Development and Applications of Microfluidic Devices for Liver-on-a-Chip Studies

Abstract

Modeling the human physiology in vitro is a challenging task, yet one of importance for the development of drugs and the study of diseases. To better address the complexities of human organs, microfluidic systems called organs-on-chips are being developed to emulate organ-specific environments for cell culture. Such systems are designed to recapitulate one or several key physiological features from the organ in question within an in vitro culture platform. This thesis describes the work performed in order to develop, refine and validate organs-on-chips designed specifically to mimic physiological cues from the human liver. Designed to allow perfusion, low levels of shear stress, and three-dimensional culture, the livers-on-chips described herein are intended for culture of induced pluripotent stem cell-derived hepatocytes as well as cocultures between hepatocytes and hepatic non-parenchymal cells, such as hepatic stellate cells. The work performed as part of this thesis involves three different iterations of such livers-on-chips and the steps taken to validate and refine them. Methodologies used in this process, ranging from flow simulations, microfabrication, and cell culture to microscopy, immunofluorescence, and more, are summarized in detail. First, a partly validated liver-on-a-chip is described, which I applied for on-chip differentiation of induced pluripotent stem cells into hepatocytes. Responding to the shortcomings of said device, I designed, developed and optimized two further refined variants of such devices. This thesis describes the work involved in fabricating, testing and optimizing these devices for use with cell lines and induced pluripotent stem cell alike. The final iteration of microfluidic device produced as part of this project, termed LC-v3, was found to support culture of HepG2 cells in monoculture as well as in coculture with LX-2 cells, and functionality was demonstrated through albumin secretion assays. The device was also proven to support on-chip differentiation of induced pluripotent stem cells into hepatocytes, validated through the cells’ expression of albumin as observed through immunofluorescence. Further validation studies are currently ongoing. Based on the successful cultures demonstrated with the device, it is believed that the LC-v3 may in the future serve as part of a more complete multi-organ model system, and/or be implemented in drug development studies in vitro.