P005 Establishing a porcine model to translate anorectal stem cell organoid models to elucidate the aetiology of perianal Crohn's fistulae

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Abstract

Background: Perianal fistulising Crohn's disease remains an extremely challenging medical problem as many fistulas do not respond to available treatments. A regenerative medicine approach is showing promise: injecting allogeneic adipose-derived mesenchymal stem cells in and around complex fistulas improves healing similar to surgery in a recent Phase 3 randomised trial (TiGenix, Cx601) [1].

Organoid-based technologies may expand the understanding of ineffective endogenous stem cell response to tissue damage in Crohn's fistulas and offer a range of new therapeutic targets. Organoids are in vitro 3D cellular structures derived from primary tissue stem cells and capable of self-renewal and self-organization [2]. Anorectal organoids provide accessible and physiologically relevant models to elucidate the inherent properties of stem cells outside a tightly regulated in vivo environment [3]. In this study, we used the porcine model to establish anorectal organoid methodology.

Methods: Anal tissue, including the anorectal transition zone (ATZ), was resected from healthy Landrace/Cross pigs (females, aged 4–6months) within one hour of termination. Biopsies taken from anal, ATZ and rectal tissue were transferred to petri-dishes, where they were washed/minced. Stem cells were isolated from tissues using a modified protocol developed for mice [4]. Tissues were exposed to enzymatic digestion (collagenase/dispase, Sigma) at 37C for 1–2 hours on a shaker to release non-adherent cells from the mucosal tissue layer; tissue fragments were then removed by sequential filtering and centrifugation. Porcine cells were cultured in human organoid medium [5].

Results: Ring structures, characteristic of developing 3D in vitro organoid were derived from anal, ATZ and rectal tissue over period of 7–14 days. Rectal organoids formed crypt-like structures, similar to the phenotype of small intestine organoids. In contrast, non-adherent cells were produced by organoids derived from anal and ATZ derived tissues and formed a monolayer in culture. All anorectal organoids can be serially passaged for extended periods for characterisation.

Conclusions: Here we describe for the first time the ability to establish porcine anorectal organoid models using modifications of established techniques. The porcine model provides a valuable model to establish methodologies and characterise anorectal organoid biology to translate to Crohn's patients

References:

[1] Panés J, García-Olmo D, Van Assche G, Colombel JF, Reinisch W, Baumgart DC, et al. (2016), Expanded allogeneic adipose-derived mesenchymal stem cells (Cx601) for complex perianal fistulas in Crohn's disease: a phase 3 randomised, double-blind controlled trial, Lancet, 1281–1290

[2] Van De Wetering M, Francies HE, Francis JM, Bounova G, Iorio F, Pronk A, et al. (2015), Prospective derivation of a living organoid biobank of colorectal cancer patients, Elsevier, Cell, 933–945

[3] Fatehullah A, Tan SH, Barker N. (2016), Organoids as an in vitro model of human development and disease, Nature Publishing Group, 246–254

[4] Booth C, O'Shea JA. (2002), Isolation and Culture of Intestinal Epithelial Cells, Wiley-Liss, 303–335, 10, Book: Freshness, R. I., & Freshney, M. G. (eds.), Culture of epithelial cells, 2nd ed. Hoboken, NJ

[5] Sato T, Stange DE, Ferrante M, Vries RGJ, Van Es JH, Van Den Brink S, et al, (2011), Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium, Elsevier, Gastroenterology, 1762–1772

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