Studying Cryptosporidium Infection in 3D Tissue-derived Human Organoid Culture Systems by Microinjection
Studying Cryptosporidium Infection in 3D Tissue-derived Human Organoid Culture Systems by Microinjection
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DOI：
10.3791/59610
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发表时间：
2019-09-01
影响因子：
1.2
通讯作者：
O'Connor, Roberta
中科院分区：
文献类型：
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作者：
Dutta, Devanjali;Heo, Inha;O'Connor, Roberta
微小隐孢子虫是引起人类生殖道疾病的主要病原之一。为了了解寄生虫的病理学并开发有效的药物，需要一种能够重现宿主条件的体外培养系统。类器官与其起源组织非常相似，是研究宿主-寄生虫相互作用的理想选择。类器官是三维（3D）组织衍生的结构，其衍生自成体干细胞并且在培养物中生长延长的时间段而不经历任何遗传畸变或转化。它们具有明确的极性，顶侧和基底侧表面都有。类器官在药物测试、生物银行、疾病建模和宿主-微生物相互作用研究中具有各种应用。在这里，我们提出了一个循序渐进的协议，如何准备卵囊和孢子的隐孢子虫感染人类肠道和气道类器官。然后，我们展示了如何显微注射可以用来注入微生物到类器官腔。类器官用于宿主-微生物相互作用研究的方法主要有三种：显微注射法、机械剪切法和平板法，以及制作单分子膜法。显微注射能够维持3D结构，并允许精确控制寄生虫体积和微生物的直接顶侧接触。我们提供了用于成像或卵囊生产的类器官的最佳生长的细节。最后，我们还展示了如何从类器官中分离新生成的卵囊，以进行进一步的下游处理和分析。
Cryptosporidium parvum is one of the major causes of human diarrheal disease. To understand the pathology of the parasite and develop efficient drugs, an in vitro culture system that recapitulates the conditions in the host is needed. Organoids, which closely resemble the tissues of their origin, are ideal for studying host-parasite interactions. Organoids are three-dimensional (3D) tissue-derived structures which are derived from adult stem cells and grow in culture for extended periods of time without undergoing any genetic aberration or transformation. They have well defined polarity with both apical and basolateral surfaces. Organoids have various applications in drug testing, bio banking, and disease modeling and host-microbe interaction studies. Here we present a step-by-step protocol of how to prepare the oocysts and sporozoites of Cryptosporidium for infecting human intestinal and airway organoids. We then demonstrate how microinjection can be used to inject the microbes into the organoid lumen. There are three major methods by which organoids can be used for host-microbe interaction studies-microinjection, mechanical shearing and plating, and by making monolayers. Microinjection enables maintenance of the 3D structure and allows for precise control of parasite volumes and direct apical side contact for the microbes. We provide details for optimal growth of organoids for either imaging or oocyst production. Finally, we also demonstrate how the newly generated oocysts can be isolated from the organoid for further downstream processing and analysis.