Influence of the Hypercapnic Tumor Microenvironment on the Viability of Hela Cells Screened by a CO2-Gradient-Generating Device

Carbon dioxide (CO 2 ) levels outside of the physiological range are frequently encountered in the tumor microenvironment and laparoscopic pneumoperitoneum during clinical cancer therapy. Controversies exist regarding the biological effects of hypercapnia on tumor proliferation and metastasis concerning time frame, CO 2 concentration, and cell type. Traditional control of gaseous microenvironments for cell growth is conducted using culture chambers that allow for a single gas concentration at a time. In the present paper, Hela cells were studied for their response to varying levels of CO 2 in an aerogel-based gas gradient-generating apparatus capable of delivering a stable and quantitative linear CO 2 profile in spatial and temporal domains. Cells cultured in the standard 96-well plate sandwiched in between the device were interfaced with the gas gradient generator, and the cells in each row were exposed to a known level of CO 2 accordingly. Both the ratiometric pH indicator and theoretical modeling have confirmed the efficient mass transport of CO 2 through the air-permeable aerogel monolith in a short period of time. Tumor cell behaviors in various hypercapnic microenvironments with gradient CO 2 concentrations ranging from 12 to 89% were determined in terms of viability, morphology, and mitochondrial metabolism under acute exposure for 3 h and over a longer cultivation period for up to 72 h. A significant reduction in cell viability was noticed with increasing CO 2 concentration and incubation time, which was closely associated with intracellular acidification and elevated cellular level of reactive oxygen species. Our modular device demonstrated full adaptability to the standard culture systems and high-throughput instruments, which provide the potential for simultaneously screening the responses of cells under tunable gaseous microenvironments.