Protection from cell death in cultured human fetal pancreatic cells

Endocrine cells from the human fetal pancreas will proliferate in vitro on extracellular matrix but lose hormone expression, and redifferentiation req uires removal of the expanded cells from the matrix and reaggregation into cell aggregates. However, extensive cell death occurs during manipulation a nd culture. The mechanism of cell death was examined at each stage througho ut the process under different experimental conditions to determine optimal protocols to increase cell viability. During shipment, the addition of tre halose to the media to prevent necrosis increased yield 17-fold, while duri ng culture as islet-like cell clusters the apoptosis inhibitor Z-VAD increa sed yield 1.8-fold. Following disruption of cell-matrix interactions and re aggregation, there was marked evidence of apoptotic bodies by the TUNEL ass ay. Addition of nicotinamide or Z-VAD, or removal of arginine from the medi a during reaggregation, reduced the number of apoptotic bodies and the effe ct was additive. However, a combination of treatments was necessary to sign ificantly increase the yield of viable cells. We conclude that cell death o f human fetal pancreatic tissue in culture results from both necrosis and a poptosis and that understanding the mechanisms at the cellular level will l ead to protocols that will improve cell viability and promote beta-cell gro wth.