Department of Clinical Laboratory, Osaka University Dental Hospital, Suita, Osaka, Japan.
Department of Clinical Laboratory and Biomedical Sciences, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Curr Protoc. 2022 Sep;2(9):e543. doi: 10.1002/cpz1.543.
The salivary glands produce saliva and are important in maintaining oral health. Saliva keeps the mouth moist, cleanses the oral cavity, aids digestion, and has antibacterial properties. Saliva also helps in swallowing and speech. Investigating the development of the salivary glands is thus relevant in the context of both health and disease. Various cell culture methods have been used to study salivary gland development, including culturing cells in two dimensions (2D). Under physiological conditions, cells constantly interact with other cells and the extracellular matrix, which controls complex biological functions such as cell migration and apoptosis, and can modulate gene expression. Since many of these functions are not accurately represented or reproduced in 2D culture, the results of in vitro experiments using such culture methods are often not reflected in vivo. The use of 3D cultures, such as organ cultures, has helped address this issue and has emerged as a model that better reflects the in vivo physiological environment. Here, we describe a protocol for establishing submandibular salivary gland organ culture that is more concise and simpler than previous methods and includes the separation and dissection of the salivary glands. We also describe the use of environmental stress (hypoxic stimulation) and inhibitors (U0126, LY294002, and rapamycin) to elucidate signaling pathways involved in salivary gland development. This protocol can provide researchers with a simpler and more robust method of salivary gland organ culture, enabling analysis of organ-based signaling pathways to advance developmental biology research. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Submandibular salivary gland organ culture Basic Protocol 2: Analysis of salivary gland development in the presence of hypoxia and signaling pathway inhibitors Basic Protocol 3: Western blotting using submandibular salivary gland organ culture.
唾液腺产生唾液，对于维护口腔健康非常重要。唾液保持口腔湿润，清洁口腔，有助于消化，并具有抗菌作用。唾液还有助于吞咽和言语。因此，研究唾液腺的发育对于健康和疾病都具有重要意义。已经使用了各种细胞培养方法来研究唾液腺的发育，包括在二维（2D）环境中培养细胞。在生理条件下，细胞不断与其他细胞和细胞外基质相互作用，细胞外基质控制着细胞迁移和凋亡等复杂的生物学功能，并能调节基因表达。由于这些功能在 2D 培养中不能准确地表现或重现，因此使用这种培养方法进行的体外实验结果往往不能在体内得到反映。3D 培养（如器官培养）的使用有助于解决这个问题，并且已经成为一种更好地反映体内生理环境的模型。在这里，我们描述了一种更简洁、更简单的下颌下腺器官培养方法，该方法包括唾液腺的分离和解剖。我们还描述了使用环境应激（缺氧刺激）和抑制剂（U0126、LY294002 和雷帕霉素）来阐明参与唾液腺发育的信号通路。本方案可为研究人员提供一种更简单、更稳健的唾液腺器官培养方法，使基于器官的信号通路分析能够推进发育生物学研究。© 2022 威立出版社
