Comparative analysis of select 3D human mesenchymal stromal cell-derived spheroid models for adipogenic differentiation

In vitro modelling of adipose tissue (AT) commonly employs 2D systems, which do not fully capture the complexity of AT, lacking cell-cell interactions and extracellular matrix (ECM) remodelling. Despite the innovations in 3D culture systems offering physiologically relevant AT platforms, the field lacks consensus and cross-protocol method comparison. We performed a comparative analysis of three scaffold-free spheroid-generation techniques: Ultra Low Attachment plates (ULA), Hanging Drops (HD), and Microwell Culture Plates (MCP), utilizing human AT-derived mesenchymal stromal cells. Each method produced viable spheroids of similar size and morphology within 48 hours. However, MCP demonstrated greater uniformity and a significant increase in mass density when compared to ULA and HD. Adipogenic media increased spheroid size in ULA and MCP, but not in HD spheroids. Notably, the diameter of ULA spheroids was 2- and 6-times larger compared to HD and MCP ones, respectively, regardless of adipogenic induction. Gene expression analysis indicated that all techniques led to PPARγ and ADIPOQ expression during adipogenesis. All methods supported adipocyte differentiation, as evidenced by lipid droplet accumulation, PLIN1, CD36, and HSL expression, and ECM rearrangement. These findings suggest that all the described techniques constitute a robust platform for producing adipogenesis-competent spheroids, while method-specific differences suggest an application-dependent choice.