Influence of RGD in Elastin-Based Coatings on Morphology and Differentiation of Three-Dimensional Adipocyte Spheroids

Three-dimensional (3D) in vitro adipocyte models provide physiologically relevant platforms for studying adipogenesis and obesity-related metabolic dysfunction. However, long-term adipocyte culture is often hindered by limited cell–matrix adhesion and spheroid detachment. Previously, we demonstrated that elastin-like polypeptide (ELP)–polyethyleneimine (PEI) coatings functionalized with a trivalent RGD motif enhanced spheroid retention during frequent media changes. The present study investigates the long-term functional consequences of RGD incorporation over a 28-day culture period. 3T3-L1 preadipocytes were seeded, differentiated, and matured on ELP-PEI or ELP-(RGD)3-PEI coatings. Spheroid morphology, triglyceride content, expression of PPAR-γ, adiponectin, HIF-1α genes, and insulin-stimulated glucose uptake were assessed. Both coatings supported initial spheroid formation, but only ELP-PEI maintained the 3D architecture and supported adipogenic maturation and insulin responsiveness. ELP-(RGD)3-PEI promoted early retention but led to spheroid disassembly by mid-culture; notably, by day 28, cells reaggregated into abnormally large spheroids with impaired metabolic function, likely due to continued proliferation. These findings highlight the critical role of extracellular matrix-mediated cell–cell versus cell–substrate interactions in maintaining 3D culture fidelity. While RGD enhances adhesion, it disrupts spheroid integrity and compromises adipogenic and metabolic maturation. Taken together, ELP-PEI coatings offer a more conducive microenvironment for long-term 3D adipocyte culture and hold promise for modeling obesity-associated metabolic dysfunction in vitro.