Department of Biomedical Engineering, Tufts University, Medford, Massachusetts.
Curr Protoc. 2024 Apr;4(4):e1027. doi: 10.1002/cpz1.1027.
The development of patient-derived intestinal organoids represents an invaluable model for simulating the native human intestinal epithelium. These stem cell-rich cultures outperform commonly used cell lines like Caco-2 and HT29-MTX in reflecting the cellular diversity of the native intestinal epithelium after differentiation. In our recent study examining the effects of polystyrene (PS), microplastics (MPs), and nanoplastics (NPs), widespread pollutants in our environment and food chain, on the human intestinal epithelium, these organoids have been instrumental in elucidating the absorption mechanisms and potential biological impacts of plastic particles. Building on previously established protocols in human intestinal organoid culture, we herein detail a streamlined protocol for the cultivation, differentiation, and generation of organoid-derived monolayers. This protocol is tailored to generate monolayers incorporating microfold cells (M cells), key for intestinal particle uptake but often absent in current in vitro models. We provide validated protocols for the characterization of MPs/NPs via scanning electron microscopy (SEM) for detailed imaging and their introduction to intestinal epithelial monolayer cells via confocal immunostaining. Additionally, protocols to test the impacts of MP/NP exposure on the functions of the intestinal barrier using transendothelial electrical resistance (TEER) measurements and assessing inflammatory responses using cytokine profiling are detailed. Overall, our protocols enable the generation of human intestinal organoid monolayers, complete with the option of including or excluding M cells, offering crucial techniques for observing particle uptake and identifying inflammatory responses in intestinal epithelial cells to advance our knowledge of the potential effects of plastic pollution on human gut health. These approaches are also amendable to the study of other gut-related chemical and biological exposures and physiological responses due to the robust nature of the systems. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Human intestinal organoid culture and generation of monolayers with and without M cells Support Protocol 1: Culture of L-WRN and production of WRN-conditioned medium Support Protocol 2: Neuronal cell culture and integration into intestinal epithelium Support Protocol 3: Immune cell culture and integration into intestinal epithelium Basic Protocol 2: Scanning electron microscopy: sample preparation and imaging Basic Protocol 3: Immunostaining and confocal imaging of MP/NP uptake in organoid-derived monolayers Basic Protocol 4: Assessment of intestinal barrier function via TEER measurements Basic Protocol 5: Cytokine profiling using ELISA post-MP/NP exposure.
肠道类器官的发展代表了模拟天然人肠道上皮的宝贵模型。与通常使用的 Caco-2 和 HT29-MTX 细胞系相比，这些富含干细胞的培养物在分化后更能反映天然肠道上皮细胞的细胞多样性。在我们最近研究聚苯乙烯（PS）、微塑料（MPs）和纳米塑料（NPs）这些广泛存在于我们的环境和食物链中的污染物对人肠道上皮的影响时，这些类器官在阐明塑料颗粒的吸收机制和潜在生物学影响方面发挥了重要作用。基于先前建立的人肠道类器官培养方案，我们在此详细介绍了一种简化的方案，用于培养、分化和生成类器官衍生的单层。该方案旨在生成包含微褶皱细胞（M 细胞）的单层，这些细胞对于肠道颗粒的摄取至关重要，但目前的体外模型中通常不存在。我们提供了经过验证的方案，通过扫描电子显微镜（SEM）对 MPs/NPs 进行特征分析，并通过共聚焦免疫染色将其引入肠道上皮单层细胞。此外，还详细介绍了使用跨内皮电阻（TEER）测量来测试 MPs/NP 暴露对肠道屏障功能的影响，并通过细胞因子谱分析来评估炎症反应的方案。总之，我们的方案能够生成具有或不具有 M 细胞的人肠道类器官单层，提供了观察颗粒摄取和识别肠道上皮细胞炎症反应的关键技术，以推进我们对塑料污染对人类肠道健康潜在影响的认识。这些方法还适用于研究其他与肠道相关的化学和生物暴露以及生理反应，因为这些系统具有很强的稳健性。
