CN104164404A - Application of serum-free culture system for efficiently culturing human umbilical cord mesenchymal stem cells in vitro - Google Patents
Application of serum-free culture system for efficiently culturing human umbilical cord mesenchymal stem cells in vitro Download PDFInfo
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- CN104164404A CN104164404A CN201410394545.5A CN201410394545A CN104164404A CN 104164404 A CN104164404 A CN 104164404A CN 201410394545 A CN201410394545 A CN 201410394545A CN 104164404 A CN104164404 A CN 104164404A
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Abstract
The invention relates to an application of a serum-free culture system for efficiently culturing human umbilical cord mesenchymal stem cells in vitro. The serum-free culture system comprises the following components: a basal culture medium a-MEM, cell factors, hormones and proteins, unsaturated fatty acids, antioxidants, energy substances, an amino acid additive, vitamins and metal additives. The serum-free culture system comprises simple and clear components, is free from harms of pathogens, does not have difference between batches, has good repeatability, can obtain plenty of high-quality human umbilical cord mesenchymal stem cells in a short time and has high passage stability, so that the serum-free culture system can be applied to scientific researches and can provide high-purity vibrant cells for cell therapy as a mating system in cell therapy.
Description
Technical field
The present invention relates to a kind of purposes of biological medium, specifically, relate to a kind of purposes of serum free culture system of efficient vitro culture of human umbilical cord mesenchymal stem cells.
Background technology
In recent years, animal cell culture technology has expanded to the many aspects such as research and commercial exploitation from simple experimental implementation field.Many biotechnology research and product all be unable to do without cell cultures.In cell cultures development history, the development of substratum occupies critical positions.Mescenchymal stem cell is to derive from the mesoblastic adult stem cell with height self-renewal capacity and multi-lineage potential, is extensively present in whole body reticular tissue and organ interstitial.Experimental results demonstrate, the mescenchymal stem cell after long-term cultivation has differentiation potential widely, and the tissue of end eventually of formation comprises brain, retina, liver, intestines, spleen, marrow, blood, skin, cartilage etc.Illustrate that mescenchymal stem cell has the ability across differentiation of germinal layers, and under suitable culture environment, can induce various kinds of cell differentiation.
For the cultivation of mescenchymal stem cell, also there is no at present harmonized programme.Cheng Fanjun etc. adopt low sugar DMEM substratum, are divided into 2% and 10% serologic group according to the difference of adding foetal calf serum concentration, and the conclusion drawing is to adopt lower concentration serum free culture system system can effectively avoid mescenchymal stem cell to lose proliferation and differentiation potentiality.In a word, the serum of different culture media, different batches, different pH values, different planting densities all can affect the growth of mescenchymal stem cell.
At present, in the cellar culture system of mescenchymal stem cell, all added a certain proportion of animal serum, that conventional is foetal calf serum (FBS).Serum is absolutely necessary in the culture system in vitro of cell, though it can provide somatomedin, in conjunction with albumen, hormone, and play a protective role, but it has following defect: 1. complicated component, and contain some factor that is unfavorable for Growth of Cells or toxicants, may affect result of study; 2. also may there is pathogenic agent, clinical application is constituted a threat to; 3. between different batches, there are differences, cause cultivation results to occur difference.
