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Method for isolation and serum gradient switching culture of human umbilical cord mesenchymal stem cells

A technology of mesenchymal stem cells and culture methods, which is applied in the field of separation of human umbilical cord mesenchymal stem cells and serum gradient switching culture, can solve the problems of high price of fetal bovine serum, unclear serum components, limited number of cells, etc., and achieve culture cost and The effect of reducing the risk of clinical use, not being restricted by ethics, and having a wide range of sources

Inactive Publication Date: 2012-07-04
肖扬
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  • Abstract
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Problems solved by technology

Many research institutions have found pluripotent stem cells in other tissues, including mobilized peripheral blood, cord blood, deciduous teeth, and umbilical cord mesenchymal cells, however, the number of cells obtained from these tissues is very limited
[0004] At present, the medium supplemented with fetal bovine serum is considered to be the most effective medium for the expansion of mesenchymal stem cells in vitro. However, the composition of the serum is not clear, and it may contain known or unknown pathogens carried by animals. In clinical applications Potentially dangerous, and fetal bovine serum is expensive

Method used

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  • Method for isolation and serum gradient switching culture of human umbilical cord mesenchymal stem cells
  • Method for isolation and serum gradient switching culture of human umbilical cord mesenchymal stem cells
  • Method for isolation and serum gradient switching culture of human umbilical cord mesenchymal stem cells

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Embodiment 1

[0021] 1. Isolation of human umbilical cord mesenchymal cells:

[0022] The umbilical cord tissues of healthy male infants born at term were collected under sterile conditions, and were approved by the Hospital Ethics Committee. Soak in α-MEM medium, take out the umbilical cord in the ultra-clean bench, fully wash the residual blood with D-Hank's balance solution, remove the umbilical vein, umbilical artery and umbilical cord adventitia, peel off the Wharon glue, and cut it into 1mm× 1mm × 1mm size tissue block. Move the tissue block into 1.0g / L collagenase II (according to 10ml of enzyme solution to digest 3 grams of tissue block), add 2ml α-MEM medium, and digest in a constant temperature oscillator at 37°C until Wharon glue is completely digested , and then use a stainless steel filter to make a single-cell suspension from the digested cells, transfer the cell suspension to a sterile centrifuge tube, and repeatedly blow and dilute it with α-MEM medium 20 times the volume o...

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Abstract

The invention discloses a method for isolation and serum gradient switching culture of human umbilical cord mesenchymal stem cells. The method comprises the following steps: digesting human umbilical cord with collagenase I until Wharton's jelly is fully digested, collecting digested single cells, inoculating the single cells into alpha-MEM medium, carrying out suspension culture until 80-90% of cells are fused, and digesting with trypsin; inoculating the suspension of trypsin-digested cells into alpha-MEM medium, carrying out suspension culture until 80-90% of cells are fused, digesting with trypsin, subcuturing and inoculating the subcultured cells into alpha-MEM medium, and carrying out subculture repeatedly. The concentration of fetal bovine serum in alpha-MEM medium in the steps of subculture decreases with the increase of the passage number until fetal bovine serum disappears. According to the method, the material human umbilical cords are easily accessible; the preparation of human umbilical cord mesenchymal stem cells is rapid and safe, and is not subject to ethical restrictions; and the subsequent culture is independent on the characteristics of fetal calf serum, resulting in greatly reduced culture cost and risk in clinical use. The method of the invention has a good application prospect.

Description

Technical field: [0001] The invention belongs to the field of biomedicine, and in particular relates to a method for separating and switching culture of human umbilical cord mesenchymal stem cells. Background technique: [0002] In recent years, stem cell research has made major breakthroughs. In 1999 and 2000, the world's most authoritative American magazine "Science" listed stem cells and the Human Genome Project as the top 10 scientific breakthroughs of the year for two consecutive years, especially in 1999, it even listed stem cell research as the first. Stem cells are likely to lead to revolutionary progress in the medical field, and thus have immeasurable medical value and have attracted widespread attention and research around the world. The American "Time" magazine believes that stem cells and the Human Genome Project will simultaneously become the most promising development and application in the new century. field. [0003] Mesenchymal stem cells (MSCs for short)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 李力肖扬
Owner 肖扬
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