Reaggregation of rat dissociated myenteric plexus in extracellular matrix gels

The aim of this study was to investigate the growth behavior of freshly dis sociated myenteric plexus in a three-dimensional extracellular matrix (ECM) environment with and without stimulation of glial cell line-derived neurot rophic factor (GDNF). Therefore, cell suspensions of the dissected myenteri c plexus of newborn rats were cultured in freshly prepared gels of commerci ally 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 polylysine-coated glass cover slips se rved as controls. Dissociated myenteric plexus grown on polylysine formed d ense clusters of neurons with radially outgrowing nerve fibers, while the n eurons cultured in the gel reaggregated to much smaller clusters. These con tained, depending on the culture conditions, 2-10 neurons. The morphology o f 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 myenter ic plexus in hypoganglionic children. Electron microscope investigations sh owed a high degree of organization with fiber bundles and vesicle-containin g varicosities and growth cones. Independent of the method of culturing, GD NF 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 de velopment, especially considering pathological conditions such as Hirschspr ung's disease or other dysganglionic diseases.