Speaker
Description
Introduction: Retinal pathologies affect more than 350 million people worldwide yearly, impairing visual acuity and quality of life [1-3]. Despite therapy procedures for these pathologies already existing, more effective and patient-friendly ones are the subject of research. Animal models, mainly used in this field, are expensive, time-consuming and present regulatory issues [4]. On the other hand, existing in vitro models lack controlled and reproducible vascularization and micro-macro scale interface [5-6]. For this reason, we propose a biomimetic in vitro model able to mimic the blood-retinal barrier filtration properties and that allows the user to perform drug testing via standard administration routes.
Methods: The model relies on a microfluidic circuit laser-engraved into PDMS, featuring 70-800µm wide and 300µm depth channels functionalized using APTES and gelatin to facilitate cell attachment. It faces electrospun membranes of PLGA or gelatin [7,8], which have already shown good properties for retinal pigmented epithelium cell culture, to mimic choroidal vasculature and Bruch’s membrane. These two components are fixed together using an acetic acid-gelatin-based solution. Two 23G cannulas are inserted into the microvascular network input and output, and the assembled component is placed inside a cylindrical PDMS culture chamber. On the top part of the chamber, an alginate-gelatin membrane mimics the corneal stroma, allowing drug injection [9]. Experiments were conducted seeding ARPE-19 cells on the membrane and HUVECs inside the microfluidic channels. Immunostaining techniques were used to address the cell layer proper formation. Antibodies used include ZO-1 to identify RPE tight junctions and CD31 and VE-cadherin to identify HUVECs junctions and specific endothelial markers. Specific drugs as anti-VEGF, were used to perform permeability tests across the membrane to verify the correct functioning of the biological barrier [10].
Results: The device showed the ability of hosting both RPE and HUVECs in both static and dynamic conditions. Moreover, the junctions between cells proved the formation of the biological barrier. The use of specific substances to test permeability of the formed barrier confirmed the active transport performed by the cells.
Discussion: The developed device merges the microstructure of the outer blood-retinal barrier with the macroscopic dimension of the eye. With a reproducible microchannel network mimicking the choroidal vasculature, it overcomes common in vitro model problems. Thanks to its geometry, the device allows the drug testing using standard procedures, like injection, systemic and topical administration, resulting in promising results for the development of in vitro models that can effectively substitute animal models.
Acknowledgements: We acknowledge the support of the European Union by the Next Generation EU project ECS00000017 ‘Ecosistema dell’Innovazione’ Tuscany Health Ecosystem (THE, PNRR, Spoke 4: Nanotechnologies for diagnosis and therapy).
[1] Stein JD. JAMA. 2021; [2] Teo ZL. Ophthalmology. 2021; [3] Stahl A. Dtsch Arztebl Int. 2020; [4] Scott A. Eye. 2010; [5] Filiz Y. Progress in Biomedical Engineering. 2024; [6] Lieto K. Int J Mol Sci. 2022 ; [7] Warnke PH. Acta Biomater. 2013; [8] Lai JY. Int J Mol Sci. 2009; [9] K. Tonsomboon. J Mech Behav Biomed Mater. 2013; [10] L. Liu. Adv Exp Med Biol. 2019
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