Method and system for separating, culturing and amplifying human umbilical cord mesenchymal stem cells

Abstract

The invention provides a method and system for separating, culturing and amplifying human umbilical cord mesenchymal stem cells. The method comprises the following steps: step I, pretreating a sample; step II, obtaining Wharton's jelly, cutting the obtained Wharton's jelly into small tissue, and adding a filtered red blood cell lysis buffer solution of which the volume is 3.5 times that of the small tissues; step III, digesting collagenase, wherein a digestion solution is a balanced salt solution containing 1% of IV type collagenase, 0.5% of hyaluronidase, 300U / ml DNA enzyme and 2% of a serum substitute; step IV, performing primary cell isolated culture: adding a serum-free culture medium of mesenchymal stem cells to obtain a cell suspension, and adding a special primary cell culture solution for umbilical cord mesenchymal stem cells for primary culture; step V, subculturing the cells, and inoculating the collected cells according to 5000 cells / cm < 2 > by using trypsin with the mass percent concentration of 0.215%; and step VI, collecting the umbilical cord mesenchymal stem cells, and performing osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation experiments.

Description

technical field [0001] The invention belongs to the technical field of stem cells, and in particular relates to a method for separating, culturing and expanding human umbilical cord mesenchymal stem cells and a system thereof. Background technique [0002] Mesenchymal stem cells (MSCs) refer to a type of adult stem cells with self-renewal and multilineage differentiation potential, mainly derived from mesoderm tissue. Since MSCs were first discovered in bone marrow and successfully isolated and cultured in the 1960s, subsequent researchers have isolated MSCs from fat, placenta, umbilical cord, dental pulp and other tissues. MSCs can differentiate into multiple germ layers and types of cells, such as epithelial cells of the ectoderm; adipocytes, chondrocytes, and bone cells of the mesoderm; insulin-secreting cells of the endoderm. MSCs come from various sources, are easy to isolate and expand, have low immunogenicity, strong renewal and differentiation capabilities, and a wi...

AI Technical Summary

Problems solved by technology

Although the enzymatic digestion method can obtain a large number of primary cells for the first time, due to the double damage of mechanical shear force and digestive enzymes during the extraction process, the cell adhesion effect and morphology are poor, and the digestion time of tissue blocks is limited in the large-scale production process. It is difficult to quantify uniformly, and the cost of separation and extraction is high, and the processing time is relatively long

The conventional tissue block culture method generally takes a long time for primary culture, which takes about 15 days, mainly because the size of the shredded Wharton's jelly tissue block and the first adherence treatment affect its separation and extraction efficiency.

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