Nathalie E Zeitouni, Joachim Fandrey, Hassan Y Naim, Maren von Köckritz-Blickwede

Measuring oxygen levels in three different systems of Caco-2 cell culture. Caco-2 cells were cultured in three different systems, using conventional polystyrene 24-well plates, special 24-well gas permeable plates, or on membrane inserts in conventional plates. Optical sensor spots were used to measure dissolved O2 levels in these cultured cells over the course of 6 days under normoxia (143 mmHg) and for 6 hours under hypoxia (7 mmHg). Western blot analysis was used to determine the protein levels of hypoxia-inducible factor 1α (HIF-1α) in the different cultures. All culture systems displayed lower O2 levels over time than expected when cultured under normoxia conditions. On average, O2 levels reached as low as 25 mmHg in 24-well plates but remained at 97 and 117 mmHg in gas permeable plates and membrane inserts, respectively. Under hypoxia, 1 mL cell cultures equilibrated to 7 mmHg O2 within the first 60 minutes and dropped to 0.39 and 0.61 mmHg O2 in 24-well and gas permeable plates, respectively, after the 6-hour incubation period. Cultures in membrane inserts did not equilibrate to 7 mmHg by the end of the 6-hour incubation period, where the lowest O2 measurements reached 23.12 mmHg. Western blots of HIF-1α protein level in the whole cell lysates of the different Caco-2 cultures revealed distinct stabilization of HIF-1α after hypoxic incubation for 1, 2, and 4 hours in 24-well plates as well as gas permeable plates. For membrane inserts, notable HIF-1α was seen after 4 hours of hypoxic incubation. Cellular oxygen depletion was achieved in different hypoxic Caco-2 culture systems. However, different oxygen levels comparing different culture systems indicate that O2 level should be carefully considered in oxygen-dependent experiments.