Toxic nephropathy is one of the major diseases that cause renal failure in humans. It can be caused by drugs such as cephalopsporins, aminoglycosides, cyclosporins, and certain anti-cancer drugs. Therefore, drug-induced toxic nephropathy is a relevant and important issue for the pharmaceutical industry.An in vim screen than can identify potential nephrotoxins &y in drug development would therefore be very usehl.Within the kidney, proximal tubule cells are a major target for toxins, and therefore, provide an appropriate cell system for an in vitro model. Cultures of primary renal tubule epithelial cells (RTC) have been established in wed a n i d species (1). It would be extremely valuable for the understanding of species differences and for the predictive value of such in vitro screens if human RTC could also be used.It is possible to isolate human RTC from nephrwtomy specimens removed during tumour resection (Z), but the availability of normal human renal tissue is very poor. A possible source of such n o d RTC could be kidneys donated for transplantation but which cannot be used because of technical or logistical problems. Such kidneys would have been stored for several hours at hypothermia before they could be considered for RTC isolationI1 has so far been uncertain whether viable RTC could be isolated and cultured from such tissues. To investigate this, we have used porcine kidneys, which are similar in size and anatomy to human organs, to develop a method for RTC isolation and culture after hypothermic preservation. Kidneys were. rem6ved fkom Large White pigs under terminal anaesthesia, and flushed via the renal artery with 25Oml of icecold citrate preservation solution (3). The organs were packed in fresh citrate solution in sterile bags and placed in ice for storage. M e r approximately l8h, the kidneys were removed and a 2-step digestion method was used to isolate RTC. The kidneys were bisected laterally, the capsule removed and they were injected randomly with Hanks buffer containing 25mM EGTA to remove any remaining blood. The cortex was then removed from the medullary tissue, chopped and placed into the digestion solution of Hanks buffer with 0.2%w/v dispase and 0.125% w/v collagenase. This mixture was incubated at 37°C on an orbital shaker (1 50rpm). After 70min the digested cortex was washed twice in Hanks (centrihgation at 350rpm for 2 min). This procedure leads to isolated tubular fragments and glomeruli. These were then re-incubated with Hanks containing EGTA for 15 min, followed by 2 washes in Hanks buffer. The second digestion step was canied out in fresh digestion solution (as described above) for 2.31, after which time the tubular fragments were broken down into cells and small clumps of cells. The cells were washed twice as above and the final pellet resuspended in Dh4EMlFlZ media. The cells were then seeded in collagencoated 6. 12 and 24 well plates at 5x10' celldml, and incubated at 37°C for 2 days. These cultures of porcine RTC were assessed by both light and scanning electron microscopy (...