Method for separating umbilical cord mesenchymal stem cell effectively

A technology of mesenchymal stem cells and umbilical cords, which is applied in the field of separation and preparation of human umbilical cord mesenchymal stem cells, can solve the problems of isolated cell contamination, inability to completely remove bacteria and other microorganisms, and achieve the goal of less cell damage, less pollution, and lower costs Effect

Active Publication Date: 2013-12-04
BEIJING HEALTH & BIOTECH STEM CELL INST CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the umbilical cord is cleaned before the process of isolating umbilical cord mesenchymal stem cells, this cannot completely remove bacteria and other microorganisms carried on the surface of the umbilical cord, and the process of mechanically breaking the umbilical cord will lead to contamination of the isolated cells

Method used

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  • Method for separating umbilical cord mesenchymal stem cell effectively
  • Method for separating umbilical cord mesenchymal stem cell effectively
  • Method for separating umbilical cord mesenchymal stem cell effectively

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Isolation, culture and cryopreservation of human umbilical cord mesenchymal stem cells

[0024] (1) Separation of umbilical cord mesenchymal stem cells: The umbilical cord tissue ligated at both ends was washed with sterile isotonic solution, and the umbilical cord ligated at both ends can basically ensure that the inner wall of the umbilical vein is not polluted. Cut off 1-2 cm inside the ligation at one end, insert a sterile tube of appropriate length and diameter from the umbilical vein at the cut end, and leave a tube of more than 2 cm outside the umbilical cord (the length and diameter of the tube can be adjusted according to the umbilical vein, Make sure that it can be firmly inserted into the umbilical cord vein, and leave a tube of more than 2cm outside the umbilical cord), and fix the umbilical cord and tube at about 1-2 cm inside the umbilical cord end (for the method of intubation, see figure 1 ), inject 10-25 ml of sterile isotonic solution from th...

Embodiment 2

[0027] Example 2 Expansion and Preparation of Human Umbilical Cord Mesenchymal Stem Cells

[0028] (1) Take 3×10 cells from qualified primary passage cell bank 6 Cell cryopreservation tubes, thaw and recover cells at 37°C, (2) press 2×10 with serum-free medium (LONZA) 4 / cm 2 The density was inoculated into culture flasks, and cultured in a 37°C, saturated humidity, 5% CO2 incubator. When the cells reached 80-90% confluence, they were passaged at a ratio of 1:3. After 3 passages, (3) Use trypLE when the confluency of the third passage reaches 90% TM Digest the cells, count them, resuspend the cells with the prepared cell protection solution at an appropriate concentration, divide them into sterile containers, seal them, and store them in a suitable environment. (4) Take part of the preparation and use the methods known to professionals to conduct pathogenic microorganism inspection of bacteria, fungi and viruses, cell purity inspection, cell viability detection, endotoxin d...

Embodiment 3

[0030] Example 3 Effect of separation method on contamination of umbilical cord mesenchymal stem cells obtained by natural delivery

[0031] The method of this patent and the method of mechanically crushing the umbilical cord and digesting them with collagenase and trypsin were used to separate 80 normal umbilical cords. The sterility test was performed on the umbilical cord collection fluid and the primary culture fluid supernatant cultured after isolation, and the success rate of cell rescue was compared. The calculation formula is as follows:

[0032] Cell rescue success rate = (1-primary supernatant contamination / umbilical cord collection fluid contamination) × 100% Result: Using this patented method: 17 cases of umbilical cord collection fluid contamination, no primary supernatant contamination after separation, and cell rescue The success rate was 100%. However, the method of mechanically crushing the umbilical cord and digesting it with collagenase trypsin: 15 cases of ...

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Abstract

The invention provides a method for separating umbilical cord mesenchymal stem cell simply and effectively, which comprises the steps of separating umbilical cord mesenchymal stem cell with cannulas, digesting umbilical cord mesenchymal stem cell with trypLETM and cultivating umbilical cord mesenchymal stem cell with a serum-free medium. According to the method, an umbilical cord mesenchymal stem cell from a mother's body completely can be obtained relatively fast, and damage on the cell is relatively low; tests show that according to the method, mesenchymal stem cell can be protected to the most extent, the motility rate as well as the activity is relatively high, and adipogenesis and osteogenic induced differentiation can be carried out effectively.

Description

technical field [0001] The invention belongs to a method for isolating and culturing mesenchymal stem cells, in particular to a method for isolating and preparing human umbilical cord mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (mesenchymal stem cells, MSCs) are a type of pluripotent stem cells derived from the mesoderm and ectoderm in early development. They were originally found in the bone marrow because of their following characteristics: 1) Hematopoietic support. As the main cellular component of the hematopoietic microenvironment—the stem / progenitor cells of the stromal cell line, co-transplantation of MSCs and hematopoietic stem cells can promote the engraftment of hematopoietic stem and progenitor cells. 2) Immune regulation. MSCs do not express CD80, CD86, HLA-DR and other co-stimulatory molecules, and can inhibit mixed lymphocyte reaction in vitro, which is of great significance for the prevention and treatment of graft-versus-host...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K35/44A61P37/00
Inventor 韩之海王涛
Owner BEIJING HEALTH & BIOTECH STEM CELL INST CO LTD
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