Creation of an In-Vitro Generated Colonic Stem-Cell Niche Using Gradient-Generating Microdevices Public Deposited

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  • March 19, 2019
Creator
  • Ahmad, Asad
    • Affiliation: School of Medicine, UNC/NCSU Joint Department of Biomedical Engineering
Abstract
  • The limitations of existing cell culture and animal studies have provided an impetus for the development of alternative cell based in vitro models that better mimic the complex structures and functions of living organs. This thesis lays the groundwork for the development of an in vitro model of the colonic epithelium by focusing on the development of microdevices to recreating the colonic stem-cell niche. New advances enable long-term organotypic culture of colonic epithelial stem cells that develop into structures known as colonoids. Colonoids represent a primary tissue source acting as a potential starting material for development of an in vitro model of the colon. However for that to be possible, there needs to an improved crypt isolation and 3-D colonoid protocols. In the first chapter, an incubation buffer and time are outlined, along with the finding that 50% Matrigel resulted in the highest colonoid formation efficiency. In the second chapter, threshold concentrations of the key Wnt-signaling factors are discovered. While critically important to homeostatic renewal, the threshold concentrations of factors such as Wnt-3a and R-spondin1 that promote stem cell renewal are unknown. A simple, linear gradient-generating device was used to screen a wide range of Wnt-3a and R-spondin1 concentrations for their impact on a large number of colonoids. A Wnt-3a concentration of 60 ng/mL and R-spondin1 concentration of 88 ng/mL were identified as the critical concentrations required for stem-cell renewal and colonoid expansion. The lower factor concentrations yielded the added benefit of a more morphologically appropriate colonoid possessing columnar cells surrounding a central lumen with active crypt-like bud formation. In the final chapter, a gradient-generating device was used to introduce variable concentrations of the two key Wnt-signaling proteins along the length of a single colonoid. After 5 days in culture under a combination of Wnt-3a and R-spondin gradients, novel image analysis techniques leveraged the intrinsic fluorescence of the mouse model to quantify the levels of stem cell polarity across a colonoid. The microenvironment able to create a stem cell niche within a colonoid by applying external growth factors in a graded fashion across the colonoid.
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  • In Copyright
Advisor
  • Allbritton, Nancy
  • Bultman, Scott
  • Loboa, Elizabeth
  • Magness, Scott
  • Ramsey, J. Michael
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2015
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Place of publication
  • Chapel Hill, NC
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