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 incubator gas. Many modern incubators maintain these concentrations by monitoring the level of CO2 and injecting the gas when the value drops below a certain set point. This feedback system results in small oscillations in the incubator CO2 concentration of about 0.5%, resulting in oscillating pH values in the medium bathing the cells. These oscillations are very small and cannot be detected using a standard pH combination electrode, and until now no one has noticed any effect on the cells. In this study, however, we demonstrate that these periodic changes can result 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 cellular changes that when analyzed with the Fast Fourier Transform have a periodicity that matches those of the CO2 addition. This effect becomes particularly prominent when the culture medium has been acidified by cell metabolism reducing the liquid's buffer capacity. We have further analyzed the cyclical morphological changes employing a model of cell-electrode interactions in the ECIS measurement. We conclude that: (1) the periodic changes in cell morphology are most apparent if the medium is partially spent and if the surface to volume ratio of the culture vessel is high and (2) for confluent WI-38/VA13 fibroblasts and bovine pulmonary endothelial cells (B3B3), the main periodic morphological changes manifest themselves primarily as alterations in paracellular resistance.
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Department of Physics and Biology, School of Science, Rensselaer Polytechnic Institute, Troy, New York 12180-3590.
Keese
C R
CR
Giaever
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I
D000818:Animals; D002245:Carbon Dioxide / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured / Q000166:cytology* / Q000378:metabolism; D003470:Culture Media; D005583:Fourier Analysis; D006863:Hydrogen-Ion Concentration
D003470:Culture Media; D002245:Carbon Dioxide