Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A complex human gut microbiome cultured in an anaerobic intestine-on-a-chip

Nature Biomedical Engineering volume 3, pages 520–531 (2019)Cite this article

Subjects

An Author Correction to this article was published on 18 June 2019

This article has been updated

Abstract

The diverse bacterial populations that comprise the commensal microbiome of the human intestine play a central role in health and disease. A method that sustains complex microbial communities in direct contact with living human intestinal cells and their overlying mucus layer in vitro would thus enable the investigation of host–microbiome interactions. Here, we show the extended coculture of living human intestinal epithelium with stable communities of aerobic and anaerobic human gut microbiota, using a microfluidic intestine-on-a-chip that permits the control and real-time assessment of physiologically relevant oxygen gradients. When compared to aerobic coculture conditions, the establishment of a transluminal hypoxia gradient in the chip increased intestinal barrier function and sustained a physiologically relevant level of microbial diversity, consisting of over 200 unique operational taxonomic units from 11 different genera and an abundance of obligate anaerobic bacteria, with ratios of Firmicutes and Bacteroidetes similar to those observed in human faeces. The intestine-on-a-chip may serve as a discovery tool for the development of microbiome-related therapeutics, probiotics and nutraceuticals.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Oxygen-sensitive human Intestine Chip microfluidic culture device.
Fig. 2: Coculture of human intestinal epithelium and B. fragilis on-chip.
Fig. 3: Analysis of the diversity and relative abundance of microbiota cocultured in Intestine Chips under aerobic and anaerobic conditions.
Fig. 4: Anaerobic conditions in the Intestine Chip enhance the growth of multiple genera compared to the aerobic chip and conventional liquid culture.
Fig. 5: Anaerobic coculture of a gut microbiome obtained from fresh human patient-derived stool with primary human ileal epithelium in the Intestine Chip.

Similar content being viewed by others

Data availability

The main data supporting the findings of this study are available in the Article and Supplementary Information. The raw data generated in this study are available from the corresponding author on reasonable request.

Change history

  • 18 June 2019

    In the version of this Article originally published, the authors mistakenly cited Fig. 5d in the sentence beginning ‘Importantly, the microbiome cultured in these primary Intestine Chips...’; the correct citation is Supplementary Table 2. This has now been amended.

References

  1. Cho, I. & Blaser, M. J. The human microbiome: at the interface of health and disease. Nat. Rev. Genet. 13, 260–270 (2012).

    Article  CAS  Google Scholar 

  2. Donaldson, G. P., Lee, S. M. & Mazmanian, S. K. Gut biogeography of the bacterial microbiota. Nat. Rev. Microbiol. 14, 20–32 (2016).

    Article  CAS  Google Scholar 

  3. Pickard, J. M., Zeng, M. Y., Caruso, R. & Núñez, G. Gut microbiota: role in pathogen colonization, immune responses, and inflammatory disease. Immunol. Rev. 279, 70–89 (2017).

    Article  CAS  Google Scholar 

  4. Sommer, F. & Bäckhed, F. The gut microbiota—masters of host development and physiology. Nat. Rev. Microbiol. 11, 227–238 (2013).

    Article  CAS  Google Scholar 

  5. Walter, J. & Ley, R. The human gut microbiome: ecology and recent evolutionary changes. Annu. Rev. Microbiol. 65, 411–429 (2011).

    Article  CAS  Google Scholar 

  6. Sommer, M. O. Advancing gut microbiome research using cultivation. Curr. Opin. Microbiol. 27, 127–132 (2015).

    Article  CAS  Google Scholar 

  7. Eain, M. M. G. et al. Engineering solutions for representative models of the gastrointestinal human-microbe interface. Engineering 3, 60–65 (2017).

    Article  Google Scholar 

  8. Fritz, J. V., Desai, M. S., Shah, P., Schneider, J. G. & Wilmes, P. From meta-omics to causality: experimental models for human microbiome research. Microbiome 1, 14 (2013).

    Article  Google Scholar 

  9. Arrieta, M.-C., Walter, J. & Finlay, B. B. Human microbiota-associated mice: a model with challenges. Cell Host Microbe 19, 575–578 (2016).

