Growth arrest induction of 3T3-L1 preadipocytes by serum starvation and their differentiation by the hormonal adipogenic cocktail

The current methods for differentiation of 3T3-L1 preadipocytes start with growth arrest by contact inhibition. However, on day 6, 3T3-L1 preadipocytes reliably detach and differentiation could no longer proceed. In the present study, we used serum starvation to induce growth arrest in 3T3-L1 preadipocytes and investigated their differentiation by the modified hormonal adipogenic cocktail. About 85% of 3T3-L1 cells were in G0/G1 stage under serum deprivation. There were no significant difference between the percentage of cells in G0/G1 phase under these serum deprivation condition and that of post-confluent cells. Growth assays indicated that cells under serum starvation condition grew slow, two-days later than growing and post-confluent cells. We determined that 0.5% FBS for 48 h was the optimal condition to arrest 3T3-L1 cells at the G0/G1 phase. The grow-arrested 3T3-L1 preadipocytes were induced to differentiation by hormonal adipogenic cocktail. After 19 days of differentiation, over 90% 3T3-L1 cells exhibited adipocyte morphology with ring-like lipid droplet in cytoplasm. The mRNA levels of critical transcriptional factor CCAAT/enhancer binding proteins-α and peroxisome proliferator-activated receptor-γ were determined to validate the differentiation. These results indicated that serum starvation effectively arrested the growth of 3T3-L1 preadipocytes and prevented cells from detachemnt caused by contact inhibition at confluence. Furthermore, growth arrested 3T3-L1 preadipocytes by serum starvation successfully differentiated into adipocytes by the hormonal adipogenic cocktail.