title: Embryonic Stem Cell-Derived Neurons Grown on Multi-Electrode Arrays as a NovelIn vitroBioassay for the Detection ofClostridium botulinumNeurotoxins.
abstract: Clostridium botulinumneurotoxins (BoNTs) are the most poisonous naturally occurring protein toxins known to mankind and are the causative agents of the severe and potentially life-threatening disease botulism. They are also known for their application as cosmetics and as unique bio-pharmaceuticals to treat an increasing number of neurological and non-neurological disorders. Currently, the potency of biologically active BoNT for therapeutic use is mainly monitored by the murine LD50-assay, an ethically disputable test causing suffering and death of a considerable number of mice. The aim of this study was to establish anin vitroassay as an alternative to the widely usedin vivomouse bioassay. We report a novel BoNT detection assay using mouse embryonic stem cell-derived neurons (mESN) cultured on multi-electrode arrays (MEAs). After 21 days in culture, the mESN formed a neuronal network showing spontaneous bursting activity based on functional synapses and express the necessary target proteins for BoNTs. Treating cultures for 6 h with 16.6 pM of BoNT serotype A and incubation with 1.66 pM BoNT/A or 33 Units/ml of Botox®for 24 h lead to a significant reduction of both spontaneous network bursts and average spike rate. This data suggests that mESN cultured on MEAs pose a novel, biologically relevant model that can be used to detect and quantify functional BoNT effects, thus accelerating BoNT research while decreasing animal use.
authors.affiliation: Neuroinfection Laboratory, Institute for Infectious Diseases, University of BernBern, Switzerland; Biology Division, Spiez Laboratory, Swiss Federal Office for Civil ProtectionSpiez, Switzerland; Cluster for Regenerative Neuroscience, Department for Clinical Research, University of BernBern, Switzerland; Graduate School for Cellular and Biomedical Sciences, University of BernBern, Switzerland.
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Jenkinson
authors.affiliation: Neuroinfection Laboratory, Institute for Infectious Diseases, University of BernBern, Switzerland; Cluster for Regenerative Neuroscience, Department for Clinical Research, University of BernBern, Switzerland.
authors.forename: Denis
authors.initials: D
authors.lastname: Grandgirard
authors.affiliation: Department of Physiology, University of Bern Bern, Switzerland.
authors.forename: Martina
authors.initials: M
authors.lastname: Heidemann
authors.affiliation: Department of Physiology, University of Bern Bern, Switzerland.
authors.forename: Anne
authors.initials: A
authors.lastname: Tscherter
authors.affiliation: Biology Division, Spiez Laboratory, Swiss Federal Office for Civil Protection Spiez, Switzerland.
authors.forename: Marc-André
authors.initials: MA
authors.lastname: Avondet
authors.affiliation: Neuroinfection Laboratory, Institute for Infectious Diseases, University of BernBern, Switzerland; Cluster for Regenerative Neuroscience, Department for Clinical Research, University of BernBern, Switzerland.
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Leib
keywords: BoNT; MEA; botulinum neurotoxins; botulism; embryonic stem cell-derived neurons; in vitro bioassay; multi-electrode array; neuronal network