FG 16: Mycotic and Parasitic Agents and Mycobacteria, Robert Koch-Institute, Berlin, Germany.
Front Cell Infect Microbiol. 2021 Feb 22;10:610368. doi: 10.3389/fcimb.2020.610368. eCollection 2020.
The small intestinal epithelium is the primary route of infection for many protozoan parasites. Understanding the mechanisms of infection, however, has been hindered due to the lack of appropriate models that recapitulate the complexity of the intestinal epithelium. Here, we describe an platform using stem cell-derived intestinal organoids established for four species that are important hosts of Apicomplexa and other protozoa in a zoonotic context: human, mouse, pig and chicken. The focus was set to create organoid-derived monolayers (ODMs) using the transwell system amenable for infection studies, and we provide straightforward guidelines for their generation and differentiation from organ-derived intestinal crypts. To this end, we reduced medium variations to an absolute minimum, allowing generation and differentiation of three-dimensional organoids for all four species and the subsequent generation of ODMs. Quantitative RT-PCR, immunolabeling with antibodies against marker proteins as well as transepithelial-electrical resistance (TEER) measurements were used to characterize ODM's integrity and functional state. These experiments show an overall uniform generation of monolayers suitable for infection, although robustness in terms of generation of stable TEER levels and cell differentiation status varies from species to species. Murine duodenal ODMs were then infected with and/or , two parasites that temporarily co-inhabit the intestinal niche but have not been studied previously in cellular co-infection models. alone did not alter TEER values, integrity and transcriptional abundance of tight junction components. In contrast, in -infected ODMs all these parameters were altered and had no apparent influence on the -triggered phenotype. In conclusion, we provide robust protocols for the generation, differentiation and characterization of intestinal organoids and ODMs from four species. We show their applications for comparative studies on parasite-host interactions during the early phase of a infection but also its use for co-infections with other relevant intestinal protozoans.
小肠上皮是许多原生动物寄生虫感染的主要途径。然而，由于缺乏能够重现肠道上皮复杂性的合适模型，对于感染机制的理解一直受到阻碍。在这里，我们描述了一个使用源自干细胞的肠类器官建立的平台，该平台适用于四种在人畜共患背景下是重要的顶复门寄生虫和其他原生动物宿主的物种：人、鼠、猪和鸡。我们的重点是使用 Transwell 系统创建类器官衍生的单层（ODM），用于感染研究，并为它们从肠隐窝衍生的器官中生成和分化提供了简单的指南。为此，我们将培养基的变化降至最低，允许所有四种物种生成和分化三维类器官，并随后生成 ODM。定量 RT-PCR、针对标记蛋白的抗体免疫标记以及跨上皮电阻（TEER）测量用于表征 ODM 的完整性和功能状态。这些实验表明，尽管在生成稳定的 TEER 水平和细胞分化状态方面的稳健性因物种而异，但总体上生成适合感染的单层是一致的。然后用 和/或 感染鼠十二指肠 ODM，这两种寄生虫暂时共同栖息在肠道生态位中，但以前在细胞共感染模型中没有研究过。单独感染 不会改变 TEER 值、紧密连接成分的完整性和转录丰度。相比之下，在感染的 ODM 中，所有这些参数都发生了改变，而 对 -触发的表型没有明显影响。总之，我们提供了从四个物种生成、分化和表征肠类器官和 ODM 的稳健方案。我们展示了它们在 感染早期阶段进行寄生虫-宿主相互作用的比较研究中的应用，但也展示了它们在与其他相关肠道原生动物的共感染中的应用。
