Dnase

Lewis rats were overdosed with isoflurane immediately prior to pancreas harvesting. Pancreas were perfused through the pancreatic duct with 9 mL CIzyme RI collagenase (Vitacyte) and 0.2 µg/mL DNAse (dornase alfa; Genentech) in Hanks balanced salt solution (HBSS; Gibco) with 10 mM HEPES (Gibco). Digestion was performed via static incubation in a water bath at 37 °C for 19 min and 40 s. Next, enzymatic activity was quenched by adding 20 mL of 20% fetal bovine serum (FBS; Gibco) in HBSS and mechanical dissociation performed via vigorous shaking for 8 s. Tissue digest was washed 3 times with ice cold HBSS and strained through a 500 µm wire mesh. The tissue pellet was re-suspended in 15 mL 1.096 g/cm³ OptiPrep (Sigma) and layered with 10 mL 1.068 g/cm³ and 1.037 g/cm³ OptiPrep and centrifuged at 480 × g for 20 min with no break. Islets were collected at the 1.096 g/cm³ and 1.068 g/cm³ interphase, washed with HBSS 3 times and cultured in RPMI-1640 media supplemented with 10% FBS, 1% penicillin/streptomycin, 20 mM HEPES, 5.5 mM glucose and 1 mM sodium pyruvate (all from Gibco) in a cell culture incubator set at 25 °C and 5% CO₂.

Freshly isolated human islets were extracted from deceased donor pancreata by the UCSF Islet Production Core (San Francisco, CA). The islets were cultured overnight after isolation at 5% CO2 and 37°C in Connaught Medical Research Laboratories (CMRL) 1066 medium (Nucleus Biologics, CA) NIH CIT supplemented with the addition of 0.5% Human Serum Albumin (Grifols, Spain), 10 U/mL of Heparin (Fresenius Kabi, Germany), 2 μg/mL DNAse (Genentech, CA) and 20 μg/mL Ciprofloxacin (Hospira, IL). Human islets were also sourced from Prodo Laboratories, Inc (Aliso Viejo, CA) and cultured with their proprietary medium (PIM(S) supplemented with Human AB Serum).