An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation.

Short Bowel Syndrome (SBS) is characterized by insufficient functional intestinal tissue capable of nutrient transport and absorption. Tissue engineering offers a promising strategy to restore intestinal function by reconstructing a bioengineered muscle layer. In this exploratory study, we investigated the feasibility of co-culturing rat smooth muscle cells (SMCs) with enteric nervous system (ENS) cells in layered three-dimensional (3D) scaffolds. Three culture conditions were compared: SMC monocultures, paracrine signaling through a semipermeable membrane, and direct ENS-SMC co-culture. Although some effects in this reductionist model may reflect in vitro artifacts rather than true developmental processes, our results demonstrate that ENS and SMCs can form structural and potentially functional (synaptic-like) connections. Electron microscopy and immunofluorescence revealed native-like smooth muscle organization and synaptic contacts between ENS cells and SMCs. Observed spontaneous contractile activity could reflect functional interaction between these two cell types. These findings establish proof-of-concept that functional, innervated smooth muscle fibers can be generated in vitro through ENS-SMC co-culture in 3D scaffolds. This work provides a foundation for the development of bioengineered intestinal tissue and highlights the need for future studies addressing epithelial integration, neuronal diversity, and detailed functional characterization.