Autofluorophores Assessed by Hyperspectral Microscopy Indicate Perturbation and Transplant Viability in Pancreatic Islets

Abstract

Abstract Background Islets prepared for transplantation into type 1 diabetes patients are exposed to compromising factors that contribute to early graft failure necessitating repeated islet infusions for clinical insulin independence. Lack of reliable pre-transplant measures to determine islet viability severely limits the success of islet transplantation. We imaged cell autofluorophores using hyperspectral microscopy to characterise loss of viability in islets and define a non-invasive methodology for predicting transplant outcomes in mice. Methods Islet viability was compromised by exposure to oxidative stress (menadione), hypoxia (dimethyloxalylglycine), cytokine injury (TNF-α, IFNγ, and IL-1β), or warm ischemia (30- and 60-minute delayed pancreas collection). The impact of islet encapsulation in a conformal coating of hydrogen-bonded poly(N-vinylpyrrolidone)/ tannic acid (PVPON/TA) multilayer film was investigated. LED illumination produced excitation at 358 to 476 ± 5 nm in 18 steps, emission was detected using filters at 414, 451, 575, 594, and 675 ± 20 nm. Syngeneic (C57BL/6Ausb) mice with diabetes (alloxan tetrahydrate) were used to test viability on transplantation. Discriminative analysis and unsupervised principal component analysis were used to differentiate groups. Unmixing of spectral signals to identify component fluorophores was carried out using the unsupervised algorithm Robust Dependent Component Analysis (RoDECA). Findings: The autofluorophores NAD(P)H, flavins, collagen-I and cytochrome-C were successfully unmixed. Redox ratio (NAD(P)H/flavins) was significantly increased in islets exposed to ROS, hypoxia, cytokine injury and warm ischemia, typically driven by elevated NAD(P)H. Receiver operating characteristic assessment showed that our models were able to detect; oxidative stress (ROS) (AUC = 1.00) hypoxia (AUC = 0.69), cytokine exposure (AUC = 0.94), or warm ischemia (AUC = 0.94) compared to islets harvested from pristine anesthetised heart beating mouse donors. Significantly, we defined an unsupervised autofluorescent score for ischemic islets that accurately predicted restoration of glucose control in diabetic recipients. Similar results were obtained for islet single cell suspensions, suggesting translational utility in the context of emerging beta cell replacement strategies. Conclusions Hyperspectral microscopy of autofluorescence has the potential to give a non-invasive indication of islet viability, prior to transplantation. This would inform clinical decision making and enable patients to be spared transplantation attempts with no potential to reduce their dependence on exogenous insulin.