The Development of Ovine Gastric and Intestinal Organoids for Studying Ruminant Host-Pathogen Interactions.
The Development of Ovine Gastric and Intestinal Organoids for Studying Ruminant Host-Pathogen Interactions.
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DOI：
10.3389/fcimb.2021.733811
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发表时间：
2021
影响因子：
5.7
通讯作者：
McNeilly TN
中科院分区：
文献类型：
--
作者：
Smith D;Price DRG;Burrells A;Faber MN;Hildersley KA;Chintoan-Uta C;Chapuis AF;Stevens M;Stevenson K;Burgess STG;Innes EA;Nisbet AJ;McNeilly TN
羊的胃肠道（GI）感染对动物健康、福利和生产力具有重要意义，也是人畜共患病病原体的来源。病原体和粘膜表面上皮细胞之间的相互作用在确定胃肠道感染的结果中起着关键作用;然而，体内胃肠道的不可及性显著限制了详细研究这种相互作用的能力。因此，我们开发了绵羊上皮类器官，代表生理上重要的胃和肠感染部位，特别是皱胃（类似于单胃中的胃）和回肠。我们发现，皱胃和回肠类器官形成自组织的三维结构，具有单一的上皮层和中央管腔，在连续传代的培养中是稳定的。我们对来自多个动物的皱胃和回肠组织以及跨多个传代的类器官进行了RNA-seq分析，并显示在相同条件下培养的皱胃和回肠类器官的转录谱反映了它们所来源的组织，并且类器官中的转录谱在至少五个连续传代中是稳定的。此外，我们证明了类器官可以成功地冷冻保存和复苏，允许长期储存类器官系，从而减少了作为组织来源所需的动物数量。我们还报告了第一次发表的观察蠕虫感染胃和肠类器官的挑战与绵羊寄生线虫Teladorsagia circumcincta，证明这些类器官的病原体共培养实验的效用。最后，皱胃和回肠类器官中的极性可以反转，以使顶端表面直接接近病原体或其产物，这里通过用人畜共患肠道细菌病原体肠道沙门氏菌鼠伤寒血清型感染顶端外类器官来显示。总之，我们报告了一个简单可靠的体外培养系统，用于小反刍动物肠和胃类器官的产生和维持。根据3Rs原则，使用此类类器官将减少并取代宿主-病原体研究中的动物。
Gastrointestinal (GI) infections in sheep have significant implications for animal health, welfare and productivity, as well as being a source of zoonotic pathogens. Interactions between pathogens and epithelial cells at the mucosal surface play a key role in determining the outcome of GI infections; however, the inaccessibility of the GI tract in vivo significantly limits the ability to study such interactions in detail. We therefore developed ovine epithelial organoids representing physiologically important gastric and intestinal sites of infection, specifically the abomasum (analogous to the stomach in monogastrics) and ileum. We show that both abomasal and ileal organoids form self-organising three-dimensional structures with a single epithelial layer and a central lumen that are stable in culture over serial passage. We performed RNA-seq analysis on abomasal and ileal tissue from multiple animals and on organoids across multiple passages and show the transcript profile of both abomasal and ileal organoids cultured under identical conditions are reflective of the tissue from which they were derived and that the transcript profile in organoids is stable over at least five serial passages. In addition, we demonstrate that the organoids can be successfully cryopreserved and resuscitated, allowing long-term storage of organoid lines, thereby reducing the number of animals required as a source of tissue. We also report the first published observations of a helminth infecting gastric and intestinal organoids by challenge with the sheep parasitic nematode Teladorsagia circumcincta, demonstrating the utility of these organoids for pathogen co-culture experiments. Finally, the polarity in the abomasal and ileal organoids can be inverted to make the apical surface directly accessible to pathogens or their products, here shown by infection of apical-out organoids with the zoonotic enteric bacterial pathogen Salmonella enterica serovar Typhimurium. In summary, we report a simple and reliable in vitro culture system for generation and maintenance of small ruminant intestinal and gastric organoids. In line with 3Rs principals, use of such organoids will reduce and replace animals in host-pathogen research.