June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Freeze-thaw Decellularization with Preserved Trabecular Meshwork Scaffold in an Ex Vivo Eye Perfusion Model
Author Affiliations & Notes
  • Susannah Waxman
    University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Yalong Dang
    University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Ralitsa Loewen
    University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Chao Wang
    University of Pittsburgh, Pittsburgh, Pennsylvania, United States
    Xiangya School of Medicine, Central South University, Changsha, Hunan, China
  • Adrianna Jensen
    University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Nils Loewen
    University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Footnotes
    Commercial Relationships   Susannah Waxman, None; Yalong Dang, None; Ralitsa Loewen, None; Chao Wang, None; Adrianna Jensen, None; Nils Loewen, None
  • Footnotes
    Support  NIH Grant K08EY022737-01
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3469. doi:
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      Susannah Waxman, Yalong Dang, Ralitsa Loewen, Chao Wang, Adrianna Jensen, Nils Loewen; Freeze-thaw Decellularization with Preserved Trabecular Meshwork Scaffold in an Ex Vivo Eye Perfusion Model. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3469.

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

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Abstract

Purpose : Repopulation of diseased trabecular meshwork (TM) with new, functional cells may provide a new approach to treating open angle glaucomas. An ideal ex vivo model to establish transplantation techniques requires TM architecture and cellularity similar to glaucomatous eyes and the option to remove existing TM cells. This study's objective was to develop a simple decellularization technique that preserves an extracellular matrix (ECM) scaffold without the requirement of chemicals or viral ablation agents.

Methods :
Anterior segments of porcine eyes were mounted and cultured in constant-rate perfusion units. After 72 hours of perfusion to stabilize IOP, eyes that received freeze-thaw treatment (F) were thrice cycled from to -80°C for 120 minutes to room temperature for 60 minutes while eyes that received saponin treatment (S) were exposed to 0.02% saponin for 15 minutes followed by an anterior chamber fluid exchange. Eight eyes were assigned each to F, S, and control group (Co). Post-treatment, eyes were perfused with culture media for 180 hours and IOP was measured at two minute intervals. Calcein and PI co-labelling was utilized to evaluate TM viability. H&E stained sections were used for gross histological analysis. After eGFP-FIV transduction of two additional eyes, real-time TM ablation via S or F was visualized with a dissecting fluorescence microscope.

Results : Similar baseline IOPs were obtained in F and S (14.75+/- 2.24 mmHg and 14.37+/- 1.14 mmHg, P=0.288). Shown in Figure 1, F and S experienced IOP reductions at 12 hours and reached similar endpoint IOPs at 180 hours (9.51+/- 0.98 mmHg and 11.65+/- 1.34 mmHg, P=0.151) while Co IOP did not decrease. Viability staining assay revealed numerous PI-labeled dead cells and Calcein-labeled viable cells in both the S and Co groups. In contrast, sparse PI-labeled and no Calcein-labeled TM cells were seen in F. Histology revealed that TM cells were absent in F while the angle architecture and ECM were preserved. Shown in Figure 2, TM cells that expressed eGFP were entirely ablated by F but only partially by S.

Conclusions : We developed a chemical-free, freeze-thaw method to produce decellularized TM scaffold with well preserved TM excellular matrix in organotypic perfusion culture.

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

 

 

Red arrows indicate fluorescent TM regions pre-treamtent (1A-F) while white arrows indicate the same regions within the corresponding sample post-treament (2A-F).

Red arrows indicate fluorescent TM regions pre-treamtent (1A-F) while white arrows indicate the same regions within the corresponding sample post-treament (2A-F).

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