Fig. 1
Influence of cultivation interval on growth kinetics and cell cycle distribution. A Adherent 2D culture-derived human pluripotent stem cells (hPSCs; hHSC_1285) were once detached and seeded as single cells at 5 × 105 cells/mL to stirred tank bioreactors (STBRs). After 24 h, cultivation medium was exchanged via perfusion, while a porous glass filter was installed to retain hPSC aggregates inside the bioreactor. Throughout the culture, pH and dissolved oxygen concentration (DO) were controlled via triggered clock. The pH level was controlled to 7.1 initially by reduction of CO2 in the gas supply and afterwards by addition of 1 M NaHCO3. The DO was similarly regulated at 40% (of air saturation) by adaption of the O2 concentration in the supply gas. In two separate cultures, hPSC aggregates were dissociated inside the bioreactor after 3 or 4 process days and single cells were seeded back into the bioreactor at 5 × 105 cells/mL. This was repeated for a total of 5 passages, resulting in the consecutive passages p1 (grey), p2 (red), p3 (green), p4 (blue) and p5 (orange) (n = 3–11). B–D Exemplary viable cell density, specific growth rate µ and cell cycle analysis for the 7 day lasting historic process (D7) averaged for three independent cell lines. E–J Viable cell density, specific growth rate µ and cell cycle analysis (5 passage average) for a cultivation interval of 4 days (D4; E–G) and 3 days (D3; H–J). K Comparison of cell yield between the historic process (D7, black) and the shortened processes of 3 days (D3, grey) and 4 days (D4, red). Cumulative cell yield was calculated based on average fold expansions. L Cumulative cell yield for a total of 5 passages calculated based on average fold expansion for each individual passage in both approaches D3 and D4. Results are presented as mean ± standard error fo the mean (SEM)