Culture of human renal proximal tubule cells and renal cortical fibroblasts
1. The method for primary culture of human renal proximal tubule cells (PTC) and renal cortical fibroblasts (CF) is described in detail.
2. Renal cortical tissue was dissected from the medulla, minced, digested with collagenase, and passed through a 100-μm mesh.
3. Filtered tissue was resuspended in 45% Percoll and separated into four distinct bands by isopyknic ultracentrifugation.
4. The uppermost band was removed for CF culture and the lowermost band for PTC culture.
5. CFs were grown in primary cell system with 10% fetal calf serum, whereas PTC were cultured in primary cell system with 5 μg/ml human transferrin, 5 μg/ml (0.87 μM) bovine insulin, 0.05 μM hydrocortisone, 10 ng/ml (1.64 nM) epidermal growth factor, 50 μM prostaglandin E1, 50 nM selenium, and 5 pmol/l tri-iodothyronine.
6. Cytologic examination of cell preparations from all donors failed to reveal any evidence of cellular atypia.
7. The morphological, biochemical, and transport characteristics of these cells have been studied.
8. PTC stained positively for cytokeratin and negatively for vimentin, generated cAMP in response to parathyroid hormone but not vasopressin, and displayed typical polarity with apical microvilli, apical Na+-glucose and Na+-phosphate cotransporters, organic anion and cation transport systems, and pharmacologically distinct apical and basolateral sodium-hydrogen exchange (NHE) activities.
9. CF exhibited typical fusiform morphologies, positive staining for vimentin and 5′-ectocytonucleotidase, and negative staining for desmin and α-smooth muscle actin.
