Advancing the Use of Three-Dimensional Spheroids for Quantitative, Live-Cell, Fluorescence Microscopy in a High-Throughput Format

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Overview

Title
Advancing the Use of Three-Dimensional Spheroids for Quantitative, Live-Cell, Fluorescence Microscopy in a High-Throughput Format
Contributors
Leary, Elizabeth A. (creator)
Morgan, Jeffrey (Advisor)
Boekelheide, Kim (Reader)
Coulombe, Kareen (Reader)
Creton, Robbert (Reader)
Mende, Ulrike (Reader)
Ruch, Randall (Reader)
Brown University. Biology and Medicine: Biomedical Engineering (sponsor)
Copyright Date
2018
Abstract
All disciplines of biomedical research utilize in vitro models to answer a range of biological questions. Traditional in vitro models, two-dimensional (2D) cell monolayers, fail to recapitulate the in vivo environment. To increase physiological relevance, three-dimensional (3D) spheroids can be utilized. Spheroids, with their multiple cell layers, yield numerous advantages, such as increased cellular communication and interaction, barriers to transport/diffusion, formation of gradients (oxygen, nutrient, carbon dioxide, pH, compounds). Despite increasing physiological relevance, multiple cell layers also increase the difficulty associated with extracting accurate, quantitative information from spheroids. This dissertation aimed to advance the use of spheroids for live cell, fluorescence microscopy, both widefield epifluorescence and confocal, through developing quantitative analysis strategies. To determine the best means of fluorescent quantitation, spheroids of variable radii were labeled with CellTrackerTM and calcein cytoplasmic dyes via two different methods: uniformly staining monolayers prior to spheroid formation (pre-labeling), or adding dye after spheroid formation (diffusion-labeling). For widefield epifluorescence, accurate fluorescent normalization was dependent upon how the spheroid was labeled: as fluorescent signal from pre-labeled dyes was best normalized by spheroid volume, while fluorescent signal from diffusion-labeled dyes was best normalized by surface area. For confocal microscopy, as spheroid radius increased, fluorescent signal loss also increased. Furthermore, the curved nature of spheroids yielded non-uniform fluorescent signal loss across each confocal image. This spheroid-specific complex staining pattern was ameliorated with ratio imaging. Finally, we applied this understanding of the unique quantitative limitations of spheroids towards developing and optimizing an assay to assess gap junction intercellular communication (GJIC). Overall, this dissertation advanced our understanding of how to accurately quantify and normalize fluorescent signal from spheroids. Utilizing proper normalization techniques will enhance the sensitivity and robustness of assays. Furthermore, we developed and optimized a biologically-based assay that quantified fluorescent changes as a function of 3D radius. Although we chose to assess GJIC, this tool can assess a wide range of biological functions.
Keywords
three-dimensional culture
self-assembled spheroids
Image analysis
Fluorescence microscopy
3-D
live-cell
Notes
Thesis (Ph. D.)--Brown University, 2018
Extent
xvii, 227 p.

Citation

Leary, Elizabeth A., "Advancing the Use of Three-Dimensional Spheroids for Quantitative, Live-Cell, Fluorescence Microscopy in a High-Throughput Format" (2018). Biomedical Engineering Theses and Dissertations. Brown Digital Repository. Brown University Library. https://repository.library.brown.edu/studio/item/bdr:792828/

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