title: Best practice for passaging murine embryonic enteric neuronal cell line before differentiation.
abstract: The enteric nervous system (ENS) is a complex network of neurons in the gut, regulating many local, vital functions of the gastro-intestinal tract. The ENS is also part of the bidirectional gut-brain axis. The murine immorto fetal enteric neuronal (IM-FEN) cell line was chosen as a model to study enteric neurons. This cell line can be differentiated into cells with a neuronal phenotype, although they do not produce action potentials in vitro. It was concluded that the differentiation process in our laboratory was successful, based on positive staining for neuronal proteins. Proliferating IM-FEN cells have an unstable growth rate in our laboratory. An indicator of growth rate was calculated, and this indicator was found to be related to seeding density and number of days in culture, and was unrelated to person culturing, previous overconfluency or passage number. The indicator of growth rate was also unrelated to successful use of differentiated cells in follow-up experiments. We recommend the following conditions for optimal culture of IM-FEN cells. Keep cells in culture until 80 % confluent before passaging, seed cells at a density of 0.0133 million cells per cm2, and anticipate on unstable growth rates and the risk for overconfluency.
authors.affiliation: Division of Pharmacology, Faculty of Science, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584 CG, Utrecht, Netherlands.
authors.forename: Carmen D
authors.initials: CD
authors.lastname: Rietdijk
authors.affiliation: Division of Pharmacoepidemiology and Clinical Pharmacology, Faculty of Science, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584 CG, Utrecht, Netherlands.
authors.forename: Lydia
authors.initials: L
authors.lastname: de Haan
authors.affiliation: Department of Biomedical Signals and Systems, MIRA, University of Twente, Drienerlolaan 5, 7522 NB, Enschede, Netherlands.|Department of Biophysics, Donders Institute of Brain, Cognition, and Behaviour, Radboud University Nijmegen, 6525 EZ, Nijmegen, Netherlands.
authors.forename: Richard J A
authors.initials: RJ
authors.lastname: van Wezel
authors.affiliation: Division of Pharmacology, Faculty of Science, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584 CG, Utrecht, Netherlands.|Nutricia Research, Utrecht Science Park, Uppsalalaan 12, 3584 CT, Utrecht, Netherlands.
authors.forename: Johan
authors.initials: J
authors.lastname: Garssen
authors.affiliation: Division of Pharmacology, Faculty of Science, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584 CG, Utrecht, Netherlands. A.D.Kraneveld@uu.nl.
authors.forename: Aletta D
authors.initials: AD
authors.lastname: Kraneveld
keywords: Best practice; Enteric nervous system; Growth rate; IM-FEN; Murine immorto fetal enteric neuronal cell line; Neurons; Seeding density