CN105200009A - Human umbilical cord mesenchymal stem cell serum-free medium - Google Patents
Human umbilical cord mesenchymal stem cell serum-free medium Download PDFInfo
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- CN105200009A CN105200009A CN201510666332.8A CN201510666332A CN105200009A CN 105200009 A CN105200009 A CN 105200009A CN 201510666332 A CN201510666332 A CN 201510666332A CN 105200009 A CN105200009 A CN 105200009A
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- umbilical cord
- mesenchymal stem
- human umbilical
- cord mesenchymal
- free medium
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- 210000003954 umbilical cord Anatomy 0.000 title claims abstract description 24
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 23
- 239000012679 serum free medium Substances 0.000 title claims abstract description 19
- 238000004113 cell culture Methods 0.000 claims abstract description 10
- 239000000654 additive Substances 0.000 claims abstract description 7
- 239000011573 trace mineral Substances 0.000 claims abstract description 6
- 235000013619 trace mineral Nutrition 0.000 claims abstract description 6
- 239000006143 cell culture medium Substances 0.000 claims abstract description 4
- 210000004102 animal cell Anatomy 0.000 claims abstract description 3
- KGWDUNBJIMUFAP-KVVVOXFISA-N Ethanolamine Oleate Chemical compound NCCO.CCCCCCCC\C=C/CCCCCCCC(O)=O KGWDUNBJIMUFAP-KVVVOXFISA-N 0.000 claims description 8
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 claims description 8
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims description 8
- 102000004877 Insulin Human genes 0.000 claims description 8
- 108090001061 Insulin Proteins 0.000 claims description 8
- 102000002070 Transferrins Human genes 0.000 claims description 8
- 108010015865 Transferrins Proteins 0.000 claims description 8
- 150000002505 iron Chemical class 0.000 claims description 8
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical group [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 claims description 7
- 230000002401 inhibitory effect Effects 0.000 claims description 7
- 208000032839 leukemia Diseases 0.000 claims description 7
- 230000000996 additive effect Effects 0.000 claims description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 5
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims description 4
- 102000013275 Somatomedins Human genes 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 3
- 239000011669 selenium Substances 0.000 claims 1
- 229910052711 selenium Inorganic materials 0.000 claims 1
- 125000003748 selenium group Chemical group *[Se]* 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 16
- 230000010261 cell growth Effects 0.000 abstract description 5
- 230000004898 mitochondrial function Effects 0.000 abstract description 4
- 239000003102 growth factor Substances 0.000 abstract 1
- 210000002966 serum Anatomy 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 5
- 239000004615 ingredient Substances 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 4
- 238000012258 culturing Methods 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 241000204031 Mycoplasma Species 0.000 description 1
- 241000233855 Orchidaceae Species 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000008288 physiological mechanism Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a human umbilical cord mesenchymal stem cell serum-free medium which comprises a serum-free animal cell culture medium, growth factors, cell culture additives, and a trace element, and has the characteristics of short population doubling time, excellent cell growth form and high cell mitochondrial function.
Description
Technical field
The present invention relates to biological technical field, refer to a kind of human umbilical cord mesenchymal stem cells serum free medium especially.
Background technology
Existing Human umbilical cord mesenchymal stem cell culture medium contains the exogeneous animal serum of 5-10%, should use following weak point:
1) there are differences between batches in application, need a large amount of checking work;
2) contain the activeconstituents of growth promoting effects and the composition of Developing restraint in animal serum, the clean effect just knowing cell growth need be tested;
3) in serum, protein content is high, complicated component, it is reported as many as 150 kinds, is unfavorable for the separation and purification in downstream, hinders cell clinical application;
4) exogeneous animal serum is often by virus and mycoplasma contamination.
More than comprehensive, animal serum substratum brings many unfavorable factors to production and scientific research.
Summary of the invention
In view of this, the present invention proposes a kind of human umbilical cord mesenchymal stem cells serum free medium, have the advantages that population doubling time is short, Growth of Cells form is good, mitochondrial function is strong.
For solving the problems of the technologies described above, the present invention is achieved by the following technical solutions:
A kind of human umbilical cord mesenchymal stem cells serum-free basic medium, comprises serum-free animal cell culture medium, somatomedin, cell cultures additive, trace element.
