June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Preparation of Human Choroidal ECM Scaffolds to Study Cell Replacement Strategies
Author Affiliations & Notes
  • Kathleen R Chirco
    Stephen A Wynn Institute for Vision Research, Iowa City, Iowa, United States
    Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Kristan Sorenson Worthington
    Stephen A Wynn Institute for Vision Research, Iowa City, Iowa, United States
    Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Miles Flamme-Wiese
    Stephen A Wynn Institute for Vision Research, Iowa City, Iowa, United States
    Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Megan Riker
    Stephen A Wynn Institute for Vision Research, Iowa City, Iowa, United States
    Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Allison E Songstad
    Stephen A Wynn Institute for Vision Research, Iowa City, Iowa, United States
    Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Malia M Collins
    Stephen A Wynn Institute for Vision Research, Iowa City, Iowa, United States
    Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Joshua Andrade
    Proteomics Laboratory, New York University School of Medicine, New York, New York, United States
  • Beatrix Ueberheide
    Proteomics Laboratory, New York University School of Medicine, New York, New York, United States
    Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, New York, United States
  • Edwin M Stone
    Stephen A Wynn Institute for Vision Research, Iowa City, Iowa, United States
    Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Budd Tucker
    Stephen A Wynn Institute for Vision Research, Iowa City, Iowa, United States
    Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Robert F Mullins
    Stephen A Wynn Institute for Vision Research, Iowa City, Iowa, United States
    Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Footnotes
    Commercial Relationships   Kathleen Chirco, None; Kristan Worthington, None; Miles Flamme-Wiese, None; Megan Riker, None; Allison Songstad, None; Malia Collins, None; Joshua Andrade, None; Beatrix Ueberheide, None; Edwin Stone, None; Budd Tucker, None; Robert Mullins, None
  • Footnotes
    Support  NIH Grant EY024605, The Elmer and Sylvia Sramek Charitable Foundation, Research to Prevent Blindness, The Carver Chair in Ocular Cell Biology
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 4586. doi:
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      Kathleen R Chirco, Kristan Sorenson Worthington, Miles Flamme-Wiese, Megan Riker, Allison E Songstad, Malia M Collins, Joshua Andrade, Beatrix Ueberheide, Edwin M Stone, Budd Tucker, Robert F Mullins; Preparation of Human Choroidal ECM Scaffolds to Study Cell Replacement Strategies. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4586.

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

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Abstract

Purpose : Endothelial cells (ECs) of the choriocapillaris are lost very early during the pathogenesis of age-related macular degeneration (AMD), and cell replacement therapy is currently the most promising option for patients with advanced AMD. Here, we sought to develop a reliable method for the production of human choroidal extracellular matrix (ECM) scaffolds, which will allow for the study of choroidal EC (CEC) replacement therapies in an environment that closely resembles the native tissue.

Methods : Using unfixed RPE/choroid punches from human donor eyes, four different protocols were evaluated for their ability to remove cells from the tissue. After decellularization, immunohistochemistry (IHC) was employed to compare treated to untreated punches, confirm cell removal, and assess the localization and relative abundance of the remaining ECM proteins. To further validate CEC removal, decellularized tissue ultrastructure was visualized using transmission electron microscopy (TEM), and mass spectrometry (MS) was performed to evaluate protein composition. Finally, the acellular choroid scaffold was co-cultured with RF/6A CECs or human endothelial cells to assess the ability to recellularize the tissue.

Results : Sequential treatment with 1% Triton X-100, 0.1% SDS, and DNase solutions is most effective at removing all native cells. Decellularized RPE/choroid tissue shows a loss of CD31 immunolabeling and endothelial cell-specific lectin labeling (UEA-I), as well as complete absence of the nuclear DAPI stain compared to native RPE/choroid. In contrast, the decellularized tissue exhibits preserved collagen IV, elastin, and laminin in both IHC and MS analyses. TEM micrographs reveal complete removal of CECs within the choriocapillaris, with retained basement membrane surrounding the vessel walls. Both RF/6A and human ECs are able to successfully migrate into the choriocapillary tubes.

Conclusions : Our data demonstrate the ability to remove all cells from human RPE/choroid punches while keeping the ECM largely intact. Furthermore, intact vascular walls with basal laminae are visible in the remaining ECM, suggesting that these methods are gentle enough to preserve tissue structure and allow for the optimization of cell replacement strategies.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

 

RF/6A cells (phalloidin; green) are shown recellularizing the choroid scaffold (collagen IV; red). Cell nuclei are shown in blue. Scale bar = 50µm.

RF/6A cells (phalloidin; green) are shown recellularizing the choroid scaffold (collagen IV; red). Cell nuclei are shown in blue. Scale bar = 50µm.

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