Although existing a lot of serum free mediums at present, but contain a small amount of animal serum.On market, can buy a few human mesenchymal stem cell serum free medium, but be substantially all external import, its expensive (>6 unit/mL), culture effect is undesirable, and only, for research application, can not be used for people's diagnosis and treatment.Chinese patent literature CN201210350602.0, open day 2012.12.19, disclose a kind of mesenchymal stem cell serum-free culture medium, its necessity consists of IMDM, L-glutaminate, sodium bicarbonate, Hepes, recombinant human insulin, recombinant human Transferrins,iron complexes, rHA, 2 mercapto ethanol, Protocatechuic Acid, lipid, amino acid, VITAMIN, trace element, hydrocortisone, vitamins C, bonding amine or recombinant human fibronectin polypeptide, Progesterone, putrescine, heparin, thrombotonin, EGF, b-FGF, PDGF-BB, IGF-I.The described serum free medium specific chemical components of this invention, non-animal derived, serum-free, culturing cell is safe, desirable, avoided doping animal component and batch unstable, cultivation mescenchymal stem cell result shows that its total cellular score, cell phenotype and secrete cytokines are all normal.But, on the one hand, above-mentioned medium component is more, and amino acid comprises L-arginine, CYSTINE, L-Histidine, ILE, L-Leu, 1B, METHIONINE, L-Phe, L-threonine, L-Trp, TYR, Valine, ALANINE, L-Aspartic acid, altheine, Pidolidone, glycine, L-PROLINE, Serine; VITAMIN comprises calcium pantothenate, choline chloride 60, folic acid, meso-inositol, niacinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride; Lipid comprises arachidonic acid, cholesterol, DL-alpha-tocopherol acetic ester, linolic acid, linolenic acid, tetradecanoic acid, oleic acid, Zoomeric acid, palmitinic acid, stearic acid; Trace element comprises Cu, Zn, Se, Fe, Sn, Ni, Ag, Al, Cr, Ge, Zr, Rb, Co, Cd, Ba, K, and composition is too complicated, causes the inconvenience in preparation; On the other hand, substratum wants to ensure the stability of passage, and to meet the needs of scientific research, the stability going down to posterity about above-mentioned substratum it be unclear that.
In sum, need the interstital stem cell serum free medium that a kind of composition is simple and clear, mitotic stability is good badly.
Summary of the invention
The object of the invention is, for deficiency of the prior art, provides a kind of purposes of serum free culture system of efficient vitro culture of human umbilical cord mesenchymal stem cells.
For achieving the above object, the technical scheme that the present invention takes is:
Serum free culture system is used for a purposes for vitro culture of human umbilical cord mesenchymal stem cells, each composition that described serum free culture system comprises following mass ratio:
Preferably, each composition that described serum free culture system comprises following mass ratio:
。
More preferably, each composition that described serum free culture system comprises following mass ratio:
。
Preferably, described basic medium a-MEM is OriCell
tMmEM minimum essential medium.
Preferably, the composition of described non-essential amino acid is:
。
More preferably, the composition of described non-essential amino acid is:
。
Described serum free culture system pH:7.0-7.4; Osmotic pressure: 260-320mOsm/kg; Bacterium, fungi detect: feminine gender; Chlamydozoan, detection of mycoplasma: feminine gender; Intracellular toxin: <0.5EU/mL.
The invention has the advantages that the purposes of a kind of serum free culture system for vitro culture of human umbilical cord mesenchymal stem cells is provided, described serum free culture system possesses following advantage:
1, composition is simply clear, bioclean harm;
2, without batch between difference, reproducible;
3, can obtain in the short period of time human umbilical cord mesenchymal stem cells of a large amount of high-qualitys, and mitotic stability is high.
Visible culture system of the present invention can be applied in scientific research, also can be used as the matching system in cell therapy, for cell therapy provides high purity and great-hearted cell.
Brief description of the drawings
Accompanying drawing 1 is the propagation multiple that A group and B organize per generation cell.
Accompanying drawing 2 is that A group and B organize the theoretical sum of per generation cell.
Accompanying drawing 3 is A group and B group cell doubling time.
Accompanying drawing 4 is A group and B group cellular form.
Embodiment
Below in conjunction with accompanying drawing, embodiment provided by the invention is elaborated.
The content of the each component of serum free culture system of the present invention and each component can be in the scope shown in following table:
。
Described basic medium a-MEM is the conventional basic medium of animal cell culture, and in following examples, that concrete use is the product of match industry (Guangzhou) bio tech ltd (Cyagen): OriCell
tMminimum Essential Medium, OriCell
tMmEM minimum essential medium, Cat. No. ME-10001-500.
product description:oriCell
tMminimum Essential Medium (MEM) minimum essential medium, is one of conventional substratum in animal cell culture, is mainly used in the cultivation of attached cell, also can be applicable to the cultivation of suspension cell through improvement.OriCell
tMmEM minimum essential medium can be used for cultivating various kinds of cell system, comprises Hela, BHK-21, HEP-2 and some primary cell etc.OriCell
tMmEM minimum essential medium includes the required amino acid of multiclass cell cultures, VITAMIN, inorganic salt and other compositions; But not containing protein, or any somatomedin; This product needed collocation serum or serum-free additive use.This product, only for non-clinical research purposes, is not used in diagnosis, treatment, clinical or other purposes.
product performance:
OriCell
tMmEM minimum essential medium formula is as shown in the table:
。
But those skilled in the art should know, every a-MEM basic medium all can be used for the present invention, is not limited to OriCell
tMmEM minimum essential medium.
