Kidney proximal tubular cells isolated by collagenase perfusion grow in defined media in the absence of growth factors

Abstract

Cells from kidney proximal tubules have been successfully isolated, characterized, and cultured from male Fischer 344 rats between 150-400 g using a two-step collagenase perfusion. The cells undergo high levels of DNA synthesis and mitosis in both serum free media (with an without hormone supplementation) and media containing 10% fetal bovine serum. Confluent monolayers were observed between 5 to 7 days after seeding 2 X 10(5) cell/35 mm collagen-coated plate. Approximately 50% of the total kidney and 70% of the cortex was isolated using this technique. The viability of the isolated tubules was 75 +/- 8% and the estimated number of viable cells was 12 +/- 3 X 10(6) cells. At the time of isolation greater than 90% of the isolated tubules and cells were positive for gamma glutamyltransferase (GGT), periodic acid-schiff (PAS), and glucose-6-phosphatase (G-6-Pase). Both GGT and G-6-Pase decreased rapidly during the first 3 days in primary culture as assessed by histochemistry. Ultrastructurally the isolates consisted of cells with numerous microvilli and mitochondria. The size and number of microvilli decrease rapidly in primary culture. The morphologic and biochemical evidence suggests that the primary isolates and cultures are proximal tubular in origin.