A reliable method for isolation of viable porcine islet cells.
OBJECTIVE
Mechanical injury and oxidative stress caused by reoxygenation of isolated porcine islet cells result in their unresponsiveness to glucose stimulation.


METHODS
Adult pigs (weighing 25-30 kg) were anesthetized, and following intra-arterial infusion of ice-cold University of Wisconsin solution, a complete pancreatectomy was performed. The pancreatic duct was cannulated for infusion of digestion medium containing collagenase type P, 1.5 mg/mL; deoxyribonuclease I, 10 000 U; and a water-soluble analogue of vitamin E (Trolox), 1 mmol/L. After 20-minute incubations on ice, and at 37 degrees C, the pancreas was hand shaken for 1 minute, followed by filtration and separation on an automatic cell separator (COBE 2991). Islet cells, identified by dithizone staining, were perifused at 37 degrees C.


RESULTS
The mean +/- SEM yield of intact purified islet cells (50-200 microm in diameter), and mostly present in clusters, was 2398 +/- 143 cells per gram (n = 12). Glucose stimulation caused a significant increase in biphasic insulin secretion in the perifusion experiments.


CONCLUSIONS
We have developed a simple, reproducible, and reliable procedure for isolating intact and viable porcine islet cells suitable for xenotransplantation.
Ching
C D
CD
Department of Surgery, Duke University Medical Center, Campus Box 3065, Durham, NC 27710, USA.
Harland
R C
RC
Collins
B H
BH
Kendall
W
W
Hobbs
H
H
Opara
E C
EC
D000818:Animals; D001786:Blood Glucose / Q000378:metabolism*; D002470:Cell Survival / Q000502:physiology*; D046508:Culture Techniques; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology*; D016381:Islets of Langerhans Transplantation; D013552:Swine; D020858:Tissue and Organ Harvesting; D014183:Transplantation, Heterologous
D001786:Blood Glucose; D007328:Insulin