Novel role of extracellular carbon dioxide in lymphocyte proliferation in culture

AbstractCO2/HCO3− buffering system is indispensable to maintain the pH of culture media for long‐term cell culture. Now‐a‐days, the zwiterionic hydrogen buffer HEPES is widely used as an additional buffer in the commonly used culture media. There are reports on the successful use of HEPES‐buffered media, under CO2/HCO3− free conditions, for long‐term cell cultures. However, still CO2/HCO3− buffering system is widely used. We aimed at investigating the reason for this. We found that lymphocytes proliferate in response to concanavalin A only in HCO3−‐buffered medium in the presence of 5% CO2, but not in the HEPES‐buffered medium in the absence of CO2. However, lymphocyte proliferation was observed in HEPES‐buffered medium in the presence of 5% CO2 and in the absence of HCO3−. On the other hand, a low level proliferation was observed in HEPES‐buffered medium supplemented with HCO3− in the absence of CO2. Supplementation of the culture medium with TCA cycle intermediates and the precursors for the salvage pathway of nucleotide synthesis did not support the lymphocyte proliferation at all. Based on these findings and other reports, we suggest that extracellular CO2 plays a novel role in