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G. M. Grobe, S. Reichl; Analyzing Cell Viability of the Stromal Bioequivalent of a Human Cornea Construct for Drug Absorption Studies After Riboflavin/UVA-Treatment. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5467.
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To establish a tissue-engineered cornea construct which is used as an in-vitro-model for drug absorption studies, this construct is to be of adequate mechanical stability. In order to strengthen the collagen matrix by crosslinking, the riboflavin/UVA-method is applied to the stromal bioequivalent. Afterwards cell viability of the keratocytes incorporated in the stromal matrix is analyzed depending on the dose of irradiation.
SV40-immortalized human corneal keratocytes (HCK-Ca) were dispersed in a collagen gel. After 7 days of cultivation the stromal bioequivalents were incubated with 0.1 % riboflavin in cell culture media over night. The following day each side of the stromal bioequivalent was irradiated at 365 nm and a dose of 0 - 5 J/cm2 . Oscillation rheology was used to determine the improvement of viscoelastic properties of the collagen gels. Analyzing cell viability an MTT-Assay, which determines the activity of mitochondrial dehydrogenases, was performed 24 hours and several days after irradiation.Ongoing studies are carried out to clarify whether the decrease of cell viability after irradiation is the consequence of an apoptotic process or necrosis. For that reason different cell assays are performed. On the one hand the activity of caspase 3 and 7, playing key effector roles in apoptosis, are measured. On the other hand a distinct dead-cell protease activity is determined which is a marker of cytotoxicity.
As expected, increasing the dose of irradiation leads to a considerable decrease of cell viability within 24 hours which even proceeds within the next days. After irradiating both sides of the stromal equivalents with a dose of 1 J/cm2 , cell viability is already reduced about 50 % within 24 hours. The improvement of the elastic components of the stromal equivalents is weak but statistically significant.
The decrease in cell viability is extensive and enduring. Consequently, in contrast to clinical applications, the riboflavin/UVA-treatment seems to be no suitable method to obtain a sufficiently firm stromal matrix including vital keratocytes.
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