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Efficient method for isolating and culturing mesenchymal stem cells from umbilical cord

A technology for separating and culturing mesenchymal stem cells, applied in the field of isolating and culturing mesenchymal stem cells, which can solve the problems of difficult to separate mesenchymal stem cells, limited number of primary cells, difficult to separate stem cells, etc., to reduce pancreatic enzyme activity, operate Simple and easy to use, avoid the effect of the use of serum

Active Publication Date: 2013-08-28
AFFILIATED HOSPITAL CHINA ACADEMY OF MILITARY MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is difficult to isolate 100% mesenchymal stem cells from umbilical cord tissue by endothelial digestion; it takes a long time (about 15 days) to obtain primary cells by tissue block attachment method, and it is difficult to completely isolate stem cells from umbilical cord tissue ; The number of primary cells obtained by simple enzyme digestion is limited, and 10 cells can be obtained in about 1 month 9 -10 10 , so there is an urgent need for a more efficient method for obtaining umbilical cord-derived mesenchymal stem cells

Method used

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  • Efficient method for isolating and culturing mesenchymal stem cells from umbilical cord
  • Efficient method for isolating and culturing mesenchymal stem cells from umbilical cord
  • Efficient method for isolating and culturing mesenchymal stem cells from umbilical cord

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: Isolation and culture of human umbilical cord-derived mesenchymal stem cells

[0064] (1) Rinse the collected umbilical cord specimen with normal saline to remove residual blood. Weigh, measure the length, bluntly dissect the arteries, cut the rest of the umbilical cord tissue into 0.5-1cm segments, and put them in a new tissue processor (automatic tissue processor produced by Miltenyi, model: gentleMACSTM).

[0065] (2) Add type II collagenase (Sigma, C6885) at a final concentration of 0.1%, add hyaluronidase (Sigma, H4274) at a final concentration of 0.0005%, digest at 37°C for 60 min, and then stop digestion.

[0066] (3) Naturally settle for 8 minutes, transfer the supernatant cell suspension to a centrifuge tube, dilute with 2 times the volume of normal saline, centrifuge at 2000 rpm, 15°C for 20 minutes, and obtain a cell pellet; at the same time, dilute the undigested tissue pieces with normal saline Finally, centrifuge at 2000rpm at 15°C for 5min, di...

Embodiment 2

[0070] Example 2: Isolation and cultivation of human umbilical cord-derived mesenchymal stem cells

[0071] (1) Rinse the collected umbilical cord specimen with normal saline to remove residual blood. Weigh, measure the length, and bluntly dissect the arteries, cut the rest of the umbilical cord tissue into 0.5-1cm segments, and put them in a new tissue processor.

[0072] (2) Add type II collagenase at a final concentration of 0.05%, add hyaluronidase at a final concentration of 0.001%, digest at 37°C for 90min, and then stop digestion.

[0073] (3) Naturally settle for 10 minutes, transfer the supernatant cell suspension to a centrifuge tube, dilute with 5 times the volume of normal saline, centrifuge at 2500 rpm, 15°C for 15 minutes to obtain cell pellets; at the same time, dilute the undigested tissue pieces with normal saline Finally, centrifuge at 2000rpm at 15°C for 5min, discard the supernatant, and obtain undigested and complete tissue pieces for use.

[0074] (4) R...

Embodiment 3

[0077] Example 3: Isolation and cultivation of human umbilical cord-derived mesenchymal stem cells

[0078] (1) Rinse the collected umbilical cord specimen with normal saline to remove residual blood. Weigh, measure the length, and bluntly dissect the arteries, cut the rest of the umbilical cord tissue into 0.5-1cm segments, and put them in a new tissue processor.

[0079] (2) Add type II collagenase to a final concentration of 0.2%, add hyaluronidase to a final concentration of 0.001%, digest at 37°C for 60 minutes, and then stop digestion.

[0080] (3) Naturally settle for 10 minutes, transfer the supernatant cell suspension to a centrifuge tube, dilute with 5 times normal saline, centrifuge at 2500rpm, 15°C for 25 minutes to obtain cell pellets; meanwhile, dilute undigested tissue pieces with normal saline , 2000rpm, 15°C, centrifuge for 5min, discard the supernatant, and obtain undigested and complete tissue pieces for use.

[0081] (4) Resuspend the cell pellet obtained...

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Abstract

The present invention relates to an efficient method for isolating and culturing mesenchymal stem cells from umbilical cords, i.e. a staged suspension explant culture method, comprising the following steps: (1) digesting an umbilical cord in vitro by using a tissue digestive enzyme; (2) culturing the digested cells with a mesenchymal stem cell culture medium for culturing to obtain mesenchymal stem cells; (3) culturing the tissue being not fully digested after digestion by a mesenchymal stem cell culture medium to obtain the mesenchymal stem cells. According to the present invention, the mesenchymal stem cells obtained by the method are in line with mesenchymal stem cell characteristics in surface markers, growth characteristics, multi-directional induced differentiation and other aspects. The method is simple, a lot of mesenchymal stem cells could be obtained at the same time, and the method provides a new way for obtaining mesenchymal stem cells.

Description

technical field [0001] The invention relates to an efficient method for isolating and culturing mesenchymal stem cells, in particular to a method for isolating and culturing mesenchymal stem cells from umbilical cords. Background technique [0002] Mesenchymal stem cells (MSCs) are a kind of pluripotent stem cells with high self-renewal ability and multi-lineage differentiation potential derived from early mesoderm and ectoderm. It can be continuously subcultured and frozen under suitable conditions in vitro, and its morphology, phenotype and differentiation ability have not changed before and after freezing. Mesenchymal stem cells do not express CD80, CD86, HLA-DR and other co-stimulatory molecules [1] It can inhibit mixed lymphocyte reaction in vitro and induce immune tolerance in vivo. It has been clinically proven to have a good therapeutic effect on the prevention and treatment of graft-versus-host disease (GVHD) caused by allogeneic hematopoietic cell transplantation;...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 陈虎张斌陈晓颖盛宏霞徐曼
Owner AFFILIATED HOSPITAL CHINA ACADEMY OF MILITARY MEDICAL SCI
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