An Integrated Microfluidic Platform for Online Analyses of Hepatic Metabolism
Adams, Anna Georgia (author)
Roper, Michael Gabriel (professor directing dissertation)
Chadwick, Brian P. (university representative)
Miller, Brian G. (committee member)
Stagg, Scott (committee member)
Florida State University (degree granting institution)
College of Arts and Sciences (degree granting college)
Department of Chemistry and Biochemistry (degree granting department)
In this dissertation, the work presented describes an integrated microfluidic system composed of pressure driven perfusion and online measurement techniques for analyses of hepatic metabolism. The liver is primarily made up of hepatocytes, which undergo anabolic and catabolic pathways in response to hormonal stimuli from the pancreas in order to maintain blood glucose homeostasis; therefore, faults in the mechanisms involving hepatocytes can have a detrimental effect on glucose metabolism. Type II diabetes, which is now a worldwide pandemic, is a result of defective insulin secretion from the pancreas and insulin resistance in surrounding tissues, ultimately leading to abnormal glucose levels in the blood. It is important to investigate the secretory patterns of pancreatic hormones such as insulin, to determine how their dynamic profiles contribute to effective glucose handling, which is gradually lost in patients with Type II diabetes, and as early as pre-diabetes.1,2 This will potentially shed light on the mechanisms behind proper glucose regulation in the body, and therefore facilitate therapeutic approaches to managing glucose levels. To investigate this, an in vitro system was developed to enable automated perfusion of pancreatic hormones to hepatocytes, coupled with real-time detection of metabolic output. With an in vitro system, it is necessary to mimic as best as possible cellular architecture in order to depict accurate structure and function of an organ. For this purpose, a microfluidic bioreactor was developed in order to generate an environment for liver cells to grow in 3D, similar to in vivo. This liver-on-a-chip allowed for successful growth, viability, and metabolic function of hepatocytes over time. Fluorescence detection was directly interfaced with the liver-on-a-chip and used for highly sensitive, continuous online measurement of extracellular secretions in order to quantify various components of metabolism, such as glucose, during long-term culture and in near realtime. Glucose was measured via an online Amplex Red based assay, which was optimized for direct on-chip analysis incorporated into the output of the liver-on-a-chip. The combined extracellular samples and assay reagents were encapsulated into microfluidic nanoliter droplets, resulting in a quick reaction time due to rapid mixing, while maintaining accurate readings. In order to determine whether the newly developed online assay could be used for in vitro measurements, glucose consumption rates were measured in the absence and presence of insulin. The liver-on-a-chip device, which accurately mimicked the structure and metabolic function of the liver, was perfused with various solutions of pancreatic hormones in order to generate different aspects of glucose metabolism using the automated pressure-driven flow system. During perfusion, the extracellular output from the bioreactor was coupled with enzymatic assay reagents to determine extracellular glucose levels. The combined solutions were encapsulated into the droplet based microfluidic system, and the intensity of the resulting fluorescent product in the droplets was measured and directly correlated with glucose concentration. This allowed for measurement and quantification of rapid changes in glucose output from hepatocytes in response to pancreatic hormone perfusion online and in near real-time. Since healthy pancreatic hormone secretions that are pulsatile and out of phase are gradually lost at the onset of diabetes, this suggests that inter-islet synchronization that occurs in the pancreas is also lost, and ultimately results in improper glucose production and storage by the liver. Dynamic stimulation of insulin is more effective in maintaining healthy blood glucose concentrations overall, but how the normal and altered dynamic profiles affect glucose metabolism in the liver specifically is not completely understood. Using the coupled microfluidic system for perfusing hepatocytes with pancreatic hormones and detecting their metabolic output, dynamic insulin stimulation was recreated in vitro, allowing for the effect of pulsed insulin characteristics on glucose consumption in hepatocytes to be observed and quantified online and in near real-time. In the future, the perfusion system can be expanded to deliver additional dynamic hormone perfusion profiles in further investigation. The detection system can also be expanded to incorporate multi-detection of additional components involved in hepatic glucose metabolism using similar Amplex Red based assays.
droplets, hepatocyte, microfluidics, online assay
June 24, 2020.
A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Includes bibliographical references.
Michael G. Roper, Professor Directing Dissertation; Brian Chadwick, University Representative; Brian Miller, Committee Member; Scott Stagg, Committee Member.
Florida State University