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Method for preparing human umbilical cord mesenchymal stem cells

A technology of mesenchymal stem cells and human umbilical cord, applied in the field of preparation of umbilical cord mesenchymal stem cells, can solve the problem of not being able to obtain MSCs for multiple passages, and achieve the effect of simple operation

Active Publication Date: 2009-11-11
TIANJIN AMCELLGENE ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But using the endothelial digestion method, only 30% of the umbilical cord samples can get MSCs, and 70% of the samples can't get MSCs that can be passaged multiple times

Method used

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  • Method for preparing human umbilical cord mesenchymal stem cells
  • Method for preparing human umbilical cord mesenchymal stem cells
  • Method for preparing human umbilical cord mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Cell isolation and culture

[0026] (1) Wash the human umbilical cord repeatedly with phosphate buffered solution (PBS) to remove residual blood, and cut it into pieces to about 1mm 3 Add 0.1% collagenase by weight of raw materials, and digest at 37°C for 45 minutes; (2) Add trypsin (Sigma, USA) with 0.125% by weight of raw materials to digest for 45 minutes at 37°C, stir with a rotor during the digestion process, add An appropriate amount of serum terminates the action of pancreatic enzymes; (3) Filter the digestive juice with a 100-mesh and 200-mesh filter successively to remove undigested tissue; (4) Dilute the filtered digestive juice with phosphate buffer , the ratio of digestive solution to phosphate buffer is 1:10; (5) centrifuge to obtain cells, and the speed of centrifuge is 1500rpm / min to obtain cells; (6) cells are obtained by 1~2×10 4 / cm 2 Inoculate in a 25ml plastic culture bottle, use 10ml serum-free medium (Jiangxi Xiehe Stem Cell Company), place at 37...

Embodiment 2

[0028] (1) Wash the human umbilical cord repeatedly with phosphate buffered solution (PBS) to remove residual blood, and cut it to about 0.05mm 3 Add 0.05% collagenase by weight of raw materials, and digest at 37°C for 60 minutes; (2) Add 0.05% by weight of raw materials of trypsin (Sigma, USA) to digest for 60 minutes at 37°C, stir with a rotor during the digestion process, add An appropriate amount of serum terminates the action of pancreatic enzymes; (3) Filter the digestive juice with a 100-mesh and 200-mesh filter successively to remove undigested tissue; (4) Dilute the filtered digestive juice with phosphate buffer , the ratio of digestive solution to phosphate buffer is 1:8; (5) centrifuge to obtain cells, and the speed of the centrifuge is 1000rpm / min for 20min to obtain cells; 6 / cm 2 Inoculate in a 75ml plastic culture bottle, use 30ml serum-free medium (Stemcell company, Germany), put 37 ℃, 5% CO 2 Culture in an incubator, change the medium after 4-5 days, discard...

Embodiment 3

[0030] (1) Wash the human umbilical cord repeatedly with phosphate buffered solution (PBS) to remove residual blood, and cut it to about 1.5mm 3 Add 0.2% collagenase by weight of raw materials, and digest at 37°C for 30 minutes; (2) Add 0.2% by weight of raw materials of trypsin (Sigma, USA) to digest for 30 minutes at 37°C, stir with a rotor during the digestion process, add An appropriate amount of serum terminates the action of pancreatic enzymes; (3) Filter the digestive juice with a 100-mesh and 200-mesh filter successively to remove undigested tissue; (4) Dilute the filtered digestive juice with phosphate buffer , the ratio of digestion solution to phosphate buffer is 1:12; (5) centrifuge to obtain cells, and the speed of the centrifuge is 2000rpm / min for 10min to obtain cells; 6 / cm 2 Inoculate in 25ml plastic culture bottle, use 15ml serum-free medium (Gibco company, USA), set 37 ℃, 5% CO 2 Culture in an incubator, change the medium after 4-5 days, discard non-adhere...

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Abstract

The invention discloses a method for preparing human umbilical mesenchymal stem cells, which comprises the following steps: (1) removing residual blood from the human umbilical cord, shearing the umbilical cord and digesting the umbilical cord by collagenase; (2) adding pancreatin into the umbilical cord to digest the umbilical cord; (3) filtering the digestive solution by using a screen, and removing tissues which are not digested; (4) diluting the filtered digestive solution by using phosphate buffer solution; (5) centrifugating the solution to obtain cells; and (6) inoculating the cells to a culture medium to culture so as to obtain large amount of human umbilical cord mesenchymal stem cells. The obtained cells can meet the requirements of clinical and experimental study, and lay a foundation for the future application.

Description

technical field [0001] The invention relates to a preparation method of umbilical cord mesenchymal stem cells. 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. They were initially found in the bone marrow and have attracted increasing attention because of their following characteristics: (1) Multiple differentiation potential. It can differentiate into fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, myocardium, endothelial and other tissue cells under specific induction conditions in vivo / in vitro, and it still has multi-directional differentiation potential after continuous subculture and cryopreservation , can be used as ideal seed cells for tissue engineering. (2) Hematopoietic support and implantation promotion. As the main cell component of the hematopoietic microenvironment—the stem / progenitor cells of the stromal cell line, MSCs r...

Claims

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Application Information

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IPC IPC(8): C12N5/08
Inventor 韩忠朝刘拥军吕璐璐孟磊龚伟
Owner TIANJIN AMCELLGENE ENG
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