Background: The transplantation of porcine islets into man might soon become reality for patients with type 1 diabetes mellitus. Therefore, porcine islets of high quality and quantity, and a scalable isolation process with strict quality control will be an unconditional prerequisite to enable the best possible transplantation graft. In this study, we provide a comparative study evaluating islet isolation outcome and in vitro survival based upon donor age, organ preservation solution (OPS), and cold ischemia time (CIT). Methods: Goettingen minipigs of younger age (1 year) and retired breeder animals (3.5 years) were studied. Pancreata were harvested according to the standards of human organ retrieval including in situ cold perfusion with either Custodiol®-HTK or Belzer® UW solution. Pancreatic tissue was characterized by quantification of apoptotic cells. Islet isolations were performed according to a modified Ricordi method, and isolation outcome was assessed by determining islet particle numbers (IP), islet equivalents (IEQ), and isolation factor (IF). Isolated islets were cultured for 24 and 48 h for the assessment of in vitro survival. Results: Islet viability was significantly higher in Custodiol®-HTK preserved pancreas organs compared to Belzer® UW. Furthermore, organs harvested from retired breeder preserved in Custodiol®-HTK resulted in stable islet isolation yields even after prolonged CIT and showed superior survival rates of islets in vitro compared to the Belzer® UW group. Younger porcine donor organs resulted generally in lower islet yield and survival rates. Conclusions: In summary, Custodiol®-HTK solution should be preferred over Belzer® UW solution for the preservation of pancreata from porcine origin. Custodiol®-HTK allows for maintaining islet viability and promotes reproducible isolation outcome and survival even after longer CIT. The usage of retired breeder animals over young animals for islet isolation is highly advisable to yield high quality and quantity.