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

Abstract

Background and Objectives:Botulinum 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, thecurrent methods for measuring the potency of BoNT involve using two-dimensional neuroblastoma cell line-based methods. In thisstudy, we aimed to develop a new method to measure the potency of BoNT using a three-dimensional organoid culture system.

Materials and Method:We established the optimal conditions for coculturing N2a neuronal cells with murine salivarygland organoids (SGOs). After determining the appropriate chemical concentrations, we treated the SGOs cocultured with N2acells with BoNT type A (BoNT/A). We confirmed the expression of salivary gland-related genes and proteins using real-timepolymerase chain reaction (PCR) and immunofluorescence staining.

Results:The SGOs cocultured with N2a cells showed that the dendrites or axons of neuronal cells were in contact withthe outermost layer of the SGOs. When we applied acetylcholine and neostigmine to the coculture systems, the mRNA expres-sion ofAqp5andBhlha15, associated with salivary gland secretory cells, increased. However, this effect was reversed whenBoNT/A was applied, as confirmed through real-time PCR.

Conclusion:We found that the coculture system of SGOs and N2a neuronal cells can potentially serve as a potency test-ing platform for BoNT