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* = Presenting author

P037 Patient-derived colonic epithelial cultures as a valuable tool for personalized medicine

W. Vanhove*, S. De Schepper, D. Staelens, I. Arijs, G. Van Assche, M. Ferrante, S. Vermeire, K. Nys

KU Leuven, Translational Research Center for Gastrointestinal Disorders (TARGID), Department of Clinical and Experimental Medicine, Leuven, Belgium

Background

Inflammatory bowel diseases (IBD) are caused by an aberrant immune response towards intestinal microbiota in genetically predisposed persons, most likely facilitated by intestinal epithelial defects. The intestinal epithelium has an important role in the intestinal immune response by conserving host-microbial interactions and tissue homeostasis.

The imminent introduction of new therapeutic classes for IBD patients emphasizes the need for personalized medicine for which the epithelial resistance or response to cytotoxic agents, therapeutics, dietary components, etc. may be essential.

However, a simple epithelial model with the potential for personalized determination has not been tested. Therefore we developed and validated a short term culture system for IBD patient-derived intestinal epithelial cells (IECs).

Methods

Endoscopically-derived mucosal biopsies were obtained from both inflamed and non-inflamed regions from the colon of IBD patients. Colonic crypts from biopsies were isolated through chemical and mechanical separation (adapted intestinal organoid protocol by Sato et al. [1]. Intact intestinal crypts were immediately plated in collagen-coated wells containing in-house designed medium, resulting in a monolayer of IECs. The epithelial character of the cells was confirmed by ICC for epithelial tight junction protein E-cadherin and positive detection of cytokeratin (CK) 18 and 20 mRNA at 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.

Conclusion

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.

References:

[1] Sato, Toshiro Stange, Daniel E Ferrante, Marc Vries, Robert G J Van Es, Johan H Van den Brink, Stieneke Van Houdt, Winan J Pronk, Apollo Van Gorp, Joost Siersema, Peter D Clevers, Hans, (2011), Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium, Gastroenterology, 1762-1772