Date of Thesis

Spring 2025

Description

Mechanobiology is an emerging field that aims to study the relationships between mechanical forces and cellular behavior. Such mechanobiological relationships are especially critical in the cardiovascular system, where endothelial cells lining the vasculature align in response to fluid shear stresses from blood flow (Sinha, 2016). Implanted flow devices, diabetes, chronic hypertension, and various other diseases and pathophysiologies alter vessel geometries and flow dynamics. Such alterations impact fluid shear stress and endothelial cell behavior, leading to microcirculatory dysfunction, vessel leakage, and organ failure (Poredos, 2021; Papadaki, 1999; Leitschuh, 1987). To simulate dysfunctional endothelial cell behavior in vitro and evaluate novel therapeutic interventions, such as therapeutic ultrasound, to recover normal endothelial cell behavior, platforms must be developed to precisely expose endothelial cells to a wide range of fluid shear stresses while accommodating a multitude of experimental configurations and imaging techniques. This thesis represents the initial development efforts of such a tool through the creation of an inexpensive, reusable device that exposes cells cultured on a removable coverslip to laminar flow and homogeneous shear at physiological and supraphysiological magnitudes. Specific objectives of this thesis include development of design considerations, evaluation of the features and limitations of current commercial and research technologies, design and manufacturing of a custom device capable of meeting design goals, and initial validation of the device with in vitro testing of live endothelial cells at physiological and supraphysiological conditions.

A unique, modular (58 mm x 32 mm x 13 mm), parallel plate flow chamber with a live-cell imaging area of 200 mm2 was designed in Onshape CAD software, and laminar flow and shear homogeneity in the chamber were validated in COMSOL Multiphysics computational fluid dynamics software. Through an extensive development and revision process, simulation and low-fidelity prototypes were used to evaluate performance and identify necessary areas of improvement, culminating in a final prototype machined from stainless steel 316L. To evaluate flow dynamics and imaging capabilities of the device, human umbilical vein endothelial cells (HUVECs) were seeded on a coverslip and placed within the device, exposing them to flow with plate shear stresses at physiological (4 Pa) and mildly supraphysiological (8 Pa) magnitudes. Brightfield imaging was performed throughout flow experiments to evaluate cell morphology. Additional experiments were performed at 4 Pa of shear, using fluorescence microscopy to visualize detailed alignment at various timepoints throughout flow and demonstrate imaging capabilities of the device. Experimental results show evidence of cellular alignment in the direction of flow, mimicking in vivo behavior and suggesting laminar and homogeneous flow at a physiologically relevant shear stress magnitude within the device. Observations of alignment occurred within 12-18 hours at 4 Pa of shear, and 8 hours at 8 Pa of shear, consistent with literature observations (Noria, 2004). To validate repeatability of the work, five devices were manufactured, and experiments were repeated at 4 Pa of shear. Initial reproducibility testing revealed device-to-device variability, prompting the development of optimal, standardized cell-handling procedures for the device to improve experimental consistency. Preliminary work into cell-handling protocol optimizations revealed strong alignment in three of the four devices tested, demonstrating promise for a reproducible platform with continual work to mitigate device-to-device variability by refining manufacturing and biological protocols. The development and performance of this device under physiological and supraphysiological shear provides a robust and versatile platform for future studies to investigate shear-dependent cellular responses and assess novel therapeutic treatments, ultimately advancing understanding and treatment of vascular disease.

Keywords

Microfluidics, Mechanobiology, Shear Stress, Endothelial Cells, Parallel Plate Flow Chamber, Vascular Disease

Access Type

Honors Thesis

Degree Type

Bachelor of Science in Biomedical Engineering

Major

Biomedical Engineering

Minor, Emphasis, or Concentration

Computer Science

First Advisor

Olivia Boerman

Second Advisor

James W. Baish

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