Accelerated cell death produced by a cholera cytotoxin on isolated epithelial cells from rabbit ileum

Villus and crypt cells were removed from rabbit ileum by a modification of a method previously described. Villus cells were isolated by vibration of everted bowel segments in citrate buffer, and crypt cells were removed by the expansion of these segments with air and simultaneous vibration in citrate buffer. Several culture media were tested for the maintenance and/or culture of these cells. Two defined media, Eagle's minimum and medium 199, were unsatisfactory and led to rapid cell death. The presence of whole serum was beneficial and both cell types could be maintained for several hours when homologous rabbit serum was employed. Initially 70-80% of villus and crypt cells were viable in homologous serum, and the effect of cholera toxin on cell viability was studied by incubating the cells with peptone dialysate supernatant (PSUP) toxin from Vibrio cholerae for 4 h. PSUP toxin reduced the viability of both villus and crypt cells compared with control preparations, as measured by the uptake of trypan blue; cell death was accelerated with time. A purified, diarrhoea-inducing cholera toxin also reduced the viability of these cells and the results were comparable to those with PSUP toxin. The effect was usually immediate, and a significant reduction in viability occurred within 1 hour of the incubation of cells with toxin. The toxin was heat-labile (60 degrees C/30 min) and nondialysable, and its cytotoxic activity could be completely neutralized with cholera antitoxin. Dose-response studies indicated that as little as 0.0275 equivalent units of ligated ileal loop toxin were active in this system.