Method for separating and culturing umbilical cord blood mesenchymal stem cells

Abstract

The invention provides a method for separating and culturing umbilical cord blood mesenchymal stem cells. The method comprises the steps of carrying out secondary separation on umbilical cord blood mononuclear cells by using a separating medium, and then inoculating the umbilical cord blood mononuclear cells into a culture bottle in which fibronectin and CD90 monoclonal antibodies are coated; and culturing for 4-5 days by using a serum-free culture system and simulating the in-vivo low-oxygen growth environment of the mesenchymal stem cells, then, removing suspension cells, further culturing adherent cells, and subculturing after the fusion rate of primary cells is up to 60%. By using the method for separating and culturing umbilical cord blood mesenchymal stem cells, provided by the invention, the problems of adherence infirmness, low culture success rate, low purity and the like caused in the extraction and culture processes of the umbilical cord blood mesenchymal stem cells are effectively solved, and the safety in clinical application is improved. In addition, the application ranges of the umbilical cord blood mesenchymal stem cells are widened, the utilization values of the umbilical cord blood mesenchymal stem cells are developed, and the clinical application prospects of the umbilical cord blood mesenchymal stem cells are widened.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a method for separating and culturing umbilical cord blood mesenchymal stem cells. Background technique [0002] Stem cells are primitive cells with certain self-renewal ability and multi-directional differentiation potential in a suitable microenvironment, which can differentiate into fat, cartilage, bone, muscle, tendon, nerve, liver, cardiac muscle and other tissue cells. Mesenchymal stem cells (mesenchymal stem cells, MSC) are non-terminal differentiated cells derived from mesoderm, which have the characteristics of mesenchymal cells, endothelial cells and epithelial cells, and are an important source of seed cells for cell therapy and tissue engineering . In addition to bone marrow, peripheral blood, fat, and umbilical cord, human umbilical cord blood is also an important source of mesenchymal stem cells. In addition to the above-mentioned differentiation ability, umbilical cor...

AI Technical Summary

Problems solved by technology

In the process of use, if the amount of samples to be separated is small, the use of cell separation medium can form a better mononuclear cell layer, and it is also convenient to collect; but if the amount of samples to be separated is large, not only the amount of separation liquid used is large, And after centrifugation, some red blood cells may still be suspended in the separation medium, resulting in the collection of cells mixed with more red blood cells, and the red blood cell layer is also mixed with nucleated cells, resulting in a cell yield between 20% and 40%.

The second category is the method of separating cells with different antigenicities on the cell surface, such as magnetic bead method, immunofluorescence labeling method, flow cytometry, etc. The common problems of these methods are: first, there is no recognized stem cell marker ; The second is that the cost is too expensive; the third is that the cells may be damaged during the labeling process

The patent (CN102002479A) uses flow cytometry to sort CD105-positive cells for tissue engineering seed cells, but uses cultured adherent cells and then sorts them. The problem is that the specificity of a single marker is not high. The difficulty of long culture time of umbilical cord blood mesenchymal stem cells has not been solved

However, due to the complex composition of fetal bovine serum, the use of serum-containing medium to culture cells has potential cytotoxicity, foreign virus and pathogenic factor contamination, which makes the standardization of cell culture and the purification of final products more difficult, and the residue of heterogeneous serum clinically allergic reactions

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