PDF(Pubmed)
Abstract：
The luminal surface of the intestinal epithelium is protected by a vital mucus layer, which is essential for lubrication, hydration, and fostering symbiotic bacterial relationships. Replicating and studying this complex mucus structure in vitro presents considerable challenges. To address this, we developed a hydrogel-integrated millifluidic tissue chamber capable of applying precise apical shear stress to intestinal models cultured on flat or 3D structured hydrogel scaffolds with adjustable stiffness. The chamber is designed to accommodate nine hydrogel scaffolds, 3D-printed as flat disks with a storage modulus matching the physiological range of intestinal tissue stiffness (~3.7 kPa) from bioactive decellularized and methacrylated small intestinal submucosa (dSIS-MA). Computational fluid dynamics simulations were conducted to confirm a laminar flow profile for both flat and 3D villi-comprising scaffolds in the physiologically relevant regime. The system was initially validated with HT29-MTX seeded hydrogel scaffolds, demonstrating accelerated differentiation, increased mucus production, and enhanced 3D organization under shear stress. These characteristic intestinal tissue features are essential for advanced in vitro models as they critically contribute to a functional barrier. Subsequently, the chamber was challenged with human intestinal stem cells (ISCs) from the terminal ileum. Our findings indicate that biomimicking hydrogel scaffolds, in combination with physiological shear stress, promote multi-lineage differentiation, as evidenced by a gene and protein expression analysis of basic markers and the 3D structural organization of ISCs in the absence of chemical differentiation triggers. The quantitative analysis of the alkaline phosphatase (ALP) activity and secreted mucus demonstrates the functional differentiation of the cells into enterocyte and goblet cell lineages. The millifluidic system, which has been developed and optimized for performance and cost efficiency, enables the creation and modulation of advanced intestinal models under biomimicking conditions, including tunable matrix stiffness and varying fluid shear stresses. Moreover, the readily accessible and scalable mucus-producing cellular tissue models permit comprehensive mucus analysis and the investigation of pathogen interactions and penetration, thereby offering the potential to advance our understanding of intestinal mucus in health and disease.
摘要：
肠上皮的腔表面受到重要粘液层的保护，这对润滑至关重要，水合作用,促进共生细菌关系。在体外复制和研究这种复杂的粘液结构提出了相当大的挑战。为了解决这个问题，我们开发了一种水凝胶集成的微流体组织室，能够将精确的根尖剪切应力施加到在具有可调节刚度的扁平或3D结构化水凝胶支架上培养的肠模型上。该室设计用于容纳九个水凝胶支架，3D打印为平盘，其储能模量与生物活性脱细胞和甲基丙烯酸酯化小肠粘膜下层（dsIS-MA）的肠组织硬度的生理范围（〜3.7kPa）相匹配。进行计算流体动力学模拟以确认生理相关方案中平坦和3D绒毛包含支架的层流分布。该系统最初用HT29-MTX接种的水凝胶支架进行了验证，表现出加速分化，增加粘液的产生，增强了剪应力下的三维组织。这些特征性的肠组织特征对于先进的体外模型是必不可少的，因为它们对功能屏障至关重要。随后，用回肠末端的人肠干细胞（ISC）攻击该室。我们的研究结果表明，生物模拟水凝胶支架，结合生理剪切应力，促进多谱系分化，在没有化学分化触发因素的情况下，对基本标记的基因和蛋白质表达分析以及ISC的3D结构组织证明了这一点。碱性磷酸酶（ALP）活性和分泌的粘液的定量分析表明，细胞在功能上分化成肠上皮细胞和杯状细胞谱系。毫流体系统，它已经开发和优化了性能和成本效率，能够在生物模拟条件下创建和调节先进的肠道模型，包括可调基体刚度和变化的流体剪切应力。此外，容易获得和可扩展的粘液产生细胞组织模型允许全面的粘液分析和病原体相互作用和渗透的研究，从而提供了促进我们对健康和疾病中肠道粘液的理解的潜力。
