Reaggregation of rat dissociated myenteric plexus in extracellular matrix gels

The aim of this study was to investigate the growth behavior of freshly dissociated myenteric plexus in a three-dimensional extracellular matrix (ECM) environment with and without stimulation of glial cell line-derived neurotrophic factor (GDNF). Therefore, cell suspensions of the dissected myenteric plexus of newborn rats were cultured in freshly prepared gels of commercially available mixtures of collagen, laminin, and hepatoglycans as a first step towards mimicking the natural environment of the myenteric plexus. The cultures were kept either in chemically defined serum-free medium alone or supplemented with GDNF. Cultures on polylysinc-coated glass cover slips served as controls. Dissociated myenteric plexus grown on polylysine formed dense clusters of neurons with radially outgrowing nerve fibers, while the neurons cultured in the gel reaggregated to much smaller clusters. These contained, depending on the culture conditions, 2-10 neurons. The morphology of the network that was seen in the gels after a few days in vitro resembled very closely the in situ situation of the submucous plexus and the myenteric plexus in hypoganglionic children. Electron microscope investigations showed a high degree of organization with fiber bundles and vesicle-containing varicosities and growth cones. Independent of the method of culturing, GDNF obviously influenced the growth behavior of the dissociated plexus. The size of the ganglia was larger, and the secondary network denser when GDNF was supplemented. Moreover, the enteric neurons in the gel cultures tended to be larger in size when treated with GDNF. Three-dimensional cultures of dissociated myenteric plexus in an ECM gel might be a valuable tool towards the understanding of the formation of the enteric nervous system during development, especially considering pathological conditions such as Hirschsprung's disease or other dysganglionic diseases.