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M. R. Hahne, S. Reichl; Development of a Serum Free Human Cornea Construct for in vitro Drug Absorption Studies. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1942.
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The increased use of ophthalmologicals over the past decades has lead to an enlarged demand of transcorneal drug absorption studies in vitro and in vivo. Due to the lack of alternatives animal experiments are widely-used for such studies as well as ocular cytotoxicity tests. Models of the human cornea based on tissue engineering could avoid several disadvantages of animal experiments such as ethical concerns, high costs, poor standardisation and problematic transferability to humans. Despite these numerous disadvantages so far no available in vitro model of the human cornea is generally accepted by international authorities. In the context of prevalidating a human cornea construct (HCC) for in vitro drug diffusion experiments, this study describes the analysis of its barrier characteristics and compares it with that of excised rabbit and porcine cornea.
Human cornea constructs were cultivated under serum free conditions on permeable polycarbonate filters (Transwells®, Costar) using SV 40 immortalised human keratocytes (HCK-Ca) and immortalised human epithelial cells (HCE-T). Briefly, using Keratinocyte Growth Medium (KGM, Lonza, USA) and an advanced cultivation schedule the HCC showed a suitable corneal barrier. Its equivalence to native tissue was analysed by comparative diffusion experiments with isolated rabbit and porcine cornea. For this purpose model substances with a wide range of molecule characteristics were used, including hydrophilic dye sodium fluorescein, lipophilic dye rhodamine B, macromolecule FITClabeled dextran (FD-4), β-blocker timolol and steroid hormone dexamethasone.Diffusion experiments with human cornea constructs were performed in Transwells® according to a standardised protocol, while permeation studies with excised corneas were accomplished in the vertical Ussing chamber system (Havard Apparatus).
Reconstructed human cornea constructs showed permeability in the same range as excised corneas. Resulting from the standardised cultivation procedure HCCs showed a high reproducibility and tended to show a lower standard deviation than excised tissue.
The introduced human corneal construct for drug diffusion studies could turn out to be a promising in vitro alternative to the use of ex vivo tissue since present results show an extensive equivalence of barrier characteristic with isolated cornea within a wide scope of molecule attributes.
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