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) .
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 . Anorectal organoids provide accessible and physiologically relevant models to elucidate the inherent properties of stem cells outside a tightly regulated in vivo environment . 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 . 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 .
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
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