Further, described somatomedin is Basic Fibroblast Growth Factor (bFGF), leukaemia inhibitory factor (LIF), and wherein, content is bFGF2-15ng/ml, preferred 5ng/ml, LIF5-15ng/ml, preferred 7ng/ml.
Further, described cell cultures additive its comprise: Regular Insulin, Transferrins,iron complexes, thanomin, wherein, content is Regular Insulin 5-20mg/L, preferred 10mg/L, Transferrins,iron complexes 2-15mg/L, preferred 5.5g/L, thanomin 0.5-5mg/L, preferred 2mg/L.
Further, described trace element is selenium powder, and content is selenium powder 2-10ng/L, preferred 6.7gn/L.
Further, described serum-free basic medium is the low sugar culture-medium of L-DMEM().
The invention provides a kind of human umbilical cord mesenchymal stem cells serum free medium of improvement, the features such as it is short that this substratum has (a) population doubling time, and (b) Growth of Cells form is good, and (c) mitochondrial function is strong.And he can 1. avoid differences between batches and not clear serum component on the impact of cell cultures; 2. avoid serum exogenous pollution and to cytotoxic effect; 3. be that product is easy to purifying, improve the rate of recovery, accelerate the process of cell therapy clinical application; 4. definite ingredients, is conducive to the Physiological Mechanism in Regulation studying cell.
Accompanying drawing explanation
Fig. 1 is that substratum in the present invention and commercially available serum free medium contrast at the propagation number of times often for cell.
Fig. 2 be substratum in the present invention to after human mesenchyme's cell cultures, taking pictures under inverted microscope;
Fig. 3 be commercially available serum free medium to after human mesenchyme's cell cultures, taking pictures under inverted microscope.
Embodiment
For the understanding the present invention making those skilled in the art more clear and intuitive, below in conjunction with accompanying drawing, the present invention is further illustrated.
Embodiment 1
According to content disclosed by the invention, according to following component and proportioning configuration substratum 1.
L-DMEM, Basic Fibroblast Growth Factor 5ng/ml, leukaemia inhibitory factor 7ng/ml, Regular Insulin 10mg/L, Transferrins,iron complexes 5.5mg/L, thanomin 2mg/L, selenium powder 6.7gn/L.
Embodiment 2
L-DMEM, Basic Fibroblast Growth Factor 2ng/ml, leukaemia inhibitory factor 15ng/ml, Regular Insulin 5mg/L, Transferrins,iron complexes 15mg/L, thanomin 0.5mg/L, selenium powder 2gn/L.
Embodiment 3
L-DMEM, Basic Fibroblast Growth Factor 15ng/ml, leukaemia inhibitory factor 5ng/ml, Regular Insulin 20mg/L, Transferrins,iron complexes 2mg/L, thanomin 5mg/L, selenium powder 10gn/L.
Result detection of the present invention and contrast is carried out by following contrast.
1. different culture media culturing cell population doubling time compares
The human umbilical cord mesenchymal stem cells getting umbilical cord source is Object of Development, adopts the substratum of different ingredients to cultivate it, and its multiplication capacity is all from the research of the 5th generation to the 15th generation.When often growing to about 100% fusion for umbilical cord mesenchymal stem cells, 0.25% trysinization, is prepared into but cell suspension, adds 0.4% Trypan Blue 2-3 minute.Calculate total cellular score and dead cell number (dead cell is dyed blueness by placenta orchid) in each culture dish, average, continuous 10 generations, and the average cell population doublings number PD calculating per generation, draw accumulative cell population doublings curve, as shown in Figure 1.Cell population doublings number P D is drawn by following formulae discovery: PD=(lgNt-lgN0)/lg2, wherein Nt: collecting cell number; N0: inoculating cell number; T: incubation time, what this adopted is the proportioning of embodiment 1;
2. after different culture media culturing cell, Growth of Cells form compares;
Adopt the substratum of different ingredients to cultivate human umbilical cord mesenchymal stem cells, take pictures under inverted microscope and compare, as shown in Figures 2 and 3, wherein Fig. 2 is the product of embodiment 1 to acquired results, and Fig. 3 is commercially available serum free medium;
3. mitochondrial function compares;
Profit CellCountingKit(CCK-8) to compare the cell mitochondrial after different ingredients culture medium culturing active for test kit, ability of cell proliferation;
1) inoculating cell suspension (100ul/ hole) in 96 orifice plates, adds different ingredients substratum in culture plate, is placed on preculture in incubator (37 DEG C, 5%CO2);
2) 10ulCCK-8 solution is added to every hole;
3) culture plate is placed in incubator hatches 1-4 hour;
4) absorbancy at 450nm place is determined at by microplate reader.
