Date of Award

Spring 1-1-2017

Document Type

Thesis

Degree Name

Master of Science (MS)

First Advisor

Kristi S. Anseth

Second Advisor

Leslie Leinwand

Third Advisor

Stephanie J. Bryant

Abstract

An automated image analysis method was developed to characterize and quantify the fibroblast and myofibroblast phenotypes of Valvular Interstitial Cells (VICs) when embedded in hydrogel scaffolds. A spinning disk confocal microscope was used for imaging the 3D cell-laden gels, and the imaging analysis used the instrument’s Harmony Software. VICs cells were encapsulated in poly(ethylene glycol) (PEG) hydrogel matrices with varying properties, specifically varying; degradability and stiffness. 10 μL/gel were spotted with the liquid handling system (EpMotion M5073, Eppendorf) to achieve a thickness of 0.9mM. For the 2D experiments different hydrogel compositions were used to analyze the cell in different hydrogel formations. 8-arm PEG-nb hydrogels with different linkers, ester and amide, were used as the culture platforms for the 2D study. 2D study of VICs with different concentrations of Endothelin 1 (ET-1) was also made. 3D culture platforms were designed with different stiffness by varying the PEG molecular weight (20-40 kDa) and varying the presence or not of TGFβ. At various time points (3 and 5 days for the 2D and the 3D respectively) images were collected, and a protocol was developed to differentiate the activated myofibroblast VIC phenotype from the quiescent fibroblast phenotype. First the analytical method, identifies the nuclei and cytoplasm regions of the VICs and calculates the fluorescence intensity of F-actin, as a measurement of stress-fiber formation, and alpha-smooth muscle actin (αSMA), an activated myofibroblast marker. Next by calculating the ratio of αSMA expression relative to F-actin expression, the method is able to identify the coexpression of the αSMA myofibroblast marker with actin, thereby providing a measure of ; αSMA organization into stress-fibers. Based upon αSMA to F-actin, a threshold can be set, here a values between 0.7-1.7 have been used, that provides a quantitative, objective, and automated method to determine whether or not the VICs are activated myofibroblasts. To validate this image analysis method, results were compared to a series of well-established data sets that used the same hydrogel formation, but manually quantified VIC myofibroblast formation by counting αSMA stress fiber positive cells. The method here developed show a high accuracy in characterizing activation of VICs. The analytical method validation present the same results as the published data within a 5±5% of difference when quantifying myofibroblast activation for the 3D culture platforms and a 9±5% of difference when quantifying myofibroblast activation in the 2D culture platform studies. The software and protocols that were developed as part of this thesis should provide a general methods that will be useful for analyzing intracellular protein expression co-localized with a cells actin cytoskeleton.

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