    Article  CAS  Google Scholar 

  10. Nguyen, T. L. A., Vieira-Silva, S., Liston, A. & Raes, J. How informative is the mouse for human gut microbiota research? Dis. Model. Mech. 8, 1–16 (2015).

    Article  CAS  Google Scholar 

  11. Sadabad, M. S. et al. A simple coculture system shows mutualism between anaerobic Faecalibacteria and epithelial Caco-2 cells. Sci. Rep. 5, 17906 (2015).

    Article  Google Scholar 

  12. Dutta, D. & Clevers, H. Organoid culture systems to study host–pathogen interactions. Curr. Opin. Immunol. 48, 15–22 (2017).

    Article  CAS  Google Scholar 

  13. Fatehullah, A., Tan, S. H. & Barker, N. Organoids as an in vitro model of human development and disease. Nat. Cell Biol. 18, 246–254 (2016).

    Article  Google Scholar 

  14. Williamson, I. A. et al. A high-throughput organoid microinjection platform to study gastrointestinal microbiota and luminal physiology. Cell. Mol. Gastroenterol. Hepatol. 6, 301–319 (2018).

    Article  Google Scholar 

  15. Bein, A. et al. Microfluidic organ-on-a-chip models of human intestine. Cell. Mol. Gastroenterol. Hepatol. 5, 659–668 (2018).

    Article  Google Scholar 

  16. Van den Abbeele, P. et al. Incorporating a mucosal environment in a dynamic gut model results in a more representative colonization by lactobacilli. Microb. Biotechnol. 5, 106–115 (2012).

    Article  Google Scholar 

  17. Marzorati, M. et al. The HMITM module: a new tool to study the Host-Microbiota Interaction in the human gastrointestinal tract in vitro. BMC Microbiol. 14, 133 (2014).

    Article  Google Scholar 

  18. Van de Wiele, T., et al. in The Impact of Food Bioactives on Health: In Vitro and Ex Vivo Models (eds Verhoeckx, K. et al.) 305–317 (Springer, 2015).

  19. Van den Abbeele, P. et al. Microbial community development in a dynamic gut model is reproducible, colon region specific, and selective for Bacteroidetes and Clostridium cluster IX. Appl. Environ. Microbiol. 76, 5237–5246 (2010).

    Article  Google Scholar 

  20. Kim, H. J., Huh, D., Hamilton, G. & Ingber, D. E. Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow. Lab Chip 12, 2165–2174 (2012).

    Article  CAS  Google Scholar 

  21. Kim, H. J., Li, H., Collins, J. J. & Ingber, D. E. Contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a human gut-on-a-chip. Proc. Natl Acad. Sci. USA 113, E7–E15 (2016).

    Article  CAS  Google Scholar 

  22. Park, G.-S. et al. Emulating host-microbiome ecosystem of human gastrointestinal tract in vitro. Stem Cell Rev. 13, 321–334 (2017).

    Article  CAS  Google Scholar 

  23. Chung, H. et al. Gut immune maturation depends on colonization with a host-specific microbiota. Cell 149, 1578–1593 (2012).

    Article  CAS  Google Scholar 

  24. Surana, N. K. & Kasper, D. L. Moving beyond microbiome-wide associations to causal microbe identification. Nature 552, 244–247 (2017).

    Article  CAS  Google Scholar 

  25. Jalili-Firoozinezhad, S. et al. Modeling radiation injury-induced cell death and countermeasure drug responses in a human Gut-on-a-Chip. Cell Death Dis. 9, 223 (2018).

    Article  Google Scholar 

  26. Kasendra, M. et al. Development of a primary human Small Intestine-on-a-Chip using biopsy-derived organoids. Sci. Rep. 8, 2871 (2018).

    Article  Google Scholar 

  27. Zheng, L., Kelly, C. J. & Colgan, S. P. Physiologic hypoxia and oxygen homeostasis in the healthy intestine. A review in the theme: cellular responses to hypoxia. Am. J. Physiol. Cell Physiol. 309, C350–C360 (2015).

    Article  CAS  Google Scholar 

  28. Jiang, B. H., Semenza, G. L., Bauer, C. & Marti, H. H. Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O2 tension. Am. J. Physiol. Cell Physiol. 271, C1172–C1180 (1996).

