Maintenance of human islets in long term culture

The long-term maintenance of human islets in culture has remained a challen ge. Despite advancements in culture techniques, human islets proved to have a short life span in vitro. For the first time, we have succeeded in maint aining human islets in a defined culture medium for more than 12 months. Fr eshly isolated islets from a 38-year-old donor were cultured in M3:5(TM) me dium and placed on a rocker for 14 days to remove contaminated exocrine and mesenchymal cells which attached to the bottom. The floating islets were p urified by daily hand-picking and transfer into fresh medium. After 14 days , purified islets were allowed to attach to the bottom of the flasks and to expand. At various time points, islets were examined immunohistochemically and electron microscopically, and the secretion of islet hormones and thei r mRNA were determined by radioimmunoassay and reverse transcriptase polyme rase chain reaction, respectively. Within seven days of culture, ductular a nd acinar cells developed within the initially normal islets. With time, ex ocrine cell types expanded while the number of the endocrine cells and thei r secretion decreased. At day 60, only a few endocrine cells were identifia ble, whereas most of the cells appeared undifferentiated and expressed cyto keratin 7 and 19, neuron specific enolase, tomato lectin, phaseolus leucoag glutinin, laminin, and vimentin. After 60 days, the culture consisted entir ely of undifferentiated cells which could be maintained in culture for 270 days before they became senescent. This is the first report on the long-ter m maintenance of human islet cells in culture and allows an insight into th e complex process of endocrine cell differentiation.