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Isolation and characterization of metabolically competent pulmonary epithelial cells from pig lung tissue

: Blickwede, M.; Borlak, J.


Xenobiotica 35 (2005), Nr.10/11, S.927-941
ISSN: 0049-8254
Fraunhofer ITEM ()
porcine pulmonary epithelial cell; alveolar epithelial type II cell; primary culture; cell culture methodology

Administration of drugs by inhalation opens new possibilities for entry into the systemic circulation and cultures of porcine pulmonary epithelial cells (PECs) may prove to be valuable in the prediction of pulmonary metabolism of drugs in humans. This paper, therefore, reports a method for the routine isolation and cultivation of PECs from slaughterhouse animals. On average 1.5 x 10(6) cells g(-1) tissue were isolated by discontinuous density-gradient centrifugation. Cells were subsequently cultivated on collagen-coated plates and characterized by staining for alkaline phosphatase, by tannic acid staining of lamellar bodies and by surfactant protein (SP) expression at days 0, 3 and 6 in culture. Over 70% of purified cells were positive for SP-C and tannic acid staining and thus defined as epithelial cells of alveolar origin (AECs). The AEC phenotype was also confirmed by specific binding of marker lectins (Maclura pomifera and Helix pomatia) and by studying gene expression and activity of cytochrome P450 monooxygenases. Testosterone, ethoxyresorufin, benzyloxyresorufin and verapamil mere used as substrates for cytochrome P450-catalysed oxidations and cultured cells were found to be differentiated as well as metabolically competent during cultivation. Therefore, this culture system enables in depth pulmonary biotransformation and toxicity studies.