    Article  CAS  Google Scholar 

  29. Chilov, D. et al. Induction and nuclear translocation of hypoxia-inducible factor-1 (HIF-1): heterodimerization with ARNT is not necessary for nuclear accumulation of HIF-1alpha. J. Cell Sci. 112, 1203–1212 (1999).

    CAS  PubMed  Google Scholar 

  30. Surana, N. K. & Kasper, D. L. The yin yang of bacterial polysaccharides: lessons learned from B. fragilis PSA. Immunol. Rev. 245, 13–26 (2012).

    Article  CAS  Google Scholar 

  31. Patrick, S., Reid, J. H. & Larkin, M. J. The growth and survival of capsulate and non-capsulate Bacteroides fragilis in vivo and in vitro. J. Med. Microbiol. 17, 237–246 (1984).

    Article  CAS  Google Scholar 

  32. Hudak, J. E., Alvarez, D., Skelly, A., von Andrian, U. H. & Kasper, D. L. Illuminating vital surface molecules of symbionts in health and disease. Nat. Microbiol. 2, 17099 (2017).

    Article  CAS  Google Scholar 

  33. Shin, W. & Kim, H. J. Intestinal barrier dysfunction orchestrates the onset of inflammatory host–microbiome cross-talk in a human gut inflammation-on-a-chip. Proc. Natl Acad. Sci. USA 115, E10539–E10547 (2018).

    Article  CAS  Google Scholar 

  34. Kuo, J. C.-H. et al. Detection of colorectal dysplasia using fluorescently labelled lectins. Sci. Rep. 6, 24231 (2016).

    Article  CAS  Google Scholar 

  35. Kim, H. J. & Ingber, D. E. Gut-on-a-Chip microenvironment induces human intestinal cells to undergo villus differentiation. Integr. Biol. 5, 1130–1140 (2013).

    Article  CAS  Google Scholar 

  36. Rozee, K. R., Cooper, D., Lam, K. & Costerton, J. W. Microbial flora of the mouse ileum mucous layer and epithelial surface. Appl. Environ. Microbiol. 43, 1451–1463 (1982).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Lock, J. Y., Carlson, T. L., Wang, C.-M., Chen, A. & Carrier, R. L. Acute exposure to commonly ingested emulsifiers alters intestinal mucus structure and transport properties. Sci. Rep. 8, 10008 (2018).

    Article  Google Scholar 

  38. Villmones, H. C. et al. Species level description of the human ileal bacterial microbiota. Sci. Rep. 8, 4736 (2018).

    Article  Google Scholar 

  39. The Human Microbiome Project ConsortiumStructure, function and diversity of the healthy human microbiome. Nature 486, 207–214 (2012).

  40. Stearns, J. C. et al. Bacterial biogeography of the human digestive tract. Sci. Rep. 1, 170 (2011).

    Article  CAS  Google Scholar 

  41. Guarner, F. & Malagelada, J.-R. Gut flora in health and disease. Lancet 361, 512–519 (2003).

    Article  Google Scholar 

  42. Wang, M., Ahrné, S., Jeppsson, B. & Molin, G. Comparison of bacterial diversity along the human intestinal tract by direct cloning and sequencing of 16S rRNA genes. FEMS Microbiol. Ecol. 54, 219–231 (2005).

    Article  CAS  Google Scholar 

  43. Fujio-Vejar, S. et al. The gut microbiota of healthy Chilean subjects reveals a high abundance of the phylum Verrucomicrobia. Front. Microbiol. 8, 1221 (2017).

    Article  Google Scholar 

  44. Schneeberger, M. et al. Akkermansia muciniphila inversely correlates with the onset of inflammation, altered adipose tissue metabolism and metabolic disorders during obesity in mice. Sci. Rep. 5, 16643 (2015).

    Article  CAS  Google Scholar 

  45. Everard, A. et al. Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity. Proc. Natl Acad. Sci. USA 110, 9066–9071 (2013).

    Article  CAS  Google Scholar 

  46. Shah, P. et al. A microfluidics-based in vitro model of the gastrointestinal human–microbe interface. Nat. Commun. 7, 11535 (2016).

