## 3 Results
### Cell Analytics
#### 3.4.1 Flow Cytometry Analysis of ASC52 telo Cells Cultured in Static 2D or Dynamic 3D Conditions
Dynamic 3D Conditions  
Since some polypeptides are only produced and secreted during certain precise stages of development or maturation, they can be used to diagnose a specific metabolic state or a specific differentiation degree of the cells under investigation. To assess the presence of 58 human adipokines, we used a commercially available \"Proteome Profiler Human Adipoking Array\" (see Table 5, Figure 11, and Supplementary Material Figure S14 and Table S8). Interpreting and finding a biological sense for a proteome profiler array is always very difficult, especially if many polypeptides are detected. In this case, which fortunately is not too complex, we notice some trends that allow us to interpret the obtained results. First, the detected polypeptides were already associated by other reports with the adipose tissue secretome (for a review, see [50]). Second, to facilitate the data's comprehension, it is possible to group the secreted polypeptides according to some characteristic traits that unite them. Two signature adipokines (adiponectin and leptin) for differentiated cells were undetectable in the test membranes, and the spot corresponding to Nidogen/Entactin was very weak. These results prove that most ASC52 telo cells remain undifferentiated and immature. We detected three Cathepsin family members. These proteins are proteases that play different crucial roles in mammalian cellular turnover. Cathepsin S promoted human preadipocyte differentiation [51] and was found only in the culture medium of cells grown under 2D conditions. A critical role of the Insulin-like growth factor-binding proteins (IGFPs) is to regulate Insulin-like growth factors (IGF-I and IGF-II) action by controlling the accessibility of these peptide hormones to their receptors [52]. Both IGF-I [53] and IGF  
Figure 10: Expression ratio measured by RT-qPCR of some essential genes which regulate positively or negatively the adipocyte differentiation process. (**A**) The illustration schematically shows the relationship between different factors that positively or negatively controls adipogenesis. Two of them are particularly important: PPREF1 is known to inhibit adipogenesis and, PPARG, considered the master regulator of adipose tissue development and differentiation. The blue dotted line represents the various differentiation stages that lead to a mature adipocyte starting from a progenitor cell. Further information and references about the marker genes used can be found in Supplementary Material, Tables S6 and S7. (**B**). Representative experiment showing differences in marker genes expression between ASC52 telo cells grown in static 2D or defined dynamic 3D conditions. The normalized expression ratio of the different markers for the defined dynamic 3D tests is related to the static 2D condition values. II [54,55] are pro-adipogenic. It has been shown that IGFBP-4 binds IGF-I and its expression negatively correlates with adipose tissue growth [56]. IGFBP-6 is not well characterized but is likely to be anti-adipogenic, too, as it can bind IGF-II [57]. Pregnancy-associated plasma protein-A (PAPP-A) is a zinc metalloproteinase regulating local insulin-like growth factor (IGF) action through cleavage of inhibitory IGFBPBPs, in particular IGFBP-4. This is the primary mechanism by which PAPP-A enhances local IGF bioavailability for receptor activation. PAPP-A is expressed primarily by pre-adipocytes in fat tissue [58]. So, IGFBP-4, IGFBP-6, and PAPP-A belong to a circuit that regulates IGFs' availability, which in turn positively regulates adipogenesis. IGFBP-4 and IGFBP-6 were detected in the supernatants of cells cultured in 2D and 3D/PNF MC conditions, but not when the _BR44_ MC was used, whereas PAPP-A only in 2D condition. The proteome profiler array also highlighted the presence of some cytokines [59] and chemokines [60] in the cell-conditioned culture media: IL-6, CXCL/IL-8, CCL2/MCP-1, MIF, and CCL5/RANTES. Interestingly, IL-6, CCL2/MCP-1, and CCL5/RANTES were detected only in cells grown in 2D conditions.