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M. R. Hahne, S. Reichl; Prevalidation of a Human Cornea Construct (HCC): Optimising Barrier Function and Comparing With Excised Cornea. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4606.
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© ARVO (1962-2015); The Authors (2016-present)
In recent years a couple of human cornea equivalents based on tissue engineering have been established with the objective of having an in vitro model for transcorneal drug absorption studies and ocular cytotoxicity avoiding the disadvantages of the widely-used animal experiments. In some cases they have been accepted by national authorities but none of them have been validated yet. This study describes in the context of prevalidating a HCC experiments with immortalised human corneal epithelial cells (HCE-T) to optimise barrier function (tight junctions) and to compare the barrier of cultivated HCC with that of excised porcine and rabbit cornea.
Corneal epithelium was cultivated on collagen-coated transwells (Costar) and transepithelial electrical resistance (TEER) was measured using the EndOhm/EVOM system (WPI) to analyse epithelial tightness. The influence of initial cell number was investigated in the range of 60,000 to 140,000 HCE-T cells per transwell and both culture media containing FBS as well as serum free media each with varying supplements were used. For inducing cell differentiation the epithelium was lifted to the air-liquid-interface (ALI) between day 7 and 19 of cultivation. Absorption studies were performed using different permeation methods and fluorescein sodium as marker substance.
Varying the initial cell number did not influence epithelial barrier function significantly, the epithelium tended to show the highest TEER values basing on 100,000 cells. By reducing FBS content of the standard culture medium from 5 % gradually till 0.2 % serum, epithelial barrier function remained in submerse cultures at almost the same level. However, when the epithelium was lifted to the air liquid interface higher FBS concentrations lead to higher and more stable TEER levels as well as to lower permeation coefficients being in the same range as excised cornea.
FBS is an important but not the only decisive factor for generating the epithelial barrier in tissue culture and its importance depends from further cultivation conditions. By choosing an appropriate serum free milieu a tight barrier can be generated under these conditions as well, whereas a slightly higher permeability must be accepted.
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