In Vitro Maturation of Viable Islets from Partially Digested Young Pig Pancreas

Abstract

Isolation of islets from market-sized pigs is costly, with considerable islet losses from fragmentation occurring during isolation and tissue culture. Fetal and neonatal pigs yield insulin unresponsive islet-like cell clusters that become glucose-responsive after extended periods of time. Both issues impact clinical applicability and commercial scale-up. We have focused our efforts on a cost-effective scalable method of isolating viable insulin-responsive islets. Young Yorkshire pigs (mean age 20 days, range 4–30 days) underwent rapid pancreatectomy (<5 min) and partial digestion using low-dose collagenase, followed by in vitro culture at 37°C and 5% CO2 for up to 14 days. Islet viability was assessed using FDA/PI or Newport Green, and function was assessed using a glucose-stimulated insulin release (GSIR) assay. Islet yield was performed using enumeration of dithizonestained aliquots. The young porcine (YP) islet yield at dissociation was 12.6 ± 2.1 × 103 IEQ (mean ± SEM) per organ and increased to 33.3 ± 6.4 × 103 IEQ after 7 days of in vitro culture. Viability was 97.3 ± 7% at dissociation and remained over 90% viable after 11 days in tissue culture ( n = ns). Glucose responsiveness increased throughout maturation in culture. The stimulation index (SI) of the islets increased from 1.7 ± 2 on culture day 3 to 2.58 ± 0.5 on culture day 7. These results suggest that this method is both efficient and scalable for isolating and maturing insulin-responsive porcine islets in culture.