The biggest paradigm shift in medicine I have experienced in my career is the unexpected discovery of the central role that the microbiome plays in human health and disease. But given the huge diversity of commensal microbes that comprise the microbiome, it is exceedingly difficult to study how they influence human pathophysiology, or how the human organ microenvironment affects their behavior. In fact, almost all we know about the importance of the microbiome for human health is based on genomic or metagenomic studies in which variations in microbial community composition appear to correlate with changes in human biology. Microbiome studies have been carried out in animal models; however, the relevance of these findings is often questionable given that there are huge differences in microbiome composition between different species. To gain deeper understanding into human host-microbiome interactions, it is therefore crucial that we develop experimental models in which complex populations of living commensal microbes can be co-cultured in close apposition to living human cells in an organ-relevant context. Unfortunately, placing living bacteria in contact with mammalian cells rapidly leads to ‘contamination’ and cell death in conventional cultures.
We set out to confront this challenge by leveraging vascularized human organ-on-a-chip (Organ Chip) microfluidic culture technology (1,2), and focusing on the gut microbiome because it plays such a central role in health and disease. Our Organ Chips contain two parallel microchannels separated by a porous extracellular matrix-coated membrane; human organ-specific epithelial cells are cultured on top of the membrane while microvascular endothelial cells are grown on the bottom to recreate an organ-level tissue-tissue interface, and medium is perfused through the lower channel to mimic vascular perfusion. We previously developed two types of human Intestine Chips one lined by the Caco-2 intestinal epithelial cells (3-5) and the other containing primary human intestinal epithelial cells isolated from patient-derived organoids (6); we used both in our new study. Our goal was to co-culture highly complex human gut microbiome in direct apposition to a mucus-producing human intestinal epithelium by placing samples of living gut microbiome obtained from human stool within the lumen of the upper intestinal channel. But we faced a major problem because the gut microbiome contains many different types of bacteria, including both aerobes and obligate anaerobes that cannot survive at the high oxygen concentrations required to sustain the viability of cultured mammalian cells.
We overcame this challenge and now are able to co-culture a complex human gut microbiome containing over 200 unique operational taxonomic units (i.e., different types of microbes) from 11 different genera in direct contact with living human intestinal epithelium for extended times (at least 5 days) in vitro. We did this by engineering an anaerobic chamber in which the Intestine Chips are cultured while perfusing oxygenated medium only through the endothelium-lined vascular channel. This configuration creates a physiologically relevant oxygen gradient across the human intestinal epithelium-endothelial interface such that the viability of the human tissues is maintained, while oxygen levels can be held below 0.5% in the upper reaches of the top channel (Fig. 1), which enables survival and growth of both aerobes and obligate anaerobes. Interestingly, we found that intestinal barrier function actually improves under these more physiological conditions. This ability to coculture of living human intestinal epithelium with stable communities of aerobic and anaerobic human gut microbiota for extended times should help to provide new insights into host-microbiome interactions. The Intestine Chips also may be used to model microbiome-related intestinal diseases and serve as discovery tools for the development of microbiome-related diagnostics and therapeutics.
Sasan Jalili-Firoozinezhad, Francesca S. Gazzaniga, Elizabeth L. Calamari, Diogo M. Camacho, Cicely W. Fadel, Amir Bein, Ben Swenor, Bret Nestor, Michael J. Cronce, Alessio Tovaglieri, Oren Levy, Katherine E. Gregory, David T. Breault, Joaquim M. S. Cabral, Dennis L. Kasper, Richard Novak & Donald E. Ingber. Nature Biomedical Engineering (2019)
1. Huh D, Matthews BD, Mammoto A, Montoya-Zavala M, Hsin HY, and Ingber DE. Reconstituting organ-level lung functions on a chip. Science 2010 328: 1662-8.
2. Ingber DE. Reverse Engineering Human Pathophysiology with Organs-on-Chips. Cell 2016 164:1105-9. doi: 10.1016/j.cell.2016.02.049.
3. Kim HJ, Huh D, Hamilton G, and Ingber DE. Human Gut-on-a-Chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow. Lab on a Chip 2012; 12: 2164-74.
4. Kim H-J, Ingber DE. Gut-on-a-Chip Microenvironment Reprograms Human Intestinal Cells to Undergo Villus Differentiation. Integrative Biology 2013; 5:1130-40.
5. Kim HJ, Collins JJ, Ingber DE. Contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a human gut-on-a-chip. PNAS 2016; 113:E7-E15.
6. Kasendra M, Tovaglieri A, Sontheimer-Phelps A, Jalili-Firoozinezhad S, Bein A, Chalkiadaki A, Scholl W, Zheng C, Rickner H, Dinis ALM, Richmond CA, Li H, Cartwright M, Super M, Breault DR and Ingber DE. Development of primary human small intestine-on-a-chip using patient-derived organoids. Sci. Rep. 2018; 8:2871 DOI:10.1038/s41598-018-21201-7