Umbilical cord mesenchymal stem cell (MSC) separation culture and amplification method

A technology for the isolation and culture of mesenchymal stem cells, which is applied in the field of isolation, culture and expansion of umbilical cord mesenchymal stem cells. It can solve the problems of long culture time and the influence of tissue block adhesion on the separation efficiency, etc., and achieves the simple method, low cost, fast and efficient separation. Effect

Pending Publication Date: 2019-12-06
孟明耀 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The conventional tissue block culture method takes a long time for the primary culture, which takes 14-20 days, mainly because the size and adhesion of the tissue block affect its separation efficiency

Method used

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  • Umbilical cord mesenchymal stem cell (MSC) separation culture and amplification method
  • Umbilical cord mesenchymal stem cell (MSC) separation culture and amplification method
  • Umbilical cord mesenchymal stem cell (MSC) separation culture and amplification method

Examples

Experimental program
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Effect test

Embodiment 1

[0046] 1) Cleaning, under aseptic conditions, obtain fresh umbilical cord, rinse the surface of the umbilical cord with a cleaning solution, and clean and remove blood and other sundries remaining in blood vessels on the umbilical cord;

[0047] 2) Treatment, remove the amniotic layer of the umbilical vein and the surface of the umbilical cord with a scalpel after cleaning, then cut the umbilical cord into thin strips, and then cut the thin strips of umbilical cord into 2mm 3 The umbilical cord tissue block; this operation method is relatively simple and saves time, only the umbilical cord is Wharton's glue, and Wharton's glue is the main source of umbilical cord mesenchymal stem cells;

[0048] 3) culturing, laying the umbilical cord tissue block on a cell culture dish, adding the water into a culture solution containing 10% fetal bovine serum for culturing; It is beneficial for cells to climb out of the tissue block and obtain more primary cells;

[0049] 4) inoculation and...

Embodiment 2

[0056] A method for isolating, culturing and expanding umbilical cord mesenchymal stem cells, comprising the following steps:

[0057] 1) Cleaning, under aseptic conditions, obtain fresh umbilical cord, rinse the surface of the umbilical cord with a cleaning solution, and clean and remove blood and other sundries remaining in blood vessels on the umbilical cord;

[0058] 2) Treatment, remove the amniotic layer of the umbilical vein and the surface of the umbilical cord with a scalpel after cleaning, then cut the umbilical cord into thin strips, and then cut the thin strips of umbilical cord into 3mm 3 The umbilical cord tissue block; this operation method is relatively simple and saves time, only the umbilical cord is Wharton's glue, and Wharton's glue is the main source of umbilical cord mesenchymal stem cells;

[0059] 3) culturing, laying the umbilical cord tissue block on a cell culture dish, volatilizing the water and adding it into a culture solution containing 20% ​​fet...

Embodiment 3

[0067] After being screened for infectious diseases, and after signing the informed consent and donation agreement, the umbilical cord was aseptically collected after delivery. Remove residual blood and other impurities from blood vessels.

[0068] Remove the umbilical artery, remove the umbilical vein and the amniotic layer on the surface of the umbilical cord with a scalpel, and cut the umbilical cord into thin strips (see figure 1 ), finally cut into 1-3mm 3 The size of the organization block.

[0069] Spread the tissue on a cell culture dish, and add complete culture solution containing 10-20% fetal bovine serum after the water evaporates.

[0070] Change the medium for 3 days of cell culture, change the medium every 2-3 days, adhere to the wall (see figure 2 ) After 4-5 days, the cells can be seen climbing out of the tissue block, and after 7-10 days, the umbilical cord mesenchymal stem cells can reach 80-90% fusion;

[0071] Digest cells with 0.0625% trypsin / EDTA, p...

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Abstract

The invention discloses an umbilical cord mesenchymal stem cell (MSC) separation culture and amplification method. The umbilical cord MSC separation culture and amplification method comprises the steps of (1) clearing, (2) treatment, (3) culture, and (4) inoculation and passage. The umbilical cord MSC method obtained by adopting a tissue mass separation method is simple, the cost is low, cell activity is good, purity is high, the final harvested cell concentration is high, the obtained MSC method is simple, MSCs can be quickly and efficiently separated, the culture time is shortened, after culture is conducted for 4-5 days, it can be seen that the cells climb out from tissue masses, the final harvested cell concentration is high, and the entire umbilical cord MSC culture process can be standardized, programmed and normalized.

Description

technical field [0001] The invention relates to the technical field of stem cell isolation and culture, in particular to a method for the isolation, culture and expansion of umbilical cord mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are cell populations with self-renewal, high proliferation and multi-lineage differentiation potential, and are the origin cells of animal organisms and various tissues and organs. Recent studies have shown that mesenchymal stem cells exist in many adult tissues (such as bone marrow, muscle, gastrointestinal tract, skin, retina, peripheral and central nervous system, etc.). MSCs have a wide range of sources, are easy to isolate and expand in vitro, and have the potential to differentiate into three germ layer cells; they have low immunogenicity, cannot stimulate allogeneic immune responses, and are not killed by cytotoxic T cells and NK cells. "Homing" repair, tumor tropism, and immune regulation. [0003...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0668C12N2509/00C12N2500/40
Inventor 孟明耀侯宗柳王文举高慧赵旖旖李琳解燕华唐维伟
Owner 孟明耀
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