June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Recreation of a 3-D Physiological Model of Conventional Outflow Tract in vitro
Author Affiliations & Notes
  • Karen Yud Torrejon
    College of Nanoscale Science and Engineering, State University of New York Polytechnic Institute, Albany, NY
    State University of New York Eye Institute, Syracuse, NY
  • Cula Dautriche
    College of Nanoscale Science and Engineering, State University of New York Polytechnic Institute, Albany, NY
  • Ellen Papke
    College of Nanoscale Science and Engineering, State University of New York Polytechnic Institute, Albany, NY
  • W Daniel Stamer
    Ophthalmology, Duke University, Durham, NC
  • John Danias
    State University of New York Eye Institute, Syracuse, NY
    Ophthalmology, State University of New York Downstate Medical Center, Brooklyn, NY
  • Magnus Bergkvist
    College of Nanoscale Science and Engineering, State University of New York Polytechnic Institute, Albany, NY
    State University of New York Eye Institute, Syracuse, NY
  • Susan T Sharfstein
    College of Nanoscale Science and Engineering, State University of New York Polytechnic Institute, Albany, NY
  • Yubing Xie
    College of Nanoscale Science and Engineering, State University of New York Polytechnic Institute, Albany, NY
    State University of New York Eye Institute, Syracuse, NY
  • Footnotes
    Commercial Relationships Karen Torrejon, None; Cula Dautriche, None; Ellen Papke, None; W Stamer, None; John Danias, None; Magnus Bergkvist, None; Susan Sharfstein, None; Yubing Xie, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 3544. doi:
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      Karen Yud Torrejon, Cula Dautriche, Ellen Papke, W Daniel Stamer, John Danias, Magnus Bergkvist, Susan T Sharfstein, Yubing Xie; Recreation of a 3-D Physiological Model of Conventional Outflow Tract in vitro. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3544.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: To explore the feasibility of engineering a physiologically relevant, 3D human conventional outflow tract utilizing cellular co-culture and differentiation approaches along with microfabrication techniques. This 3D tissue will provide normal and disease-relevant, in vitro model systems for understanding the trabecular outflow physiology and its impact on glaucoma pathology.

Methods: We established an “Artificial HTM” on microfabricated porous scaffolds, which demonstrated an in vivo-like phenotype of HTM cells and responded to pharmacological agents in a similar fashion to that seen ex vivo. In order to better mirror the biological and physiological characteristics of the trabecular outflow tract in vivo, we co-cultured human Schlemm’s canal (HSC) cells or differentiated HSC-like cells with HTM to mimic the Schlemm’s canal inner wall. The effects of pore size, biomolecule coating, and cultivation period for co-culture were investigated. Samples were assayed for expression of HTM and HSC markers. Scanning electron microscopy (SEM) was used to reveal the morphology of the co-cultured tissue. In parallel, the responsiveness of these models to glucocorticoids (PA), rho kinase inhibitor (Y-27632) and TGF-β2 was investigated by perfusion studies. The “outflow facility” of the bioengineered 3D HTM/HSC construct was determined by the ratio of Δ(flow rate)/Δ(pressure).

Results: We successfully established 3D co-cultured HTM/HSC constructs to mimic the conventional outflow tract. Co-cultured samples expressed the HTM marker of αβ-crystalline on one side of the construct while the other side expressed VE-cadherin, fibulin-2 and PECAM-1, markers of HSCs. Both cell layers expressed myocilin; greater expression was observed on the HTM layer. The co-culture decreased outflow facility compared to HTM cell culture alone. Additionally, PA and TGF-β2 treatments increased myocillin and ECM expression/secretion compared to untreated controls. The outflow facility of the co-culture model decreased after 300nM PA treatment, and to a greater extent after TGF-β2 treatment. Exposure to Y-27632 decreased α-smooth muscle actin expression and disrupted F-actin fibers, along with increasing outflow facility.

Conclusions: This work established a bioengineered HTM/HSC model mimicking the conventional outflow tract and demonstrated physiologically-relevant responses to biological agents.

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