Standard 2D cultures inadequately mimic the natural microenvironment of mesenchymal stromal cells (MSCs), compromising their properties. This study investigated the impact of 3D cultures in spheroids, alginate microspheres (AMSs), and blood plasma scaffolds on human-adiposederived MSC behavior. The cell morphology, viability/apoptosis (6-CFDA/Annexin-Cy3.18), actin filament development (phalloidin-FITC), and metabolic activity (Alamar Blue) were assessed on the 3rd day of the generated 3D construct cultures. The abilities for adipogenic and osteogenic differentiation were evaluated after 21 days of culture in media with inducers by Nile Red and Alizarin Red staining, respectively. The 3D culture supported closer-to-physiological cell interactions and morphology and resulted in F-actin reduction compared with the 2D culture. While the metabolic activity was elevated in the scaffolds, it was significantly reduced in the spheroids and AMSs, which reflected natural-like quiescence. The differentiation was maintained across all the 3D constructs. These findings highlight the essential influence of 3D construct design on MSC function, underscoring its potential for advancing both in vitro models and cell-based therapies.
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