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Presenter: Jose, Oberholzer, Chicago, United States
Authors: Jose Oberholzer
Beta-cell replacement through islet transplantation can achieve tight glycemic control, prevent hypoglycemia and diabetic complications. Manufacturers have to ensure the safety, purity and potency of isolated human islets or surrogates before release as a cellular product to treat diabetic patients. Potency testing should include the measurement of a clinically relevant function, be beta-cell specific, reproducible, and easy to apply. Ideally, in vitro potency tests should have a good predictive value for in vivo islet transplant function. At present, there is no reliable potency test that meets all requirements. Given the limited pool of cadaveric islets, it is imperative to develop unlimited islet cell sources, either stem cell derived or xenogeneic, and new immuno-protective strategies. It would be desirable to possess new in vitro systems that could facilitate screening methods for improved protocols in stem cell research for islet derivatives and in material science for cell encapsulation. Similar to pharmacokinetics in drug development, future cell-biomaterial combination products will need precise characterization of cellular sensing and secretion kinetics. One of the challenges is to have comprehensive in vitro tools for better understanding the physiology of surrogate cells as compared to isolated human islets, as well as describe their behavior within devices. While some of the current assays such as static glucose incubation and dynamic perifusion assays provide important information, they give limited insight into the glucose-insulin coupling mechanism and the kinetics.
Over the last decade, our laboratory has developed a set of biochip-based micro- and nanofluidic, multiparametric perifusion assays designed specifically for studying beta-cell physiology and phenotyping islet surrogates from various sources and with or without microdevices. The biochips integrate islet mico- and nanoperifusion with multiparametric imaging technology that measures not only insulin secretion kinetics but also the property of key insulin-stimulator coupling factors such as calcium influx, mitochondrial potentials, KATP and calcium channel activity, NAD(P)H, and ROS. Furthermore, integrated high-throughput islet arrays and multiplexing have significantly increased the analytical power and better understanding of the heterogeneity of stem cell derived islet surrogates. To date, we have tested stem cell derived beta-like cells originating from various protocols, showing functional heterogeneity and varying responsive profiles to insulin stimulators.
It would also be important to understand insulin granule biogenesis, sorting, trafficking, fusion, and exocytosis in primary and stem cell derived islets. Thought the field has already significant insights into these processes, there are numerous areas of these signals and molecular pathways that remain to be investigated, especially for stem cell derived islets. More recently, our laboratory has developed dynamic, label-free, live-cell electron microscopy imaging technology for quantification of insulin granule mass and size distribution, as well as visualization of insulin granule trafficking, insulin granule fusion with plasma membrane, and exocytosis. This novel technology may help further characterize stem cell derived islets.
In summary, we developed several in vitro, biochip based micro- and nanofluidic technologies that can be applied to study the physiology, function and maturity of pancreatic islets and its stem cell derived surrogates.
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