Subculture at 1:3 ratio for up to eight passages with the cells maintaining proximal tubular phenotype.

1. Aspirate media from monolayer culture, and rinse the cells with HBSS

2. Aspirate HBSS, and pipette trypsin-EDTA onto the monolayer. Incubate at 37°C for 4-5 min. Bump the side of the plate against the palm of the hand to help detach the cells.

3. Transfer cells to a centrifuge tube. Rinse the culture vessel with HBSS to collect remaining cells, and add to centrifuge tube. Neutralize trypsin action by adding FCS, approximately one-half the volume of trypsin used.

4. Centrifuge cells at 150g, 7 min

5. Resuspend cell pellet in fresh growth media and seed the cells onto collagen coated plates.

Note: This protocol was taken from "Primary Culture of Human Proximal Renal Tubular Epithelial Cells" by Paul A. Glynne. From: Methods in Molecular Medicine, Vol.36: Septic Shock. Humana Press Inc., Totowa, NJ.