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Method for separating and amplifying mesenchymal stem cells from umbilical cord

A technology of mesenchymal stem cells and umbilical cords, 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 practical and simple method for isolating and expanding mesenchymal stem cells from umbilical cords in vitro

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1, the method for isolating and expanding mesenchymal stem cells from umbilical cord

[0056] (1) Disinfection and cleaning: The treatment of umbilical cord tissue should be carried out in a biological safety cabinet. Disinfect the surface of fresh umbilical cord tissue with alcohol, cut the umbilical cord from the middle, and spread it on a sterile 10cm cell culture plate. Wash the tissue with PBS to minimize red blood cells on the tissue.

[0057] (2) Adherence treatment of umbilical cord tissue: transfer the tissue to another 10cm cell culture plate, cut the tissue into 1cm in size 3In the square shape of the cell culture dish, the number of tissue blocks in each plate was maintained at 10-15 pieces. Allow the tissue block to air dry for 10-15 minutes until the tissue adheres to the plate.

[0058] (3) Umbilical cord tissue culture: Slowly add mesenchymal stem cell special medium (15%FBS+1%L-Glutamine+0.05%Gentamicin+84%DMEM-F12) along the edge of the ...

Embodiment 2

[0062] Embodiment 2, the method for isolating and expanding mesenchymal stem cells from umbilical cord

[0063] Carried out with reference to the method of Example 1. Adherent cells began to crawl out of the umbilical cord tissue on the 7th day of culture, and the cell fusion rate reached 60% on the 13th day of culture. After passage to T25 culture flasks, the fusion rate reached 90% on day 19. After 3 passages, the cell purity was greater than 95%.

Embodiment 3

[0064] Embodiment 3, the method for isolating and expanding mesenchymal stem cells from umbilical cord

[0065] Carried out with reference to the method of Example 1. Adherent cells began to crawl out of the umbilical cord tissue on the 8th day of culture, and the cell fusion rate reached 60% on the 15th day of culture. After passage to T25 culture flasks, the fusion rate reached 90% on day 18. 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 mesenchymal stem cells from an umbilical cord. The method comprises the following steps of: disinfecting and cleaning an umbilical cord tissue; cutting the tissue into pieces, and paving in another cell culture plate to ensure that the tissue pieces are air-dried until the tissue is attached to the plate; culturing the umbilical cord tissue in a culture medium special for the mesenchymal stem cells, supplementing / replacing liquid and clearing all umbilical cord tissue pieces when the umbilical cord tissue is cultured for 11 to 13 days, and continuing to culture; completely replacing liquid every 1 to 3 days later on; separating wall attaching cells from the bottom of the plate by using digestive enzyme when the fusion rate of the wall attaching cells in the plate reaches about 50-70 percent; centrifuging and removing a supernatant, adding into the culture medium special for the mesenchymal stem cells, suspending the cells again, and inoculating in a T25 cell culture bottle for subculture and amplification culture; and replacing liquid once every 1 to 3 days later on until the fusion rate reaches 70-90 percent to obtain the mesenchymal stem cells. The method can be effectively used for separating and amplifying the mesenchymal stem cells.

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] Studies have found that bone marrow is rich in mesenchymal stem cells (MSCs), which have gradually attracted people's attention because of their multi-lineage differentiation potential, immune regulation and self-replication. Mesenchymal stem cells can be induced to differentiate into fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelial and other tissue cells. After continuous subculture and cryopreservation, it still has multi-directional differentiation potential, and has great clinical application value for the treatment of aging and the repair of tissue and organ damage. [0003] However, with aging, the number of stem cells in the bone marrow is also significantly ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 周丹林卓衡朱业峰陈俊峯
Owner BOYALIFE
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