Effects of media buffer systems on growth and electrophysiologic characteristics of cultured sweat duct cells

Summary Primary cultures of human reabsorptive sweat duct cells were grown in MCDB 170 medium buffered with either HEPES, bicarbonate, or a mixture of HEPES and bicarbonate buffers. Cultures grown in MCDB media containing bicarbonate seemed to differentiate into a multilayered, keratinized epithelium and began senescing after 1 wk in culture. In contrast, cultures grown in media containing HEPES as the only buffer seemed to undergo a selection process, resulting in the outgrowth of cells that did not multilayer or keratinize extensively for up to 3 or 4 wk in culture. Despite marked differences in growth, cells grown in both bicarbonate and HEPES-buffered media retained electrophysiologic characteristics appropriate to the progenitor. Mean resting potentials were −21.8±0.8 mV (n=82), −23.3±1.3 mV (n=70) and −18.2±0.8 mV (n=82) for duct cells grown in HEPES, bicarbonate, and HEPES-bicarbonate media, respectively. Substitution of Cl⁻ with the impermeant anion gluconate in the bathing medium caused membrane potential depolarization in all media, revealing the presence of a Cl⁻ conductance. Administration of the Na⁺ conductance inhibitor amiloride hyperpolarized the mean resting potential of cells grown in HEPES medium (−6.8±0.6 mV,n=68), bicarbonate medium (−6.9±0.5 mV,n=60), and HEPES-bicarbonate medium (−5.9±0.6 mV,n=69), demonstrating expression of a Na⁺ conductance. We observed some but minimal variation with age in any of these conditions. This work was supported by grant DK41329-02 from the National Institute of Health, Bethesda, MD, and a Postdoctoral Fellowship to Dr. Bell from the National Cystic Fibrosis Foundation.