2022, Vol. 26 ›› Issue (31): 5002-5007

Modified primary culture and identification of human adipose-derived mesenchymal stem cells

Wang Hui1, Wang Cuiju2, Zheng Junyuan1, Wang Hua3, Guo Baojuan1, Chen Pei1, Yang Xufang1   

Abstract: BACKGROUND: Human adipose-derived mesenchymal stem cells were extracted by conventional enzymatic digestion. The long-term digestion resulted in poor cell viability. To meet the demand for the number of seed cells in tissue engineering, this experiment aimed to find a modified human adipose-derived mesenchymal stem cell culture method.  
OBJECTIVE: Human adipose-derived mesenchymal stem cells were obtained by collagenase I digestion combined with tissue block culture method and the relevant biological characteristics were identified.
METHODS: The adipose tissue was cut up and cultured with collagenase I digestion combined with tissue block culture and conventional collagenase digestion method. Cells were counted by trypan blue exclusion method. The yield, viability and proliferation ability of cells obtained by different methods were compared. The expression of cell surface antigens CD105, CD73 and CD44 was tested by flow cytometry in the modified group. After osteogenic or adipogenic induction, alkaline phosphatase staining and oil red O staining were used to identify cell differentiation ability.
RESULTS AND CONCLUSION: (1) The number of cells obtained by collagenase I digestion combined with tissue block culture method was far more than that obtained by conventional enzyme digestion method. (2) The cells obtained by collagenase I digestion combined with tissue block culture had the strong proliferation ability and good cell viability. They entered the plateau phase 1 day earlier than the conventional collagenase digestion method, and each passage took about 5 days. (3) The expression of CD105, CD73 and CD44 was positive in the modified group of P3 generation, and the purity was over 93%. (4) Human adipose-derived mesenchymal stem cells obtained by the modified method had good osteogenic and adipogenic differentiation capabilities. (5) The results showed that the collagenase I digestion method combined with tissue block culture method could obtain a large number of human adipose-derived mesenchymal stem cells with better viability and proliferation ability than the conventional enzymatic digestion method, and they could be passaged stably with a good differentiation ability, which met the needs of tissue engineering.
Key words: human adipose-derived mesenchymal stem cells, primary culture, collagenase I digestion method, tissue block culture method, modification, yield, proliferation, vitality, differentiation