3D in vitro morphogenesis of human intestinal epithelium in a gut-on-a-chip or a hybrid chip with a cell culture insert.
3D in vitro morphogenesis of human intestinal epithelium in a gut-on-a-chip or a hybrid chip with a cell culture insert.
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DOI：
10.1038/s41596-021-00674-3
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发表时间：
2022-03
期刊：
影响因子：
14.8
通讯作者：
Kim, Hyun Jung
中科院分区：
文献类型：
--
作者：
Shin, Woojung;Kim, Hyun Jung
人类肠道形态发生建立了3D上皮微结构和空间组织的隐窝绒毛特征。这种独特的结构是必要的，以维持肠道内稳态，保护干细胞龛在基底隐窝从外源性微生物抗原及其代谢产物。此外，肠绒毛和分泌粘液呈现功能分化的上皮细胞，在肠粘膜表面具有保护屏障。因此，重建3D上皮结构对于构建体外肠模型至关重要。值得注意的是，器官模拟肠芯片可以诱导肠上皮的自发3D形态发生，具有增强的生理功能和生物力学。在这里，我们提供了一个可重复的协议，在微流控肠芯片以及在Transwell嵌入式混合芯片中稳健地诱导肠道形态发生。我们描述了设备制造的详细方法，Caco-2或肠类器官上皮细胞在传统的设置以及微流控平台上的培养，诱导3D形态发生和使用多种成像模式建立的3D上皮的表征。该方案通过在5 d内控制基底外侧流体流动使功能性肠微结构再生成为可能。我们的体外形态发生方法采用生理学相关的剪切应力和机械运动，并且不需要复杂的细胞工程或操作，这可能优于其他现有技术。我们预计我们提出的方案可能会对生物医学研究界产生广泛影响，为生物医学、临床和制药应用提供一种体外3D肠上皮层再生的方法。
Human intestinal morphogenesis establishes 3D epithelial microarchitecture and spatially organized crypt–villus characteristics. This unique structure is necessary to maintain intestinal homeostasis by protecting the stem cell niche in the basal crypt from exogenous microbial antigens and their metabolites. Also, intestinal villi and secretory mucus present functionally differentiated epithelial cells with a protective barrier at the intestinal mucosal surface. Thus, re-creating the 3D epithelial structure is critical to building in vitro intestine models. Notably, an organomimetic gut-on-a-chip can induce spontaneous 3D morphogenesis of an intestinal epithelium with enhanced physiological function and biomechanics. Here we provide a reproducible protocol to robustly induce intestinal morphogenesis in a microfluidic gut-on-a-chip as well as in a Transwell-embedded hybrid chip. We describe detailed methods for device fabrication, culture of Caco-2 or intestinal organoid epithelial cells in conventional setups as well as on microfluidic platforms, induction of 3D morphogenesis and characterization of established 3D epithelium using multiple imaging modalities. This protocol enables the regeneration of functional intestinal microarchitecture by controlling basolateral fluid flow within 5 d. Our in vitro morphogenesis method employs physiologically relevant shear stress and mechanical motions, and does not require complex cellular engineering or manipulation, which may be advantageous over other existing techniques. We envision that our proposed protocol may have a broad impact on biomedical research communities, providing a method to regenerate in vitro 3D intestinal epithelial layers for biomedical, clinical and pharmaceutical applications.
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