In vitro characterization of neonatal, juvenile, and adult porcine islet oxygen demand, ß-cell function, and transcriptomes.

BACKGROUND: There is currently a shortage of human donor pancreata which limits the broad application of islet transplantation as a treatment for type 1 diabetes. Porcine islets have demonstrated potential as an alternative source, but a study evaluating islets from different donor ages under unified protocols has yet to be conducted. METHODS: Neonatal porcine islets (NPI; 1-3 days), juvenile porcine islets (JPI; 18-21 days), and adult porcine islets (API; 2+ years) were compared in vitro, including assessments of oxygen consumption rate, membrane integrity determined by FDA/PI staining, ß-cell proliferation, dynamic glucose-stimulated insulin secretion, and RNA sequencing. RESULTS: Oxygen consumption rate normalized to DNA was not significantly different between ages. Membrane integrity was age dependent, and API had the highest percentage of intact cells. API also had the highest glucose-stimulated insulin secretion response during a dynamic insulin secretion assay and had 50-fold higher total insulin content compared to NPI and JPI. NPI and JPI had similar glucose responsiveness, ß-cell percentage, and ß-cell proliferation rate. Transcriptome analysis was consistent with physiological assessments. API transcriptomes were enriched for cellular metabolic and insulin secretory pathways, while NPI exhibited higher expression of genes associated with proliferation. CONCLUSIONS: The oxygen demand, membrane integrity, ß-cell function and proliferation, and transcriptomes of islets from API, JPI, and NPI provide a comprehensive physiological comparison for future studies. These assessments will inform the optimal application of each age of porcine islet to expand the availability of islet transplantation.

Antegrade persufflation of porcine kidneys improves renal function after warm ischemia.

Introduction: Transplantation of kidneys from expanded criteria donors (ECD), including after circulatory death (DCD), is associated with a higher risk of adverse events compared to kidneys from standard criteria donors. In previous studies, improvements in renal transplant outcomes have been seen when kidneys were perfused with gaseous oxygen during preservation (persufflation, PSF). In the present study, we assessed ex-vivo renal function from a Diffusion Contrast Enhanced (DCE)-MRI estimation of glomerular filtration rate (eGFR); and metabolic sufficiency from whole-organ oxygen consumption (WOOCR) and lactate production rates. Methods: Using a porcine model of DCD, we assigned one kidney to antegrade PSF, and the contralateral kidney to static cold storage (SCS), both maintained for 24 h at 4°C. Post-preservation organ quality assessments, including eGFR, WOOCR and lactate production, were measured under cold perfusion conditions, and biopsies were subsequently taken for histopathological analysis. Results: A significantly higher eGFR (36.6 ± 12.1 vs. 11.8 ± 4.3 ml/min, p < 0.05), WOOCR (182 ± 33 vs. 132 ± 21 nmol/min*g, p < 0.05), and lower rates of lactate production were observed in persufflated kidneys. No overt morphological differences were observed between the two preservation methods. Conclusion: These data suggest that antegrade PSF is more effective in preserving renal function than conventional SCS. Further studies in large animal models of transplantation are required to investigate whether integration with PSF of WOOCR, eGFR or lactate production measurements before transplantation are predictive of post-transplantation renal function and clinical outcomes.

Accelerated absorption of regular insulin administered via a vascularizing permeable microchamber implanted subcutaneously in diabetic Rattus norvegicus.

In Type 1 diabetes patients, even ultra-rapid acting insulins injected subcutaneously reach peak concentrations in 45 minutes or longer. The lag time between dosing and peak concentration, as well as intra- and inter-subject variability, render prandial glucose control and dose consistency difficult. We postulated that insulin absorption from subcutaneously implantable vascularizing microchambers would be significantly faster than conventional subcutaneous injection. Male athymic nude R. norvegicus rendered diabetic with streptozotocin were implanted with vascularizing microchambers (single chamber; 1.5 cm2 surface area per side; nominal volume, 22.5 µl). Plasma insulin was assayed after a single dose (1.5 U/kg) of diluted insulin human (Humulin®R U-100), injected subcutaneously or via microchamber. Microchambers were also implanted in additional animals and retrieved at intervals for histologic assessment of vascularity. Following conventional subcutaneous injection, the mean peak insulin concentration was 22.7 (SD 14.2) minutes. By contrast, when identical doses of insulin were injected via subcutaneous microchamber 28 days after implantation, the mean peak insulin time was shortened to 7.50 (SD 4.52) minutes. Peak insulin concentrations were similar by either route; however, inter-subject variability was reduced when insulin was administered via microchamber. Histologic examination of tissue surrounding microchambers showed mature vascularization on days 21 and 40 post-implantation. Implantable vascularizing microchambers of similar design may prove clinically useful for insulin dosing, either intermittently by needle, or continuously by pump including in "closed loop" systems, such as the artificial pancreas.

Oxygen Perfusion (Persufflation) of Human Pancreata Enhances Insulin Secretion and Attenuates Islet Proinflammatory Signaling.

BACKGROUND: All human islets used in research and for the clinical treatment of diabetes are subject to ischemic damage during pancreas procurement, preservation, and islet isolation. A major factor influencing islet function is exposure of pancreata to cold ischemia during unavoidable windows of preservation by static cold storage (SCS). Improved preservation methods may prevent this functional deterioration. In the present study, we investigated whether pancreas preservation by gaseous oxygen perfusion (persufflation) better preserved islet function versus SCS. METHODS: Human pancreata were preserved by SCS or by persufflation in combination with SCS. Islets were subsequently isolated, and preparations in each group matched for SCS or total preservation time were compared using dynamic glucose-stimulated insulin secretion as a measure of ß-cell function and RNA sequencing to elucidate transcriptomic changes. RESULTS: Persufflated pancreata had reduced SCS time, which resulted in islets with higher glucose-stimulated insulin secretion compared to islets from SCS only pancreata. RNA sequencing of islets from persufflated pancreata identified reduced inflammatory and greater metabolic gene expression, consistent with expectations of reducing cold ischemic exposure. Portions of these transcriptional responses were not associated with time spent in SCS and were attributable to pancreatic reoxygenation. Furthermore, persufflation extended the total preservation time by 50% without any detectable decline in islet function or viability. CONCLUSIONS: These data demonstrate that pancreas preservation by persufflation rather than SCS before islet isolation reduces inflammatory responses and promotes metabolic pathways in human islets, which results in improved ß cell function.