Purpose: The in vitro study of human islets is often limited by the availability of specific tools. Microfluidic technology has emerged as a hugely promising platform to effectively study insulin secretion kinetics as well as screen for potential drugs and chemicals that may be used to target diabetes. For this project, we have developed a microfluidic perifusion multiplexer, designed exclusively to study the effects of numerous chemicals on viable islets in a dynamic culture environment. Methods: Four identical three-layer PDMS microfluidics were designed, based on our first generation single perifusion device, and integrated with a chaotic mixer and distributor to generate various chemical gradients. The top layer is composed of an inlet and outlet; the middle layer acts as a perifusion chamber; and the bottom layer is used to trap islets. Without the need for tissue fixing and/or islet dissociation, islets were simultaneously cultured and optical imaged of key insulin stimulator-secretion pathways. Results: (i) Chemical gradients were generated and verified by using various concentrations of glucose and bovine albumin and indicated very high consistence (R 2 = 0.997) and flexibility. (ii) Rodent islets were dynamically cultured for an extended period of time in a square-wave cycle (20 min 5 mM -20 min 12 mM; up to 72 hours) at a flow rate of 60¯o;l/min and displayed normal morphology and viability (95% ± 2.5). Additionally, the post-cultured islets demonstrated normal glucose-stimulated insulin secretion kinetics, mitochondrial potential changes, and intracellular calcium signaling. (iii) Rodent islets exposed to various concentrations of sirolimus (0, 12.5, 37.5, and 50 nM) for 72 hrs, displayed a dose–dependent inhibition of mitochondrial energetic status (65-100%) and calcium influx (8-18%) were observed. (iv) Sirolimus also inflicts an acute reduction of NAD(P)H in a dose-dependent manner (35-90%) stimulated by 14mM glucose. This reduction in NAD(P)H was linked to NAD(P)H oxidase upregulation. Conclusion: This Microfluidic-Based Multiplexer is the first microfluidic device used to dynamically study islet physiology and immunosuppressants. This novel technology provides immense potential for understanding underlying mechanisms associated with immunosuppression toxicity on islets and in the area of drug screening.

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