Phenotypic Characteristic of Cell Spreading, Migration, Traversing and Intracellular Cytoskeleton Behavior Utilizing Microengineered Structure

利用微結構的細胞鋪展,遷移,穿梭和細胞內骨架行為的表型特徵研究

Student thesis: Doctoral Thesis

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Award date24 Nov 2021

Abstract

Cellular and subcellular behaviors of primary and immortalized cells play a critical role in many diseases such as atherosclerosis, hypertension and tumor metastasis. Such behaviors including cell spreading, cell migration and cytoskeletal dynamics are essential and fundamental for tissue and organ development. For instance, cancer cell migrate from primary tumor site to adjacent or distant tissue, such migrate properties and preference will eventually lead to different tumor metastasis status. Tremendous techniques and methodologies had been utilized and applied in cellular behavior studies in the past decades. However, it still unclear that how cell behaviors and morphologies of the specific cell lines are affected by the biomimetic microstructures in vitro. Besides, microtopographical factors have been reported to be the vital factor that affect cellular behaviors in vitro. Herein, I proposed three approaches with micro/nanoengineered substrate and one microfluidic chip to investigate cellular and subcellular behaviors of different types of cell lines. At the first step, I elaborated the spreading and migration characteristics of normal and cancerous nasopharyngeal cells on micro-grating substrates mimicking the microtopography of myotubes of the pterygoid muscles. Specifically, I first utilize an interference reflection microscopy (IRM) to visualize the cell-substrate adhesion dynamics, afterwards, bright field microscopic images of each labeled cell are taken to investigate cell migration properties. As I am trying to have better understanding of cell initial spreading behaviors on more substrates with different scale. In the second work, I investigated cell initial spreading of breast cancer and epithelial cell lines on micro-/nano-scale and flat PDMS substrate, respectively. IRM is a very sensitive and accurate method to quantify cell spreading dynamics. However, the IRM is strictly limited by the optical theory and applicable on flat or nanorough surface. To capture the cell spreading area on microengineered curved surfaces such as microgroove. I applied a refractive microlens to modify the ray trace generated by monochrome laser source. Microlens strategy could provide customized substrate configurations to meet different curved microstructures such as convex and concave microgroove/well. Considering the microstructure factor play important role in cell morphology and subcellular behavior, such as cytoskeleton behavior. In the third work, I further extended the research by utilizing primary vascular cell lines on microwell and microgroove for cell morphology and intracellular cytoskeleton studies. Human umbilical vein endothelial cells (HUVECs) and primary human vascular smooth muscle cells (HVSMCs) were seeded on the substrate with microwells and microgrooves array to observe the cell morphology characteristics and actin microfilaments dynamics. The physical model was proposed to describe the dynamic process of such cell behavior. This work reveals the arrangement and dynamics of intracellular cytoskeleton on blood vessel like microtopologies, aims to provide valuable insights for further clinical investigation in vascular diseases such as atherosclerosis and hypertension. During the cancer metastasis process, circulating tumor cells (CTCs) traversing through the blood vessel to the new colony. To evaluate the CTCs traverse ability, I utilized microfluidic device containing parallel straight channel to mimic the blood capillaries to exam the tendency of cell diameter versus traverse velocity. Further, I establish a theoretical model to reveal the specific relation between cell diameter and traverse velocity. The reported cell traversing velocity analyzing method can also be applied in straight microchannels with various dimensions and may further provide a simple way to evaluate cancer cell metastasis ability in human capillaries.