Isolation and Dispersion of Neonatal Porcine Islet-Like Cell Clusters

NPICCs were isolated from pancreata (n = 12) of 2–5 days-old piglets by collagenase digestion as described previously (22 (link), 23 (link)). Cell clusters were cultured in RPMI 1640 (PAN-Biotech, Aidenbach, Germany), 2% human serum albumin (Takeda, Konstanz, Germany), 10 mM nicotinamide, 20 ng/ml exendin-4 (Merck, Darmstadt, Germany) and 1% antibiotic-antimycotic (Thermo Fisher Scientific, Germering, Germany) (basal islet culture [B-IC] medium). On day 4, NPICCs were harvested and islet equivalents (IEQ) were determined under a stereomicroscope. NPICCs were either re-cultivated in B-IC medium (control group) or washed with PBS and incubated in TrypLE Express solution (Thermo Fisher Scientific) for 8–12 min at 37°C with gentle mixing every 60 s until dispersion into single cells was observed under a stereomicroscope. Then, the cells were filtered through a 40-µm filter (Corning, Wiesbaden, Germany) to remove debris.