Institute of Chemistry and Biochemistry, Freie Universität Berlin, Takustr. 3, 14195 Berlin, Germany.
Department of Gastroenterology, Infectious Diseases and Rheumatology (Including Nutrition Medicine), Charité-Universitätsmedizin Berlin, Hindenburgdamm 30, 12203 Berlin, Germany.
Cells. 2024 Jun 21;13(13):1080. doi: 10.3390/cells13131080.
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.
肠上皮的腔面被一层重要的黏液层所保护，这层黏液对于润滑、水合作用以及促进共生细菌关系至关重要。在体外复制和研究这种复杂的黏液结构存在很大的挑战。为了解决这个问题，我们开发了一种水凝胶集成的微流控组织室，能够对在平面或 3D 结构化水凝胶支架上培养的肠模型施加精确的顶端剪切应力，这些水凝胶支架的硬度可调。该室设计用于容纳 9 个水凝胶支架，这些支架是通过 3D 打印制成的，其存储模量与肠组织硬度的生理范围相匹配（~3.7 kPa），使用的是生物活性脱细胞和甲基丙烯酰化的小肠黏膜下基质（dSIS-MA）。进行了计算流体动力学模拟，以确认在生理相关范围内，平面和包含 3D 绒毛的支架都具有层流剖面。该系统最初使用 HT29-MTX 接种的水凝胶支架进行了验证，结果表明在剪切应力下，细胞加速分化，黏液产生增加，3D 组织更加有序。这些特征性的肠组织特征对于先进的体外模型至关重要，因为它们对于功能性屏障至关重要。随后，该室对来自回肠末端的人肠干细胞（ISCs）进行了挑战。我们的研究结果表明，仿生水凝胶支架与生理剪切应力相结合，促进了多谱系分化，这可以通过对基本标志物的基因和蛋白质表达分析以及在没有化学分化触发的情况下 ISCs 的 3D 结构组织来证明。碱性磷酸酶（ALP）活性和分泌黏液的定量分析表明，细胞分化为肠上皮细胞和杯状细胞谱系。该微流控系统已经针对性能和成本效率进行了开发和优化，可在仿生条件下创建和调节先进的肠模型，包括可调基质硬度和变化的流体剪切应力。此外，这种易于获取和可扩展的黏液产生细胞组织模型允许进行全面的黏液分析以及研究病原体相互作用和穿透，从而有可能提高我们对健康和疾病中肠黏液的理解。
