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A three-dimensional expansion method with high activity of hepatic stem cells in vitro to maintain cell phenotype

A technology of liver stem cells and high activity, applied in the field of stem cells, can solve the problems of harsh three-dimensional culture conditions, difficult industrialization promotion, high technical requirements, etc., and achieve the effect of good stem cell characteristics, uncomplicated components, and simple culture and expansion methods

Active Publication Date: 2020-10-30
绍兴橡树企业管理咨询合伙企业(有限合伙)
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AI Technical Summary

Problems solved by technology

[0003] As far as liver (stem) cells are concerned, the general culture method is difficult to maintain the function of cells, and three-dimensional culture can partially solve this problem
However, the conditions of the existing three-dimensional culture are relatively harsh, and good results can only be obtained in a state of weightlessness. The technical requirements are high, and it is difficult to promote industrialization

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Rat liver stem cell line WB-F344 was purchased from Beijing Yuhengfeng Technology Co., Ltd.

[0054] Prepare medium, which consists of the following components:

[0055] DMEM / F12 1L

[0056] Fibroblast Growth Factor 18 mg / L

[0057] Insulin 13mg / L

[0058] Glutamic acid 0.125mg / L

[0059] Niacinamide 0.7mg / L

[0060] Selenite 0.15mg / L

[0061] β-mercaptoethanol 0.08 mg / L

[0062] α-actin 0.3mg / L

[0063] Sodium acetate 0.25mg / L

[0064] Sodium pyruvate 0.002mg / L

[0065] Fetal bovine serum accounted for 2.5% of the total medium weight.

[0066] Inoculum size 1×10 4 Liver stem cells / ml were seeded in chitosan porous gel with a pore size of 500 microns, placed in the above medium, the volume ratio of the gel to the medium was 2.5:1, at 37°C, the volume fraction was 5% CO 2 Cultured under the conditions of 21 days, the medium was changed on the 5th day of culture, and the medium was changed every 4 days thereafter.

[0067] During the culture process, the LDH c...

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Abstract

The invention provides a phenotype maintaining and in-vitro high-activity three-dimensional amplification method of hepatic stem cells. The method comprises the following steps: 1, preparing a medium, wherein the medium comprises 1L of DMEM / F12, 15-20 mg / L of a fibroblast growth factor, 10-15 mg / L of insulin, 0.1-0.2 mg / L of glutamic acid, 0.5-0.8 mg / L of nicotinamide, 0.1-0.2 mg / L of seleninic acid, 0.05-0.1 mg / L of beta-mercaptoethanol, 0.1-0.6 mg / L of alpha-actin, 0.2-0.3 mg / L of sodium acetate, 0.001-0.003 mg / L of sodium pyruvate and 2-3 wt% of fetal calf serum; and 2, immersing sterilized porous gel in the medium, inoculating the porous gel with hepatic stem cells, and culturing the hepatic stem cells. Stem cells with stable phenotype and high activity are obtained in the invention, and the good characteristics of the stem cells are kept.

Description

technical field [0001] The invention belongs to the technical field of stem cells, and in particular relates to a method for in vitro high-activity three-dimensional expansion of hepatic stem cells maintaining cell phenotype. Background technique [0002] With the increasing attention of stem cells in the medical field, the expansion and culture of stem cells has begun to attract people's attention. However, the current stem cell culture technology faces the problem of stem cell differentiation, making the expansion and acquisition of stem cells a technical difficulty. In addition, how to maintain the activity of stem cells during the culture and expansion process is also one of the technical difficulties. [0003] As far as liver (stem) cells are concerned, it is difficult to maintain the function of cells in general culture methods, but three-dimensional culture can partially solve this problem. However, the existing three-dimensional culture conditions are relatively ha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/074
CPCC12N5/067C12N2500/12C12N2500/30C12N2500/32C12N2500/44C12N2500/46C12N2500/84C12N2501/113C12N2501/115C12N2501/119C12N2501/33C12N2501/998C12N2513/00C12N2533/70C12N2533/80
Inventor 向红先
Owner 绍兴橡树企业管理咨询合伙企业(有限合伙)
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