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Surface Modifications for Cell Culture Systems

  • Author(s): Reese, Willie M.
  • Advisor(s): Healy, Kevin E
  • et al.
Abstract

This dissertation focuses on methods to understand and improve surface modifications for 2D and 3D cell culture platforms. In this work, novel methods are investigated for their ability to alter cell adhesion and block small molecule absorption, events that have the ability to alter cell behavior and phenotype.

Chapter 1 is an introduction to surface modification techniques, the mechanisms within the cell that are currently understood to probe these alterations of surface chemistry, topography, and other attributes, and the application of these methods. This chapter serves to tie together the investigations done in this dissertation.

Chapter 2 presents an investigation on the effects of change of topography using multi-photon ablation lithography to fabricate arrays of nanoscale craters in quartz substrates with a variety of geometries and spacing (i.e. pitch). This direct-write patterning method developed by a collaborator induced directed NIH 3T3 cell migration. In this work, I focused on understanding the mechanism of action for this phenomena. Briefly, the direct write patterning produced sub-micron sized pits in quartz that could be arranged in any number of spacial patterns. I investigated both isometrically spaced pits (2-8µm) as well as gradient spaced patterns, where pits were spaced at 1µm spacings toward the center of the pattern up to 10µm toward the perimeter of the pattern. Immunofluorescent labeling of intracellular focal adhesion Vinculin was used to quantify focal adhesion size and revealed significant differences based on pattern spacing. In isometric patterns, 2 and 4 µm patterns showed significantly smaller focal adhesion size distributions leading to the conclusion that the nanocraters restricted focal adhesion size and also maturity leading to higher turnover of these adhesion. Higher turnover in regions where the pits were densely arranged led to an observation of cells migrating away. With motion tracking on gradient patterns, cells were seen to migrate areas where the nanopits were spaced closer together along directions of the highest rate of increase in surface area, confirming that the pits were restricting the area available for cells to form larger stable focal adhesions. Other researchers have shown that area is a critical attribute for focal adhesion formation that can be overcome by intercellular adhesion protein activation. To investigate this, NIH3t3 cells were transfected with a plasmid containing the DNA for the first 405 amino acids of the Talin-1 protein, which has shown to perform as the full Talin molecule in intracellular signaling. After transfection, cells showed no significant difference on any of focal adhesion size on any of the isometric patterns and also lost their directed migration function on gradient patterns. This data supports our hypothesis that the physical restriction of area for adhesion causes cells to statistically migrate from regions of high pit density (low pitch) to regions where more area is available, but with intracellular signaling of talin this can be overcome by the cell. This study helps to elucidate the mechanism through which cells probe their substrate topography and if employed on more biologically relevant surfaces can be used as a way to decrease cell adhesion.

Chapter 3 presents a novel method using a macrocylic polyphenol coating to block drug absorption in Poly(dimethysilane) (PDMS) based microfluidic Microphysiological Systems (MPS). MPS can be used to combine genetically relevant cell lines in micro-environments that recapitulate not only genetically relevant organ specific structure, but also organ system relationships to access on and off-target toxicities and efficacies. Most MPSs are easily manufactured via soft lithography techniques using poly-dimethylsiloxane (PDMS), which has shown to be biologically compatible and amenable to many standard cell culture techniques due to its high transparency, high oxygen permeability, and low auto-fluorescence. Although PDMS has several positive attributes, many have shown that due to its hydrophobicity, small lipophilic molecules can be absorbed into PDMS creating unpredictable drug concentrations in MPSs.

To address this limitation, we designed and developed a novel macrocyclic polyphenol, which is able to reduce the absorbance of the model drug compounds rhodamine B, C1-BODIPY-C12, Brilliant Green, and Metanil Yellow into PDMS by 87, 96, 93, and 95 percent, respectively. This outperforms established polyphenol coatings such as polydopamine or pyrogallol. Initial experiments have shown low cytotoxicity allowing for the culture of iPSC-derived cardiomyocytes for at least one month. Preliminary experiments also show good coating stability not only on PDMS but also on other biologically relevant polymeric materials such as tissue culture polystyrene, polycarbonate, and Teflon. This coating is also advantageous over other solutions of drug absorption such as Sol-Gel methods or other glass like coatings, due to its ease of use, low cost, and high oxygen permeability.

Chapter 4 gives an overall conclusion and details future directions for investiga- tions in Chapter 2 and 3.

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