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Method for separating and amplifying mesenchymal stem cell from fresh tissue of umbilical cord

A technique for stem cells and umbilical cord tissue, applied in the field of isolation and expansion of stem cells

Inactive Publication Date: 2012-09-12
BOYALIFE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the defects of existing methods for obtaining umbilical cord mesenchymal stem cells, and provide a simple and effective method for isolating and expanding mesenchymal stem cells from isolated umbilical cords

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1, the method for isolating and expanding mesenchymal stem cells from umbilical cord

[0065] (1) Disinfection and cleaning: In a biological safety cabinet, disinfect the surface of the umbilical cord tissue with alcohol, cut the umbilical cord from the middle, spread it on a sterile 10cm cell culture plate, and clean the tissue with PBS buffer to minimize Red blood cells on the tissue;

[0066] (2) Digestion treatment: Add 0.1 g of type I collagenase to 100 ml of DMEM-F12, then filter with a 20 μm filter to obtain the digestion solution, transfer the umbilical cord tissue obtained in step (1) to another 10 cm cell culture dish, Cut the umbilical cord tissue into 1cm 3 Tissue pieces of different sizes, put the tissue pieces into the prepared digestive solution, digest in a constant temperature shaker at 37°C for 1.5 hours, use a 100μm filter to remove the remaining tissue pieces, and add intervenant at a volume ratio of 1:1. Stem cell medium (15%FBS+1%L-Gl...

Embodiment 2

[0073] Embodiment 2, the method for isolating and expanding mesenchymal stem cells from umbilical cord

[0074] Carried out with reference to the method of Example 1. The whole cells of the umbilical cord tissue obtained after digestion began to have adherent cells climbing out on the 8th day of culture, and the fusion rate reached 90% on the 20th day. After 3 passages, the cell purity was greater than 95%.

Embodiment 3

[0075] Embodiment 3, the method for isolating and expanding mesenchymal stem cells from umbilical cord

[0076] Carried out with reference to the method of Example 1. The whole cells of the umbilical cord tissue obtained after digestion began to have adherent cells climbing out on the 7th day of culture, and the fusion rate reached 90% on the 18th day. After 3 passages, the cell purity was greater than 90%.

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Abstract

The invention relates to a method for separating and amplifying a mesenchymal stem cell from a fresh tissue of an umbilical cord. The method comprises the following steps of: sterilizing and cleaning an umbilical cord tissue; shearing the tissue into a block shape; carrying out digestion treatment for 1-2.5 hours and ending the digestion; cleaning the cell obtained by digesting to obtain a cell suspension; adding the cell suspension into a T25 cell culture bottle for culturing; supplementing after culturing the cell suspension for 3-6 days and continuously culturing; carrying out full liquid change for the first time when the cell suspension is cultured for 8-10 days and then carrying out one full medium change every one to three days; when the fusion rate of anchorage-dependent cells in a plate reaches about 50-70 percent, enabling the anchorage-dependent cells to be separated from the bottom of the T25 cell culture bottle by using digestive enzyme; removing supernate by centrifuging, adding a mesenchymal stem cell culture medium for re-suspending the cell, inoculating the cell to the T25 cell culture bottle for carrying out passage and amplifying culture; and then changing liquid once every 1-3 days until the fusion rate reaches 70-90 percent and obtaining the umbilical cord mesenchymal stem cell. The method disclosed by the invention can be effectively used for separating and augmenting the mesenchymal stem cell.

Description

technical field [0001] The invention relates to a method for isolating and expanding stem cells from fresh umbilical cord tissues, in particular to a method for isolating and expanding mesenchymal stem cells from fresh umbilical cord tissues. Background technique [0002] Mesenchymal stem cells (MSCs) originate from mesoderm and ectoderm in the early stages of development, and have the characteristics of multilineage differentiation potential, immune regulation and self-replication, and have attracted increasing attention. Under specific induction conditions in vivo or in vitro, mesenchymal stem cells can differentiate into fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelial and other tissue cells. It has multi-directional differentiation potential and can be used as an ideal seed cell for the repair of tissue and organ damage caused by aging and disease, especially for the treatment of aging and the repair of tissue and organ damage. It...

Claims

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Application Information

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IPC IPC(8): C12N5/0775G06F19/28
Inventor 陈俊峯林卓衡朱业峰周丹
Owner BOYALIFE
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