Establishment of a novel in vitro co-culture system of enteric neurons and Caco-2 cells for evaluating the effect of enteric nervous system on transepithelial transport of drugs

The gastrointestinal tract is innervated by extrinsic autonomic nerves and intrinsic enteric nervous system (ENS). However, the role of ENS in drug absorption has remained to be clarified. To investigate the effect of ENS on drug transport across the intestinal epithelial cells, we established a novel co-culture system of Caco-2 cells and enteric neurons differentiated from neural crest stem (NCS)-like cells isolated from mouse longitudinal muscle/myenteric plexus (LMMP). Immunostaining analysis revealed that the proportions of neuron, glia, and NCS-like cells were only <5 % at population in the primary culture of LMMP cells. Therefore, we proliferated NCS-like cells and differentiated them into neuronal cells and successfully increased the neuronal cell population upto about 40 %. Then, the differentiated neuronal cells were co-cultured with Caco-2 cell monolayers, and we found that the co-culture significantly decreased the transepithelial electrical resistance and enhanced the transport of fluorescein isothiocyanate-labeled dextran-4 across Caco-2 cell monolayers, suggesting that the enteric neurons would function to open the tight junction and facilitate the drug transport via the paracellular route. On the other hand, no changes in the permeability of antipyrine were observed, suggesting that the enteric neurons would not affect the passive transport via the transcellular pathway.