Recapitulation of the accessible interface of biopsy-derived canine intestinal organoids to study epithelial-luminal interactions
Recapitulation of the accessible interface of biopsy-derived canine intestinal organoids to study epithelial-luminal interactions
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DOI：
10.1371/journal.pone.0231423
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发表时间：
2020-04-17
期刊：
影响因子：
3.7
通讯作者：
Kim, Hyun Jung
中科院分区：
文献类型：
--
作者：
Ambrosini, Yoko M.;Park, Yejin;Kim, Hyun Jung
犬肠道类器官的最新进展为建立更好的体外模型以研究人与动物之间的肠道生理学和病理学的翻译科学提供了更好的选择。然而，犬肠道器官的三维几何形状和封闭的管腔严重阻碍了进入上皮顶端的途径，以研究营养和药物吸收、宿主-微生物组串扰和药物毒性测试。因此，创建一个从根尖或基底外侧可达的极化上皮界面是至关重要的。在这里，我们演示了使用犬结肠活检衍生的结肠器质(结肠样物)产生肠上皮单分子层。我们优化了培养条件，使用犬结肠样体在纳米孔膜上形成了完整的犬结肠上皮单层，持续14天。透射电子显微镜和扫描电子显微镜显示了一个生理性的刷状边缘界面，被微绒毛覆盖，并有粘蛋白颗粒、紧密连接和桥粒的存在。通过免疫荧光染色和RNA原位杂交验证干细胞和分化的谱系依赖的上皮细胞的数量。P-糖蛋白外排泵在根尖膜上呈极化表达。此外，上皮单层在4天内形成紧密和粘连连接屏障，其中跨上皮电阻值与表观通透性呈负相关。因此，我们验证了犬肠道上皮屏障的稳定建立、维持、分化和生理功能，这对药物和生物医学研究是有用的。
Recent advances in canine intestinal organoids have expanded the option for building a better in vitro model to investigate translational science of intestinal physiology and pathology between humans and animals. However, the three-dimensional geometry and the enclosed lumen of canine intestinal organoids considerably hinder the access to the apical side of epithelium for investigating the nutrient and drug absorption, host-microbiome crosstalk, and pharmaceutical toxicity testing. Thus, the creation of a polarized epithelial interface accessible from apical or basolateral side is critical. Here, we demonstrated the generation of an intestinal epithelial monolayer using canine biopsy-derived colonic organoids (colonoids). We optimized the culture condition to form an intact monolayer of the canine colonic epithelium on a nanoporous membrane insert using the canine colonoids over 14 days. Transmission and scanning electron microscopy revealed a physiological brush border interface covered by the microvilli with glycocalyx, as well as the presence of mucin granules, tight junctions, and desmosomes. The population of stem cells as well as differentiated lineage-dependent epithelial cells were verified by immunofluorescence staining and RNA in situ hybridization. The polarized expression of P-glycoprotein efflux pump was confirmed at the apical membrane. Also, the epithelial monolayer formed tight- and adherence-junctional barrier within 4 days, where the transepithelial electrical resistance and apparent permeability were inversely correlated. Hence, we verified the stable creation, maintenance, differentiation, and physiological function of a canine intestinal epithelial barrier, which can be useful for pharmaceutical and biomedical researches.