LONGTERM 3D COLLAGEN SPONGE MATRIX ISLET CULTURE-THE EFFECT OF HEPATOCYTE COCULTURE

Abstract

119 We believe that a stable or growing culture of beta islets would provide advantages in availability, immune response, and numbers of islets that could improve the results in islet transplantation. Loss of function and mass of beta islets in cultures is a barrier to utilizing islets in transplantation. We hypothesized that islets would have improved survival and function when co-cultured with hepatocytes(HC). Methods: Islets were isolated with collagenase and differential (ficoll) centrifugation. HC were isolated using collagenase and percoll centrifugation. Routine viability was >95% by trypan blue. Islets were cultured in a collagen sponge matrix with HC at a ratio of 1:50, 1:25, or 0 HC. Insulin response is measured by static glucose stimulation (300mg/dl for 1 hr) and stimulation index (SI) calculated (stimulated insulin secretion/basal insulin secretion). All insulin was measured by RIA and reported as ng/ml/islet. Results: The 3-D sponge collagen matrix culture environment resulted stable viability and SI for 14wks. The benefit of HC co-culture in the insulin production and islet survival was seen in the 1:50 HC groups (see graph). The ratio of insulin release to static glucose stimulation at week 12 compared to week 1 p=0.038 1:50 HC(0.82) vs no HC(.064). The stimulation index did not change over the 14 weeks (see graph), but the absolute amount of insulin released during static glucose stimulation per islet decreased steadily in all culture groups. We have interpreted this as loss of islet mass even though BrDU uptake in the islets does indicate cell division. We conclude from this study that 3 dimensional collagen matrix allows long-term in vitro islet viability, HC co-culture does result in improved islet response to glucose stimulation during long-term culture, but that loss of islet mass is not halted by co-culture with HC.Figure