George Wahba1, Anne-Emilie Hebert2, David Grynspan3, William Staines1, Sarah Schock4. 1. University of Ottawa, Department of Cellular and Molecular Medicine, Ottawa, ON, Canada. 2. Children's Hospital of Eastern Ontario, Research Institute, Ottawa, ON, Canada. 3. Children's Hospital of Eastern Ontario, Department of Pathology, Ottawa, ON, Canada. 4. Children's Hospital of Eastern Ontario, Research Institute, Ottawa, ON, Canada. Electronic address: sschock@uottawa.ca.
Abstract
BACKGROUND: The 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 METHOD: Here 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. RESULTS: This 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 METHODS: Unlike 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. CONCLUSION: We 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.
BACKGROUND: The 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 METHOD: Here 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. RESULTS: This 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 METHODS: Unlike 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. CONCLUSION: We 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.
Authors: Simone L Schonkeren; Tara T Küthe; Musa Idris; Ana C Bon-Frauches; Werend Boesmans; Veerle Melotte Journal: Neurogastroenterol Motil Date: 2021-07-08 Impact factor: 3.960