Method for separating and culturing human placental-derived mesenchymal stem cell

Abstract

The invention relates to a method for separating and culturing a human placental-derived mesenchymal stem cell. Aiming at problems and defects in an existing common PMSCs separation method, the technical scheme for solving the problems comprises the following steps: 1, taking out placenta by big tweezers, and cutting the placenta; 2, peeling the maternal surface and the infant surface of the placenta; 3, digesting placental villus; 4, diluting the digestive juice with 1000ml of PBS, filtering and centrifuging to collect cells for later use; 5, resuspending the cells with PBS, and centrifugally precipitating the cells; 6, subculturing the cells; and 7, freezing the cells. According to the method, PMSCs is separated by a one step digestive treatment with collagenase I, the operation steps can be reduced, cell damage can be lightened, the proportion of target cells can be effectively improved, and pollution of other cells can be reduced, so that the time for cell primary culture can be shortened, the cell purity can be improved, the cost can be greatly reduced, the original state of PMSCs can be well maintained, and a firm foundation is laid for massive production and wide clinical application of PMSCs in the future.

Description

technical field [0001] The invention relates to the separation and cultivation of stem cells, in particular to a method for the separation and cultivation of human placenta-derived mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) have high clinical application value due to their characteristics such as self-renewal, multi-directional differentiation, hematopoietic support, and immune regulation. In recent years, they have become a research hotspot in the field of stem cells. Major breakthroughs have been made in various blood system diseases, cardiovascular diseases, liver cirrhosis, nervous system diseases, autoimmune diseases, etc. [0003] MSCs were first discovered in bone marrow. At present, bone marrow is still the main source of MSCs, but the acquisition method is invasive and the source is limited, and the number and differentiation ability of bone marrow-derived MSCs (bone marrow derived MSCs, BMSCs) will vary with the age of the d...

AI Technical Summary

Problems solved by technology

[0005] The separation methods of PMSCs are different, and the separation effect is also very different. Among them, the number of primary cells cultured by the tissue block method grows slowly, and only a small number of cells in this mixed cell population conform to the surface antigen expression characteristics of MSCs; the method of mixed enzyme digestion requires tissue The block is digested multiple times, the operation is complicated, the chance of contamination is increased, and it is easy to cause cell damage; because MSCs lack specific surface markers, a large number of cells will be lost by flow cytometry or magnetic bead sorting, and the cost is high

For the in vitro culture of PMSCs, fetal bovine serum (FBS) is mostly added at present, but the use of animal-derived products is not allowed in clinical applications, and only expensive serum-free special media can be selected, resulting in a substantial increase in the cost of cell culture