Figure 3.

Culture Density Affects the Cells' Metabolism and Culture Medium

(A) AnnexinV/PI analysis did not detect any difference between the culture densities for the fractions of live, apoptotic, or dead cells (n = 4; two-way ANOVA).

(B) Cell cycle profile of hESC growing in the four culture densities. We detected a constant fraction of cells in S phase, while the fraction of cells in G2/M phase decreases and the G1 fraction increases significantly (n = 4; two-way ANOVA).

(C) ROS concentration was measured by flow cytometry after staining with 2′,7′-dichlorodihydrofluorescein diacetate. No difference could be detected between the four groups (n = 4; ANOVA). Concentration of aspartate (D), glutamine (E), glucose (F), and lactate (G) on day 5 after 24 hr incubation differs between the different culture densities and on mouse embryonic fibroblast feeders (MEF) only (n = 9, p < 0.05; ANOVA).

(H) pH of the culture medium on day 5 after 24 hr incubation on the different culture densities or on feeders only decreases significantly (n = 5, p < 0.05; ANOVA).

(I and J) Evolution of lactate concentration and pH of the culture medium over the 5 days, after overnight incubation on the four culture densities and on feeders only (n = 3).

Results are presented as mean ± SEM. Asterisk marks differences with p ≤ 0.05.