Abstract：
OBJECTIVE: 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, 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.
METHODS: We 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.
RESULTS: The 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.
CONCLUSIONS: We found that the coculture system of SGOs and N2a neuronal cells can potentially serve as a potency testing platform for BoNT.
METHODS: NA Laryngoscope, 134:2697-2704, 2024.
METHODS: We 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.
RESULTS: The 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.
CONCLUSIONS: We found that the coculture system of SGOs and N2a neuronal cells can potentially serve as a potency testing platform for BoNT.
METHODS: NA Laryngoscope, 134:2697-2704, 2024.
摘要：
目的：肉毒杆菌神经毒素（BoNT）是一种用于治疗慢性流涎的物质，肌肉张力障碍,并用于化妆品应用。测量BoNT的效力至关重要，因为它即使少量也起作用。然而,目前用于测量BoNT效力的方法涉及使用基于二维神经母细胞瘤细胞系的方法。在这项研究中,我们旨在开发一种使用三维类器官培养系统测量BoNT效力的新方法。
方法：我们建立了将N2a神经元细胞与鼠唾液腺类器官（SGO）共培养的最佳条件。在确定合适的化学浓度后，我们用BoNTA型(BoNT/A)处理与N2a细胞共培养的SGO。我们使用实时聚合酶链反应（PCR）和免疫荧光染色证实了唾液腺相关基因和蛋白质的表达。
结果：与N2a细胞共培养的SGO表明，神经元细胞的树突或轴突与SGO的最外层接触。当我们将乙酰胆碱和新斯的明应用于共培养系统时，与唾液腺分泌细胞相关的Aqp5和Bhlha15的mRNA表达，增加。然而,当应用BoNT/A时，这种效果被逆转，通过实时PCR证实。
结论：我们发现SGO和N2a神经元细胞的共培养系统可以潜在地充当BoNT的效力测试平台。
方法：NA喉镜，2024.
方法：我们建立了将N2a神经元细胞与鼠唾液腺类器官（SGO）共培养的最佳条件。在确定合适的化学浓度后，我们用BoNTA型(BoNT/A)处理与N2a细胞共培养的SGO。我们使用实时聚合酶链反应（PCR）和免疫荧光染色证实了唾液腺相关基因和蛋白质的表达。
结果：与N2a细胞共培养的SGO表明，神经元细胞的树突或轴突与SGO的最外层接触。当我们将乙酰胆碱和新斯的明应用于共培养系统时，与唾液腺分泌细胞相关的Aqp5和Bhlha15的mRNA表达，增加。然而,当应用BoNT/A时，这种效果被逆转，通过实时PCR证实。
结论：我们发现SGO和N2a神经元细胞的共培养系统可以潜在地充当BoNT的效力测试平台。
方法：NA喉镜，2024.
