Method for culturing human umbilical cord mesenchymal stem cells through combination of adherence density gradient method and EGF (Epidermal Growth Factor)

A density gradient, quality stem cell technology, applied in the biological field, can solve the problems of unsatisfactory cell volume and low content, and achieve the effect of an effective separation and purification method and simple operation.

Inactive Publication Date: 2013-04-03
陆华
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Problems solved by technology

Because the content of UC-MSCs in umbilical cord Walton jelly tissue is low, it cannot meet the cell quantity required as seed cells for cell therapy

Method used

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  • Method for culturing human umbilical cord mesenchymal stem cells through combination of adherence density gradient method and EGF (Epidermal Growth Factor)

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Embodiment Construction

[0015] Specifically, such as figure 1 The flow chart of the method for culturing human umbilical cord mesenchymal stem cells by the adherent density gradient method combined with epidermal growth factor (EGF) in an embodiment of the present invention is shown, and the method specifically includes the following steps:

[0016] S1, the separation of human umbilical cord Walton jelly mesenchymal stem cells (UC-MSCs):

[0017] 1) Under sterile conditions, 10cm of the umbilical cord of a healthy fetus with cesarean section and full-term pregnancy was immersed in 0.01mol / L PBS and stored at 4°C; the umbilical cord was taken out of the culture medium in the ultra-clean bench, containing 1% double antibody and 250ug Rinse the outer surface of the umbilical cord with D-Hank's solution of / mL amphotericin, and insert a 20mL syringe into two umbilical veins and one umbilical artery for repeated washing to remove the residual blood in the umbilical vein and artery;

[0018] 2) Tear off t...

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Abstract

The invention provides a method for culturing human umbilical cord mesenchymal stem cells through combination of an adherence density gradient method and as EGF (Epidermal Growth Factor). The method comprises the following steps of: seperation of the UC-MSCs (Umbilical Cord Mesenchymal Stem Cells), primary culture of the UC-MSCs, subculture and augmentation of the UC-MSCs, cryopreservation and resuscitation of the UC-MSCs and detection of UC-MSCs immunophenotyping. According to the method, 4ng/ml of EGF is added by adopting the adherence density gradient method, adherent culture and digestion time control are combined, so that the UC-MSCs with higher purity and activity is obtained; and the method combining the adherence density gradient method and the EGF has the advantages of simple operation, rapidness, practicability and the like and is an effective method for separating and purifying the UC-MSCs.

Description

technical field [0001] The invention relates to a method for cultivating human umbilical cord mesenchymal stem cells in the field of biotechnology, in particular to a method for cultivating human umbilical cord mesenchymal stem cells using an adherent density gradient method combined with epidermal growth factor (EGF). Background technique [0002] The umbilical cord is a cord-like structure connected between the umbilicus of the embryo and the placenta. It is surrounded by amniotic membrane and contains differentiated mucous connective tissue. In addition to the atresia yolk sac and allantois, there are umbilical arteries and umbilical veins in the connective tissue. Umbilical vessels are surrounded by mucin-like tissue, called umbilical jelly or Wharton's jelly (Wharton's jelly, WJ), which is rich in hyaluronic acid, forms a hydrogel structure around fibroblasts, and contains hematopoietic , mesenchymal, neural and endothelial stem / progenitor cells. Walton jelly mesenchym...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 陆华
Owner 陆华
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