## 3 Results
### Cell Analytics  
#### 3.4.1 Flow Cytometry Analysis of ASC52 telo Cells Cultured in Static 2D or Dynamic 3D Conditions  
It was desirable to verify whether the ASC52 telo cells growing on _BR44_ (3D conditions) express the same standard surface markers and maintain the same stemness level as a batch of the same cells cultivated in traditional cell culture vessels (2D conditions). Flow cytometry analysis of some common surface antigens suggested no significant differences between cells grown in 2D and those grown in 3D on _BR44_. Essential markers for stemness are preserved (CD73, CD90, and CD105), and adipogenic markers (CD34, CD36, and CD146) upregulated during adipogenic differentiation are not expressed in 3D conditions \begin{table}
\begin{tabular}{c c c c} \hline  & **Parameter** & _BR44_ & **ProNectin-F** \\ \hline \(\mu\) & (d\({}^{-1}\)) & 0.35 & 0.56 \\ \(t_{d}\) & (d) & 2.01 & 1.24 \\ \(Att_{off}\) & (\%) & 38.7 & 78.2 \\ \(X_{max}\) & (10\({}^{5}\) cells/mL) & 1.2 & 7.5 \\ _EF_ & (-) & 7.0 & 21.3 \\ \(Y_{Lac,Gcl}\) & (mmol/mmol) & 6.35 & 5.29 \\ \(qGlc\) & (pmol/cell/d) & 21.74 \(\pm\) 9.12 & 7.78 \(\pm\) 1.83 \\ \(qLac\) & (pmol/cell/d) & 137.88 \(\pm\) 40.64 & 41.24 \(\pm\) 19.39 \\ \(qAmm\) & (pmol/cell/d) & 7.32 \(\pm\) 3.71 & 1.61 \(\pm\) 0.74 \\ \hline \end{tabular} \(\mu\): Specific cell growth rate. \(t_{d}\): Doubling time of cell population. \(Att_{off}\): Attachment efficiency. \(X_{max}\): Maximum cell concentration on planar growth surface. EF: Expansion factor. \(Y_{Lac,Gcl}\): Lact yield per glucose equivalent. \(qGlc\): Specific glucose consumption rate. \(qLac\): Specific lactate production rate (growth-dependent). \(qAmm\): Specific ammonium production rate (growth-dependent).  
\end{table} 
Table 4: Overview of main growth-dependent parameters. (see Figure 9 and Supplementary Material Figures S12 and S13) [48]. CD61 is a control marker that is usually not expressed by human fibroblasts [49]. 4.2 Expression Levels of Some Essential Genes Involved in Cell Stemness or Cell Differentiation Measured by RT-qPCR  
Several genes have been discovered to seal the undifferentiated status of hMSCs and other genes required for their differentiation (Figure 10A). Further information about a selection of these genes, including a short description and references, can be found in Supplementary Material Tables S6 and S7. Some of them are useful as markers in RT-qPCR assays to check the in vitro expanded cells' differentiation status. In these experiments, ASC52 telo cells were grown in standard cell culture vessels (2D static) or spinner flasks on _BR44_ (3D dynamic). RT-qPCR measured the expression levels of some of the selected marker genes, and the values were compared to those obtained in static 2D conditions. _PREF1_, _WISP2_, _ZFP521_, and _SOX9_ are among the essential genes linked with the undifferentiated status and against differentiation of adipose tissue progenitor cells. Interestingly, an increase in the expression of these markers was observed in 3D cultures. _PPARG_, _ZFP423_, _DKK1_, and _RUNX2_, are related to the commitment to an adipogenic (chondrogenic for _RUNX2_) lineage and differentiation. In 3D cultivations, a down-regulation of two of these important genes (_PPARG_ and _DKK1_) was observed (see Figure 10B), meaning better preservation of the undifferentiated status of the cells compared to the 2D static situation. Figure 9: Flow cytometry data were summarized and depicted using a “Radar Plot” for ASC52 telo cells cultured in xeno- and serum-free conditions (_UrSuppe_). The diagram shows the percentage of positive cells for the respectively indicated surface marker. Cells were cultured either in 2D (T25 flask) or on the _BR44_ (3D condition). 4.3 Comparing the Secretetome Profile of ASC52 telo Cells Grown in Standard Static 2D vs.