Immune System On-a-Chip: Microfluidic-approaches to Study Cellular Interactions under In Vivo Mimetic Environment

Abstract

The human immune system is a complex network of molecules, organelles, cells, tissues, and organs - there are an uncountable number of interactions and transformations interconnecting all the system components. In addition to such biochemical components, pressure, flow, morphology and location of all of such biophysical components also play an important role in the human immune system. While technical difficulties have frequently limited consideration of the immune system component interactions, some state-of-the-art analytical techniques have revealed distinct cellular behaviors that occur only in vivo or in vivo-mimetic environments. These types of findings inspire analytical/bioanalytical chemists to provide new tools to mimic such interactions occurring in the human body and facilitate better understanding of the human immune system. Use of microfluidics in analytical/bioanalytical science is a relatively recent development but provides significant potential for such purpose. Well-designed microfluidic approaches can reveal both biophysical and biochemical aspects of human in vivo systems with quick and temporally, spatially, and chemically resolved analyses of in vitro assays. The aim of this thesis is to enable the study of cellular behaviors in physiologically relevant environments using microfluidics. Chapter 1 of this thesis provides a brief review of microfluidics-based innovations in cellular biology studies, and the following chapters focus on biophysical (e.g. flow, Chapter 2), biochemical (e.g. multiple chemical signals, Chapter 3), and higher level biological networks (e.g. cell-cell interaction, Chapter 4) and describe how microfluidics-based approaches advance our current understanding of cellular behaviors. Furthermore, this thesis suggests a potential future direction of microfluidics-based assay platforms by incorporating a powerful detection scheme, surface-enhanced Raman scattering (SERS), into a microfluidic platform (Chapter 5). Fluid flow is ubiquitous in the human body, and thus, the first part of the thesis focuses on the flow-induced biophysical impact on cellular behaviors. The biological model herein is the human blood vessel, and the cytotoxicity of nanoparticles under varying flow conditions is investigated in the context of its impact on cellular viability and adhesion/aggregation behaviors. Mesoporous silica nanoparticles are used as the model nanoparticles and human endothelial cells and platelets are used as the model cell types to mimic human blood vessels. Compared to traditional cytotoxicity assays performed under static conditions, mesoporous silica nanoparticles show higher and shear stress-dependent toxicity to endothelial cells under flow conditions. Furthermore, nanoparticles slightly compromise platelet adhesion to endothelial cells at low nanoparticle doses; however, high nanoparticle doses significantly increase the number of platelet adhesion events, leading to higher probability for uncontrolled platelet actions (e.g. clot formation in vivo). Interestingly, despite the altered level of platelet adhesion and aggregation, in no case did nanoparticle exposure result in significant loss of platelet viability. This work clearly demonstrates that 1) biophysical aspects such as fluid flow have to be considered in studying cellular behaviors and 2) aspects besides viability, such as cellular adhesion and interaction with other cell types, have to be considered in the context of nanotoxicology. The simple and highly adaptable microfluidic analytical platform used in this chapter will be useful for further studies involving other nanoparticles, different cell types, and different cell functions. Besides biophysical aspects such as flow, cells in the human body always interact with numerous chemical signals and other cell types around them - this thesis also investigates these interactions. By using a model of chemotaxis, chemically directed cellular migration, the dynamics of cellular behaviors under multiple chemical signals (Chapter 3) and in the context of cell-cell interactions (Chapter 4) were quantitatively studied using a microfluidic platform. The target cells used in these chapters are neutrophils, the most abundant and motile leukocyte in humans. The microfluidic platform establishes a stable and dynamic gradient of chemicals across a cell culture chamber and enables the quantitative investigation of cellular migration in the presence of multiple chemical signals. This study leads to interesting findings such as a hierarchy of neutrophil response to chemical signals, the involvement of an enzyme in regulating neutrophils to decide which signal to follow, and the involvement of endothelial cells in regulating neutrophil chemotaxis through surface marker expression. In addition, this study also reveals the impact of neutrophil activation in neutrophil chemotaxis, providing insights into the potential of neutrophil chemotaxis regulation for therapeutic purposes. This study brings new knowledge about neutrophil chemotaxis by replicating physiologically relevant environments for neutrophils (e.g. multiple chemical signals in the context of cell-to-cell communications), and yields both fundamental and therapeutically relevant insight. In parallel to above mentioned biochemical studies with in vivo mimetic microfluidic platforms, this thesis also aims to achieve microfluidics-based analytical platforms with powerful chemical identification capability. Microfluidics facilitates parallel, low volume sample manipulation and provides opportunities for various optical signal transduction mechanisms. Chapter 5 of this thesis provides proof-of-concept microfluidic sensor platforms that fully utilize such advantages of microfluidics. The developed microfluidic sensor platforms are equipped with gold-based SERS substrates that enable highly sensitive chemical fingerprinting, and the microfluidic device enables serial dilution of introduced analyte solution that terminates in five discrete sensing elements. Chapter 5 demonstrates the utility of the developed sensor platforms to create a calibration curve or do a limit of detection study in a single experiment. Overall, the thesis herein advances our current understanding of cellular behaviors occurring inside the human body, and demonstrates the utility and a potential future direction of microfluidics-based experimental platforms. All findings described in this thesis will facilitate better understanding of the human immune system and lead to new development of medical and pharmacological applications.