Large-scale and innervated functional human gut tissues for transplantation via transient spheroid confinement

nature biomedical engineering Article https://doi.org/10.1038/s41551-026-01688-6 Large-scale and innervated functional human gut tissues for transplantation via transient spheroid confinement Received: 15 June 2025 Accepted: 18 April 2026 Published online: xx xx xxxx Check for updates Holly M. Poling1,2,3, Théo Noël 4, Akaljot Singh1,3, Garrett W. Fisher1, Konrad Thorner5, Praneet Chaturvedi5, Kalpana Nattamai3, Kalpana Srivastava3, Matthew R. Batie6, Taylor Hausfeld1, Amy L. Pitstick3, Nicole E. Brown1,3, Séverine Ménoret7,8, Ignacio Anegon7, Riccardo Barrile2,3, Christopher N. Mayhew3,5, Takanori Takebe 3,5,9, James M. Wells 3,5, Michael A. Helmrath 1,3 & Maxime M. Mahe 1,3,4 Key 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 neuro muscular 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. The ability to culture and manipulate human pluripotent stem cells (hPSC) has advanced the generation of specific tissues1. Translational embryology has enabled ‘organogenesis in a dish’ in three-dimensional (3D) environments, fostering more physiologically relevant models such as organoids2,3. Defined by their self-assembly and renewal, organoids mimic native tissue, include correct cell types and replicate some organ functions4. These systems have been developed for many organs, including the gastrointestinal tract5–10, offering real-time visualization of organogenesis and bridging the gap between animal models and humans. hPSC-derived organoids hold immense potential for studying development, disease processes, cell-based therapies and personalized drug discovery. A full list of affiliations appears at the end of the paper. Nature Biomedical Engineering Despite these advantages, current organoid technologies face limitations in mimicking physiological environments in vitro, often requiring in vivo transplantation for continued tissue differentiation. Some hPSC-derived gastrointestinal tissues exhibit incomplete maturation, limiting their transplantation11. We hypothesized that methods to promote cellular complexities would be required to support both in vitro and in vivo maturation that will approximate native developing tissues11,12. hPSC-derived cells undergo normal maturation, thus initially have embryonic or fetal-like characteristics12,13. Methods to further develop these tissues are required to achieve the much-desired mature tissues to model human bowel. Using existing protocols, human intestinal organoids (HIOs) grow individually, reaching ~1 mm after 28 days e-mail: ; Article in culture, expanding to ~1 cm with transplantation14–16. However, their spherical shape differs from the intestinal tube. We introduced strain into transplanted HIOs to induce maturation and enterogenesis, producing elongated tissues resembling the postnatal gut12. Recent methods to generate hindgut (human colonic organoids, HCO) and foregut (human gastric organoids, HGO; human antral gastric organoids, HaGO) organoids have furthered understanding of gastrointestinal development and disease, although traditional culturing results in low engraftment rates and small sizes compared to HIOs7,11. Initial attempts to correct this were achieved in an assembloid approach by adding both splanchnic mesenchyme and neural crest cells11. In this study, we focused on simplifying the methods required to achieve robust engraftment using a confined culture system (CCS). We designed a specialized scaffolding tray to provide a permissive environment for spheroid fusion (the 3D rounded structures that form from 2D monolayers upon differentiation, which are typically collected and re-plated for maturation into organoids). Loading a critical mass of spheroids into the scaffolding trays yielded longitudinal organoid structures. This device combined with 3D culture techniques to prime organoids successfully enhanced tissue growth and maturation, yielding organoids ten times larger than those in previous protocols. Notably, a de novo enteric nervous system (ENS) emerged, displaying selective neuronal excitation and contractile functions. Similar results were observed in colonic and gastric organoids. These structures readily engrafted, exhibited enhanced maturation and developed a functional ENS. Here we present a scalable organoid generation method emphasizing cellular confinement, advancing the production of complex organs for clinical applications. Results Implementation of a scaffolding tray for organoid generation To impart a defined geometry on intestinal organoids, a specialized scaffolding tray was designed to culture mid-hindgut spheroids, and introduced into the traditional workflow (Fig. 1a). The scaffolding tray was made by 3D-printing a mould with longitudinal confinement lanes to culture a critical spheroid mass (Supplementary Fig. 1a). Biocompatible polydimethylsiloxane (PDMS) was then cured within the mould and removed for use (Supplementary Fig. 1b). This approach, termed the CCS, restricted spheroid growth within a defined space while enhancing scalability. Typically, a single spheroid forms a single HIO, but here, ~4,000 spheroids from spontaneous morphogenesis were used (Supplementary Fig. 1c). They began as discrete spheroids at d0 that developed into a unified construct by d5 (Fig. 1b). By d6, these structures were manually removed and re-plated in Matrigel for continued culture until d14 (Supplementary Fig. 1d). When comparing small intestinal CCS (SI CCS) to conventional HIOs at d6 and d14, the geometry was maintained, and both simple columnar epithelium and supporting mesenchymal populations persisted (Fig. 1c). Reduction in time to engraftment and enhanced morphometric features After d14 in culture, SI CCS structures displayed robustness in size and cellular integrity, suggesting sufficient maturation for transplantation. This contrasts with previous findings requiring HIOs to be cultured (...truncated)