1. Collect the tissue immediately into HBSS containing penicillin-streptomycin (P/S) and place on ice during transfer to the laboratory. Collect the specimen as soon after resection as possible as there is a clear decline in the amount of successfully cultured material the longer the tissue remains ex vivo.

2. Place the specimen in a shallow Petri dish in the flowhood and add HBSS containing P/S. Pre-warmed HBSS to 37°C.

3. Using sterile dissecting equipment strip the fibrous capsule away from the cortex. The capsule should peel off using forceps only. Be sure to remove capsule completely.

4. Dissect away the very outer cortex from the inner cortex and medulla. Use only the extreme outer cortex or the cell culture may become very heterogeneous.

5. Cut the sample with scissors and forceps into pieces approx 1mm3.

6. Transfer the tissue fragments to a sterile 50 ml tube and wash with HBSS three times. 5 min spins at 200g, pouring off supernatant and resuspending in HBSS.

7. Add collagenase (type II from Clostridium histolyticum 1 mg/ml (sigma), -20 °C) 1mg/ml to the 50-ml tube so that all the fragments are covered by the solution. Incubate at 37°C for 60 min with agitation of the tube.

8. Transfer the tube to the flow hood and add HBSS to make a final volume of 30 ml.

9. Force the tissue fragments within the 30-ml volume of HBSS through a 100 mm sieve into a 50-ml tube. Then pass sieved material through a 40 mm sieve into another 50 ml tube. This removes tubular fragments and glomeruli respectively.

10. Sediment the sieved cells by centrifugation (200g, 5min at RT)

11. Resuspend and initiate the primary culture by seeding the cells in HAM's F12:DMEM (1:1) at an approximate density of 2 x 105 cell/cm2 . The medium is supplemented with 5 mg/ml transferrin, 10 mg/ml insulin, 5 ng/ml selenium, 36ng/ml hydrocortisone, 10 ng/ml epidermal growth factor and 4 pg/ml triodothyronine.

12. The remaining tissue fragments, excluded by sieving, can then be further digested by incubating with trypsin-EDTA for a further 45 min at 37°C. Trypsin activity is terminated after this time by adding a equal volume of 0.1% soybean trypsin inhibitor. Following this, proceed through step 9-11 and plate cells out at the recommended density.

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.