A comprehensive microbiological safety approach for agarose encapsulated porcine islets intended for clinical trials

BACKGROUND: The use of porcine islets to replace insulin‐producing islet β‐cells, destroyed during the diabetogenic disease process, presents distinct challenges if this option is to become a therapeutic reality for the treatment of type 1 diabetes. These challenges include a thorough evaluation of the microbiological safety of the islets. In this study, we describe a robust porcine islet‐screening program that provides a high level of confidence in the microbiological safety of porcine islets suitable for clinical trials. METHODS: A four‐checkpoint program systematically screens the donor herd (Large White – Yorkshire × Landrace F1 hybrid animals), individual sentinel and pancreas donor animals and, critically, the islet macrobeads themselves. Molecular assays screen for more than 30 known viruses, while electron microscopy and in vitro studies are employed to screen for potential new or divergent (emergent) viruses. RESULTS: Of 1207 monthly samples taken from random animals over a 2‐year period, only a single positive result for Transmissible gastroenteritis virus was observed, demonstrating the high level of biosecurity maintained in the source herd. Given the lack of clinical signs, positive antibody titers for Porcine reproductive and respiratory syndrome virus, Porcine parvovirus, and Influenza A confirm the efficacy of the herd vaccination program. Porcine respiratory coronavirus was found to be present in the herd, as expected for domestic swine. Tissue homogenate samples from six sentinel and 11 donor animals, over the same 2‐year period, were negative for the presence of viruses when co‐cultured with six different cell lines from four species. The absence of adventitious viruses in separate islet macrobead preparations produced from 12 individual pancreas donor animals was confirmed using validated molecular (n = 32 viruses), in vitro culture (cells from four species), and transmission electron microscopy assays (200 cell profiles per donor animal) over the same 2‐year period. There has been no evidence of viral transmission following the implantation of these same encapsulated and functional porcine islets into non‐immunosuppressed diabetic cynomolgus macaques for up to 4 years. Isolated peripheral blood mononuclear cells from all time points were negative for PCV (Type 2), PLHV, PRRSV, PCMV, and PERV‐A, PERV‐B, and PERV‐C by PCR analysis in all six recipient animals. CONCLUSION: The four‐checkpoint program is a robust and reliable method for characterization of the microbiological safety of encapsulated porcine islets intended for clinical trials.