Mesenchymal stem cell subculture method

A technology of quality stem cells and subculture, applied in the field of stem cell culture, can solve the problems of long cell growth cycle, weak proliferation ability and high price.

Pending Publication Date: 2021-04-09
广东佰鸿干细胞再生医学有限公司
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  • Abstract
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  • Claims
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Problems solved by technology

[0004] The existing culture technology has the following problems: using the existing serum-containing medium to cultivate human umbilical cord mesenchymal stem cells has problems such as immune rejection, which cannot meet the needs of future clinical trials; using the existing serum-free subculture medium to cultivate human umbilical cord mesenchymal stem cells Stem cells have problems such as long cell growth cycle, poor stability, weak proliferation ability, and unstable differentiation ability
The method for isolating and culturing adipose-derived mesenchymal stem cells disclosed in patent CN108300690A has made some technical progress in cultivating adipose-derived mesenchymal stem cells, but the cell proliferation ability is still limited, and the number of cells obtained after 6 days of adipose-derived mesenchymal stem cells is only 63× 10 3 ,refer to figure 1 , which leads to a large amount of samples and high cost. In addition, the patent CN108300690A uses human serum albumin, which is derived from human blood and cannot be artificially synthesized. The source is difficult and the price is high, which leads to the separation of adipose-derived mesenchymal stem cells Cultivation is difficult to industrialize and cannot meet the needs of large-scale cultivation

Method used

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Embodiment 1

[0032] This embodiment provides a method for subculture of human umbilical cord mesenchymal stem cells, comprising the following steps:

[0033] 1) Prepare serum-free subculture medium. The components of the serum-free subculture medium are: α-MEM medium, platelet-derived factor, fibroblast growth factor and ascorbic acid; the concentration of the platelet-derived factor is 10 ng / mL , in other preferred embodiments, the concentration of the platelet-derived factor is 11ng / mL, 12ng / mL, 13ng / mL, 14ng / mL, or 15ng / mL; the concentration of the fibroblast growth factor is 10ng / mL ; In other preferred embodiments, the concentration of the fibroblast growth factor is 11ng / mL, 12ng / mL, 13ng / mL, 14ng / mL or 15ng / mL; the concentration of ascorbic acid is 50 μg / mL, in other preferred In an embodiment, the ascorbic acid concentration is 25 μg / mL, 28 μg / mL, 30 μg / mL, 32 μg / mL, 35 μg / mL, 38 μg / mL, 40 μg / mL, 42 μg / mL, 45 μg / mL, 48 μg / mL or 49 μg / mL.

[0034] 2) Take primary human umbilical ...

Embodiment 2

[0036] This embodiment provides a serum-free subculture medium. The components of the serum-free subculture medium are: α-MEM medium, platelet-derived factor, fibroblast growth factor, and ascorbic acid; the concentration of the platelet-derived factor is 10ng / mL, in other preferred embodiments, the concentration of the platelet-derived factor is 11ng / mL, 12ng / mL, 13ng / mL, 14ng / mL or 15ng / mL; the concentration of the fibroblast growth factor is 10ng / mL / mL, in other preferred embodiments, the concentration of the platelet-derived factor is 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL or 15 ng / mL. The concentration of the ascorbic acid is 50 μg / mL, and in other preferred embodiments, the concentration of the ascorbic acid is 25 μg / mL, 28 μg / mL, 30 μg / mL, 32 μg / mL, 35 μg / mL, 38 μg / mL, 40 μg / mL , 42 μg / mL, 45 μg / mL, 48 μg / mL or 49 μg / mL.

[0037] The above-mentioned serum-free subculture medium added low concentrations of platelet-derived factor, fibroblast growth factor and ascorbic ...

Embodiment 3

[0040] This embodiment provides a serum-free subculture medium. The components of the serum-free subculture medium are: α-MEM medium, platelet-derived factor, fibroblast growth factor, ascorbic acid and melatonin; the platelet-derived The concentration of the factor is 15ng / mL, and in other preferred embodiments, the concentration of the platelet-derived factor is 10ng / mL, 11ng / mL, 12ng / mL, 13ng / mL or 14ng / mL; the fibroblast growth factor The concentration of the platelet-derived factor is 15ng / mL, and in other preferred embodiments, the concentration of the platelet-derived factor is 10ng / mL, 11ng / mL, 12ng / mL, 13ng / mL, 14ng / mL or 15ng / mL; Concentration is 25 μg / mL, in other preferred embodiments, the concentration of described ascorbic acid is 28 μg / mL, 30 μg / mL, 32 μg / mL, 35 μg / mL, 38 μg / mL, 40 μg / mL, 42 μg / mL, 45 μg / mL , 48 μg / mL, 49 μg / mL or 50 μg / mL; the content of the melatonin is 10 nM, and in other preferred embodiments, the content of the melatonin is 2 nM, 5 nM, 15 n...

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Abstract

The invention discloses a mesenchymal stem cell subculture method. The method is characterized by comprising the following steps of 1) preparing a serum-free subculture medium which comprises the following components of an alpha-MEM medium, a platelet-derived factor, a fibroblast growth factor and ascorbic acid, wherein the concentration of the platelet-derived factor is 10-15 ng / mL, and the concentration of the fibroblast growth factor is 10-15 ng / mL; and 2) adding primary mesenchymal stem cells into a T-75 porous culture bottle, adding the serum-free subculture medium, performing culture in a 5% CO2 incubator at 37 DEG C, performing digesting by using a trypsin-EDTA solution when the cell fusion degree reaches 80%-90%, and carrying out subculture according to a ratio of 1:(4-5) after the digestion is finished. According to the culture method, the human umbilical cord mesenchymal stem cells with high purity, strong differentiation capacity, stable stemness and strong multiplication capacity can be obtained in a short time.

Description

technical field [0001] The invention relates to the technical field of stem cell culture, in particular to a mesenchymal stem cell subculture method and a serum-free subculture medium for mesenchymal stem cell subculture. Background technique [0002] Mesenchymal stem cells (Mesenchymal stem cells, MSC) are a type of stem cells with self-renewal, proliferation and multi-directional differentiation potential. , no allogeneic rejection, avoid ethical controversy, stimulate tissue regeneration, regulate immune function and many other advantages. [0003] At present, umbilical cord mesenchymal stem cells (UC-MSC, Umbiliacl cord mesenchymal stem cells) have good therapeutic effects in cartilage regeneration, cerebral ischemic diseases, diabetes, rheumatoid arthritis and other animal models and clinical studies, showing Broad application prospects. At home and abroad, there is no unified standard for the isolation, culture and expansion of umbilical cord mesenchymal stem cells. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0668C12N2500/38C12N2500/90C12N2501/135
Inventor 程蕊苹高迎凤蒲锋星
Owner 广东佰鸿干细胞再生医学有限公司
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