Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), University Medical Center Utrecht; Oncode Institute, Hubrecht Institute.
Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), University Medical Center Utrecht.
J Vis Exp. 2023 Feb 3(192). doi: 10.3791/65040.
The lacrimal gland is an essential organ for ocular surface homeostasis. By producing the aqueous part of the tear film, it protects the eye from desiccation stress and external insults. Little is known about lacrimal gland (patho)physiology because of the lack of adequate in vitro models. Organoid technology has proven itself as a useful experimental platform for multiple organs. Here, we share a protocol to establish and maintain mouse and human lacrimal gland organoids starting from lacrimal gland biopsies. By modifying the culture conditions, we enhance lacrimal gland organoid functionality. Organoid functionality can be probed through a "crying" assay, which involves exposing the lacrimal gland organoids to selected neurotransmitters to trigger tear release in their lumen. We explain how to image and quantify this phenomenon. To investigate the role of genes of interest in lacrimal gland homeostasis, these can be genetically modified. We thoroughly describe how to genetically modify lacrimal gland organoids using base editors-from guide RNA design to organoid clone genotyping. Lastly, we show how to probe the regenerative potential of human lacrimal gland organoids by orthotopic implantation in the mouse. Together, this comprehensive toolset provides resources to use mouse and human lacrimal gland organoids to study lacrimal gland (patho)physiology.
泪腺是眼表面稳态的重要器官。它通过产生泪膜的水液部分，保护眼睛免受干燥应激和外部刺激。由于缺乏足够的体外模型，人们对泪腺（病理）生理学知之甚少。类器官技术已被证明是一种用于多种器官的有用的实验平台。在这里，我们分享了一个从泪腺活检开始建立和维持小鼠和人泪腺类器官的方案。通过修改培养条件，我们增强了泪腺类器官的功能。可以通过“哭泣”测定法来探测类器官的功能，该测定法涉及将泪腺类器官暴露于选定的神经递质中，以触发其管腔中的泪液释放。我们解释了如何对这种现象进行成像和定量。为了研究感兴趣的基因在泪腺稳态中的作用，可以对其进行基因修饰。我们详细描述了如何使用碱基编辑器对泪腺类器官进行基因修饰——从向导 RNA 设计到类器官克隆的基因分型。最后，我们展示了如何通过在小鼠中进行原位移植来探测人泪腺类器官的再生潜力。总之，这个全面的工具集为使用小鼠和人泪腺类器官研究泪腺（病理）生理学提供了资源。
