Beamer Maria A, Furuta Saori
Division of Pediatric Rheumatology, Department of Pediatrics, University of Michigan, Ann Arbor, Michigan.
MetroHealth Medical Center, Case Western Reserve University School of Medicine, Case Comprehensive Cancer Center, Cleveland, Ohio.
Curr Protoc. 2025 Feb;5(2):e70107. doi: 10.1002/cpz1.70107.
Gut mucosae are composed of stratified layers of microbes, a selectively permeable mucus, an epithelial lining, and connective tissue homing immune cells. Studying cellular and chemical interactions between the gut mucosal components has been limited without a good model system. We have engineered a three-dimensional (3D) multi-cellular co-culture system we coined "3D Flipwell system" using cell culture inserts stacked against each other. This system allows an assessment of the impact of a gut mucosal environmental change on interactions between gut bacteria, epithelia, and immune cells. As such, this system can be utilized in examining the effects of exogenous stimuli, such as dietary nutrients, bacterial infection, and drugs, on the gut mucosa that could predetermine how these stimuli might influence the rest of body. Here, we describe the methods of construction and application of the new 3D Flipwell system we utilized previously in assessing the crosstalk between the gut mucosa and macrophage polarization. We demonstrate the physiological responses of different components of the co-cultures to Sepiapterin (SEP), the precursor of the nitric oxide synthase cofactor tetrahydrobiopterin (BH). We reported previously that SEP induces a pro-immunogenic shift of macrophages having acquired an immune suppressive phenotype. We also showed that SEP induces a defense mechanism of commensal gut bacteria. The protocol describing the assembly and use of the 3D Flipwell co-culture system herein would grant its utility in evaluating the concurrent effects of pharmacologic and microbiologic stimuli on gut mucosal components. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: 3D Flipwell construction, assembly, and collagen coating Basic Protocol 2: Flipwell cell seeding and cell culture Basic Protocol 3: Addition of bacterial culture to the Flipwell system Basic Protocol 4: Flipwell disassembly for scanning electron microscopy (SEM) studies Basic Protocol 5: Immunofluorescence antibody staining for confocal microscopy.
肠道黏膜由多层微生物、具有选择性渗透性的黏液、上皮衬里以及归巢免疫细胞的结缔组织组成。在没有良好模型系统的情况下，对肠道黏膜各组成部分之间细胞和化学相互作用的研究一直受到限制。我们利用相互堆叠的细胞培养插入物构建了一种三维（3D）多细胞共培养系统，我们将其命名为“3D Flipwell系统”。该系统能够评估肠道黏膜环境变化对肠道细菌、上皮细胞和免疫细胞之间相互作用的影响。因此，该系统可用于研究外源性刺激，如饮食营养、细菌感染和药物，对肠道黏膜的影响，而这些影响可能预先决定这些刺激如何影响身体的其他部位。在此，我们描述了我们之前用于评估肠道黏膜与巨噬细胞极化之间相互作用的新型3D Flipwell系统的构建和应用方法。我们展示了共培养物不同组分对一氧化氮合酶辅因子四氢生物蝶呤（BH）的前体蝶酰三谷氨酸（SEP）的生理反应。我们之前报道过，SEP会诱导已获得免疫抑制表型的巨噬细胞发生促免疫原性转变。我们还表明，SEP会诱导共生肠道细菌的防御机制。本文描述3D Flipwell共培养系统组装和使用的方案将使其在评估药理和微生物刺激对肠道黏膜组分的联合作用方面具有实用性。© 2025作者。由Wiley Periodicals LLC出版的《当前方案》。基本方案1：3D Flipwell构建、组装和胶原包被；基本方案2：Flipwell细胞接种和细胞培养；基本方案3：向Flipwell系统中添加细菌培养物；基本方案4：拆卸Flipwell用于扫描电子显微镜（SEM）研究；基本方案5：用于共聚焦显微镜的免疫荧光抗体染色。
