Maintenance of human islets in long term culture

Abstract

The long-term maintenance of human islets in culture has remained a challenge. Despite advancements in culture techniques, human islets proved to have a short life span in vitro. For the first time, we have succeeded in maintaining human islets in a defined culture medium for more than 12 months. Freshly isolated islets from a 38-year-old donor were cultured in M3:5 medium and placed on a rocker for 14 days to remove contaminated exocrine and mesenchymal cells which attached to the bottom. The floating islets were purified 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 their mRNA were determined by radioimmunoassay and reverse transcriptase polymerase chain reaction, respectively. Within seven days of culture, ductular and acinar cells developed within the initially normal islets. With time, exocrine cell types expanded while the number of the endocrine cells and their secretion decreased. At day 60, only a few endocrine cells were identifiable, whereas most of the cells appeared undifferentiated and expressed cytokeratin 7 and 19, neuron specific enolase, tomato lectin, phaseolus leucoagglutinin, laminin, and vimentin. After 60 days, the culture consisted entirely of undifferentiated cells which could be maintained in culture for 270 days before they became senescent. This is the first report on the long-term maintenance of human islet cells in culture and allows an insight into the complex process of endocrine cell differentiation.

Influence of heme oxygenase-1 gene transfer on the viability and function of rat islets inin vitroculture

“…Short-term islet culture indeed has some benefits, such as purification of the islet preparation, immunomodulation [6] , and possibly improved allograft survival. However, cultured islets are known to degrade rapidly [19,20] , and lose viability and functional responsiveness to glucose stimulation with the extension of culturing time [7,8] . Islet loss as high as 30% to 50% has been reported after 48 hours of culture [21] .…”

“…It offers advantages over immediate infusion post-isolation, enabling assessment of islet quality and safety, and reducing islet immunogenicity as well as recipient travel to the transplantation site and immunosuppression before transplantation [6] . But in vitro culture of islets has been shown to result in loss of viable tissue over time, and a decrease in glucose responsiveness has been observed in those islets which survive [7,8] . Several phenomena, including activation of free radicals, apoptosis and necrosis, may be responsible for these effects.…”

Improved survival of microencapsulated islets during in vitro culture and enhanced metabolic function following transplantation

“…Currently islets from multiple donors are required to effectively establish normoglycaemia in diabetic recipients. Long-term culture has been considered as a potential way of accruing a suitable mass of islets for transplantation from multiple donors [2,3]. Unfortunately, long-term islet culture often results in a significant loss of islet mass and viability.…”

Are islet cells the gatekeepers of the pancreas?

“…In an attempt to locate these 'stem cells' within islets, we established a long-term culture of human and hamster islets [49][50][51]. Strikingly, the growth and differentiation patterns of human and hamster islets in culture were identical.…”

“…At day 21 in culture, many cells within the islets expressed carbonic anhydrase II, CA 19-9 and/or DU-PAN-2. We were unable to identify any cell that could be regarded as stem cells although more than 50 human and hamster islets were subjected to electron-microscopic examination [49][50][51].…”