Adipocyte differentiation is affected by media height above the cell layer

BACKGROUND: 3T3-L1 cells have been widely used as a model for adipogenesis. However, despite its popularity, differentiation of this cell line has been reported to be inconsistent with low efficiency. OBJECTIVE: To investigate the effect of media height during adipocyte differentiation on lipid accumulation and adipokine secretion in mature adipocytes. METHODS: Three cell lines (3T3-L1, OP9 and ChubS7) were used to test the influence of media volume on adipogenesis. Total lipid content and lipid droplet size and number were quantified. Adipocyte related gene expressions were quantified during the course of differentiation. Secretion of leptin and adiponectin from mature adipocytes were measured using enzyme-linked immunosorbent assays. The influence of oxygen partial pressure on adipogenesis was investigated using three oxygen percentages: 5, 21 and 30%. Insulin sensitivity was measured by insulin inhibition of isoproterenol-induced lipolysis and phosphorylation of insulin receptor substrate-1. RESULTS: A lower media height during adipogenesis increased total lipid accumulation, NEFA release and leptin and adiponectin secretion in mature adipocytes. Insulin sensitivity was not affected by media height during differentiation. CONCLUSION: Media height during adipogenesis was inversely correlated with lipid content in mature adipocytes. To achieve a high lipid content and greater adipokine secretion, it is best to use a low media volume during differentiation.