Continuous on-line hydrogen ion monitoring to study flow dynamics of perifusion systems and cellular metabolism

Time-dependent concentration profiles of input signals and feedback of metabolic products can strongly influence cellular responsiveness. To study these parameters, we developed a perifusion system that can deliver biological signals to cells with minimal dispersion, monitor real time responses, and remove waste products continuously. By monitoring pH with miniature hydrogen ion-selective electrodes at intervals of 1 s, effects of dispersion, flow rate, pumping system, and changes in cellular metabolism were demonstrated. Dynamic responses of a human cell line to a series of 10-min pulses of the metabolic uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) were monitored. A rapid 1-min increase in acid release occurred on exposure to CCCP, followed by a decrease in acidification and then a gradual return to a baseline slightly more acidic than before administration of CCCP. These observations demonstrate that this perifusion system can reveal small changes in pH (+/- 0.0005 units) induced by metabolic perturbations and has the potential to reveal the dynamics of cellular responsiveness to a wide range of hormonal, metabolic, and other chemical signals.