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Bioengineered human trabecular meshwork membrane constructs: opportunities and challenges

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Abstract:

Purpose: To review our data from using well-defined, porous scaffolds for supporting human trabecular meshwork (HTM) and Schlemm’s canal (HSC) cells to mimic the structure and outflow physiology of the trabecular outflow tract and explore future applications for this technology in the development of novel glaucoma treatment strategies.
Methods: The microporous SU-8 scaffolds were fabricated with specific pore patterns and beam dimensions, and coated with a thin layer of gelatin or hyaluronic acid. HTM and HSC cells were cultured on the scaffolds for 14 days. Bioanalysis of relevant extracellular matrix (ECM) and HTM- or HSC-specific markers on these 3-D tissue constructs was performed to confirm their proper expression. To further simulate aqueous humor fluid dynamic across both in-vitro models, the 3-D HTM and HSC constructs were secured in a perfusion apparatus which controls microfluidic flow and is integrated with a pressure transducer. Pressure measurements at different flow rates allowed for calculation of the hydraulic conductivity, equivalent to the outflow facility (ΔF/ΔP), of the bioengineered HHTM constructs. Glaucomatous tissue constructs were induced using steroids and transforming growth
factor-beta 2 (TGFβ2). Furthermore, the feasibility to screen genes using the HTM and HSC tissue constructs was explored using siRNA and miRNA technologies.
Results: The HTM 3-D tissue constructs express HTM markers such as α-smooth muscle actin, myocilin,
αB-crystallin, and properly secreted fibronectin and collagen type IV. On the other hand, HSC tissue constructs express HSC markers such as CD31, PROX-1, VE-cadherin, and occludin. Hydraulic conductivity of the engineered 3-D HTM was calculated to be 0.131 μl/min/mmHg/mm2, while that of the engineered in-vitro HSC inner wall was calculated to be 0.046 μl/min/mmHg/mm2. Treatments with steroids induced overexpression of myocilin, ECM proteins, junction proteins, and actin rearrangement in tissue constructs comprised of steroid-responder cells. TGFβ2 treatment elicited more pronounced responses than those described in steroid treatments. Both steroids and TGFβ2 lowered
hydraulic conductivity. To assess whether the 3-D tissue constructs are amenable to gene silencing, the
3-D HSC construct was transfected with FITC-siRNA and the efficiency of siRNA uptake was evaluated. Nearly
100% of the HSC cells on the construct incorporated FITC-siRNA 24 hours after the transfection.
Conclusions: Altogether, the studies described in this chapter demonstrate the use of bioengineered 3-D human HTM and HSC tissue constructs for understanding glaucoma pathology and potentially studying the
mechanism of action of novel IOP-lowering agents at the conventional outflow tract. In addition, these bioengineered 3-D HTM and HSC models could accelerate the development of gene-targeted therapies at the conventional outflow tract to combat glaucoma progression.

Glaucoma Research 2018-2020, pp. 85-98 #6
Edited by: John R. Samples and Paul A. Knepper
© Kugler Publications, Amsterdam, The Netherlands

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