TY - JOUR
T1 - Investigating Intestinal Barrier Breakdown in Living Organoids
AU - Bardenbacher, Marco
AU - Ruder, Barbara
AU - Britzen-Laurent, Natalie
AU - Naschberger, Elisabeth
AU - Becker, Christoph
AU - Palmisano, Ralph
AU - Stürzl, Michael
AU - Tripal, Philipp
PY - 2020/3/26
Y1 - 2020/3/26
N2 - Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques. Here we describe an assay to quantitatively determine the barrier integrity over time in living small intestinal mouse organoids. To validate our model, we applied interferon gamma (IFN-γ) as a positive control for barrier destruction and organoids derived from IFN-γ receptor 2 knock out mice as a negative control. The assay allowed us to determine the impact of IFN-γ on the intestinal barrier integrity and the IFN-γ induced degradation of the tight junction proteins claudin-2, -7, and -15. This assay could also be used to investigate the impact of chemical compounds, proteins, toxins, bacteria, or patient-derived probes on the intestinal barrier integrity.
AB - Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques. Here we describe an assay to quantitatively determine the barrier integrity over time in living small intestinal mouse organoids. To validate our model, we applied interferon gamma (IFN-γ) as a positive control for barrier destruction and organoids derived from IFN-γ receptor 2 knock out mice as a negative control. The assay allowed us to determine the impact of IFN-γ on the intestinal barrier integrity and the IFN-γ induced degradation of the tight junction proteins claudin-2, -7, and -15. This assay could also be used to investigate the impact of chemical compounds, proteins, toxins, bacteria, or patient-derived probes on the intestinal barrier integrity.
KW - Animals
KW - Humans
KW - Image Processing, Computer-Assisted
KW - Intestines/physiology
KW - Mice
KW - Models, Biological
KW - Organoids/physiology
KW - Permeability
U2 - 10.3791/60546
DO - 10.3791/60546
M3 - Original Article
C2 - 32281970
SN - 1940-087X
JO - JOURNAL OF VISUALIZED EXPERIMENTS
JF - JOURNAL OF VISUALIZED EXPERIMENTS
IS - 157
ER -