| Popis: |
different time points by qRT-PCR, whereas detection of fibroblast markers PDGFR and COL1A1 and COL1A2 mRNA remained negative. Inflammasome sensors NLPR3, AIM2 and IFI16 were assessed by ICC. Results: Crypt isolation was successful in 80%. The crypts attached to the bottom of the wells overnight. After 24 hours, normal crypt architecture switched to a monolayer culture, observed as patches of densely packed cuboidal cells. We were able to culture the IECs for a maximum of 12 days before cell death and detachment appeared. As an initial validation, we could confirm the reported cytoplasmic localization for inflammasome sensors NLRP3 and AIM2, whereas IFI16 showed nuclear staining. Conclusions: We have developed a simple ex vivo 2D IEC culture system complementary to the 3D organoid culture system. Endoscopy-derived IEC isolation will allow clinicians to evaluate patient-specific epithelial response to e.g. different or new classes of gastrointestinal therapeutics. This approach also provides a simple model for screening of drug effects at the site of the intestinal epithelium in a personalized manner. Moreover association of epithelial responses with patient-specific genetic profiles (eg. with mutations in inflammasome-related genes) may lead to further insights into disease biology of intestinal diseases such as IBD. |
