Separation and purification method of umbilical cord mesenchymal stem cells

A technology for separation and purification of mesenchymal stem cells, applied in the field of separation and purification of umbilical cord mesenchymal stem cells, can solve the problems of difficult separation of cells, difficulty in controlling the degree of digestion, unstable effect of enzymatic digestion, etc. The expansion of mesenchymal stem cells and later clinical application, no effect on cell activity, fast and efficient separation, culture and purification

Pending Publication Date: 2020-01-03
GUANGDONG VITALIFE BIOTECHNOLOGY CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The economic cost of enzymatic digestion culture method is high, generally 4 to 5 hours, and collagenase is generally used for digestion, but the degree of digestion is not easy to control. If the digestion is not enough, a sufficient number of cells cannot be obtained, but if the digestion time is too long, the enzymes used will damage the cells. If the damage is too large, the cells will die in large numbers
And because the digestive juice is a viscous liquid, it is not easy to separate the cells during centrifugation, so the effect of the enzyme digestion method is unstable
Although the process of tissue block culture method is simple and easy, and the economic cost is low, the primary culture time is too long, and it usually takes 14-20 days to obtain a small number of primary cells

Method used

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  • Separation and purification method of umbilical cord mesenchymal stem cells
  • Separation and purification method of umbilical cord mesenchymal stem cells
  • Separation and purification method of umbilical cord mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] A method for separating and purifying umbilical cord mesenchymal stem cells includes the following steps:

[0046] 1. Collection of umbilical cord tissue.

[0047] The hospital collects fetal placental tissues from full-term caesarean section and signs the customer’s informed consent before collection. The tissues are transported to the laboratory in a refrigerated aseptic environment at 4°C. During transportation, tissue protection fluid is used to protect the biological activity of placental tissues. The saline is prepared by adding 25μg / ml gentamicin sulfate and 5μg / ml amphotericin B to ensure no bacterial and fungal contamination during transportation.

[0048] 2. Separate the umbilical cord tissue.

[0049] 1. Clean and separate.

[0050] Separate the umbilical cord tissue in a sterile laboratory, wash the umbilical cord tissue 2-3 times with pre-cooled tissue cleaning solution, and clean the sticky blood stains on the tissue to make the umbilical cord tissue translucent. T...

Embodiment 2

[0075] A method for separating and purifying umbilical cord mesenchymal stem cells is basically the same as the method in Example 1, except that the content of neutral protease in the tissue digestion fluid is 1.5 mg / ml.

[0076] Among them, the cell growth curve of primary culture is as image 3 As shown, it can be seen from the figure that the time for the cells to enter the logarithmic growth phase is longer than that of Example 1.

Embodiment 3

[0078] A method for separating and purifying umbilical cord mesenchymal stem cells is basically the same as the method in Example 1, except that the content of neutral protease in the tissue digestion fluid is 2.5 mg / ml.

[0079] Among them, the cell growth curve of primary culture is as Figure 4 As shown, it can be seen from the figure that the time for the cells to enter the logarithmic growth phase is longer than that of Example 1.

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Abstract

The invention relates to a separation and purification method of umbilical cord mesenchymal stem cells and belongs to the technical field of stem cells and regenerative medicine. The method comprisesthe steps as follows: separation of umbilical cord tissue: taking the umbilical cord tissue, cleaning the umbilical cord tissue and removing blood, and shearing the tissue into tissue fragments for later use; tissue digestion: taking the tissue fragments, and adding tissue digestive juice for oscillation digestion, wherein the tissue digestive juice contains neutral protease, hyaluronidase, collagenase II and DNA enzyme; after digestion, adding a stop buffer for stop, performing filtration to obtain undigested tissue blocks and filtrate, and centrifuging the filtrate to remove supernatant to obtain cells; and cell culture: inoculating a culture flask with the tissue blocks and cells, using serum-free culture medium for culture to obtain the umbilical cord mesenchymal stem cells. The separation and purification method of umbilical cord mesenchymal stem cells overcomes the defects of an enzyme digestion method and a tissue block culture method in the prior art and can obtain enough primary cells within 3-5 days.

Description

Technical field [0001] The invention relates to the technical field of stem cells and regenerative medicine, in particular to a method for separating and purifying umbilical cord mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSC) are a type of pluripotent stem cells derived from the mesoderm with high self-renewal and multi-differentiation capabilities. They can be cultured and expanded in vitro and have multi-differentiation capabilities under specific conditions. They can differentiate into fat Cells, chondrocytes, bone cells, liver cells, and even lateral differentiation into nerve cells, epidermal cells, etc., MSC has broad clinical application prospects and is the most important seed cell in the field of regenerative medicine and tissue engineering. Mesenchymal stem cells were first found in bone marrow tissues. With the deepening of research, they have also been found in various tissues of the human body, such as bone marrow, fat, umbilical c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0665C12N2509/00
Inventor 江嘉豪何美弟李静静赵蓝
Owner GUANGDONG VITALIFE BIOTECHNOLOGY CO LTD
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