Human umbilical cord mesenchymal stem cells different culture media cultivates the plastosome vigour of rear cell, and acquired results is as shown in the table:
Result shows, the substratum in the present invention can improve the competence for added value of umbilical cord mesenchymal stem cells, maintains the normal morphology of cell.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. a human umbilical cord mesenchymal stem cells serum free medium, is characterized in that: comprise serum-free animal cell culture medium, somatomedin, cell cultures additive, trace element.
2. human umbilical cord mesenchymal stem cells serum free medium according to claim 1, is characterized in that: described somatomedin is Basic Fibroblast Growth Factor, leukaemia inhibitory factor.
3. human umbilical cord mesenchymal stem cells serum free medium according to claim 2, it is characterized in that: the content of described Basic Fibroblast Growth Factor is 2-15ng/ml, the content of described leukaemia inhibitory factor is 5-15ng/ml.
4. human umbilical cord mesenchymal stem cells serum free medium according to claim 3, it is characterized in that: the content of described Basic Fibroblast Growth Factor is 5ng/ml, the content of described leukaemia inhibitory factor is 7ng/ml.
5. human umbilical cord mesenchymal stem cells serum free medium according to claim 1, is characterized in that: described cell cultures additive its comprise Regular Insulin, Transferrins,iron complexes, thanomin.
6. human umbilical cord mesenchymal stem cells serum free medium according to claim 5, is characterized in that: the content of described cell cultures additive is Regular Insulin 5-20mg/L, Transferrins,iron complexes 2-15mg/L, thanomin 0.5-5mg/L.
7. human umbilical cord mesenchymal stem cells serum free medium according to claim 6, is characterized in that: the content of described cell cultures additive is Regular Insulin 10mg/L, Transferrins,iron complexes 5.5mg/L, thanomin 2mg/L.
8. human umbilical cord mesenchymal stem cells serum free medium according to claim 1, it is characterized in that: described trace element is selenium powder, content is selenium 2-10ng/L.
9. human umbilical cord mesenchymal stem cells serum free medium according to claim 1, is characterized in that: described trace element and content are selenium powder 6.7gn/L.
10. human umbilical cord mesenchymal stem cells serum free medium according to claim 1, is characterized in that: serum-free basic medium described in 1 is L-DMEM.
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| CN201510666332.8A CN105200009A (en) | 2015-10-15 | 2015-10-15 | Human umbilical cord mesenchymal stem cell serum-free medium |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106947737A (en) * | 2017-04-14 | 2017-07-14 | 青岛青春派生物科技有限公司 | A kind of human umbilical cord mesenchymal stem cells Serum-free complete medium |
| CN106947736A (en) * | 2017-04-14 | 2017-07-14 | 青岛青春派生物科技有限公司 | A kind of human adipose mesenchymal stem cells Serum-free complete medium |
| CN113234662A (en) * | 2021-05-08 | 2021-08-10 | 康妍葆(北京)干细胞科技有限公司 | Stem cell serum-free culture medium and preparation method and application thereof |
-
2015
- 2015-10-15 CN CN201510666332.8A patent/CN105200009A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106947737A (en) * | 2017-04-14 | 2017-07-14 | 青岛青春派生物科技有限公司 | A kind of human umbilical cord mesenchymal stem cells Serum-free complete medium |
| CN106947736A (en) * | 2017-04-14 | 2017-07-14 | 青岛青春派生物科技有限公司 | A kind of human adipose mesenchymal stem cells Serum-free complete medium |
| CN113234662A (en) * | 2021-05-08 | 2021-08-10 | 康妍葆(北京)干细胞科技有限公司 | Stem cell serum-free culture medium and preparation method and application thereof |
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Application publication date: 20151230 |
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