Permeability analyses and three dimensional imaging of interferon gamma-induced barrier disintegration in intestinal organoids

Publication date: Available online 7 February 2019Source: Stem Cell ResearchAuthor(s): Marco Bardenbacher, Barbara Ruder, Natalie Britzen-Laurent, Benjamin Schmid, Maximilian Waldner, Elisabeth Naschberger, Michael Scharl, Werner Müller, Claudia Günther, Christoph Becker, Michael Stürzl, Philipp TripalAbstractThe aberrant regulation of the epithelial barrier integrity is involved in many diseases of the digestive tract, including inflammatory bowel diseases and colorectal cancer. Intestinal epithelial cell organoid cultures provide new perspectives for analyses of the intestinal barrier in vitro. However, established methods of barrier function analyses from two dimensional cultures have to be adjusted to the analysis of three dimensional organoid structures. Here we describe the methodology for analysis of epithelial barrier function and molecular regulation in intestinal organoids. Barrier responses to interferon-γ of intestinal organoids with and without epithelial cell-specific deletion of the interferon-γ-receptor 2 gene were used as a model system. The established method allowed monitoring of the kinetics of interferon-γ-induced permeability changes in living organoids. Proteolytic degradation and altered localization of the tight junction proteins claudin-2, −7, and − 15 was detected using confocal spinning disc microscopy with 3D reconstruction. Hessian analysis was used for quantification of re-localization of claudins. In summary, we provide a novel me...
Source: Stem Cell Research - Category: Stem Cells Source Type: research