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Mesenchymal stem cell, cell culture media and culture method and application

A culture method, mesenchymal stem cell technology, applied in the field of cell culture medium and culture method and application, mesenchymal stem cells, can solve the problems of serum batch difference, heterogeneous pollution, unfavorable cell culture, etc., to avoid heterogeneity Contamination, stable and efficient immune rejection, and the effect of simple and easy-to-operate culture methods

Inactive Publication Date: 2019-03-29
TIANJIN CHANGHE BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The current production of mesenchymal stem cells mainly relies on serum culture, but too much serum will cause problems such as immune rejection, heterologous contamination, and differences between different batches of serum; however, when using serum-free culture, the growth and proliferation of cells will be slow. Significantly slower, not conducive to cell culture

Method used

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  • Mesenchymal stem cell, cell culture media and culture method and application
  • Mesenchymal stem cell, cell culture media and culture method and application
  • Mesenchymal stem cell, cell culture media and culture method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] This embodiment provides a method for culturing mesenchymal stem cells, specifically as follows:

[0092] 1. Separation of the umbilical cord

[0093] 1) Take a 5cm-long healthy donated umbilical cord, rinse it with normal saline, and remove the vascular tissue. Shredded to 1mm 3 tissue fragments;

[0094] 2) The tissue fragments were inoculated on a 150mm petri dish, and the first medium was slowly added until the tissue fragments were submerged, and incubated at 36°C, 6% CO 2 Cultivated in an incubator with saturated humidity;

[0095] 3) Change the first medium every other day until the confluence of the adherent cells crawling out of the tissue fragments reaches 90% and then subculture (the cell generation is p0 generation);

[0096] 2. Subculture of umbilical cord mesenchymal stem cells (gradual domestication)

[0097] 1) The primary cells (generation p0) were digested with 0.15% trypsin for 7 minutes to form single cells, and the trypsin reaction was terminat...

Embodiment 2

[0108] This embodiment provides a method for culturing mesenchymal stem cells, specifically as follows:

[0109] 1. Separation of the umbilical cord

[0110] 1) Take a 5cm-long healthy donated umbilical cord, rinse it with normal saline, and remove the vascular tissue. Shredded to 1mm 3 tissue fragments;

[0111] 2) The tissue fragments were inoculated in a 150mm petri dish, slowly added the first medium until the tissue fragments were covered, and placed at 38°C, 4% CO 2 Cultivated in an incubator with saturated humidity;

[0112] 3) Change the first medium every 3 days until the confluence of the adherent cells climbing out of the tissue fragments reaches 80% and then subculture (the cell generation is p0 generation);

[0113] 2. Subculture of umbilical cord mesenchymal stem cells (gradual domestication)

[0114] 1) The primary cells (generation p0) were digested with 0.35% trypsin for 3 minutes to form single cells, and the trypsin reaction was terminated with the seco...

Embodiment 3

[0124] This embodiment provides a method for culturing mesenchymal stem cells, specifically as follows:

[0125] 1. Separation of the umbilical cord

[0126] 1) Take a 5cm-long healthy donated umbilical cord, rinse it with normal saline, and remove the vascular tissue. Shredded to 1mm 3 tissue fragments;

[0127] 2) The tissue fragments were inoculated on a 150mm petri dish, and the first medium was slowly added until the tissue fragments were submerged, and incubated at 37°C, 5% CO 2 Cultivated in an incubator with saturated humidity;

[0128] 3) Change the first medium every 2 days until the confluence of the adherent cells crawling out of the tissue fragments reaches 85% and then subculture (the cell generation is p0 generation);

[0129] 2. Subculture of umbilical cord mesenchymal stem cells (gradual domestication)

[0130] 1) The primary cells (generation p0) were digested with 0.25% trypsin for 5 minutes to form single cells, and the trypsin reaction was terminated ...

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Abstract

The present invention relates to the field of biotechnology and particularly provides a mesenchymal stem cell, cell culture media, and a culture method and an application. The provided set of cell culture media comprise four culture media, each culture medium independently comprises a basic medium, serum and cytokines, wherein the basic medium comprises DMEM / F12, DMEM or alpha-MEM, the serum comprises FBS or NBCS, and the cytokines comprise EGF and bFGF. The set of the cell culture media are simple in components and low in cost, each culture medium is designed reasonably and highly efficientlycultures cells, and at the same time continuously reduces serum content, and continuous culturing of the four culture media can highly efficiently separate and culture the mesenchymal stem cell fromtissues, and avoids quality instability and heterogeneous contamination caused by batch-to-batch differences. The provided mesenchymal stem cell is low in immune rejection, good in quality, small in the batch-to-batch differences and free of heterogeneous pollution.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mesenchymal stem cell, a cell culture medium, a culture method and an application. Background technique [0002] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cells, derived from the mesoderm and ectoderm in the early stages of development, and can be isolated from various tissues such as bone marrow, fat, synovium, bone, muscle, and umbilical cord. Mesenchymal stem cells still have multi-directional differentiation potential after continuous subculture and cryopreservation, and can differentiate into osteoblasts, chondrocytes, adipocytes, bone marrow stroma, nerve cells, liver cells, islet cells and cardiomyocytes potential. [0003] The current production of mesenchymal stem cells mainly relies on serum culture, but too much serum will cause problems such as immune rejection, heterologous contamination, and differences between different batches of serum; however, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K35/51A61K35/28A61K35/14A61K35/35A61P17/06
CPCA61K35/14A61K35/28A61K35/35A61K35/51A61P17/06C12N5/0663C12N5/0665C12N5/0667C12N2501/11C12N2501/115
Inventor 李政楠徐永胜倪琳牟春琳
Owner TIANJIN CHANGHE BIOLOGICAL TECH
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