Described NEAA is non-essential amino acid, and what following examples were specifically used is the product of Cyagen (Suzhou) Biotechnology Co., Ltd., and its concrete composition is preferably:
But be not limited only to this, if in following ranges all can:
。
Other components, comprise that cytokine, hormone and albumen, unsaturated fatty acids, polyphenoils, energy matter, VITAMIN, metallic additions are the existing reagent of prior art, can buy and obtain by biological reagent company, described GlutaMAX-I 100 × buy the company in Gibco, article No. is 35050-061.
Each parameter of substratum of the present invention is as follows:
pH：7.0-7.4；
Osmotic pressure: 260-320mOsm/kg;
Bacterium, fungi detect: feminine gender;
Chlamydozoan, detection of mycoplasma: feminine gender;
Intracellular toxin: <0.5EU/mL.
embodiment 1 serum free culture system of the present invention (one)
embodiment 2 serum free culture system of the present invention (two)
Described serum free culture system formula is as shown in the table:
。
embodiment 3 serum free culture system of the present invention (three)
Described serum free culture system formula is as shown in the table:
。
comparative example 1
the effect experiment of embodiment 4 serum free culture systems of the present invention
1, test materials
1. the serum free culture system of embodiment 1, is called for short D7;
2. the human plasma of deactivation, is called for short HS(for cultivating certain patient's cell, and blood plasma just self is collected by this patient so);
3. umbilical cord stem cell perfect medium (LD), is purchased from Cyagen (Suzhou) Biotechnology Co., Ltd., and concrete composition is as follows:
；
④?PBS；
5. pancreatin surrogate;
6. people's umbilical cord mesenchymal stem cells;
7. serum-free cell culture six orifice plates, Corning
cellbind.
2, test method
Table 1 experiment grouping
Note: HS concentration is volumetric concentration.
Experimental procedure is as follows:
1. the 1 pipe people umbilical cord mesenchymal stem cells of recovering, is inoculated in T25 Tissue Culture Flask, adopts umbilical cord stem cell perfect medium (B organize substratum) cultivation;
2. after cell covers with, digest and collect centrifugally, wash twice rear sampling with PBS and count;
3. according to count results, get 2.5 × 10
5cells is inoculated into 1 hole in serum-free cell culture six orifice plates, inoculates altogether 2 holes, and every hole adopts respectively above-mentioned A group and B group substratum to cultivate;
4. cultivate next day, every porocyte changes liquid, and change once every day;
5. after cultivating 3-4d, reach more than 90% when B organizes cell density, take pictures; Adopt pancreatin surrogate digest respectively each porocyte and collect, sampling counting thereafter;
6. according to count results, all get 2.5 × 10 for every group
5cells is seeded in new serum-free cell culture six orifice plates and continues to cultivate;
7. repeat above-mentioned microorganism culture and count method, more than five generations of cultured continuously.
3, experimental result
In seven generations of cultured continuously, per generation propagation multiple, theoretical sum and the doubling time of cell the results are shown in Figure 1-3, and cellular form is shown in Fig. 4.
As can be seen from Figure 1, A group and B organize cell proliferation multiple the there was no significant difference in per generation.
As can be seen from Figure 2, A group total cellular score is stable, substantially can be consistent with the rhythm that goes down to posterity of B group; And finally also can reach 10 of clinical requirement
8cell concentration.
As can be seen from Figure 3, A group all kept relative stability with the doubling time of B group.Although A group part is greater than 24 hours, also meets the product standard lower than 72 hours.
As can be seen from Figure 4, change after seven generations of substratum, people's umbilical cord mesenchymal stem cells still can be bred in A group, and effect is approaching with B group.
4, experiment conclusion
Serum free culture system of the present invention for Growth of Cells with active compared with having blood serum medium and there was no significant difference.
the contrast experiment of embodiment 5 serum free culture system of the present invention and other serum free medium
The effect of the screening formulation cultivator umbilical cord stem cell of serum free culture system prepared by serum free culture system prepared by comparing embodiment 1-2 and comparative example 1, the serum free medium of patent documentation CN201210350602.0.
