## 3 Results
### Cell Analytics
#### 3.4.1 Flow Cytometry Analysis of ASC52 telo Cells Cultured in Static 2D or Dynamic 3D Conditions
On the other hand, IL-8 and MIF were detected in 2D and 3D (see Table 5 and Figure 11). Pentraxin 3 (PTX3), also known as tumor necrosis factor (TNF)-inducible gene 14 protein (TSG-14), is a pattern recognition molecule that functions in innate immunity and is a useful biomarker of a pro-inflammatory status [61]. Remarkably, this polypeptide was detected only in the supernatants of cells cultured in 2D and 3D/PNF MC conditions, but not when the _BR44_ MC was used. These observations suggest that the defined dynamic 3D conditions might lead the cells to secrete fewer inflammatory factors, and _BR44_ MC seems more suitable than PNF MC to induce this phenomenon. As a last interesting factor found only in the supernatant of cells grown in 2D or 3D combined with _BR44_ MC, there is Serpin E1/PAI-1. Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor family member, inhibits fibrinolysis and has complex cellular matrix interactions. PAI-1 deficiency or inhibition stimulates adipocyte differentiation. Therefore it can be considered an anti-adipogenic factor [62]. In summary, these results suggest that the secretion profile of ASC52 telo depends on the cultivation system (standard static 2D vs. dynamic 3D). Interestingly, when these cells grew on _BR44_ in a dynamic system, the secretion profile is less complex. This may suggest that the cells remain more immature, thus preserving better \"stemness\". This observation is in good agreement with the results obtained by RT-qPCR. Ultimately, culturing ASC52 telo cells on MCs in a dynamic 3D cell culture system using the _UrSuppe_ medium seems to enhance the cells' undifferentiated status and reduces the secretion of pro-inflammatory mediators. \begin{table}
\begin{tabular}{c c c c} \hline
**Analyte/Control** & **2D** & **3D** & **3D** \\  & **ASCs 52 telo** & **ASCs 52 telo** _BR44_ & **ASCs 52 telo** _ProNectin-F_ \\ \hline Cathepsin D & X & X & X \\ Cathepsin L & X & X & X \\ Cathepsin S & X & & \\ \hline IGFBP-4 & X & & X \\ IGFBP-6 & X & & X \\ Pappalysin-1/PAPP-A & X & & X \\ \hline \end{tabular}
\end{table} 
Table 5: Summary of the relevant secreted factors of ASC52 telo cells cultured in standard cell culture vessels (2D) or dynamic conditions (spinner flask, 3D). In this latter case, the cells were cultured either on _BR44_ MC or commercial ProNectin-F (PNF) MC. Interestingly, the secretion profile of cells grown in 3D is less complex than the one obtained in the 2D condition. Moreover, when the _BR44_ MC is used, the number of secreted polypeptides detected is further reduced compared to PNF MC. These results suggest that the secretome of ASC52 telo cells can be influenced by the cell culture conditions (2D vs. 3D) and the microcarrier type on which they grow (_BR44_ vs. PNF). A less complex profile of secreted adipokines in 3D conditions may also signify that the ASC52 telo better preserve their undifferentiated status when they grow on MCs in a defined dynamic system. The _UrSuppe_ medium was used in all conditions tested for these investigations. For more details about these experiments, see Supplementary Material Figure S14.