Zeitouni Nathalie E, Fandrey Joachim, Naim Hassan Y, von Köckritz-Blickwede Maren
Department of Physiological Chemistry, University of Veterinary Medicine Hannover.
Institute of Physiology, University Clinics Essen, University of Duisburg-Essen, Essen.
Hypoxia (Auckl). 2015 Oct 9;3:53-66. doi: 10.2147/HP.S85625. eCollection 2015.
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 O 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 O levels over time than expected when cultured under normoxia conditions. On average, O 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 O within the first 60 minutes and dropped to 0.39 and 0.61 mmHg O 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 O 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 O level should be carefully considered in oxygen-dependent experiments.
测量Caco-2细胞培养的三种不同系统中的氧水平。
将Caco-2细胞培养于三种不同系统中，分别使用传统聚苯乙烯24孔板、特殊的24孔透气板或传统板中的膜插入物。在常氧（143 mmHg）条件下培养6天以及在低氧（7 mmHg）条件下培养6小时的过程中，使用光学传感器点来测量这些培养细胞中的溶解氧水平。采用蛋白质免疫印迹分析来测定不同培养物中缺氧诱导因子1α（HIF-1α）的蛋白质水平。
在常氧条件下培养时，所有培养系统随时间推移显示的氧水平均低于预期。平均而言，24孔板中的氧水平低至25 mmHg，但透气板和膜插入物中的氧水平分别保持在97 mmHg和117 mmHg。在低氧条件下，1 mL细胞培养物在最初60分钟内平衡至7 mmHg的氧水平，在6小时孵育期后，24孔板和透气板中的氧水平分别降至0.39 mmHg和0.61 mmHg。膜插入物中的培养物在6小时孵育期结束时未平衡至7 mmHg，最低氧测量值达到23.12 mmHg。不同Caco-2培养物全细胞裂解物中HIF-1α蛋白质水平的蛋白质免疫印迹显示，在24孔板和透气板中低氧孵育1、2和4小时后，HIF-1α有明显的稳定。对于膜插入物，低氧孵育4小时后可见明显的HIF-1α。
在不同的低氧Caco-2培养系统中实现了细胞氧耗竭。然而，不同培养系统之间不同的氧水平表明，在依赖氧的实验中应仔细考虑氧水平。
