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

Abstract

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 analysis of low permeable substances (Fluoresceine and Desmopressin) resulted in a higher permeability for the dynamic 3D tissue model compared to the conventional 2D assay. This increased paracellular permeability of the cells within the enhanced 3D model rather equates to the physiological barrier function of the enterocytes in vivo.Based on their carcinogenic and colorectal origin, the Caco-2 cells in general are not the optimal cells to represent the intestinal barrier in vitro. Therefore an otimized isolation and cultivation technique of primary intestinal epithelial cells from neonatal piglets was established. Due to the development of a cell culture medium as well as the modification of published isolation protocols the cultivation of the primary jejunal epithelial cells was possible over a cultivation period of 5 days within the enhanced 3D model. A cell population of enterocytes and goblet cells could be proven by immonohistological staining methods. Based on the high prismatic cell growth and the mucous layer theses cells reflect the physiological barrier of the small intestine more realistically.Altogether the 3D tissue model could represent a new tool for the prediction of the intestinal permeation of new drug candidates in future. The experimental data obtained within this model showed a more physiological cell growth as well as a better in-vivo-in-vitro-correlation for low permeable substances. For the investigation, whether this model can be standardized for the screening of substances, further experimental data is necessary.