Isolation and culture of proximal tubular cells from porcine kidney

By Kruidering, M. - Prine, F.A. - Heer, E. de. - Mulder, G.J. - Nagelkerke, J.F.

Porcine proximal tubular cells (PPTC) were isolated from kidneys obtained from slaughterhouse pigs. After disruption of the connective tissue by collagenase, purification was achieved by filtration and centrifugation on a discontinuous density gradient. Single cells and clusters of 10-40 cells were obtained, having a viability of 93-99%. More than 81% of the single cells showed gamma-glutamyltranspeptidase (GGT) activity and more than 95% showed non-specific esterase (NE) activity, marker enzymes for proximal tubule cells. One kidney yielded 1 x 10(7) single cells and 3 x 10(7) cells in clusters. Cells were kept in primary culture on plastic or collagen-coated dishes. In the presence of 10% serum, confluency was reached within four days. The monolayers could be kept in culture for four days after confluency, in serum-free conditions. When seeded in serum-free conditions, PPTC did not reach confluency, but the cells could be kept in culture for at least 16 days. The cells displayed epithelial morphology, i.e. cobblestone shape, dome formation, microvilli, basal infoldings and abundant mitochondria. PPTC in primary culture still displayed NE activity, while 80% of the cells showed GGT activity. In conclusion, the isolated cells are of proximal tubular origin, reach confluency in 3-4 days in the presence of 10% serum, and can be kept as monolayers in serum-free conditions for four additional days and may provide a suitable in vitro model for long-term nephrotoxicity studies.