A rapid and efficient method for dissociated cultures of mouse myenteric neurons

⿢Myenteric neurons can be isolated from multiple mice in under 2 h.⿢A high yield of neurons and few non-neuronal cell types are present.⿢Many neuronal subtypes are present, reflecting the in vivo population.⿢Neurons are optimal at 3 weeks but can survive for at least 5 weeks.

BackgroundThe enteric nervous system controls gastrointestinal functions such as secretion and smooth muscle contraction/relaxation. Neuronal enteric dysfunction is a feature of many direct gastrointestinal disorders and can be secondary to central nervous system disorders. Research in this field has been limited and there are few published methods on dissociated enteric cultures.New methodHere we describe a quick and efficient method for culturing myenteric neurons which optimizes neuronal yield. A simplified technique is presented to easily dissect the myenteric plexus and longitudinal muscle from the outside of the intestinal wall reducing non-neuronal cell and bacterial contamination from the final culture. These segments are subjected to enzymatic dissociation and the resulting neurons are placed into an optimal growth media for long term culture.ResultsThis protocol produces a high yield of neuronal cells. Multiple neuronal subtypes reflecting the in vivo population are observed. Cultures are optimal at 3 weeks in vitro but can be sustained for at least 5 weeks.Comparison with existing methodsUnlike other protocols our method does not require a time consuming challenging dissection, long enzymatic treatment times or the use of specialized equipment. Resulting cultures are of higher quality and can be sustained longer permitting proper neuronal recovery. In addition cell attachment to culture substrates have been optimized.ConclusionWe provide a novel method for researchers to dissociate and grow high quality enteric neuronal cultures. Our method can be used for studies on gastrointestinal diseases caused by enteric neuronal dysfunction and to explore possible pharmacological interventions in vitro.