Optimization of Adipogenic Differentiation Protocol for Murine and Human Cell Culture Models

Adipogenic differentiation efficiency remains highly variable across laboratories and cellular models, underscoring a critical need for a robust and standardized protocol. Here, we describe an optimized and highly effective protocol for inducing adipogenesis in multiple models, including murine 3T3-L1 preadipocytes, stromal vascular fraction (SVF) from neonatal and adult mice, and human adipose-derived stem cells (hADSCs). Systematic optimization was performed on key parameters such as initial cell confluence, induction timing, inducer composition, and culture surface coating. We show that high cell density, rosiglitazone supplementation, and an extended primary induction phase combine to promote lipid accumulation. Notably, we introduce a crucial modification-prolonged low-dose insulin stimulation during the maintenance phase-that is essential for the efficient differentiation of adult SVF. Furthermore, when applied to hADSCs, the protocol consistently induced robust adipogenesis, confirming its cross-species applicability. Taken together, this comprehensive and reproducible protocol serves as a valuable tool for advancing in vitro adipogenesis research. Key features • Extend a robust, standardized adipogenic differentiation protocol from 3T3-L1 preadipocytes to clinically relevant models, including hADSCs and the heterogeneous SVF. • Identify key optimized parameters-cell density, induction timing, and inducer composition-enabling highly reproducible differentiation across species.