Poling, H. M.; Noel, T.; Singh, A.; Fisher, G. W.; Thorner, K.; Chaturvedi, P.; Nattamai, K.; Srivastava, K.; Batie, M. R.; Hausfeld, T.; Pitstick, A. L.; Brown, N. E.; MENORET, S.; Anegon, I.; Barrile, R.; Mayhew, C. N.; Takebe, T.; Wells, J. M.; Helmrath, M. A.; Mahe, M. M.

A confined culture system (CCS) establishes methods to generate complex functional human gastrointestinal tissues. This approach yields large-scale innervated small intestinal, colonic and gastric organoids with an elongated tubular shape for both in vitro and in vivo studies. Transcriptomic and electrophysiological data demonstrate the co-development of a functional de novo enteric nervous system, which is absent from conventional organoids. When compared to traditional methods, CCS derived small intestinal, colonic and gastric organoids reached maturation supporting transplantation in half of the time, resulting in enhanced engraftment rates and sizes. Murine luminal content exposure within CCS organoids in vivo further augmented function, supporting the potential translational benefits required to model complex intestinal diseases. In summary, the CCS methodology simplifies current protocols while adding complexity and expediting the generation of clinically relevant functional gut tissues.