## 3 Results
### Cultivation Studies
#### 3.3.2 Proof-of-Concept: Spinner Cultivation with _BR44_ and ProNectin-F MCs  
Table 4 summarizes the results of the proof-of-concept cultivation. The data shows that the ASC52 telo cells proliferated on the _BR44_ MC under dynamic conditions (growth curves can be found in Supplementary Material Figure S11). A maximum specific growth rate of up to 0.35 d\({}^{-1}\) was obtained, which corresponded to a doubling time of 2.0 d. Thus, a maximum cell density of 1.2 \(\times\) 10\({}^{5}\) cells/mL (EF 7.0) was achieved at the end of the cultivation. Cell growth on the ProNectin-F MC was 1.6-times faster (\(\mu_{max}\) = 0.56 d\({}^{-1}\) and \(t_{d}\) = 1.24 d). Thus, the maximum cell density (7.5 \(\times\) 10\({}^{5}\) cells/mL) and the expansion factor (21.3) were 7.5 and 3.1-times higher, respectively, after nine days of cultivation. This might result from the lower cell attach  
Figure 8: Confocal laser-scanning microscopy analysis of _BR44_ MCs after two days of culture. (**A**) The strong natural autofluorescence of the cells can be exploited to visualize the hASCs grown on the surface of the _BR44_ carrier. (**B**) The DAPI staining reveals the presence of several nuclei on the surface of _BR44_. (**C**) Merged. Magnification 400\(\times\), scale bar 100 \(\upmu\)m.  
Figure 7: SEM microphotographs to confirm the presence of ASC52 Telo cells on MC _BR44_ surface after four days of culture in static conditions. Fold magnification: (**A**): 50\(\times\), scale bar 2 mm (**B**,**C**): 250\(\times\), scale bar 300 \(\upmu\)m (**D**): 500\(\times\), scale bar 200 \(\upmu\)m. Interestingly, the cells are tiny and packed together, which is a feature often found with undifferentiated/immature cells. ment efficiency of the _BR44_ MC compared to the ProNectin-F MC. In both cases, the ASC52 telo cells consumed glucose (\(q_{Gcl,BR44}\): 21.7 pmol/cell/d, \(q_{Gcl,PNF}\): 7.78 pmol/cell/d) and produced lactate (\(q_{La,BRA4}\): 137.8 pmol/cell/d, \(q_{La,PNF}\): 41.2 pmol/cell/d) and ammonia (\(q_{Amm,BRA4}\): 7.3 pmol/cell/d, \(q_{Amm,PNF}\): 1.6 pmol/cell/d). In both cultivations, no critical substrate or metabolite concentrations were achieved (\(Lac_{PNF}\) = 26.87 mmol/L; \(Lac_{BR44}\) = 21.31 mmol/L, \(Amm_{PFN}\) = 0.96 mmol/L, \(Amm_{BR44}\) = 0.65 mmol/L); however, cells growing on _BR44_ MC could experience a high localized concentration of lactate, which would constantly be released from the MC core. This is suggested by the calculated amount of lactate (in pmol) released per cell and per day, which is three times higher for the _BR44_ MC (\(\sim\)138 pmol/cell/day) than for ProNectin-F MC (\(\sim\)41 pmol/cell/day). This high concentration of localized lactate may have been the reason for the slower growth rate and doubling time observed for the tests performed with _BR44_ MC, as lactate is known to inhibit cell growth [44]. Furthermore, we noticed that cultures based on the _BR44_ MC easily generate clusters, even under dynamic conditions. This phenomenon is much less relevant when ProNectin-F MC was used. Cell-MC clusters could lead to a decrease in cell density due to nutrient diffusion limitations to aggregate centers. The calculated average Y\({}_{lactate/glucose}\) values are superior to the theoretical values of 2 for both MCs type (\(Y_{Lac/Gcl,BR44}\) = 6.35; \(Y_{Lag/Gcl,PNF}\) = 5.29). This suggests that alternative substrates such as glutamine, serine, and possibly other amino acids are used for energy generation and contribute to the measured lactate concentration [45]. During these catabolic reactions, ammonia is produced, which we detected in significant quantities, especially in the presence of MC _BR44_ (\(\sim\)4.5 times higher than with MC ProNectin-F), supporting the previous suggestion. It is well known that glutamine is chemically labile in the cell culture medium, and thus ammonia could arise through spontaneous hydrolyzation of this amino acid [46; 47]. However, the amounts detected could not be explained with only the spontaneous degradation of glutamine in cell culture conditions.