[An experimental study of the culture, isolation and biological characteristics of rat adipose tissue-derived stromal cells in vitro].

ABSTRACT

OBJECTIVE: To establish a method for the isolation and culture of rat adipose tissue-derived stromal cells (ADSCs) and explore some biological characteristics of the acquired ADSCs. METHODS: Adipose tissues were isolated from the inguinal fat of SD rats. Primary ADSCs were obtained by the method of collagenase I digestion, inoculated, cultured in the Dulbecco modified Eagle medium with 10% fetal bovine serum, and subcultured at the right moment. The morphology and proliferation characteristics of the cells were observed under the inverted phase contrast microscope every day. Their growth curves were detected and experiments of freezing and resuscitation performed. The third passage ADSCs were induced into osteoblasts by osteogenic inducing fluid and into adipocytes by adipogenic inducing fluid. The osteogenic phenotypes were examined by Von Kossa staining and the adipocytes by Oil Red O staining. RESULTS: ADSCs were successfully obtained and cultured from the rat adipose tissue. They appeared fibroblast-like and could proliferate rapidly in vitro, the third passage having the most active proliferative ability. Calcium nodes characteristic of osteoblasts were observed in the ADSCs on Von Kossa staining after induction with dexamethasone, ascorbic acid and beta-sodium glycerophosphate, and red-stained fats characteristic of adipocytes were noted in the cytoplasm on Oil Red O staining after induction with IBMX, indomethacin and insulin. The ADSCs showed no significant decrease in their proliferation activity and capability of differentiating into diverse cell types after cryopreserved in liquid nitrogen for a month. CONCLUSION: A simple and effective method for the isolation and culture of rat ADSCs was successfully established. The ADSCs obtained could grow and proliferate rapidly in vitro, capable of differentiating into diverse cell types, easy to be preserved and promising to be seed cells for cell therapy and tissue engineering. The procedure of schizolysising erythrocytes with NH4Cl could be omitted in the isolation of the rat ADSCs and dexamethasone is not indispensable in the induction of ADSCs into adipocytes.