    Article  CAS  Google Scholar 

  47. Pedicord, V. A. et al. Exploiting a host-commensal interaction to promote intestinal barrier function and enteric pathogen tolerance. Sci. Immunol. 1, eaai7732 (2016).

    Article  Google Scholar 

  48. Sheridan, W. G., Lowndes, R. H. & Young, H. L. Intraoperative tissue oximetry in the human gastrointestinal tract. Am. J. Surg. 159, 314–319 (1990).

    Article  CAS  Google Scholar 

  49. He, G. et al. Noninvasive measurement of anatomic structure and intraluminal oxygenation in the gastrointestinal tract of living mice with spatial and spectral EPR imaging. Proc. Natl Acad. Sci. USA 96, 4586–4591 (1999).

    Article  CAS  Google Scholar 

  50. Ohland, C. L. & Jobin, C. Microbial activities and intestinal homeostasis: a delicate balance between health and disease. Cell. Mol. Gastroenterol. Hepatol. 1, 28–40 (2015).

    Article  Google Scholar 

  51. Flint, H. J., Scott, K. P., Louis, P. & Duncan, S. H. The role of the gut microbiota in nutrition and health. Nat. Rev. Gastroenterol. Hepatol. 9, 577–589 (2012).

    Article  CAS  Google Scholar 

  52. Albenberg, L. et al. Correlation between intraluminal oxygen gradient and radial partitioning of intestinal microbiota in humans and mice. Gastroenterology 147, 1055–1063 (2014).

    Article  Google Scholar 

  53. Baughn, A. D. & Malamy, M. H. The strict anaerobe Bacteroides fragilis grows in and benefits from nanomolar concentrations of oxygen. Nature 427, 441–444 (2004).

    Article  CAS  Google Scholar 

  54. Hsiao, E. Y. et al. Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders. Cell 155, 1451–1463 (2013).

    Article  CAS  Google Scholar 

  55. Clemente, J. C., Ursell, L. K., Parfrey, L. W. & Knight, R. The impact of the gut microbiota on human health: an integrative view. Cell 148, 1258–1270 (2012).

    Article  CAS  Google Scholar 

  56. Shin, N.-R., Whon, T. W. & Bae, J.-W. Proteobacteria: microbial signature of dysbiosis in gut microbiota. Trends Biotechnol. 33, 496–503 (2015).

    Article  CAS  Google Scholar 

  57. Goodman, A. L. et al. Extensive personal human gut microbiota culture collections characterized and manipulated in gnotobiotic mice. Proc. Natl Acad. Sci. USA 108, 6252–6257 (2011).

    Article  CAS  Google Scholar 

  58. Karhausen, J. et al. Epithelial hypoxia-inducible factor-1 is protective in murine experimental colitis. J. Clin. Invest. 114, 1098–1106 (2004).

    Article  CAS  Google Scholar 

  59. Manresa, M. C. & Taylor, C. T. Hypoxia inducible factor (HIF) hydroxylases as regulators of intestinal epithelial barrier function. Cell. Mol. Gastroenterol. Hepatol. 3, 303–315 (2017).

    Article  Google Scholar 

  60. Cirstea, M., Radisavljevic, N. & Finlay, B. B. Good bug, bad bug: breaking through microbial stereotypes. Cell Host Microbe 23, 10–13 (2018).

    Article  CAS  Google Scholar 

  61. Workman, M. J. et al. Enhanced utilization of induced pluripotent stem cell–derived human intestinal organoids using microengineered chips. Cell. Mol. Gastroenterol. Hepatol. 5, 669–677 (2018).

    Article  Google Scholar 

  62. Hooper, L. V., Littman, D. R. & Macpherson, A. J. Interactions between the microbiota and the immune system. Science 336, 1268–1273 (2012).

    Article  CAS  Google Scholar 

  63. Rimoldi, M. et al. Intestinal immune homeostasis is regulated by the crosstalk between epithelial cells and dendritic cells. Nat. Immunol. 6, 507–514 (2005).

    Article  CAS  Google Scholar 

  64. Henry, O. Y. F. et al. Organs-on-chips with integrated electrodes for trans-epithelial electrical resistance (TEER) measurements of human epithelial barrier function. Lab Chip 17, 2264–2271 (2017).

