PH CHANGES IN PULSED CO2 INCUBATORS CAUSE PERIODIC CHANGES IN CELL MORPHOLOGY

It is common in animal cell culture to utilize media that are buffered with bicarbonate ions and elevated CO2 concentrations in the incubato r gas. Many modern incubators maintain these concentrations by monitor ing the level of CO2 and injecting the gas when the value drops below a certain set point. This feedback system results in small oscillation s in the incubator CO2 concentration of about 0.5%, resulting in oscil lating pH values in the medium bathing the cells. These oscillations a re very small and cannot be detected using a standard pH combination e lectrode, and until now no one has noticed any effect on the cells. In this study, however, we demonstrate that these periodic changes can r esult in detectable changes in cell morphology and therefore do affect cell behavior. Using electric cell-substrate impedance sensing (ECIS) to detect subtle alterations in cell morphology, we have measured cel lular changes that when analyzed with the Fast Fourier Transform have a periodicity that matches those of the CO2 addition. This effect beco mes particularly prominent when the culture medium has been acidified by cell metabolism reducing the liquid's buffer capacity. We have furt her analyzed the cyclical morphological changes employing a model of c ell-electrode interactions in the ECIS measurement. We conclude that: (1) the periodic changes in cell morphology are most apparent if the m edium is partially spent and if the surface to volume ratio of the cul ture vessel is high and (2) for confluent WI-38/VA13 fibroblasts and b ovine pulmonary endothelial cells (B3B3), the main periodic morphologi cal changes manifest themselves primarily as alterations in paracellul ar resistance. (C) 1994 Academic Press, Inc.