Abstract
BackgroundOxygen consumption reflects multiple processes in pancreatic islets including mechanisms contributing to insulin secretion, oxidative stress and viability, providing an important readout in studies of islet function, islet viability and drug testing. Due to the scarcity, heterogeneity, and intrinsic kinetic properties of individual islets, it would be of great benefit to detect oxygen consumption by single islets. We present a novel method we have developed to image oxygen in single islets.Methodology/Principal FindingsUsing a microfluidics system, individual islets and a fluorescent oxygen-sensitive dye were encased within a thin alginate polymer layer. Insulin secretion by the encapsulated islets was normal. Fluorescent signal from the encased dye, detected using a standard inverted fluorescence microscope and digital camera, was stable and proportional to the amount of oxygen in the media. When integrated into a perifusion system, the sensing system detected changes in response to metabolic substrates, mitochondrial poisons, and induced-oscillations. Glucose responses averaged 30.1±7.1% of the response to a metabolic inhibitor (cyanide), increases were observed in all cases (n = 6), and the system was able to resolve changes in oxygen consumption that had a period greater than 0.5 minutes. The sensing system operated similarly from 2–48 hours following encapsulation, and viability and function of the islets were not significantly affected by the encapsulation process.Conclusions/SignificanceAn oxygen-dependent dye situated around and within a pancreatic islet encapsulated by a thin layer of alginate was sensitive to changes in oxygen consumption, and was not harmful to the function or viability of islets over the course of two days. The microcapsule-based sensing method is particularly suited to assessing the effects of compounds (dose responses and time courses) and chronic changes occurring over the course of days. The approach should be applicable to other cell types and dyes sensitive to other biologically important molecules.
Highlights
Oxygen consumption rate (OCR) is a critical parameter to assess as part of investigations of islet physiology and viability
Oxygen is the final acceptor of electrons traversing the electron transport chain (ETC), and in islets oxygen utilization associated with the ETC represents most of the total cellular OCR [1,2]
A number of microsensors have been used for measuring oxygen tension in islets [18,35], the use of microencapsulated oxygen-sensitive dye offers several advantages
Summary
Oxygen consumption rate (OCR) is a critical parameter to assess as part of investigations of islet physiology and viability. Since the rate of the ETC reflects cellular metabolism of nutrients, the ability to generate energy by the cell, and driving forces and signals for oxidative stress and apoptosis. OCR has been used for islet quality assessment in islet transplantation [3,4], tests of viability in the optimization of immunoisolation devices [5,6], studies of beta cells during differentiation from stem cells [7,8], and mechanistic studies of islet physiology [9,10,11]. Oxygen consumption reflects multiple processes in pancreatic islets including mechanisms contributing to insulin secretion, oxidative stress and viability, providing an important readout in studies of islet function, islet viability and drug testing. We present a novel method we have developed to image oxygen in single islets
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