Specific experiment method is with embodiment 4.1 group of serum free culture system+HS(5% that uses embodiment 1 to prepare of embodiment), 2 groups of serum free culture system+HS(5% that use embodiment 2 to prepare of embodiment), 1 group of serum free culture system+HS(5% that uses comparative example 1 to prepare of comparative example), CN201210350602.0 group is used the most preferably formula in patent documentation CN201210350602.0.
The cell proliferation multiple of adding up every group of per generation, result is as shown in table 2.Embodiment 1 and 2 is at the propagation multiple in the the 1st, 3,5,7 generations all higher than the serum free medium of comparative example 1 and patent documentation CN201210350602.0, and difference has statistical significance (P<0.05).Above result shows that serum free culture system of the present invention is conducive to Growth of Cells breeding more, and mitotic stability is high.
The cell proliferation multiple in every group of per generation of table 2 (
)
In addition, experiment of the present invention has repeated three times, and substratum used and all ingredients are that different batches is bought, and between the experimental result drawing, there is no difference, show that it is reproducible.
Visible in sum, serum free culture system of the present invention possesses following advantage: 1, composition is simply clear, bioclean harm; 2, without batch between difference, reproducible; 3, can obtain in the short period of time human umbilical cord mesenchymal stem cells of a large amount of high-qualitys, and mitotic stability is high.Visible serum free culture system of the present invention can be applied in scientific research, also can be used as the matching system in cell therapy, for cell therapy provides high purity and great-hearted cell.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of the inventive method; can also make some improvement and supplement, these improvement and the supplementary protection scope of the present invention that also should be considered as.
Claims (7)
1. serum free culture system, for a purposes for vitro culture of human umbilical cord mesenchymal stem cells, is characterized in that each composition that described serum free culture system comprises following mass ratio:
2. purposes according to claim 1, is characterized in that, each composition that described serum free culture system comprises following mass ratio:
3. purposes according to claim 2, is characterized in that, each composition that described serum free culture system comprises following mass ratio:
4. according to the arbitrary described purposes of claim 1-3, it is characterized in that, described basic medium a-MEM is OriCell
tMmEM minimum essential medium.
5. according to the arbitrary described purposes of claim 1-3, it is characterized in that, the composition of described non-essential amino acid is:
。
6. purposes according to claim 5, is characterized in that, the composition of described non-essential amino acid is:
。
7. according to the arbitrary described purposes of claim 1-3, it is characterized in that described serum free culture system pH:7.0-7.4; Osmotic pressure: 260-320mOsm/kg; Bacterium, fungi detect: feminine gender; Chlamydozoan, detection of mycoplasma: feminine gender; Intracellular toxin: <0.5EU/mL.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630142A (en) * | 2015-02-06 | 2015-05-20 | 广州赛莱拉干细胞科技股份有限公司 | Separation and culture method of bovine umbilical cord mesenchymal stem cells |
| CN104630143A (en) * | 2015-02-11 | 2015-05-20 | 广州赛莱拉干细胞科技股份有限公司 | Method for separating and culturing goat bone marrow mesenchymal stem cells |
| WO2021248675A1 (en) * | 2020-06-09 | 2021-12-16 | 生物岛实验室 | Inducer for differentiation of stem cells into chondrocytes and application thereof |
| WO2022077784A1 (en) * | 2020-10-16 | 2022-04-21 | 武汉睿健医药科技有限公司 | Chemical culture system and use thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2014
- 2014-08-12 CN CN201410394545.5A patent/CN104164404A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630142A (en) * | 2015-02-06 | 2015-05-20 | 广州赛莱拉干细胞科技股份有限公司 | Separation and culture method of bovine umbilical cord mesenchymal stem cells |
| CN104630142B (en) * | 2015-02-06 | 2018-06-05 | 广州赛莱拉干细胞科技股份有限公司 | A kind of isolation and culture method of ox umbilical cord mesenchymal stem cells |
| CN104630143A (en) * | 2015-02-11 | 2015-05-20 | 广州赛莱拉干细胞科技股份有限公司 | Method for separating and culturing goat bone marrow mesenchymal stem cells |
| WO2021248675A1 (en) * | 2020-06-09 | 2021-12-16 | 生物岛实验室 | Inducer for differentiation of stem cells into chondrocytes and application thereof |
| WO2022077784A1 (en) * | 2020-10-16 | 2022-04-21 | 武汉睿健医药科技有限公司 | Chemical culture system and use thereof |
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