Mesenchymal stem/stromal cells (MSC) are capable of renewing the progenitor cell fraction or differentiating in a tissue-specific manner. Adipogenic differentiation of adipose-tissue-derived MSC (adMSC) is important in various pathological processes. Adipocytes and their progenitors are metabolically active and secrete molecules (adipokines) that have both pro- and anti-inflammatory properties. Cell culturing in 2D is commonly used to study cellular responses, but the 2D environment does not reflect the structural situation for most cell types. Therefore, 3D culture systems have been developed to create an environment considered more physiological. Since knowledge about the effects of 3D cultivation on adipogenic differentiation is limited, we investigated its effects on adipogenic differentiation and adipokine release of adMSC (up to 28 days) and compared these with the effects in 2D. We demonstrated that cultivation conditions are crucial for cell behavior: in both 2D and 3D culture, adipogenic differentiation occurred only after specific stimulation. While the size and structure of adipogenically stimulated 3D spheroids remained stable during the experiment, the unstimulated spheroids showed signs of disintegration. Adipokine release was dependent on culture dimensionality; we found upregulated adiponectin and downregulated pro-inflammatory factors. Our findings are relevant for cell therapeutic applications of adMSC in complex, three-dimensionally arranged tissues.

Mesenchymal stem/stromal cells (MSC) are capable of progenitor cell fraction renewal or tissue-specific differentiation. These properties are maintained during in vitro cultivation, making them an interesting model system for testing biological and pharmacological compounds. Cell cultivation in 2D is commonly used to study cellular responses, but the 2D environment does not reflect the structural situation of most cell types. Therefore, 3D culture systems have been developed to provide a more accurate physiological environment in terms of cell–cell interactions. Since knowledge about the effects of 3D culture on specific differentiation processes is limited, we studied the effects on osteogenic differentiation and the release of factors affecting bone metabolism for up to 35 days and compared them with the effects in 2D culture. We demonstrated that the selected 3D model allowed the rapid and reliable formation of spheroids that were stable over several weeks and both accelerated and enhanced osteogenic differentiation compared with the 2D culture. Thus, our experiments provide new insights into the effects of cell arrangement of MSC in 2D and 3D. However, due to the different culture dimensions, various detection methods had to be chosen, which in principle limits the explanatory power of the comparison between 2D and 3D cultures.

Adipose tissue includes various cell types beyond the typical adipocytes. The stromal vascular fraction (SVF) contains mesenchymal stem cells (MSCs), pericytes, and endothelial cells, which can be isolated from adipose tissue by mechanical and enzymatic methods. The composition of the SVF is heterogeneous, and donor factors such as sex, age, body mass index (BMI), and harvesting site are associated with variations in cellular composition and viability. The expression of specific surface markers, which determine the immunophenotype of the cells, can also vary. In this study, we investigated the effects of donor age, BMI, and harvesting site on cell yield, viability, and size. Our results showed that BMI significantly influenced cell yield and size, with overweight and obese donors yielding more cells than normal-weight donors. Additionally, cells isolated from the adipose tissue of the thighs/legs were larger than those from other areas. Flow cytometry showed considerable variability in SVF composition among donors. These results emphasize that SVF donor characteristics have a significant impact on cell yield, viability, and cell size, with the immunophenotype being highly donor-dependent. Understanding these factors is crucial for optimizing cell yield and defining populations for therapeutic applications of SVF cells.