The Usefulness of Salivary Gland Organoids for Evaluation of the Potency of Botulinum Neurotoxin

Background and ObjectivesBotulinum neurotoxin (BoNT) is a substance used to treat chronic sialorrhea, muscle dystonia, and is used in cosmetic applications. Measuring the potency of BoNT is crucial because it acts even with a small amount. However, the current methods for measuring the potency of BoNT involve using two-dimensional neuroblastoma cell line-based methods. In this study, we aimed to develop a new method to measure the potency of BoNT using a three-dimensional organoid culture system.Materials and MethodWe established the optimal conditions for coculturing N2a neuronal cells with murine salivary gland organoids (SGOs). After determining the appropriate chemical concentrations, we treated the SGOs cocultured with N2a cells with BoNT type A (BoNT/A). We confirmed the expression of salivary gland-related genes and proteins using real-time polymerase chain reaction (PCR) and immunofluorescence staining.ResultsThe SGOs cocultured with N2a cells showed that the dendrites or axons of neuronal cells were in contact with the outermost layer of the SGOs. When we applied acetylcholine and neostigmine to the coculture systems, the mRNA expression of Aqp5 and Bhlha15, associated with salivary gland secretory cells, increased. However, this effect was reversed when BoNT/A was applied, as confirmed through real-time PCR.ConclusionWe found that the coculture system of SGOs and N2a neuronal cells can potentially serve as a potency testing platform for BoNT.Level of EvidenceNA Laryngoscope, 2024 This study introduces a salivary gland organoid-N2a coculture system enabling to evaluate the potency of botulinum neurotoxin type A. image