Abstract
A collagen vitrigel membrane (CVM) we developed can function as both a scaffold for cells and a pathway for chemicals. To extrapolate the corneal permeability of chemicals in vivo, we proposed six corneal models using the CVM. Thin and thick CVMs were used as models for Bowman's membrane (BM) and an acellular stroma (AS), respectively. Models for a corneal epithelium (CEpi), a CEpi-AS, a CEpi-endothelium (Endo), and a CEpi-AS-Endo were fabricated by culturing corneal epithelial cells and/or corneal endothelial cells on the surface of CVMs. Subsequently, the permeability coefficient (Papp) value of each model was calculated using five chemicals with different molecular radii; cyanocobalamin and four fluorescein isothiocyanate-dextrans (FD) (FD-4, FD-10, FD-20, and FD-40). The slopes of Papp versus molecular radii of those chemicals in the both BM and AS models were almost similar to data using an excised rabbit corneal stroma. The ratios of Papp values in models for BM, CEpi, and CEpi-Endo against those in data using an excised rabbit cornea were calculated as 75.4-fold, 6.4-fold, and 4.5-fold for FD-4, and 38.7-fold, 10.0-fold, and 4.2-fold for FD-10, respectively. Similarly, those in models for AS, CEpi-AS, and CEpi-AS-Endo were calculated as 26.1-fold, 2.5-fold, and 0.6-fold for FD-4, and 26.1-fold, 1.5-fold, and 0.6-fold for FD-10, respectively. These results suggest that the CEpi-AS-Endo model with both the barrier function of corneal cell layers and the diffusion capacity of chemicals in thick CVM is most appropriate for extrapolating the corneal permeability of chemicals in vivo.
Highlights
Corneal permeation studies required for ophthalmic drug development are generally performed by the test methods using laboratory animals in vivo or ex vivo (Hahne et al, 2012; Dave et al, 2015)
The corneal epithelium (CEpi), stroma, and endothelium (Endo), in order from the outside, are formed by approximately six corneal epithelial cell layers with barrier function, keratocytes scattered in high-density collagen fibrillar layers approximately 500 mm thick with diffusional inhibition, and an endothelial cell monolayer with barrier function, respectively
One clone representing excellent proliferative performance was subjected to the following experiment
Summary
Corneal permeation studies required for ophthalmic drug development are generally performed by the test methods using laboratory animals in vivo or ex vivo (Hahne et al, 2012; Dave et al, 2015). Animal experiments have disadvantages such as ethical issues on the sacrifice of life, high costs, poor reproducibility, and the questionable extrapolation of animal results to humans Et al, 2014; Pescina et al, 2015) To overcome these issues on animal experiments, a novel corneal model in cell culture systems in vitro appropriate for extrapolating the corneal permeability of chemicals in vivo is required for promoting the efficient development of eye drops.
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