Effects of cryopreservation and coculture with pancreatic ductal epithelial cells on insulin secretion from human pancreatic islets.
Long-term storage methods, such as cryopreservation and long-term in vitro culture, hinder the therapeutical application of pancreatic islet transplantation, because they decrease islet viability. Pancreatic ductal epithelial cells (DEC) are putative stem cells for islets, which may secrete specific factors supporting islet growth and function. Hence, we studied the effect of coculture with DEC on the viability of fresh and cryopreserved human pancreatic islets. Islets and DEC were isolated from the pancreas of an organ donor, and part of them were cryopreserved. Fresh and cryopreserved-thawed islets were cultured alone or in the presence of DEC for 14 days at 33 degrees C or 37 degrees C. At day 1 and day 14 of culture, insulin secretion was stimulated by two sequential 45-min exposures to low and high glucose concentrations (3.3 and 16.7 mmol/l, respectively). Insulin concentrations were measured by radio-immunoassay, and the ratio between the insulin responses to high and low glucose was calculated (insulin stimulation index, ISI). After 14 days of culture, some fresh islets were processed for scanning electron microscopy (SEM). At day 14, ISI was markedly reduced in both fresh and cryopreserved islets with respect to 1 day cultures. Cryopreservation reduced ISI at day 1 and day 14, but in the latter case only when cultures were maintained at 37 degrees C. Coculture with DEC did not affect ISI of fresh islets at day 1, and enhanced it at day 14, but only at a culture temperature of 33 degrees C. Conversely, coculture raised ISI of cryopreserved islets at both day 1 and day 14, independently of the culture temperature. SEM showed that at day 14 of culture, the morphology of fresh islets displayed the best preservation when cocultured with DEC at 33 degrees C. Our findings confirm that both long-term culture and cryopreservation decrease viability of human pancreatic islets. Moreover, they indicate that coculture of islets with DEC at 33 degrees C represents a valuable tool to improve the survival and functional activity of islets, especially in the case of cryopreserved material.
Gatto
Claudio
C
Tissue Bank of Veneto Region, Treviso Regional Hospital, I-31100 Treviso, Italy.
Callegari
Maura
M
Folin
Marcella
M
Conconi
Mariateresa
M
Paolin
Adolfo
A
Di Falco
Giuseppe
G
Bredariol
Simonetta
S
Spinazzi
Raffaella
R
Parnigotto
Pier Paolo
PP
Nussdorfer
Gastone G
GG
D002470:Cell Survival; D018920:Coculture Techniques; D003080:Cold Temperature; D015925:Cryopreservation; D004848:Epithelium / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000378:metabolism* / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning; D010183:Pancreatic Ducts / Q000378:metabolism
D007328:Insulin