Establishment and characterization of a strial marginal cell line maintaining vectorial electrolyte transport

Abstract

E6/E7 genes of human papilloma virus type 16 were used to immortalize a primary culture of marginal cells (MC) from gerbils. One of the cloned lines was selected which demonstrated preservation of the main characteristics of the MC, both morphologically and physiologically. Electron microscopic examination showed well-developed junctional complexes and apical microvilli which suggested its epithelial origin. Polymerase chain reaction (PCR) demonstrated the incorporation of E6/E7 genes with the genome. Reverse transcription PCR revealed the existence of mRNA of the I sK channel, a unique marker of MC among the inner ear cells, in this clone. Flow cytometric analysis of this cell line's DNA content was diploid. Numerous large domes formed after confluence of the cell monolayer. Electrophysiologic studies displayed evidence of apical K + and Na + channels which were blocked by Ba 2+ (2 mM) and amiloride (10 −5 M), respectively. Existence of basolateral Na,K-ATPase and Na +/Cl −/K + cotransporter was shown by blockage by ouabain (10 −3 M) and bumetanide (50 μM), individually. Injection of the cell line to nude mice failed to induce growth of tumors. This cell line was serum-, density- and anchorage-dependent when cultured in plastic dishes. In conclusion, this cell line shows characteristics of well-differentiated MC maintaining the major ionic transport processes, and provides us a good model to study the possible mechanisms and regulating factors of endolymph production.