Date of Award

5-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Bioengineering

Advisor

Burg, Karen J.L.

Committee Member

Webb , Ken

Committee Member

Laberge , Martine

Committee Member

Bridges , Billy

Abstract

Three-dimensional in vitro tissue test systems are employed to examine cell behavior, test responses to drugs and vaccines, and answer basic biological questions. These systems are more physiologically relevant than two-dimensional cell cultures, and are more relevant, easier and less expensive to maintain than animal models. However, methods used to measure cell behavior and viability have been developed specifically for two-dimensional cell cultures or animal models, and are often not optimally translated to three-dimensional in vitro test systems. The purpose of this work was to aid in the development of three-dimensional, spatially controlled in vitro test systems, and to develop the corresponding quantitative, spatial measurement methods of cell behavior and viability.
Optical widefield microscopy was selected as a measurement tool because of its ease of use, wide availability, and inherent large-scale spatial measurement capacity. Digital image analysis and processing were used to collect quantitative data. Fluorescent cellular labels were examined for use in spatial, quantitative imaging, and methods were developed to quantify cell location, morphology, and viability from either fluorescent or phase contrast images.
Maintenance of oxygen supply to cells is integral in a tissue engineered construct and in 3D in vitro test systems. Cells in the body are supplied oxygen by the vasculature, but in tissue engineered constructs, cells must be supported by oxygen diffusion alone. In addition to tracking cellular behavior, microscope digital image processing was used in conjunction with fluorescent oxygen-sensitive nanoparticles for the quantitative, spatial measurement of oxygen in a 3D in vitro test system. These methods were used to confirm the presence of oxygen gradients that occur in 3D cell cultures due to cellular oxygen consumption. Artifacts that impede quantitative fluorescence imaging were identified, and fluorescence ratio imaging was used to minimize artifacts and facilitate quantitative oxygen measurement.
In follow-up work, methods were developed to allow sterile microscope imaging and culture of cells in a 3D tissue engineered construct; the setup allowed spatial control of oxygen delivery to approximate oxygenation via vasculature in the body. An oxygen-gradient bioreactor was developed and imaging techniques were used to show that culture medium perfusion rates can be used to control the rate of distribution of a factor or gas, such as oxygen, throughout a tissue engineered construct.
Lastly, a 3D in vitro hydrogel test system with modular substrate stiffness was created and assessed using quantitative cellular imaging methods to examine cancer development. Cancer cell behavior has been shown to be strongly correlated to local stiffness variations in the extracellular matrix; however, this relationship is not well understood. Human breast cancer cells were cultured on hydrogel substrates of varying mechanical properties, and quantitative imaging and metabolic activity assays were used to examine cell behavior and viability. Phase contrast microscopy imaging and image processing were conducted to allow quantitative measurement of cell morphology. Mathematical modeling work performed by collaborators indicated both temporal and substrate-stiffness based effects on cancer cell colony size, number, and shape (perimeter). Continuing this work, hydrogel test systems with a spatial stiffness gradient were produced. Imaging methods were used to provide large-scale, quantitative measurement of cell density to estimate cell migration and growth as a function of both time and position on these spatially non-uniform substrates.
This research facilitated the development of methods for spatially controlling the mechanical properties of 3D tissue test systems as well as methods for spatial, quantitative measurement of cellular position, growth, and morphology. An oxygen gradient bioreactor was also designed and tested to simulate a more physiologically representative environment. The end goal of this research is to aid in the understanding of cancer development by creating robust, controllable cancer test systems that can be used to expose cells to predefined conditions and quantitatively measure resulting cellular behavior.

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