Speaker
Description
Introduction
A substantial body of scientific evidence demonstrates that gut microbiota plays a central role in human health and disease1,2. Understanding bacteria–host interactions in the human gut is essential for advancing microbiome research. Early 3D culture systems relied on physical membranes to separate bacteria from the intestinal epithelium model, which often limited the ability to study bacterial adhesion and invasion3. Most recent in vitro models of host–bacteria interactions rely on a 3D-culture of either the host tissue or the bacterial community, while the counterpart remains in a liquid suspension4,5. These models often fail to replicate the complex spatial organization and dynamic interactions characteristic of native tissues, limiting their physiological relevance for studying microbiota–host crosstalk6. Fabricate constructs in which both components are simultaneously incorporated into a structured 3D environment from the outset of the bioprinting, while ensuring coculture stability over time, is untrivial.
Methods
Structured cocultures fabricated by chaotic bioprinting mimic natural microenvironments by creating defined niches where bacteria and mammalian cells coexist without intermixing (Figure 1a-d). Using a printhead containing Kenics static mixer elements, we bioprinted hydrogel constructs with intercalated layers of Caco-2 cells and bacteria (either Escherichia coli or Lactobacillus rhamnosus) (Figure 1e-f). Characterization, including the analysis of Caco-2 cell dynamics and bacterial community evolution over time, was performed through fluorescence microscopy, colony forming unit counting, and live/dead assays.
Results/discussion
The microarchitecture of printed filaments significantly defines bacterial growth dynamics6. This new approach in which we incorporate Caco-2 cells offers a versatile, cost-effective, and high-throughput platform to analyze how bacterial strains influence mammalian cell behavior within a spatially organized 3D microenvironment. The printed filament preserves spatial compartmentalization over time, effectively preventing significant bacterial migration into the Caco-2 regions. Existing models often struggle to maintain both viable mammalian cells and bacteria in the same system over extended periods4,5. By providing individualized compartments and perfusable channels, our constructs improve nutrient delivery, waste removal, and overall coculture stability for at least 8 days. To our knowledge, this is the first report of the one-step biofabrication of an architected, membrane-less, gut-like construct containing both bacterial and mammalian cells.
Conclusion
We anticipate that model bacteria-host interactions using chaotic bioprinting will contribute to in vitro studies of microbiome-related therapeutics, probiotic and pathogen dynamics, dysbiosis modeling, and host immune responses in a structured, physiologically relevant environment.
References
1. Fan, Y. & Pedersen, O. Gut microbiota in human metabolic health and disease. Nature Reviews Microbiology vol. 19, (2021).
2. Hou, K. et al. Microbiota in health and diseases. Signal Transduction and Targeted Therapy vol. 7, (2022).
3. Cheng, L. et al. A 3D Bioprinted Gut Anaerobic Model for Studying Bacteria–Host Interactions. Research 6, (2023).
4. Jeong, Y. & Irudayaraj, J. Hierarchical encapsulation of bacteria in functional hydrogel beads for inter- and intra- species communication. Acta Biomater 158, (2023).
5. Puschhof, J. et al. Intestinal organoid cocultures with microbes. Nat Protoc 16, 4633–4649 (2021).
6. Ceballos-González, C. F. et al. High-Throughput and Continuous Chaotic Bioprinting of Spatially Controlled Bacterial Microcosms. ACS Biomater Sci Eng 7, 2408–2419 (2021).