Please use this identifier to cite or link to this item: http://scholarbank.nus.edu.sg/handle/10635/17968
Title: Microfluidic study of plasticity in pancreatic beta-cell heterogeneity
Authors: TAN CHERNG-WEN, DARREN
Keywords: pancreatic, beta-cell, heterogeneity, microfluidic, gradient generator
Issue Date: 31-Jul-2009
Source: TAN CHERNG-WEN, DARREN (2009-07-31). Microfluidic study of plasticity in pancreatic beta-cell heterogeneity. ScholarBank@NUS Repository.
Abstract: This dissertation is divided into two parts - a major part and a minor part. In the major part comprising five chapters, a microfluidic system is developed and applied to the study of pancreatic ß-cells. A hypothesis is proposed that the establishment of spatial gradients of extracellular glucose and promotion of ß-cell interactions are adequate for development of glucose concentration-mapped functional heterogeneity in ß-cell populations. Three main questions are posed within the context of this hypothesis and addressed with the help of two specific assays. The two assays are tested by designing an in vitro microfluidic system that allows the establishment and control of glucose gradients over the micro-scale length of islets. An immortalized insulin secreting cell line derived from transgenic mice, ß-TC-6 is applied in this test. The gradient generating section of the microfluidic system comprises a source microchannel and a sink microchannel aligned in parallel. These are connected by 24 perpendicular cross-channels, and converge at the end of the series of cross-channels to form a single eluent channel. The main and cross-channels are about 105 µm and 310 µm in width respectively. Each cross-channel is about 317 µm in length. All the microchannels are 50 µm in height. The final product includes poly(ethylene glycol) diacrylate hydrogel barriers at either end of each cross-channel. Each hydrogel has a height of 50 µm and a width of 82 µm. This microsystem is designated the hydrogel-assisted gradient generator or HAGG. The fluorophore Alexa Fluor 488 and a fluorescent glucose analog (2-NBDG) are used as probes to illustrate the generation of stable, reproducible and linear, probe concentration gradients in the absence of cells. A method is developed for estimating the diffusivity and hydrogel permeability of a solute from in situ imaging data. Concentration gradients are also generated in the presence of ß-TC-6 to demonstrate the compatibility of the system for our study. The three questions are concerned with the establishment of microscale glucose gradients over a population of ß-TC6 cells to determine the resultant mapping of (a) 2-NBDG accumulation, (b) insulin storage, and (c) insulin secretion. The two specific assays test for ß-cell response by probing for net GLUT-2/glucokinase activity that leads to intracellular 2-NBDG fluorescence, and intracellular vesicle density revealed by quinacrine fluorescence. Our results show that (a) net GLUT-2/glucokinase activity is heterogeneous and does not map to extracellular glucose gradient, (b) quinacrine uptake is homogeneous with no mapping of insulin storage to glucose gradient, and (c) insulin secretion is not influenced by the imposed glucose gradient. In the second part of this dissertation, a microfluidic competition assay valid for the case of equilibrium binding between a receptor and competing ligands is developed. A mathematical model describes the transient, convection-dispersion of solutes, undergoing equilibrium binding to immobilized receptors, while entrained in a low Reynolds number incompressible fluid flowing through a microchannel. The proposed method involves monitoring the elution profile of a reference molecule and ligand in the presence of a competitor. The time difference between the two breakthrough curves provides a measure of the unknown concentration of the competitor. Theoretical results illustrate the general method for determining the equilibrium dissociation constant (Kd) of the ligand and competitor, as well as the competitor concentration. Experimental data is presented for the binding of fluorescein-labeled insulin and unlabeled insulin to a monoclonal antibody. It is found that the unlabeled insulin binds with higher affinity ( Kd = 0.17 µM) than the labeled insulin (Kd = 0.76 µM). The potential advantages of the method and further improvements in the model are discussed.
URI: http://scholarbank.nus.edu.sg/handle/10635/17968
Appears in Collections:Ph.D Theses (Open)

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