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Matthias R. Hahne, Gesa M. Grobe, Michaela Zorn-Kruppa, Johanna Brandner, Gustavo Guzman, Eleonore Haltner-Ukomadu, Stephan Reichl; Prevalidation of a Serum Free Human Cornea Construct for Drug Diffusion Studies: Repeatability, Transferability and Comparison with Isolated Porcine and Rabbit Cornea. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1953.
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© ARVO (1962-2015); The Authors (2016-present)
The increased use of ophthalmic products 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. Models of the human cornea based on tissue engineering could avoid several disadvantages of animal experiments such as ethical concerns and poor standardisation. Despite these numerous disadvantages so far no available in vitro model is generally accepted. In the context of prevalidating a human cornea construct (HCC) for in vitro drug absorption 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®) using SV 40 immortalised human keratocytes (HCK-Ca) and immortalised human epithelial cells (HCE-T). Briefly, using Keratinocyte Growth Medium (KGM, Lonza) and an advanced cultivation schedule the HCC showed a suitable barrier. Its equivalence to native tissue was analysed by comparative absorption 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 FITC labeled dextran, β-blocker timolol, steroid hormone dexamethasone, prostaglandin analogue bimatoprost and antiviral drug aciclovir. To investigate the intra- and inter-laboratory repeatability the construct cultivation as well as the permeation studies were performed independently by different experimenters in three laboratories.
Reconstructed HCCs exhibited a diffusion barrier in the same range as excised corneas. Resulting from the standardised cultivation procedure HCCs showed high reproducibility and lower standard deviation than excised tissue.
The introduced human corneal construct for drug absorption studies could turn out to be a promising in vitro alternative to the use of ex vivo tissue since current results show an extensive equivalence of barrier characteristic with isolated corneas.
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