Method for separating MSC (Mesenchymal Stem Cell) climbing sheets of umbilical tissue

Abstract

The invention relates to a method for separating MSC (Mesenchymal Stem Cell) climbing sheets of umbilical tissue. The method comprises the following steps of: taking an umbilical cord of a newborn baby and cleaning with a phosphate buffer solution; crushing the umbilical cord; filtering the crushed umbilical cord with a rough filter screen and a filter screen of 200 meshes to obtain an umbilical tissue block with the diameter of 1-1.5mm; draining water and inoculating the umbilical tissue block in a culture dish; putting the culture dish in a 37 DEG C culture box containing 5% of CO2 for 3h; adding culture fluid D-MEM (Dulbecco-Modified Eagle Media)/F12 containing 20% of cow's serum and 10ng/ml of EGF (Epidermal Growth Factor) and putting back to the culture box for 5 days; adding trypsinase to the culture dish and putting dry cells in digestive juice; filtering the digestive juice by the filter screen of 200 meshes; and centrifugating the filtrate and removing supernatant fluid to obtain mesenchymal stem cells of the umbilical tissue. The invention has simpler and more convenient operation, keeps the original activity and the growth state of the cells, improves the yield of the MSCs and avoids the loss action of collagenase on the stem cells and prevents the pollution danger by introducing animal-based proteins and pathogens into animal original collagenase.

Description

[technical field] [0001] The present invention relates to a method for separating stem cells by using the chemotactic properties of stem cells, in particular to a method for separating umbilical cord tissue mesenchymal stem cells by climbing sheets. [Background technique] [0002] Mesenchymal stem cells (English: mesenchymal stem cells, referred to as: MSCs) is a type of stem cells, with two important characteristics of stem cells: strong self-proliferation ability and multi-differentiation potential. MSCs originated from mesoderm, and theoretically, it can differentiate to other mesoderm tissues. Recent studies have shown that under suitable in vivo or in vitro conditions, MSCs can not only differentiate into mesenchymal tissues derived from mesoderm, but also maintain the differentiation potential of endoderm, and can differentiate into nerve cells, epithelial cells, cardiomyocytes, etc. [0003] In recent years, the understanding of the biological properties and potentia...

AI Technical Summary

Problems solved by technology

However, excessive digestion of collagenase will lead to changes in some living substances on the cell surface, which will affect the vitality of the cells and have a damaging effect on the cells.

There are certain requirements for the time of enzymatic digestion of umbilical cord tissue. If the digestion time is too long, it will affect the activity of destroying the obtained cells, and if the digestion time is too short, it will affect the cell yield.

In addition, the umbilical cord tissue to be digested is not a completely uniform system, so it is difficult to ensure the uniformity of the degree of digestion of all cells

Therefore, it is also difficult to grasp the optimal digestion time, which affects the quantity and quality of the obtained cells, which in turn affects future research and use.

In addition, because the existing collagenase is derived from animal viscera, when the enzyme digestion method is used for isolation and culture, there is contamination of animal-derived protein and animal-derived pathogens, which increases the risk of clinical application of stem cells

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