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Micropost Arrays to Advance Cell Handling

  • Author(s): Sochol, Ryan Daniel
  • Advisor(s): Lin, Liwei
  • et al.
Abstract

Mechanical engineering methods and microfabrication techniques offer powerful means for meeting biological challenges. In particular, microfabrication processes enable researchers to develop technologies at scales that are biologically relevant and advantageous. In this work, microfabricated posts were employed to advance cell handling capabilities in both static and dynamic (i.e., microfluidic) systems.

Static, substrate-based biophysical properties influence diverse cellular processes. Methods for engineering micropost arrays enable microscale control over the biophysical characteristics of discrete topographic features. Here, unidirectional micropost array gradients of variable micropost stiffness and variable interpost spacing were constructed to regulate cell motility using two distinct biophysical cues: (i) gradients in substrate rigidity (i.e., via durotaxis - a subset of mechanotaxis), and (ii) variable spacing of substrate binding sites - via a phenomenon herein referred to as spatiotaxis. Micropost array stiffness gradients were designed with post-to-post differences in stiffness of 0.5 nN/µm, 2 nN/µm, 3 nN/µm, and 7.5 nN/µm. Bovine aortic endothelial cells (BAECs) seeded on micropost array gradients with variable micropost stiffness exhibited preferential cell migration in the direction of increasing micropost stiffness. Gradients of elliptical microposts further enhanced unidirectional guidance by limiting cellular movement perpendicular to the direction of increasing micropost stiffness. Micropost array spacing gradients were designed with average post-to-post differences in spacing of 10 nm, 20 nm, and 40 nm. Micropost array gradients with variable interpost spacing were found to promote BAEC migration in the direction of decreasing interpost spacing, which represents the first demonstration of unidirectional spatiotaxis. Higher gradient strengths were observed to enhance the aforementioned migratory behaviors for both biophysical cues. For substrates with simultaneous, anti-parallel stiffness and spatial stimuli, the spatial cues were found to dominate the migratory response. The micropost array gradient methodology offers a powerful technique for investigating the biophysical cellular response, while also providing the basis for new classes of passive substrates capable of directing cell motility in biological fields, such as biomaterials, tissue engineering, and regenerative medicine.

In order to create high-speed lab-on-a-chip devices for quantitative cell biology, drug discovery, and molecular diagnostics, precision hydrodynamic controls of microparticles (e.g., cells and microbeads) are in critical demand. The ability to achieve multi-stage fluidic reaction processes for microparticles is integral to diverse chemical and biological applications; however, microfluidic particulate-based systems remain limited due to particle handling issues. In contrast to suspended cells, which are experimentally complex, microbeads offer a simplified example for initial demonstrations of microfluidic particulate handling. Thus, the ability to manipulate microbeads in microfluidic systems represents a fundamental first step toward advancing microfluidic cell handling. In this dissertation, microposts (15×15 µm2) were arrayed within microfluidic architectures (18 µm in height) to enhance microparticle handling and enable multi-stage fluidic reactions and analyses for suspended particles. The presented microfluidic systems were first characterized using suspended microbeads (15 µm in diameter); thereafter, the potential of employing the platforms for cell handling applications was also investigated using suspended BAECs. A resettable, hydrodynamic microparticle trapping system - termed micropost array trapping (µPAT) - was designed and demonstrated to accomplish controlled particulate arraying and microarray resettability by trapping-and-releasing both microbeads and cells. The µPAT technique was integrated into: (i) a dynamic microarray to detect multiple bio-molecules in parallel via molecular beacon probes conjugated to microbead substrates, and (ii) a "Microfluidic Ping Pong" (MPP) system to achieve multi-stage fluidic reactions under discontinuous flow conditions. As a demonstrative example, the MPP technique was employed to detect an inflammatory cytokine at 100 pM concentrations via an 11-stage aptamer beacon-based sandwich assay performed using microbeads. Additionally, a microfluidic micropost array railing (µPAR) system was developed to rapidly transport both microbeads and cells into adjacent flow streams under continuous flow conditions. To demonstrate the µPAR technique, a multiplexed layer-by-layer (LbL) molecular synthesis process (i.e., consisting of up to 18 fluidic stages) was accomplished on microbead substrates. This work represents the first demonstration of a microfluidic platform capable of railing either microbeads or cells into adjacent flow streams. Through improved microparticle handling in microfluidic systems, the presented methodologies could further extend the efficacy of dynamic cell-based and bead-based microarrays for applications in diverse chemical and biological fields.

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