Etablierung einer 3D-Darmgewebekultur zur in-vitro-Untersuchung der Resorption potentieller Wirkstoffe auf Basis einer natürlichen Kollagenmatrix

The dissertation describes the development of a three-dimensional intestinal tissue culture which reflects the physiological barrier by the simulation of the micro environment of the small intestine in vitro. Therefore an enhanced transfer of the experimental data from absorption studies in vitro to the human organism in vivo was expected. The development of the intestinal tissue model was geared to the conventional oversimplified two-dimensional Caco-2 assay. This model is used at present for the classification of substances regarding its permeability at the intestinal barrier. For the assembling of the 2D-model the seeding of the cell line Caco-2 on a porous synthetic membrane and their static cultivation takes place over a cultivation period of 21 days. For the improvement of the artificial 2D assay, physiological parameters were integrated gradually, to establish a new 3D intestinal tissue model. At first the exchange of the synthetic membrane against an acellularised collagen scaffold from porcine jejunal segments took place. This scaffold represents the natural micro environment for intestinal epithelial cells in vitro. Exclusively the remaining villus structure of the natural scaffold sustained the barrier function of the reseeded cells during permeation studies by the use of viscous or particular substances. However no improvement of the system could be obtained concerning the cell morphology under static culture conditions. Only the development of a 2-chamber-bioreactor system led to an improvement. This system realise the dynamic cultivation of the epithelial cells and the simultaneous implementation of directional permeation studies. The mechanical stimulation of the cells, based on the media perfusion, the improved nutrient supply and the removal of toxic metabolite was analysed to be the crucial factor for the change of the cell morphology. Moreover the co-cultivation of epithelium cells with primary micro vascular endothelial cells represents the complete gut-blood-barrier and therefore the simulation of the mass transport across the intestinal lumen into the systemic circulation in vitro. Due to the immunohistological characterization (anti-Villin) of the Caco-2 cells and the analysis of efflux protein activity (P-Glycoprotein), a significant reduction of the cultivation period, from 21-to 14-day-cultivation, could be realised. Directional absorption studies were successfully accomplished by the use of the efflux substrate Rhodamine 123. The