These cells make up a subpopulation of renal cells isolated from rat kidneys by discontinuous density gradient. These cells are predominantly epithelial and enriched for the tubules and collecting duct cells.
Whole kidneys were harvested from 5-wk-old male Lewis rats and enzymatically dissociated in a buffer containing 4.0 U/ml dispase and 300 U/ml collagenase IV. Red blood cells and debris were removed from the initial cell suspension by centrifugation through 15% iodixanol. Cells were seeded onto tissue culture-treated polystyrene plates and cultured in a 1:1 mixture of high-glucose DMEM and keratinocyte serum-free medium (KSFM) containing (vol/vol) FBS (5%), EGF (2.5 μg), bovine pituitary extract (BPE, 25 mg), 1× insulin-transferrin-sodium selenite medium supplement (ITS), and antibiotic-antimycotic. Before postculture cell separation, primary cultures were transferred from atmospheric oxygen conditions (21%) to a more physiologically relevant low-oxygen (2%) environment for 24 h, which improved cell separation efficiency and enabled greater detection of hypoxia-induced markers. Postculture cell suspensions, prepared as 75 × 10^6 cells in 2 ml of unsupplemented KSFM (uKSFM), were separated in an iodixanol [60% (wt/vol) in uKSFM] density gradient of 13% iodixanol in 15-ml conical polypropylene tubes and centrifuged at 800 g for 20 min at room temperature (without brake). Cellular subfractions were collected after centrifugation and washed three times in sterile phosphate-buffered saline (PBS) before use.