Freeze-thaw decellularization of the trabecular meshwork in an ex vivo eye perfusion model
- Published
- Accepted
- Subject Areas
- Bioengineering, Ophthalmology, Statistics
- Keywords
- trabecular meshwork, decellularization, ablation, intraocular pressure, pig eyes, freeze-thaw, glaucoma
- Copyright
- © 2017 Dang et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2017. Freeze-thaw decellularization of the trabecular meshwork in an ex vivo eye perfusion model. PeerJ Preprints 5:e2736v3 https://doi.org/10.7287/peerj.preprints.2736v3
Abstract
Objective: Trabecular meshwork (TM) is the primary substrate of outflow resistance in glaucomatous eyes. Repopulating diseased TM with fresh, functional TM cells might represent a novel therapeutic breakthrough. Various decellularized TM scaffolds were developed by ablating existing cells with suicide gene therapy or saponin, but always with incomplete cell removal or dissolve the extracellular matrix. We hypothesized that a chemical-free, freeze-thaw method would be able to produce a fully decellularized TM scaffold for cell transplantation.
Materials and Methods: We obtained 24 porcine eyes from a local abattoir, dissected and mounted them in an anterior segment perfusion and pressure transduction system within two hours of sacrifice. After they stabilized for 72 hours, eight eyes each were assigned to freeze-thaw (F) ablation (-80°C×2), to 0.02% saponin (S) treatment, or the control group (C), respectively. The trabecular meshwork was transduced with an eGFP expressing feline immunodeficiency viral (FIV) vector and tracked via fluorescent microscopy to confirm ablation. Following treatment, the eyes were perfused with standard tissue culture medium for 180 hours. We assessed histological changes by hematoxylin and eosin staining. TM cell viability was evaluated with a calcein AM/propidium iodide (PI) assay. We measured IOP and modeled it with a linear mixed effects model using a B-spline function of time with 5 degrees of freedom.
Results: F and S experienced a similar IOP reduction by 30% from baseline (P=0.64). IOP reduction of about 30% occurred in F within 24 hours and in S within 48 hours. Live visualization of eGFP demonstrated that F conferred a complete ablation of all TM cells and only a partial ablation in S. Histological analysis confirmed that no TM cells survived in F while the extracellular matrix remained. The viability assay showed very low PI and no calcein staining in F in contrast to numerous PI-labeled dead TM cells and calcein-labeled viable TM cells in S.
Conclusion: We developed a rapid TM ablation method that uses cyclic freezing that is free of biological or chemical agents and able to produce a decellularized TM scaffold with preserved TM excellular matrix in an organotypic perfusion culture.
Author Comment
In this version, we have corrected several wording and grammar mistakes in the introduction and discussion. We have also uploaded all full figures files for a regular submission and believe this is our last preprint version.