An Optimized Method to Decellularize Human Trabecular Meshwork

Crouch, Devon J ORCID: 0000-0002-9782-107X, Sheridan, Carl M ORCID: 0000-0003-0100-9587, Behnsen, Julia G ORCID: 0000-0003-1511-3051 and Bosworth, Lucy A ORCID: 0000-0002-6726-4663 (2022) An Optimized Method to Decellularize Human Trabecular Meshwork. BIOENGINEERING-BASEL, 9 (5). 194-.

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Official URL: http://dx.doi.org/10.3390/bioengineering9050194

Abstract

Glaucoma is linked to raised intraocular pressure (IOP). The trabecular meshwork (TM) plays a major role in regulating IOP by enabling outflow of aqueous humor from the eye through its complex 3D structure. A lack of therapies targeting the dysfunctional TM highlights the need to develop biomimetic scaffolds that provide 3D in vitro models for glaucoma research or as implantable devices to regenerate TM tissue. To artificially mimic the TM's structure, we assessed methods for its decellularization and outline an optimized protocol for cell removal and structural retention. Using bovine TM, we trialed 2 lysing agents-Trypsin (0.05% v/v) and Ammonium Hydroxide (NH4OH; 2% v/v). Twenty-four hours in Trypsin caused significant structural changes. Shorter exposure (2 h) reduced this disruption whilst decellularizing the tissue (dsDNA 26 ± 14 ng/mL (control 1970 ± 146 ng/mL)). In contrast, NH4OH lysed all cells (dsDNA 25 ± 21 ng/mL), and the TM structure remained intact. For human TM, 2% v/v NH4OH similarly removed cells (dsDNA 52 ± 4 ng/mL (control 1965 ± 233 ng/mL)), and light microscopy and SEM presented no structural damage. X-ray computed tomography enabled a novel 3D reconstruction of decellularized human TM and observation of the tissue's intricate architecture. This study provides a new, validated method using NH4OH to decellularize delicate human TM without compromising tissue structure.

Item Type:	Article
Uncontrolled Keywords:	decellularization, trabecular meshwork, eye, glaucoma, bioengineering, X-ray computed tomography, trypsin, ammonium hydroxide, lysing agent
Divisions:	Faculty of Health and Life Sciences Faculty of Health and Life Sciences > Institute of Life Courses and Medical Sciences
Depositing User:	Symplectic Admin
Date Deposited:	16 May 2022 12:25
Last Modified:	18 Jan 2023 21:02
DOI:	10.3390/bioengineering9050194
Open Access URL:	https://www.mdpi.com/2306-5354/9/5/194
Related URLs:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3154894