An investigation of the molecular and cellular biology of human physioxic cell culture

Abstract

Researchers routinely culture cells under controlled temperature (37 °C) and CO2 (5%) conditions to mimic the temperature and pH levels of the human body. Oxygen is an essential parameter that is often neglected in the majority of cell culture and experimental procedures. Though human cells require oxygen, the atmospheric oxygen levels (21% O2; 160 mmHg; normoxia) are much higher relative to the oxygen conditions experienced in vivo (~12 – 1% O2; ~92 mmHg – 7 mmHg; physioxia). We have not only found that basic cellular functions operate differently in physioxia, but both cancerous and primary cells benefit from being cultured under physioxic conditions compared to normoxic conditions. Under physioxia, human cells utilize two cap-dependent protein synthesis machineries that act upon two discreet pools of mRNA, strongly suggesting that physioxia cultured cells have a different proteome compared to normoxia cultured cells. Additionally, under physioxia cells display increased viability, growth, and metabolism and decreased oxidative damage, oxidative stress response, DNA breaks, and mitochondrial morphology abnormalities relative to normoxia cultured cells. Each cell line was unique in its response to the different oxygen conditions, but in general all cell lines benefited from being cultured under 8% - 3% O2. These data also indicate that standard cell culture practice subjects cells to avoidable levels of oxidative damage and emphasize the importance of oxygen as a cell culture parameter when attempting to obtain physiologically relevant results.