Microfluidic device for multimodal characterization of pancreatic islets

Abstract

A microfluidic device to perfuse pancreatic islets while simultaneously characterizing their functionality through fluorescence imaging of the mitochondrial membrane potential and intracellular calcium ([Ca(2+)](i)) in addition to enzyme linked immunosorbent assay (ELISA) quantification of secreted insulin was developed and characterized. This multimodal characterization of islet function will facilitate rapid assessment of tissue quality immediately following isolation from donor pancreas and allow more informed transplantation decisions to be made which may improve transplantation outcomes. The microfluidic perfusion chamber allows flow rates of up to 1 mL min(-1), without any noticeable perturbation or shear of islets. This multimodal quantification was done on both mouse and human islets. The ability of this simple microfluidic device to detect subtle variations in islet responses in different functional assays performed in short time-periods demonstrates that the microfluidic perfusion chamber device can be used as a new gold standard to perform comprehensive islet analysis and obtain a more meaningful predictive value for islet functionality prior to transplantation into recipients, which is currently difficult to predict using a single functional assay.

Figure 1

(a) Design of the microfluidic device and image of the actual device. The schematic depicts the cross-section view and experiment setup (left part) and the isometric view of a single-chamber of PDMS microfluidic device is shown in the insert image (right part). The device contains three layers. The bottom layer consists of an array of small circular (150 µm deep, 500 µm diameter) wells that help immobilize the islets exposed to flow. The next layer is a large circular well (3 mm deep, 7 mm diameter) that encompasses the array of the tiny wells and the top-most layer is a rectangular microchannel (500 µm deep, 2 mm wide) that provides access to these wells. (b) Plots of fluorescence intensity versus time across different regions in the perfusion chamber during the perfusion (flow rate of 1 mL/min) of DI water for 30 s followed by fluorescein isothiocyanate (2 µM FITC) for 60 s, and finally flushing out the fluorescein with DI water (intensity values are area averages of n = 3 scans at a region of interest).

Figure 1

(a) Design of the microfluidic device and image of the actual device. The schematic depicts the cross-section view and experiment setup (left part) and the isometric view of a single-chamber of PDMS microfluidic device is shown in the insert image (right part). The device contains three layers. The bottom layer consists of an array of small circular (150 µm deep, 500 µm diameter) wells that help immobilize the islets exposed to flow. The next layer is a large circular well (3 mm deep, 7 mm diameter) that encompasses the array of the tiny wells and the top-most layer is a rectangular microchannel (500 µm deep, 2 mm wide) that provides access to these wells. (b) Plots of fluorescence intensity versus time across different regions in the perfusion chamber during the perfusion (flow rate of 1 mL/min) of DI water for 30 s followed by fluorescein isothiocyanate (2 µM FITC) for 60 s, and finally flushing out the fluorescein with DI water (intensity values are area averages of n = 3 scans at a region of interest).

Figure 2

Fabrication process flow for the microfluidic perfusion device. Standard SU8 lithography was used to create the masters for the array of small wells and the rectangular channels. The microfluidic device contains three layers of PDMS.

Figure 3

(a) Temporal insulin secretion profiles of mouse islets perfused with basal and different stimulatory glucose solutions, (b) Temporal insulin secretion and [Ca²⁺]_i [indicated by Fura-2 ratio of the fluorescence intensities (340/380 nm)] profiles of C57/B6 mice islets perfused with basal and stimulatory glucose, KCl solutions, (c) Temporal insulin secretion and [Ca²⁺]_i [indicated by Fura-2 ratio of the fluorescence intensities (340/380 nm)] profiles of BALB/c mice islets perfused with basal and stimulatory glucose, KCl solutions, (d) Temporal insulin secretion and mitochondrial membrane potential profiles (indicated by fluorescence intensity of Rh123) of the mouse islets perfused with basal and different stimulatory glucose solutions. In all the experiments, 25 mouse islets were loaded per perfusion chamber. These are results from single representative experiments with n = 3 replicates at each point for Fura-2 ratios and Rh123 intensities.

Figure 3

(a) Temporal insulin secretion profiles of mouse islets perfused with basal and different stimulatory glucose solutions, (b) Temporal insulin secretion and [Ca²⁺]_i [indicated by Fura-2 ratio of the fluorescence intensities (340/380 nm)] profiles of C57/B6 mice islets perfused with basal and stimulatory glucose, KCl solutions, (c) Temporal insulin secretion and [Ca²⁺]_i [indicated by Fura-2 ratio of the fluorescence intensities (340/380 nm)] profiles of BALB/c mice islets perfused with basal and stimulatory glucose, KCl solutions, (d) Temporal insulin secretion and mitochondrial membrane potential profiles (indicated by fluorescence intensity of Rh123) of the mouse islets perfused with basal and different stimulatory glucose solutions. In all the experiments, 25 mouse islets were loaded per perfusion chamber. These are results from single representative experiments with n = 3 replicates at each point for Fura-2 ratios and Rh123 intensities.

Figure 4

(a) Temporal insulin secretion profiles of human islets perfused with basal and different stimulatory glucose solutions, (b) Temporal insulin secretion profiles of human islets perfused with basal and different secretagogue (KCl, TB, KIC) solutions, (c) Temporal insulin secretion and [Ca²⁺]_i [indicated by Fura-2 ratio of the fluorescence intensities (340/380 nm)] profiles of human islets perfused with basal and stimulatory glucose, KCl solutions. In all the experiments, 100 human islets were loaded per perfusion chamber. These are results from single representative experiments with n = 3 replicates at each point for Fura-2 ratios.

Figure 4

(a) Temporal insulin secretion profiles of human islets perfused with basal and different stimulatory glucose solutions, (b) Temporal insulin secretion profiles of human islets perfused with basal and different secretagogue (KCl, TB, KIC) solutions, (c) Temporal insulin secretion and [Ca²⁺]_i [indicated by Fura-2 ratio of the fluorescence intensities (340/380 nm)] profiles of human islets perfused with basal and stimulatory glucose, KCl solutions. In all the experiments, 100 human islets were loaded per perfusion chamber. These are results from single representative experiments with n = 3 replicates at each point for Fura-2 ratios.