Fig. 6

Influence of intermediate cryopreservation of hPSCs on long-term, matrix-free suspension culture in STBRs. A–G After 3 passages of D4 cultivation (12 days), hHSC_1285 hiPS cells were cryopreserved at 100 × 10⁶ cells/mL for direct inoculation of bioreactors with these cells. Depicted here are the viable cell density (A), cell cycle analysis (averaged over 5 passages; B), metabolic conversion rates (qGlc as continuous line, qLac as dashed line; C), specific growth rate µ (D), aggregate diameter distribution (E), metabolic yield coefficient (F) and the recovery on d1 (G) for hHSC_1285 in a D4 cultivation approach over 3 passages (not shown) pre- and 5 passages post-cryopreservation. This resulted in the consecutive passages p3 + 1 (grey), p3 + 2 (red), p3 + 3 (green), p3 + 4 (blue) and p3 + 5 (orange) (n = 3). Results are presented as mean ± SEM. H Representative immunocytological stainings of cryosections of hPSC aggregates derived after 3 + 5 passages in D4 (32 days) stained for markers of an undifferentiated state TRA-1-60, OCT-3/4, SSEA-4 and SOX2 (scale bar = 100 µm). (I) Exemplary flow cytometry analysis plots for surface markers associated with an undifferentiated state TRA-1-60 and SSEA-3 as well as transcriptions factors OCT-3/4 and NANOG and proliferation marker KI67. Cells were harvested at process endpoint (3 + 5 Passages; 32 days). J Karyotype of cells cultivated for 3 + 5 passages in D4 approach (32 days). K Undirected differentiation of D4 process-derived aggregates after 5 passages revealed expression of markers representing the three germ layers ectoderm (based on TUBB3), endoderm (based on SOX17 and FOXA2) and mesoderm (based on Vimentin). Scale bar = 100 µm. (L) Exemplary flow cytometry analysis plots of cardiomyocyte-specific markers NKX2.5, MHC, SA and cTNT after directed differentiation of cells at the end of passage 3 + 5