10.1016/j.jbiotec.2017.01.004 [PubMed] [CrossRef] [Google Scholar] 27. long\term expansion of hiPSCs. Expansion of hiPSCs in the large\scale bioreactor led to a successful production of 5.4??109 hiPSCs, thereby achieving sufficient cell amounts for clinical applications. Conclusions In conclusion, the results show a significant effect of the inoculum density on cell expansion, differentiation and production of hiPSCs, emphasizing the importance of the inoculum density for downstream applications of hiPSCs. Furthermore, the bioreactor technology was successfully Rabbit polyclonal to ACTL8 applied for controlled and scalable production of hiPSCs for clinical use. for 3?minutes and incubated overnight at 37C and 5% CO2. On the following day, the formed embryoid bodies were removed from the plate using a trimmed pipette tip with a 1?mL pipette and transferred to wells of non\treated 12\well culture plates (Costar?, Corning?, NY, USA) for expression analysis or to Lumox plates (Sarstedt, Nmbrecht, Germany) for immunohistochemical staining. Also, the mTeSR medium was replaced with E6\medium,16 consisting of 96.8% DMEM\F12 (Gibco?; Thermo Fisher Scientific), 2% insulin\transferrin\selenium (Gibco?; Thermo Fisher Scientific), 1% Pen Strep (Gibco?; Thermo Fisher Scientific) and 0.2% l\Ascorbic Acid (Sigma\Aldrich/Merck). Embryoid bodies were cultured over 15?days in total; during the culture period, half of the medium was removed and replaced with fresh E6\medium three times per week. 2.7. Gene expression analysis Gene expression analysis was performed as described previously15, 17 using human\specific primers KRas G12C inhibitor 1 and probes as listed in Table ?Table2.2. Expression values of measured genes were normalized to expression values of the housekeeping gene glyceraldehyde\3\phosphate dehydrogenase (GAPDH), and fold changes KRas G12C inhibitor 1 of expression levels were calculated using the test. Gene expression data were compared between AS 10 and AS 50, corresponding 2D cultures and embryoid bodies by one\way analysis of variance (ANOVA). Slope values obtained in the CellTiter\Blue? Cell Viability Assay as well as cell quantification data, population doublings and doubling times were compared using the unpaired, two\tailed Student’s test. 3.?RESULTS 3.1. Metabolic activity of KRas G12C inhibitor 1 hiPSCs during bioreactor expansion For comparative evaluation of the hiPSC growth behaviour in the two analytical\scale bioreactors (AS) and the large\scale bioreactor (LS), glucose and lactate were measured as indicators for the energy metabolism of the cells. Time courses of glucose consumption and lactate production revealed significant differences between AS 10 and AS 50 (Figure ?(Figure2A,B).2A,B). The area under curve (AUC) of AS 50 was significantly larger KRas G12C inhibitor 1 compared with the AUC of AS?10 (and (Figure ?(Figure3A,B)3A,B) revealed only slight changes in pluripotency of bioreactor cultures KRas G12C inhibitor 1 and 2D cultures compared with the undifferentiated state. For the embryoid bodies, however, a distinct reduction in and expression was detected, which was significant for compared with 2D cultures ((Figure ?(Figure3C)3C) with highest values being detected for embryoid bodies and for AS 50. Gene expression measurements for the other two endodermal markers, (Figure ?(Figure3D)3D) and (Figure ?(Figure3E)3E) revealed an increase compared with the undifferentiated state in AS 10 and AS 50. For showed the highest value for the embryoid bodies, which was significantly higher compared with AS 10 and AS 50 ((Figure ?(Figure2F)2F) revealed a comparable increase in AS 10 and AS 50, while LS?50 had a noticeable lower increase in expression. The expression data for the second marker of the ectodermal lineage, (Figure ?(Figure3G),3G), showed the strongest increase for embryoid bodies, with expression values being significantly higher compared with AS 10 and AS 50 as well as the 2D cultures ((Figure ?(Figure3H)3H) showed a similar gene expression for all tested groups. In contrast, values for (Figure ?(Figure3I),3I),.