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Spinal cord – skeletal muscle cocultures detect muscle-relaxant action of botulinum neurotoxin A

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Veit-Simon Eckle1, Berthold Drexler1, Christian Grasshoff1, Thomas Seeger2, Horst Thiermann2 and Bernd Antkowiak1
1 Department of Anesthesiology and Intensive Care Medicine, Experimental Anesthesiology Section, Eberhard-Karls University Tübingen, Germany;
2 Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany


The mouse LD50 assay is routinely used for potency testing of botulinum toxins. Unfortunately, this test is associated with severe pain and distress in animals and consumes large numbers of mice. Here we used cocultures of spinal cord and muscle tissue to assess botulinum toxin potency. Cocultures were prepared from mouse embryonic tissue (C57/BL6J) and cultured for 24-27 days. Spontaneous muscle activity was quantified in sham- and botulinum toxin-treated cultures for up to 3 days by video microscopy. At a concentration of 58 fmol/l or higher, incobotulinumtoxin A significantly reduced the frequency of muscle contractions within 24 h after exposure. Hence, the sensitivity of nerve-muscle cultures is comparable to that of the mouse LD50 assay. The limit of detection observed in this study is close to that of the most sensitive cell-based bioassays, capable of detecting concentrations of botulinum neurotoxin A of between 30 and 50 fmol/l. However, spontaneous muscle activity of individual cultures displayed considerable fluctuations when evaluated on a day-to-day basis and, in its present form, this in vitro assay might be too laborious for botulinum toxin potency testing. Thus, methodical improvements to decrease data variability and increase throughput are the next milestones towards developing the model into an assay that can be utilized to reduce animal experimentation.


Keywords: organotypic culture, botulinum toxin A, incobotulinumtoxin A, LD50 assay, nerve-muscle culture



ALTEX (31)4: 433-440


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