Pure islets transplanted from single donors survive in outbred dogs when CsA is administered at high doses (15-20 mg/kg/day), but not report has documented prolonged function with less CsA. In this study, we investigated survival of canine islets that were immunomodulated with in vitro culture and transplanted with minimal CsA. Highly purified islets from single donors (n = 17) were cultured in vitro at 22 degrees C for 7 days. Four groups of apancreatic, outbred mongrel dogs received islets into the spleen: group 1 (n = 4), fresh islet controls; group 2 (n = 5), cultured islet controls; group 3 (9033 +/- 994 islets/kg, n = 12), culture and CsA; group 4 (9050 +/- 1091 islets/kg, n = 8), fresh islet and CsA. CsA was administered in doses of 5-8 mg/kg/day to maintain whole blood trough levels of 300-500 ng/ml. At 30 days after implant, the CsA was stopped. After 7-day culture, 80% of the endocrine mass was recovered. All grafts restored plasma glucose to < 150 mg/dl. Group 1 and 2 dogs became hyperglycemic at 5 +/- 1 (+/- SE) and 6 +/- 1 days. Three grafts in group 3 failed when CsA did not reach target blood levels. Of the 9 dogs with CsA levels of 300-500 ng/ml, 1 died at 9 days while normoglycemic, and the remainder were normoglycemic for 35.5 +/- 8.3 days (median 38 days). Seven group 4 dogs with these CsA levels achieved normoglycemia for 7.7 +/- 1.9 days (median 9 days, survival vs. group 3, P = 0.03, by Mann-Whitney test). In vitro tissue culture of mass quantities of single-donor allogeneic islets enhances their survival with low dose immunosuppression in outbred diabetic dogs.