    Article  CAS  Google Scholar 

  65. Huh, D. et al. Microfabrication of human organs-on-chips. Nat. Protoc. 8, 2135–2157 (2013).

    Article  CAS  Google Scholar 

  66. Otsu, N. A threshold selection method from gray-level histograms. IEEE Trans. Syst. Man Cybern. 9, 62–66 (1979).

    Article  Google Scholar 

  67. McMurdie, P. J. & Holmes, S. phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS ONE 8, e61217 (2013).

    Article  CAS  Google Scholar 

  68. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    Article  Google Scholar 

Download references

Acknowledgements

This research was supported by a US FDA grant (HHSF223201310079C), a DARPA THoR grant (W911NF-16-C-0050), the Bill & Melinda Gates Foundation, the Wyss Institute for Biologically Inspired Engineering at Harvard University and Fundação para a Ciência e a Tecnologia Portugal (project PD/BD/105774/2014 at the Institute for Bioengineering and Biosciences). We thank D. E. Achatz (PreSens GmbH) for providing oxygen-sensing particles and for her expert technical advice and T. Ferrante for his assistance with imaging.

Author information

Author notes

  1. These authors contributed equally: Sasan Jalili-Firoozinezhad, Francesca S. Gazzaniga, Elizabeth L. Calamari.

Authors and Affiliations

  1. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA

    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, Richard Novak & Donald E. Ingber

  2. Department of Bioengineering and Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal

    Sasan Jalili-Firoozinezhad & Joaquim M. S. Cabral

  3. Department of Immunology, Harvard Medical School, Boston, MA, USA

    Francesca S. Gazzaniga & Dennis L. Kasper

  4. Department of Health Sciences and Technology, ETH Zurich, Zurich, Switzerland

    Alessio Tovaglieri

  5. Department of Pediatric Newborn Medicine, Brigham and Women’s Hospital, Boston, MA, USA

    Katherine E. Gregory

  6. Department of Pediatrics, Harvard Medical School, Boston, MA, USA

    Katherine E. Gregory & David T. Breault

  7. Department of Endocrinology, Boston Children’s Hospital, Boston, MA, USA

    David T. Breault

  8. Harvard Stem Cell Institute, Cambridge, MA, USA

    David T. Breault

  9. Vascular Biology Program and Department of Surgery, Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA

    Donald E. Ingber

  10. Harvard John A. Paulson School of Engineering and Applied Sciences, Cambridge, MA, USA

    Donald E. Ingber

Authors
  1. Sasan Jalili-Firoozinezhad
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Francesca S. Gazzaniga
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Elizabeth L. Calamari
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Diogo M. Camacho
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Cicely W. Fadel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Amir Bein
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ben Swenor
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Bret Nestor
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Michael J. Cronce
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Alessio Tovaglieri
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Oren Levy
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Katherine E. Gregory
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. David T. Breault
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Joaquim M. S. Cabral
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Dennis L. Kasper
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Richard Novak
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Donald E. Ingber
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.J.-F., F.S.G., E.L.C., J.M.S.C., R.N. and D.E.I. designed the research. S.J.-F., E.L.C., F.S.G., B.N., C.W.F., A.T., A.B., B.S. and M.J.C. performed experiments. S.J.-F., F.S.G., D.M.C., E.L.C., B.N., D.L.K., R.N. and D.E.I. analysed and interpreted the data. K.E.G. helped to prepare infant microbiota. D.T.B. established and prepared human ileal organoids. S.J.-F., F.S.G., E.L.C., D.M.C. and D.E.I. wrote the Article with input from B.N., O.L., J.M.S.C. and R.N. All authors reviewed, discussed and edited the manuscript.

Corresponding author

Correspondence to Donald E. Ingber.

Ethics declarations

Competing interests

D.E.I. holds equity in Emulate, Inc., consults for the company and chairs its scientific advisory board.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Supplementary Information

Supplementary figures and tables.

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jalili-Firoozinezhad, S., Gazzaniga, F.S., Calamari, E.L. et al. A complex human gut microbiome cultured in an anaerobic intestine-on-a-chip. Nat Biomed Eng 3, 520–531 (2019). https://doi.org/10.1038/s41551-019-0397-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41551-019-0397-0

This article is cited by

Search

Advanced search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing: Microbiology