Design of a Multiparameter Islet-on-a-chip Device to Measure the Functional Variability of Individual Pancreatic Islets
Regeenes, Romario 1 ; Saleem, Afifa 2 ; Chang, Huntley 1 ; Rocheleau, Jonathan 1, 3, 4
1. Institute of Biomaterials and Engineering, University of Toronto; 2. Division of Engineering Science, University of Toronto; 3. Department of Physiology, University of Toronto; 4. Toronto General Research Institute, University Health Network
Rationale: Type 1 diabetes results from autoimmune destruction of pancreatic islets, which are small micro-tissues (~150 m) primarily composed of insulin secreting beta-cells. Islet transplantation is the current gold-standard for treatment; however, limited supply of useable donor tissue severely limits treatment accessibility. Tissue engineered islets could help increase the supply of viable beta-cells, providing an opportunity to treat many more people. Such type of tissue engineering however would require a robust method of measuring the quality and uniformity of islet tissue. Gold standard approaches to measure pancreatic islet function include measuring glucose-stimulated insulin secretion (GSIS), oxygen consumption rate (OCR), and extracellular acidification rate (ECAR). However, these assays are currently done on large batches of islets and thus do not reveal the full functional heterogeneity of the tissue samples.
Approach/Research Plan: To measure the functional variability of islets/engineered tissues, we are creating a microfluidic device to assay multiple readouts of function such as GSIS, OCR, and ECAR. First, we aim to measure C-peptide release as a proxy for endogenous insulin levels. This will be done by creating an on-chip competition assay based on C-peptide conjugated to 5-TAMRA (C-peptide*) and then monitoring the accompanying fluorescence anisotropy. Secondly, we aim to measure oxygen consumption rates of individual islets. Oxygen is consumed by the electron transport chain and by monitoring its consumption we can identify perturbations in oxidative phosphorylation. We are using RuII(bpy)3 as an optical sensor to measure oxygen with excitation and emission maxima at 450 and 600 nm, respectively. Lastly, we aim to measure extracellular acidification rate as a readout of glycolytic rate and/or oxidative phosphorylation. We will use HPTS, a pH sensitive dye, that will be dissolved in solution. Each of these sensors is spectrally and/or spatially resolved with respect to the other sensors, which will allow us to simultaneously measure each of these responses to fully index the function of individual islets and engineered micro-tissues.
Significance: Creating a device that can simultaneously measure multiple functional readouts from an individual islet provides an unprecedented opportunity to assess the full function and variability of both isolated islets and engineered tissues. This holds promising clinical relevance as an understanding of the quality of an islets/engineered tissue batch allows for pre-screening prior to transplantation. In terms of research, the ability to study individual islets provides information about heterogeneity that was previously unmeasurable. Our development of a novel microfluidic device provides a unique opportunity to investigate how glucotoxic levels affect islet health and whether pre-treating isolated islets can improve function before transplantation.