志贺毒素 (Stx) 由某些致病性大肠杆菌菌株产生。为了研究 Stx 对人类肠道的影响，我们利用了人类肠道类器官和在 Transwell 中生长为人类肠道单层 (HIEM) 的人类肠道类器官。为了建立最佳的实验条件，HIEM 在基底外侧孔中添加或不添加间充质细胞的情况下生长，以重现肠上皮和下面的间充质之间的相互作用。单层屏障完整性通过跨上皮电阻（TEER）读数来确定。由于生长培养基导致 TEER 发育不均匀，因此在顶端表面使用顶端盐水。用于上皮细胞的培养基含有添加的生长因子，而间充质培养基则缺乏这些生长因子。我们已经证明间充质细胞可以在缺乏生长因子的培养基中维持上皮单层，这表明它们产生这些因子。此外，间充质细胞产生的生长因子需要随着时间的推移在培养基中积累，因为每日更换培养基不如每 3 天更换一次培养基有效。我们还表明，添加生长因子对间充质细胞有毒。上皮细胞比间充质细胞对 Stx2 具有更强的抵抗力，并且间充质细胞导致上皮细胞死亡。上皮细胞对管腔暴露的耐受性优于基底外侧暴露。这些研究证明了在使用体外和体外模型时了解疾病状态下组织相互作用的重要性。这些研究巩固了在研究宿主-病原体相互作用时对复杂细胞培养模型的需求。小鼠等常见动物模型对大肠杆菌 O157:H7 感染和 Stx2 的肠道传递具有抵抗力，而人类似乎对两者都敏感。有人提出，在人类中，产生志贺毒素的大肠杆菌介导的肠道损伤会破坏肠道屏障，并允许基底外侧进入 Stx2。在小鼠中，没有上皮损伤；因此，它们对 Stx2 的上皮传递具有抵抗力，同时对 Stx2 注射保持敏感。我们的研究表明，与小鼠一样，人类上皮层对Stx2具有相当的抵抗力，正是间充质细胞的敏感性杀死了上皮细胞。我们已经证明 Stx2 通过完整的上皮转运，而不会对抵抗性上皮层造成损害。因此，了解感染期间组织的相互作用对于确定病原体对人体组织的影响至关重要。

Shiga toxin (Stx) is produced by some pathogenic strains of Escherichia coli. To study the impact of Stx on the human intestine, we utilized human intestinal organoids and human intestinal enteroids grown as human intestinal enteroid monolayers (HIEMs) in transwells. To establish optimal experimental conditions, HIEMs were grown with or without mesenchymal cells added to the basolateral wells to recapitulate the interactions between the intestinal epithelium and the underlying mesenchyme. Monolayer barrier integrity was determined through transepithelial electrical resistance (TEER) readings. Apical saline was used on the apical surface since growth medium caused uneven development of the TEER. The medium used for epithelial cells contains added growth factors, while the mesenchymal medium lacks these growth factors. We have shown that mesenchymal cells can maintain the epithelial monolayer in the medium lacking growth factors, suggesting they produce these factors. Furthermore, growth factors produced by mesenchymal cells need to build up in the medium over time, since daily medium changes were not as effective as medium changes performed every 3 days. We have also shown that addition of growth factors is toxic to mesenchymal cells. Epithelial cells were more resistant to Stx2 than the mesenchymal cells, and mesenchymal cells contributed to epithelial cell death. Epithelial cells tolerated luminal exposure better than basolateral exposure. These studies demonstrate the importance of understanding tissue interactions in a disease state when using in vitro and in vitro models.These studies have cemented the need for complex cell culture models when studying host-pathogen interactions. Common animal models such as mice are resistant to E. coli O157:H7 infections and intestinal delivery of Stx2, while humans appear to be sensitive to both. It has been proposed that in humans, shiga toxin-producing E. coli-mediated intestinal damage destroys the intestinal barrier and allows basolateral access to Stx2. In mice, there is no epithelial damage; therefore, they are resistant to epithelial delivery of Stx2 while remaining sensitive to Stx2 injection. Our studies show that like mice, the human epithelial layer is quite resistant to Stx2, and it is the sensitivity of the mesenchymal cells that kills the epithelial cells. We have shown that Stx2 is transported through the intact epithelium without causing damage to the resistant epithelial layer. Understanding tissue interactions during infections is therefore critical in determining the effects of pathogens on human tissues.