"Real-time" assessment of human islet preparations with confocal live cell imaging.

Pancreatic islet cell transplantation is a promising approach to restoring normoglycemia in diabetic patients. The outcome of islet transplantation is uncertain for two reasons: The quality of isolated islets is still poorly defined and the functional potential of transplanted islets is difficult to predict. Therefore, one of the primary challenges in islet transplantation is to identify and understand the changes taking place in islets after isolation and culture. Description of such changes in living islet cells offers insights not achievable by use of fixed-cell techniques. Three fluorescent dyes, dichlorodihydrofluorescein diacetate, tetramethylrhodamine methyl ester perchlorate (TMRM), and fluorescent wheat germ agglutinin, were used to assess either overall oxidative stress, time-dependent mitochondrial membrane potentials, or localization of oligosaccharides. Confocal microscopy was performed with a microlens-enhanced Nipkow disk-based confocal system mounted on an inverse microscope. We were able to show differences in the amount of oligosaccharides on the cell surface between endocrine and exocrine cells in freshly isolated human islet preparations. The study of the mitochondrial membrane potential via TMRM proved to be useful to early identification of damaged or stressed cells. Thus a combination of fluorescent dyes as subcellular markers, with a powerful live confocal imaging system may be of great value to isolation and culture.