Primary culture of the enteric nervous system from neonatal hamster intestine: Selection of vasoactive intestinal polypeptide-containing neurons

Abstract The enteric nervous system is a major division of the autonomic nervous system and is responsible for the regulation of gastrointestinal function. The objective of the present study was to develop a simple and effective technique for isolating and culturing neurons of the enteric nervous system that would permit characterization of their development and regulatory peptide content. This was accomplished using a dispersed intestinal cell preparation cultured under conditions designed to support the growth and differentiation of neurons and neuroendocrine cells. Newborn hamster intestine was digested in 0.1% collagenase, mechanically dispersed, and cultured in RPMI 1640 supplemented with 2.5% serum and other additives. Phase and bright-field microscopy demonstrated neuronal cells and fibers after the second day in culture. This was confirmed by immunohistochemistry using antibodies directed against neurofilament and vasoactive intestinal polypeptide. Acetic acid extracts of the culture indicated that during the first 4 days of the culture the content of vasoactive intestinal polypeptide increased, whereas the content of substance P, mammalian bombesin, and neurotensin declined. High-performance liquid chromatography and fast protein liquid chromatography confirmed that the immunoreactive vasoactive intestinal polypeptide coeluted with synthetic and iodinated forms of the peptide. This study describes a technique for primary culture of intestinal tissue that supports the survival of enteric neurons and permits analysis of the development and synthetic and secretory characteristics of the enteric nervous system.