Isolation of Cadaveric Islet Cells

hPIs used in this study were isolated from six cadaveric multiorgan donors, in particular three women and three men, donor age 52.6 ± 3.6 years, BMI 25.5 ± 2.6 kg/m², islets purity 82.5 ± 5.2%, according to the procedure previously described (Ricordi et al., 1988 (link); Petrelli et al., 2011 (link)). The overall protocol has been approved by the Niguarda Cà Granda Ethics Board. Islets were isolated using the automated method previously described (Ricordi et al., 1988 (link)). Pancreata were obtained from multiorgan cadaveric donors utilizing cold perfusion. Exclusion and inclusion criteria were applied based on the Italian Guidelines. Briefly, pancreata were digested by a cold enzymatic blend solution of collagenase and thermolysin (Liberase MTF GMP Grade kit, Roche Diagnostics, Mannheim, Germany) reconstituted in Hank’s Balanced Salt Solution (HBSS, Euroclone, Italy) with 25 mM of HEPES. Subsequently, islets were purified with discontinuous polysucrose solutions at decreasing density 1.132, 1.108, 1.096, 1.060 and 1.037 g/L (Mediatech-Cellgro, VA, United States). Islets were counted by dithizone staining islet equivalent (IEQ) method (see “Dithizone staining” section) and they were cultured at 24°C, 20% O₂, 5% CO₂ in a humidified atmosphere in MIAMI Medium #1A (Mediatech-Cellgro, VA, USA) supplemented with Ciprofloxacin (Fresenius Kabi, Verona, Italy), or in serum-free medium in the presence of basic fibroblast growth factor (bFGF, PeproTech) and epidermal growth factor (EGF, PeproTech) at final concentrations of 10 ng/ml and 20 ng/ml, respectively.