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Title: The impact of substrate texture on cell behavior
Author: Karimbabanezhadmamaghani, Pooya
ISNI:       0000 0004 8507 7030
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2020
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Traditionally, cell studies have focused mainly on the effects of biochemical signals on cellular behaviour; however, there is a growing interest in investigating the effects of physical cues on the behaviour of the cells. Development of fabrication techniques has allowed researchers to design topographical cues at the micro and nanometer scale. In this study, we fabricated fibronectin stripes, which mimic some of the frequent patterns that cells are exposed to in their natural microenvironment, and studied the effects of these patterns on 3T3 fibroblasts and human mesenchymal stem cells which are of significant importance in the field of regenerative medicine and tissue engineering. Our results show that cells align and elongate in the direction of the patterns and the morphological features of cell, nucleus and architecture of force-bearing stress fibers of the actin cytoskeleton are all highly dependent on the stripe width. 3D confocal measurements showed that the cell thickness and nuclear shape is regulated through the different arrangement of perinuclear actin stress fibers depending on the stripe sizes. We also observed that when the size of stripes (5 μm) is significantly smaller than the size of cell nuclei (subnuclear stripes), cells exhibit 3 different morphological responses to the patterns. Minimal confinement, branching on the stripes and elongation on a single stripe. The majority of cells and nuclei align themselves with the subnuclear stripes; however, a substantial number of hMSCs nuclei are elongated perpendicular to the direction of the pattern, a finding which we explained using a geometrical model based on cell size. The behaviour of 3T3 cell lines and the hMSCs were compared for the different types of pattern. We designed setups that allow us to perform Raman microspectroscopy both in 2D and 3D topographical patterns in order to be able to study their morpho-chemical effects on cells. We also genetically modified 3T3 fibroblasts to express fluorescent-tagged proteins that visualize the actin cytoskeleton and the nucleus which allow us to perform live cell imaging to study the dynamics of cell behaviour on topographical patterns.
Supervisor: Donald, Athene Sponsor: Cambridge Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Cell adhesion ; Substrate texture ; Cellular volume ; Cell thickness ; Nuclear deformation ; Perinuclear actin-cap ; Raman Microspectroscopy ; mesenchymal stem cells ; Stem cell ; Stem cell diffrentiation ; contact guidance ; micro-patterning ; Physical micro-environment ; ECM ; fibronectin ; Fibronectin patterns ; Micro-contact printing ; Soft Lithography ; Micro-fabrication ; Nano coating ; Physical cues ; Genetic engineering ; Transfection ; GFP Lamin-A ; Gold nanolayer coating ; Cellular morphology ; Electromagnetic wave prpagation ; Skin depth of a conductor ; Cell culture ; hMSCs ; Fibroblasts ; Cellular Area ; Biophysics ; Mechanotransduction ; Mechanobiology ; Apoptotic cell death ; Necrotic cell death ; Mechano-sensivity ; PDMS ; Anoikis ; Confocal microscopy ; Phase contrast microscopy ; NIH/3T3 cells ; Actin cytoskeleton ; Actin Filaments ; E-beam evaporation ; Nuclear Lamina ; Biomaterials ; CAMs