistics of fat mesenchymal stem cell after 5 times or 10 times
| CD73 | CD90 | CD105 | CD11b | CD34 | CD45 | CD19 | |
| The umbilical cord mesenchymal stem cells that goes down to posterity for 5 times | 98.2% | 96.7% | 97.6% | 3.1% | 2.5% | 4.1% | 1.4% |
| The umbilical cord mesenchymal stem cells that goes down to posterity for 10 times | 99.4% | 98.6% | 97.8% | 0.2% | 2.7% | 2.1% | 1.4% |
| The fat mesenchymal stem cell that goes down to posterity for 5 times | 98.3% | 96.4% | 96.3% | 1.1% | 3.2% | 0.4% | 0.6% |
| The fat mesenchymal stem cell that goes down to posterity for 10 times | 97.9% | 99.2% | 97.3% | 3.2% | 1.8% | 0.7% | 2.1% |
Find out from table 3; No matter adopting mesenchymal stem cell serum-free culture medium cultured umbilical mescenchymal stem cell provided by the invention and fat mesenchymal stem cell is through 5 times or the 10 times cultivations of going down to posterity, can both guarantee well that the biological characteristics of umbilical cord mesenchymal stem cells or fat mesenchymal stem cell is constant.
Claims (2)
1. a mesenchymal stem cell serum-free culture medium is characterized in that, comprises following ingredients:
Fibronectin: 25 μ g/ml;
Prostatropin: 10ng/ml;
Human epidermal growth factor: 15ng/ml;
Recombinant human insulin: 1mg/ml;
HTrf: 0.55mg/ml;
RHSA: volume ratio is 5%;
Sodium Selenite: 0.67 μ g/ml;
Left-handed Carnitine: 5mM;
Trans-resveratrol: 30 μ M.
2. mesenchymal stem cell serum-free culture medium according to claim 1 is characterized in that: also comprise basic medium, said basic medium is the DMEM/F12 substratum.
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| 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 |
| CN102876629A (en) * | 2012-06-06 | 2013-01-16 | 深圳市人民医院 | Novel method for efficiently amplifying mesenchymal stem cells |
| CN103740644A (en) * | 2013-03-20 | 2014-04-23 | 曾宪卓 | Method for amplifying hematopoietic stem cells based on 3D culture system |
| CN104830758A (en) * | 2015-04-15 | 2015-08-12 | 广州赛莱拉干细胞科技股份有限公司 | Mesenchymal stem cell osteogenic induced differentiation culture medium and preparation method thereof |
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