Abstract

Alzheimer's Disease (AD) is a leading cause of death in Canada, contributing to a significant number of annual fatalities. In 2021, it was estimated that more than 50 million Canadians had dementia, with the majority of cases attributed to AD. This condition also places a substantial burden on individuals, families, and the healthcare system. These statistics demand studying AD and developing effective and practical approaches for diagnosing, predicting, and treating the disease. Microglia cells, recognized as immune cells in the brain, have multifunctional roles and play a crucial part in brain health and AD. However, most research on microglia cells has focused on the impact of chemokines, cytokines, and cell-cell interactions. Conversely, our understanding of microglia migration and adhesion as mechanobiological properties is limited. Microfluidic chips have been increasingly used to characterize these mechanobiological properties due to their unique advantages, such as high controllability for fluid flow, portability, minimal reagent volume, and reproducibility. This dissertation focuses on developing an AD model on chip for the diagnosis and progression of AD through amyloid beta oligomers (AβO) as a biomarker using the mechanobiology study of microglia. Exploring these aspects could provide valuable insights for developing cell-based immune therapeutic strategies for AD.
Firstly, in the present thesis, a bi-functional microfluidic chip was developed to be capable of quantifying the adhesion and migration of microglial BV2 cells by combining two assays: cell adhesion assay and wound-healing migration assay. The chip could create the cell-free area (wounding) under either chemical stimuli with trypsin in chemical assays or mechanical stimuli with Phosphate-Buffered Saline (PBS) flow, as in the case of mechanical assays. As a wound-healing migration assay, this microfluidic device, with a simple microstructure and fabrication, can mimic any conditions between chemical and mechanical wound-healing migration assays using around 50 chips. During mechanical wounding, the cell removal was characterized to quantify the cell adhesion under various shear stresses of PBS flow at inlet Reynold number (Re)= 25, 50, and 100. It allowed the investigation of microglia adhesion dynamics during the wounding process and the effect of mechanical stress on cell migration. In chemical wound generation, laminar trypsin flowed within the device using gravity without any peripheral equipment (such as an external pump and microfluidic tubing), making the cell migration quantification cost-effective compared to previous approaches. The wounding study with our microfluidic chip has shown that cell migration in cell-free generated chemically with trypsin highly improved compared to mechanical cell-free creations with PBS flow and the scratch assay. The effect of shear stress variations and geometry on chemical and mechanical wounding performance is predicted in detail in this thesis.
Microglia cells, as highly active cells, always migrate in the brain and encounter various extracellular matrixes (ECMs), including various substrate elasticities (stiffness) of central nervous system (CNS) tissue in both healthy and diseased conditions of the brain. However, conventional migration assays, such as scratch assays, quantify cell migration, which removes ECMs from the substrate. Therefore, this dissertation also focuses on developing microfluidic migration assays to assess microglia BV2 without removing ECM coatings. This migration assay could generate a cell-free area by creating a laminar trypsin flow over the confluent cell monolayer. The trypsin was flown by gravity without using the syringe pump and microfluidic tubing, which makes cell migration assessment affordable and feasible in the wound-healing method compared to previous methods. Microglia BV2 migration was studied using 40 chips on the substrate coated with different ECMs, such as collagen, fibronectin, Poly-L-Lysine (PLL), and gelatin, and also on the substrate with various physical properties, including polystyrene, Polydimethylsiloxane (PDMS), and glass. It was found that PLL and gelatin had a stimulatory-migration effect compared to fibronectin, collagen, and the control condition (glass). Our findings demonstrated that among polystyrene, PDMS, and glass substrates with different physical properties, microglia BV2 cell migration improved on the petri dish coated with plasma compared to PDMS and glass. There was a migration enhancement on the substrate with lower elasticity when their migration on the PDMS was higher than glass.
In AD, Amyloid beta (Aβ) monomers are overproduced and accumulated in the brain, which can cause the formation of toxic amyloid beta oligomers (AβO). AβO are most potent for AD pathogenesis, and the variation of AβO levels is one of the major biomarkers in AD progression. This type of toxic protein can affect microglia mechanobiology properties as the cells are responsible for clearing AβO. Therefore, a quantitative method can be developed to assess microglia-substrate adhesion as a mechanobiology marker to indicate AD progression. In this dissertation, a simple microfluidic device was developed to assess microglia BV2 cells-substrate adhesion without using labeling when the cells were exposed to AβO. Microglia BV2 cells were subjected to fluid shear stresses to analyze microglia-substrate adhesion using microfluidic AD models to characterize the cell removal using time-lapse microscopy. For a comprehensive analysis of microglia cells in AD conditions, 96 chips were used to expose microglia cells in 32 conditions. Shear stresses of 3 Pa and 7.5 Pa were applied to the cells through the inlet flow of the device when the cells were exposed to various AβO concentrations of 1 µM, 2.5 µM, and 5 µM for 1 h, 6 h, 12 h, and 24 h, then compared to their control conditions. Our data showed that quantifying the cell-substrate adhesion could successfully represent various conditions created by increasing AβO concentrations exposed to the cells. It was revealed that increasing the time of the cells exposed to AβO, the binding strength of cells to the substrate reduced. Our present study demonstrated that advancing AβO concentrations caused cell-substrate interaction to lose strength. The result showed that all cells were dead after 24 h incubation of microglia BV2 cells exposed AβO 5 µM.
The quantitative information of this dissertation using the proposed microfluidic chips can provide a better understanding of microglial mechanobiology properties in the control conditions and also the diagnosis and progression of AD through AβO as a biomarker.