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Interplay of reactive oxygen species with the mechanical properties of cells and mitochondria
(2023)
Cell mechanical properties are a popular label-free method for understanding basic cellular processes. In this thesis, I used Real-time deformability cytometry (RT-DC), a high-throughput microfluidic technology, to investigate the mechanical properties of cells and mitochondria under various conditions such as increased reactive oxygen species (ROS) levels and the application of different ligand coated gold nano-particles (Au-Nps) effect on cells. Initially, we showed the possibility to measure organelles, cells, and tissue-like structures (spheroids) in a single system by constructing a virtual fluidic channel. We investigated a potential application using cytochalasin D (cyto D) treatment, which revealed increased deformation and decreased stiffness in both the normal and virtual channels. Using mechanics as a marker, I investigated the effect of excessive ROS on the mechanical properties of human myeloid precursor cells (HL60). My findings suggest that the mechanical response of HL60 cells to increased ROS levels is mediated by re-localization of microtubules toward the cell center and F-actin to the cell periphery. Interestingly, I also observed intracellular acidification, which is a largely unexplored mechanism that may have contributed to our findings. I then extended our ROS and mechanics assay to investigate cell-AuNP interactions, demonstrating that cell properties vary depending on the cell culture media and ligand coating. The results showed that dextran coated gold nano-particels (Au-Nps) had low cytotoxicity, lower ROS release, and no change in cell mechanics, indicating a potential application for dextran Au NPs. Finally, I expanded our assays to include high-throughput microfluidic characterization of isolated mitochondria. Using both exogenously and endogenously induced ROS, we found an increase in mitochondrial deformation and a decrease in their size, which could have implications on mitochondrial function, i.e., fission and fusion. We believe that advanced applications of RT-DC technology will improve the comparability of results across different sample sizes while also promoting it as a disease detection technique.