A comparison of methods for effective differentiation of the frozen-thawed 3T3-L1 cells

The differentiation efficiency of 3T3-L1 preadipocytes is an essential factor affecting studies on cellular mechanisms associated with obesity, diabetes, and related disorders. Differentiation of 3T3-L1 cells is commonly induced by an adipogenic cocktail containing insulin, dexamethasone (DEX), and 3-isobutyl-1-methylxanthine (IBMX). However, 3T3-L1 cells after freezing and thawing for many times always have a low differentiation efficiency. To solve this problem, we compared the differentiation efficiency of six commonly used adipogenic cocktails and protocols published in 2017. On this basis, we further compared 18 adipogenic cocktails with 2 μM rosiglitazone added and/or with a prolonged treatment with IBMX. The results revealed that the adipogenic cocktail containing 0.5 mM IBMX, 1 μM DEX, and 10 μg/mL insulin was the most effective for the frozen-thawed 3T3-L1 cells differentiation. Rosiglitazone, and IBMX under a prolonged treatment, could improve the differentiation efficiency of the frozen-thawed 3T3-L1 cells. However, the effect was closely related to concentrations of agents in the adipogenic cocktails. •3T3-L1 cells with repeated freeze and thaw have a low differentiation efficiency.•We compared 24 adipogenic cocktails for differentiation of the frozen-thawed 3T3-L1 cells.•The cocktail with 0.5 mM IBMX, 1 μM DEX, and 10 μg/mL insulin was the most effective differentiation mixture.•Rosiglitazone and/or prolonged IBMX treatment could improve the frozen-thawed 3T3-L1 cells differentiation.