Document Type
Article
Publication Date
2-18-2025
Abstract
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 (BH₄). 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.
Keywords
3D co-culture, caco-2, gut, immune cells, macrophages, microbiome
Language
English
Publication Title
Current Protocols
Grant
5017
Rights
© 2025 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/), which permits non-commercial copying and redistribution of the material in any medium or format, provided the original work is not changed in any way and is properly cited.
Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
Recommended Citation
Beamer, M. A., & Furuta, S. (2025). Redefining cell culture using a 3D flipwell co-culture system: A mimetic for gut architecture and dynamics in vitro. Current Protocols, 5, e70107. doi: 10.1002/cpz1.70107
Manuscript Version
Final Publisher Version