June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
An in vitro 3-D cell culture model for studying pathomechanisms in AMD
Author Affiliations & Notes
  • Audra Shadforth
    School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, Australia
    Queensland Eye Institute, Brisbane, QLD, Australia
  • Damien Harkin
    School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, Australia
    Queensland Eye Institute, Brisbane, QLD, Australia
  • Traian Chirila
    School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, Australia
  • Anthony Weiss
    School of Molecular Bioscience, University of Sydney, Sydney, NSW, Australia
  • Dietmar Hutmacher
    Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD, Australia
  • Beatrix Feigl
    Queensland Eye Institute, Brisbane, QLD, Australia
    Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD, Australia
  • Footnotes
    Commercial Relationships Audra Shadforth, None; Damien Harkin, None; Traian Chirila, None; Anthony Weiss, None; Dietmar Hutmacher, None; Beatrix Feigl, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 314. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Audra Shadforth, Damien Harkin, Traian Chirila, Anthony Weiss, Dietmar Hutmacher, Beatrix Feigl; An in vitro 3-D cell culture model for studying pathomechanisms in AMD. Invest. Ophthalmol. Vis. Sci. 2013;54(15):314.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose: To develop a novel 3-D cell culture model with the view to studying the pathomechanisms underlying the development of age-related macular degeneration (AMD). Our central hypothesis is that the silk structural protein fibroin, used in conjunction with cultured human retinal pigment epithelium cells (RPE) cells and microvascular endothelial cells, can serve as a structural analogue of Bruch’s membrane.

Methods: Co-cultures of human RPE cells (ARPE-19 cells grown in Miller’s medium) and microvascular endothelial cells (HMEC-1 cells grown in endothelial culture medium) were established on opposing sides of a 3 micron thick membrane constructed from B. mori silk fibroin. Cell attachment was facilitated by pre-coating the fibroin membrane with vitronectin (for ARPE-19 cells) and gelatin (for HMEC-1 cells) respectively. Barrier function was examined by measurement of trans-epithelial resistance (TER) using a voltohmmeter (EVOM-2). The phagocytic activity of the RPE cells cultured on silk fibroin was tested using vitronectin-coated microspheres (2 micron diameter FluoSpheres). In some cultures, membrane defects were created by puncturing with a 27 G needle to mimic breaks in Bruch’s membrane that are established factors of inducing neovascularisation. The architecture of the cultured tissue before and after wounding was examined by confocal microscopy after staining with rhodamine phalloidin and Hoechst 33342 nuclear dye.

Results: The RPE component of the 3D model developed a cobblestoned morphology as well as barrier function and displayed vitronectin-mediated phagocytic activity. Microvascular endothelial cells attached well to the gelatin-coated lower surface of the fibroin membrane and remained physically separated from the overlying RPE layer. The fibroin membranes remained intact throughout the duration of cell culture and were amenable to puncturing without collapse.

Conclusions: The Bruch’s membrane analogue constructed from silk fibroin, vitronectin and gelatin, supports the co-cultivation of RPE cells and microvascular endothelial cells. The resulting RPE layer displays functions similar to that of native RPE and the entire tri-layered structure displays potential to be used as an in vitro model for studying pathomechanisms of AMD including choroidal neovascularization.

Keywords: 412 age-related macular degeneration • 701 retinal pigment epithelium • 438 Bruch's membrane  
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×