Background: Porcine neonatal islet cell clusters (NICC) are widely accepted as a potential means for treatment of type 1 diabetes amid significant issues of low donor organ availability for allotransplantation and the poor yield of islet cells from donor pancreata. Several studies have shown NICC xenotransplantation reverses hyperglycaemia in large animals and non-human primates; however, these are varied in results and methodology. By developing an optimised method for pancreas retrieval and NICC culture, we hope to develop a universal protocol for NICC xenotransplantation to be used for clinical translation.
Aim: To optimise NICC culture and evaluate the short-term efficacy (100 days) for their use in pig-to-baboon xenotransplantation.
Methods: A retrospective audit of 9 NICC preparations from which NICC were transplanted into diabetic baboons was reviewed to determine which factors yielded the greatest NICC yield and provided good function. NICC were retrieved from piglets ranging from 3-6 days of age, digested with 25 mg/ml collagenase, and filtered through a 500um sieve. Tissue was plated in Petri dishes in Hams F10 medium (containing additives) for 6 days in a 37°C, 5% CO2 incubator. Full media changes were performed on days one and three. 10 ml of Ham’s F10 medium with additives was added on day 5 before a final count and transplant on day 6. The 9 NICC preps were transplanted into 9 baboons, each of which received 5 doses of anti-CD2 at 5 mg/kg from day -3 to day 21 along with fortnightly anti-CD154 at 20-30 mg/kg and belatacept at 20 mg/kg. With daily maintenance immunosuppression of tacrolimus at 5 mg/kg or sirolimus at 2 mg/kg. Prior to immunosuppression, blood was collected to establish baseline immunological parameters which were assessed by flow cytometry.
Results: Over the 9 NICC isolations, the most common age for pancreatectomy of the piglets was 3 days (4 preps), followed by 4 (2 preps), 5 (2 preps) and 6 days (1 prep). The difference in age, however, did not significantly alter the yield of islet equivalents (IEQ), a mean of 7,060 1,696IEQ per gram of pancreas (p-gmP) was produced for all preps combined, however, day 3 pancreatectomy demonstrated higher IEQ/ p-gmP with a mean of 7,794 1,802 IEQ/ p-gmP. In 8 of the 9 recipients, there was detectable porcine C peptide (PcPep) produced within 1 month, with an average PcPep of 97pmol/L, and all baboons achieved normoglycaemia with functional IVGTTs over the duration of the treatment protocol. In all recipients, the immunosuppressive therapy was effective in suppressing total white cell count including all key immune cells (B cells, T cells, monocytes and granulocytes) with all grafts surviving for over 100 days.  Discussion/Conclusion: The current NICC isolation technique allowed optimisation to produce the greatest IEQ yield with the resulting transplants achieving normoglycemia in less than 1 month and providing normoglycaemia and PcPep production for up-to 12 months.