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Abstract #100722 Published in IGR 23-1
An Optimized Method to Decellularize Human Trabecular Meshwork
Crouch DJ; Sheridan CM; Behnsen JG; Bosworth LABioengineering (Basel, Switzerland) 2022; 9:
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% ) and Ammonium Hydroxide (NHOH; 2% ). 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, NHOH lysed all cells (dsDNA 25 ± 21 ng/mL), and the TM structure remained intact. For human TM, 2% NHOH 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 NHOH to decellularize delicate human TM without compromising tissue structure.
Department of Eye and Vision Science, Institute of Life Course and Medical Sciences, Faculty of Health and Life Sciences, University of Liverpool, Liverpool L7 8TX, UK.
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