Figure 2.
Glycolysis is accelerated by increasing medium depth. (A) Medium glucose and (B) medium lactate amounts after 24 hours of culture in medium without cells, medium with cells at 21% atmospheric O2, or cells at 8% atmospheric O2. Decreased O2 levels increase glucose utilization and lactate production (n = 6, donor 2). Increasing medium volume also limits cellular O2 availability, increasing glucose consumption (C shows total glucose levels over time and D is the quantified consumption rate from 4–22 hours) and lactate production (E shows lactate levels over time and F is the quantified production rate from 4–22 hours) (n = 6, donors 3, 4, and 5). (G) Limited cellular O2 availability develops only after several hours in culture as steady-state O2 diffusion gradients develop. This explains why medium volume effects on glucose and lactate (C–F) develop only after 4 hours. The graph shows the drop in medium [O2] 1 mm above the cells following a media change. (H) Change in [O2] 1 mm over cells during the first 12 hours after media change, confirming marked drops in O2 availability only (i) with higher media column heights and (ii) after several hours in culture (n = 6, donor 1). (I–L) 13C6-glucose tracing demonstrates increased m+3 lactate (I–J) and m+3 pyruvate (K–L) production with higher media column heights, confirming higher glycolysis rates with higher media volumes and mimicking trends in unlabeled lactate production from E to F (n = 5–6, donors 3, 4, and 5). All cultures are hfRPE and all metabolite analyses were done from media pooled from apical and basal chambers.