Method for rapidly obtaining and purifying bone marrow stroma stem cells

Abstract

The invention relates to a method for rapidly obtaining and purifying bone marrow stroma stem cells. The method comprises the following steps: re-suspending bone marrow stroma cells (P0) obtained from a bone marrow cavity by using an anticoagulant-containing serum-free cell culture fluid; culturing a suspension of the obtained P0 cells by using a serum-containing culture solution, and obtaining a first generation of bone marrow stroma stem cells (P1) by using a composite collagenase differential digestion method; carrying out double-fluorescence discrimination on stem cell surface CD-marking and non-stem cell surface CD-marking by using a flow cytometer to rapidly increase the proportion of bone marrow stem cells; culturing by using the serum-containing culture solution again; and finally, obtaining a second generation of bone marrow stroma stem cells (P2) with high purity by using the composite collagenase differential digestion method again. The method has the benefits that the bone marrow stroma cells (P0) obtained from the bone marrow cavity are re-suspended by employing an anticoagulant-containing serum-free culture medium alpha-MEM so as to prevent hematopoietic cells from aggregating and precipitating at the bottom of a culture dish and from interfering wall-attaching of the bone marrow stroma stem cells.

Description

technical field [0001] The invention relates to a method for obtaining and purifying stem cells, in particular to a method for quickly obtaining and purifying bone marrow stromal stem cells. Background technique [0002] In recent years, studies have found that the bone marrow of adult mammals contains a type of mesenchymal cells—bone marrow stromal cells (BMSCs), which have the potential to differentiate into skeletal muscle cells, Vascular endothelial cells, central neurons and glial cells, etc. [0003] There are a variety of cell components in bone marrow tissue. In addition to differentiated cells such as stromal cells, it also contains two types of pluripotent stem cells: hematopoietic stem cells and mesenchymal stem cells. In 1987, Friedenstein et al. found that single adherent bone marrow cells cultured in plastic culture dishes could differentiate into various types of cells under certain conditions, and they could still maintain their multidirectional differentiat...

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Problems solved by technology

[0005] In the previous studies on obtaining bone marrow mesenchymal stem cells, most of them were usually prepared by immunomagnetic bead separation, density gradient centrifugation and differential attachment method, but the purity and speed of the obtained BMSCs often did not meet the requirements

In addition, since the proliferation rate of BMSCs during in vitro culture is almost the same as the division rate of fibroblasts mixed in, after several days of culture, the growth of fibroblasts reduces the purity of BMSCs, although fibroblasts tend to There is more cell matrix formed around it, which is different from the less cell matrix formed by bone marrow stromal stem cells. However, the trypsin digestion method usually used requires multiple passages to gradually reduce the interference of fibroblasts and achieve a satisfactory result. Purity of BMSCs

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