02395nas a2200409   4500000000100000000000100001008004100002260001500043653002800058653003000086653002300116100002000139700001600159700001800175700002200193700001900215700002300234700002100257700002300278700002100301700002000322700002000342700002000362700002300382700001900405700002100424700002600445700002000471700001900491700002400510700001900534245011500553856005500668300000900723520123900732022001401971       2026                            d  c2026-05-2210aGastrointestinal system10aStem-cell differentiation10aTissue engineering1 aHolly M. Poling1 aThéo Noël1 aAkaljot Singh1 aGarrett W. Fisher1 aKonrad Thorner1 aPraneet Chaturvedi1 aKalpana Nattamai1 aKalpana Srivastava1 aMatthew R. Batie1 aTaylor Hausfeld1 aAmy L. Pitstick1 aNicole E. Brown1 aSéverine Ménoret1 aIgnacio Anegon1 aRiccardo Barrile1 aChristopher N. Mayhew1 aTakanori Takebe1 aJames M. Wells1 aMichael A. Helmrath1 aMaxime M. Mahe00aLarge-scale and innervated functional human gut tissues for transplantation via transient spheroid confinement  uhttps://www.nature.com/articles/s41551-026-01688-6  a1-173 aKey limitations of current human gastrointestinal organoids include incomplete physiological maturation and the need for complex, time-consuming assembloid approaches to integrate a functional nervous system for transplantation. Here we present a confined culture system (CCS) method that generates large-scale, elongated and functional human small intestinal, colonic and gastric tissues with a de novo enteric nervous system (ENS). We use a 3D-printed scaffolding tray to restrict spheroid fusion and growth, promoting the spontaneous co-development of a functional ENS. Transcriptomic and electrophysiological data demonstrate selective neuromuscular function and the presence of excitatory and inhibitory neurons within the tissues. When compared to traditional methods, the CCS expedites tissue maturation for transplantation, yielding organoids that are up to ten times larger, reaching widths of 8 cm after 10 weeks, and exhibit enhanced engraftment rate. CCS organoids integrate and adapt to murine luminal environment while maintaining barrier integrity and functional capacity. The CCS methodology simplifies current protocols while accelerating the production of complex, functional and clinically relevant gut tissues.  a2157-846X