The porcine pancreas is a promising source for a scalable and standardised beta cell replacement therapy. A flexible supply chain will be key to optimize time and cost efficiency and to make islet transplantation available to the global diabetes patient population. Pancreas source tissue cold ischemia time (CIT) and porcine islet cryopreservation were explored.

Animal studies comply Directive 2010/63/EU. Piglets were obtained at gestational day 109 to 115 or neonatal day 1. Pancreas tissue was stored at 4-8°C with variable intervals of CIT and one hour as reference. Standard isolation medium, Institut Georges Lopez-1® Organ Preservation Solution (IGL-1), HypoThermosol® (HTS), University of Wisconsin solution (UW) and Histidine-tryptophan-ketoglutarate solution (HTK) were used as pancreas preservation media. Endocrine cell isolation was performed via collagenase digestion and counterflow elutriation. On day 1 and after 1 week of culture, samples were taken to determine cell numbers, viability and composition. Cryopreservation was performed on fresh pancreas tissue and cells were frozen in Cryostor 10 on day 2 and 7 after isolation. In vivo maturation and diabetes reversal were assessed in nude and NSG mice under the kidney capsule.

A clear negative correlation exists between CIT and beta cell yield ( p < 0.0001). Beta cell yield was stable after 3 h of CIT, but 12 h or more resulted in a 40-50% drop. No impact was seen on cell viability or in vivo maturation in nude mice, with only a minimal negative impact on beta cell purity (p = 0.03). Cell yields and quality were compared for CIT ≥ 12h in HTK, HTS, IGL-1 and UW and compared with the reference control. An average beta cell yield of 7.7 x 10⁶ beta cells per g pancreas was obtained for the 1 h CIT control, 5.0 x 10⁶ for HTS, 5.9 x 10⁶ for IGL-1 and 3.2 x 10⁶ for UW, with only 2.0 x 10⁶ beta cells per g pancreas for HTK. Beta cell purity was 36.8% for the control, 42.0% for the HTS, 37.5 for IGL-1, 31.7% for IGL-1 and 30.7% for HTK.

Cryopreservation on day 2 allows higher beta cell recoveries compared to cryopreservation after one week of culture with a viable beta cell recovery of 114±36% for fresh cells cultured for one week, 84±28% for cells frozen on day 2 after culture and 41±15% for cells frozen after one week of culture. Purity with respect to beta cell composition and in vivo graft maturation and diabetes reversal in NSG mice were comparable for fresh and cryopreserved cells, irrespective of freezing day. Nevertheless, cryopreservation still results in reduced cell yields with a drop in beta cell recovery from 11±5 x 10⁶ to 6±2 x 10⁶ per gram pancreas for fresh and day 2 cryopreserved cells respectively.

Pancreas CIT limits porcine beta cell replacement therapy flexibility, while cryopreservation does allow supply chain flexibility. Despite reduced yields, it enables cells to be produced at a centralized facility and preserved for future patient treatment in clinics worldwide.