Fig. 5
Cryopreservation of suspension-derived hPSCs. A Intermediate cryopreservation of suspension culture-derived cells was established to fully enable matrix-free cultivation of hPSCs in stirred tank bioreactors. After 3 passages of each 4 days, cells were harvested after dissociation inside the bioreactor and cryopreserved in different cell densities as single cells. After thawing, these cells were directly transferred to bioreactors for an additional 4 days of cultivation. The cryopreservation cell densities (CPDs) tested were 20 (CPD20, yellow), 40 (CPD40, purple), 60 (CPD60, dark green), 80 (CPD80, dark orange) and 100 × 106 cells/mL (CPD100, brown) (n = 3). B The recovery after thawing was calculated as a ratio of the amount of cryopreserved cells and the viable cell count after thawing. C Recovery on d1 was calculated as a ratio of the amount of the starting cell number on process day 0 (aimed to be at 0.5 × 106 cells/mL) and the viable cell count on process day 1. D Viable cell density (bar chart) and viability (line graph) throughout 4 process days of cultivation after thawing cells cryopreserved at different cell densities. E Viability of cells directly after thawing. F Viability of cells on process day 1 (corresponding to line graph in D). G Specific growth rate µ calculated based on viable cell densities. Results are presented as mean ± SEM