Figure 1

Pluripotent stem cell bioprocess optimization in small scale suspension cultures. (a) Box Behnken Experimental Design. This experiment used 14 wells of the cassette - 12 wells for points along the edges of the experimental design and 2 centrepoint replicates. This experimental design allows for the identification of higher-order effects while using fewer than the 24 wells available in the Micro-24 cassette. (b) Micro-24 Cassette. The PRC-Cell culture cassette with Type A caps is loaded into the Micro-24 system. (c) Significant parameters in quadratic regression of PSC expansion. Bold factors are significant, including seeding density, feeding, interaction, and quadratic factors of these two parameters. Block effect was also found to be significant, which indicates variability between passage number of the same cell line. (d) Surface response curve of significant parameters investigated in the bioreactor expansion of HES2 PSCs. Seeding density and feeding frequency were found to significantly influence the fold expansion of HES2 cells, with both quadratic and interaction effects between these two parameters also observed. (e) Representative FACS Plot of PSCs after six days of bioreactor expansion. In all conditions tested, >90% Oct4 Sox2 cell populations were observed. (f) Relationship between fold expansion and aggregate radius for cells seeded at 2*10⁵ cells/mL. While frequent feeding yields the highest fold expansion and is correlated to larger final aggregate radius after six days of expansion, it also results in the highest variability in aggregate size. Error bars represent one standard deviation. (g) Formation and growth of PSC aggregates spontaneously in bioreactor over six days of culture.