Human islets from normal donors were purchased from Prodo Laboratories Inc. and were cultured immediately upon arrival in CMRL complete media consisting of glutamine free CMRL supplemented with 10 mM niacinamide and 16.7 uM zinc sulfate (Sigma), 1% ITS supplement (Corning), 5 mM sodium pyruvate, 1% Glutamax, 25 mM HEPES (American Bio), 10% HI FBS and antibiotics (10,000 units mL−1 penicillin and 10 mg mL−1 streptomycin). All media components were obtained from Life Technologies unless otherwise indicated. Human donor islets were cultured as intact islets then dispersed and re-aggregated as pseudo-islets for dynamic insulin secretion studies. The formation of single pseudo-islet aggregates were performed by first dispersing the intact islets using accutase (Gibco), then the resulting cell suspension was seeded at 5000 cells per well of a 96-well V-bottom plate, lightly centrifuged (~200×g) and then incubated for 12–24 h at 37 °C 5% CO2/95%.
Rat islets were isolated from Sprague-Dawley rats and recovered in culture at 37 °C 5% CO2/95% for 12–24 h in 5 mM glucose RPMI (Sigma) supplemented with 10 mM HEPES (American Bio), 10% FBS (Gemini Bio-products) and antibiotics (Life Technologies). The islets were then dispersed and re-aggregated into pseudo islets as described above.
Rat islets were isolated from Sprague-Dawley rats and recovered in culture at 37 °C 5% CO2/95% for 12–24 h in 5 mM glucose RPMI (Sigma) supplemented with 10 mM HEPES (American Bio), 10% FBS (Gemini Bio-products) and antibiotics (Life Technologies). The islets were then dispersed and re-aggregated into pseudo islets as described above.