The Primary Culture Of Human Adipo-derived Stromal Cells And Differentiation Ionto Adipocytes

ObjectiveTo establish a method for primary culture of human adipose tissue-derievd stromal cells(hADSCs),explore their biological properties,and then induce cells into adipocytes.To observe the basical properties and adipocyte differentiation potential of cells,which derived from cryopreserved hADSCs,and to provide laboratory evidences for clinical application of hADSCs.MethodsMononuclear cells were obtained from human abdominal fat tissue by enzymatic digestion.To observed the morphological changes of cultured cells.Cells were cryopreserved in -196℃liquid nitrogen for 6 months,then thawed,to examine the growth features and the potential of differentiation into adipocytes. ADSCs were induced with dexamethasone,insulin,1-methy-3-isobutylxanthine which acted as adipocyte differentiation inducer.Induced cells were stained with Oil Red O to display intracytoplasmic lipid .The number of adipocytes were counted under a phase-contrast microscope.ResultsThe isolated ADSCs comprised a single phenotypic population and displayed a fibroblast-like morphology with active proliferation.Cells were expanded as undifferentiated cells in culture for more than 15 P.After induced with induction medium ,lipid vacuoles can be observed,which displayed a perinuclear accumulation on 3 day.Oil red O staining and morphological changes verified these cells with accumulation of lipid drops in the cytoplasm. Intracytoplasmic lipid stained with Oil Red O suggested a rapid increase in the intracytoplasmic lipid content on 9 day of inoculation,which peaked on day 17. Two weeks later ,more than 90% ADSCs differentiated into adipocytes which displayed a perinuclear accumulation of lipid vacuoles.Between cryopreserved ADSCs and fresh ADSCs,the growth features and adipocyte differentiation potential was similar(P>0.5).ConclusionsThe stromal cells are present in mature human fat tissue,possess good proliferating activity and differentiation potential into adipocytes. ADSCs after cryopresernvation can be also induced to differentiate into adipocytes,and be similar with non- cryopreserved cells.The study suggested ADSCs have enormous potentialities used as seed cells of fat tissue engineering.