Abstract: BACKGROUND: Adipose-derived mesenchymal stem cells can regulate endocrine metabolism, maintain the homeostasis of microenvironment around adipocytes, participate in the development of adipocytes, and play an important regulatory role in lipid metabolism and the occurrence and development of obesity. At present, type I collagenase digestion is mainly used for the culture of adipose-derived mesenchymal stem cells in vitro, but it has the disadvantages of long digestion time and low efficiency. How to select appropriate digestive enzymes to shorten digestion time and improve experimental efficiency is worthy of attention and exploration.  
OBJECTIVE: To establish a method for primary culture of rat adipose-derived mesenchymal stem cells in vitro, and to provide important cell carriers for clinical development of stem cell therapy and gene therapy. 
METHODS: Under aseptic condition, the adipose tissue from the bilateral groin and epididymis of rats was taken out, cut into paste with iris scissors. Totally 2 mL of 0.1% type II collagenase was added for digestion for 45 minutes. After washing and centrifugation, the adipocytes were inoculated in low-sugar DMEM complete medium containing 10% fetal bovine serum and placed in the CO2 incubator for primary culture. When the fusion rate reached 80%-90%, the cells were subcultured at the ratio of 1:3. The morphological changes of cells were observed under the inverted microscope. The fourth generation of adipose-derived mesenchymal stem cells was detected for specific cell surface marker CD molecules, and the adipogenic and osteogenic differentiation experiments were carried out. 
RESULTS AND CONCLUSION: (1) After 3 days of culture, a small number of short spindle cells crawled out. 5 days later, the cells grew like colonies or clusters, and the shape was long spindle. After 6-8 days, the colonies fused 80%-90% with each other, arranged in a vortex and then subcultured. In the third generation, the cells grew rapidly and could be subcultured again 48 hours after inoculation, and the cells had uniform morphology and showed obvious fish like growth. (2) Flow cytometry showed that CD90, CD73, and CD29 were positive, while CD34, CD45, and CD11b/c were negative. (3) At 7-10 days after adipogenic differentiation of the fourth generation of adipose-derived mesenchymal stem cells, cell morphology gradually changed from a long spindle to a polygonal or round shape, and fat droplets of various sizes accumulated in the cytoplasm. Oil red O staining exhibited that fat droplets were bright and deep red. At 21 days after osteogenic differentiation, the cells gradually changed from a long spindle shape to a polygonal shape, growing in an aggregated shape, with small black particles of varying sizes on the surface, and mineralized crystals were visible in some parts. Alizarin red staining displayed “mushroom-like” dark red spherical nodules scattered in different sizes. (4) It is concluded that this experiment successfully established a simple and efficient method for primary culture of rat adipose-derived mesenchymal stem cells.

Key words: stem cells, adipose-derived mesenchymal stem cells, chemical enzyme digestion method, primary culture, identification, rat