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 requires removal of the expanded cells from the matrix and reaggregation into cell aggregates. However, extensive cell death occurs during manipulation and culture. The mechanism of cell death was examined at each stage throughout the process under different experimental conditions to determine optimal protocols to increase cell viability. During shipment, the addition of trehalose to the media to prevent necrosis increased yield 17-fold, while during culture as islet-like cell clusters the apoptosis inhibitor Z-VAD increased yield 1.8-fold. Following disruption of cell matrix interactions and reaggregation, there was marked evidence of apoptotic bodies by the TUNEL assay. Addition of nicotinamide or Z-VAD, or removal of arginine from the media during reaggregation, reduced the number of apoptotic bodies and the effect was additive. However, a combination of treatments was necessary to significantly increase the yield of viable cells. We conclude that cell death of human fetal pancreatic tissue in culture results from both necrosis and apoptosis and that understanding the mechanisms at the cellular level will lead to protocols that will improve cell viability and promote beta-cell growth.
Beattie
G M
GM
Department of Pediatrics, the Whittier Institute for Diabetes, Cancer Center, UCSD La Jolla, CA 92037, USA.
Leibowitz
G
G
Lopez
A D
AD
Levine
F
F
Hayek
A
A
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis; D001120:Arginine / Q000494:pharmacology; D061945:Caspase Inhibitors; D016923:Cell Death; D002470:Cell Survival; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D005109:Extracellular Matrix / Q000378:metabolism; D016332:Fetal Tissue Transplantation; D017228:Hepatocyte Growth Factor / Q000494:pharmacology; D006801:Humans; D020287:In Situ Nick-End Labeling; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology* / Q000196:embryology* / Q000502:physiology; D016381:Islets of Langerhans Transplantation; D014021:Tissue Preservation; D014199:Trehalose / Q000494:pharmacology
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D003470:Culture Media; D015853:Cysteine Proteinase Inhibitors; D007328:Insulin; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D017228:Hepatocyte Growth Factor; D001120:Arginine; D014199:Trehalose