Abstract

DO.02.01

Influence of riboflavin and UVA-irradiation on cell viability and viscoelastic properties of the stromal bioequivalent of a human cornea construct for drug absorption studies

Grobe G. M., Reichl S.
Technische Universität Carolo-Wilhelmina zu Braunschweig, Institut für Pharmazeutische Technologie, Braunschweig

Objective: This study examines the suitability of the riboflavin/UVA-method for strengthening the stroma of a cornea construct, which is used as an in-vitro-model for drug permeation studies. Varying the dose of irradiation, the viability of the keratocytes incorporated in the stroma and the viscoelastic properties of the collagen matrix were researched.
Methods: SV40-immortalized human corneal keratocytes (HCK-Ca) were dispersed in a collagen gel and cultivated in 24 well multiwell plates. After seven days of cultivation the stromal equivalents were incubated with 0.1% riboflavin in cell culture media over night. The following day both sides of the stromal equivalents were irradiated with UV light of the wavelength of 365 nm and a dose of 0 to 5 J/cm². After another 24 hours cell viability was analyzed using the MTT-Assay, which determines the activity of mitochondrial dehydrogenases by regarding the metabolism of a tetrazolium salt to a colored product and following photometric measurement. Furthermore the collagen gels were examined using the method of oscillation rheology, which allows to make a statement concerning the viscoelastic properties.
Results: As expected increasing the dose of irradiation leads to a decrease of cell viability. Cell viability is already less than 50% after irradiating both sides of the stromal equivalent with dose of 1 J/cm². Rheological measurement showed an increase of elastic components with increasing dose of irradiation.
Conclusions: The treatment of the stromal equivalents with riboflavin and UVA-irradiation strengthens the collagen matrix but also leads to a considerable decrease of cell viability. Further studies concerning the ability of the HCK-Ca to regenerate have to show if this method is suitable for the construction of a sufficiently firm stroma-matrix including vital keratocytes, which allows to build up a three-layered corneal model for drug permeation studies by seeding endothelial and epithelial cells onto it.

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