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

Adegbola S.*1, Moore J.2, Sahnan K.1, Tozer P.1, Phillips R.1, Warusavitarne J.3, Faiz O.1, Hart A.4

1St Mark's Academic Institute, Surgery, London, United Kingdom 2Curileum Discovery Ltd, Northwick Park Institute of Medical Research/St Mark's Hospital Site, London, United Kingdom 3St Mark's Hospital, London, London, United Kingdom 4St Mark's Academic Institute, Gastroenterology, London, United Kingdom


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.


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].


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.

Figure 1. Anorectal tissue cell production in 3D in vitro organoids.


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


[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