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.

Microfabrication of human organs-on-chips

Abstract

'Organs-on-chips' are microengineered biomimetic systems containing microfluidic channels lined by living human cells, which replicate key functional units of living organs to reconstitute integrated human organ-level pathophysiology in vitro. These microdevices can be used to test efficacy and toxicity of drugs and chemicals, and to create in vitro models of human disease. Thus, they potentially represent low-cost alternatives to conventional animal models for pharmaceutical, chemical and environmental applications. Here we describe a protocol for the fabrication, microengineering and operation of these microfluidic organ-on-chip systems. First, microengineering is used to fabricate a multilayered microfluidic device that contains two parallel elastomeric microchannels separated by a thin porous flexible membrane, along with two full-height, hollow vacuum chambers on either side; this requires 3.5 d to complete. To create a 'breathing' lung-on-a-chip that mimics the mechanically active alveolar-capillary interface of the living human lung, human alveolar epithelial cells and microvascular endothelial cells are cultured in the microdevice with physiological flow and cyclic suction applied to the side chambers to reproduce rhythmic breathing movements. We describe how this protocol can be easily adapted to develop other human organ chips, such as a gut-on-a-chip lined by human intestinal epithelial cells that experiences peristalsis-like motions and trickling fluid flow. Also, we discuss experimental techniques that can be used to analyze the cells in these organ-on-chip devices.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Mechanically active organ-on-chip microdevice with compartmentalized 3D microarchitecture.
Figure 2: Fabrication of the upper microchannels of the lung-on-a-chip.
Figure 3: Fabrication of porous PDMS membranes.
Figure 4: Alignment, bonding and chemical etching of the lung-on-a-chip microdevice.
Figure 5: Microfabrication of gut-on-a-chip.
Figure 6: A multilayered 3D microfluidic device for the production of the human breathing lung-on-a-chip.
Figure 7: Production and microfluidic engineering of the alveolar epithelium and microvascular endothelium in the lung-on-a-chip microdevice.
Figure 8: Intestinal epithelial cell culture and spontaneous villus morphogenesis in the gut-on-a-chip microdevice.

References

  1. 1

    Huh, D., Hamilton, G.A. & Ingber, D.E. From 3D cell culture to organs-on-chips. Trends Cell Biol. 21, 745–754 (2011).

    CAS  Article  Google Scholar 

  2. 2

    Jang, K., Sato, K., Igawa, K., Chung, U.I. & Kitamori, T. Development of an osteoblast-based 3D continuous-perfusion microfluidic system for drug screening. Anal. Bioanal. Chem. 390, 825–832 (2008).

    CAS  Article  Google Scholar 

  3. 3

    Leclerc, E. et al. Study of osteoblastic cells in a microfluidic environment. Biomaterials 27, 586–595 (2006).

    CAS  Article  Google Scholar 

  4. 4

    O'Neill, A.T., Monteiro-Riviere, N.A. & Walker, G.M. Characterization of microfluidic human epidermal keratinocyte culture. Cytotechnology 56, 197–207 (2008).

    Article  Google Scholar 

  5. 5

    Chao, P.G. et al. Dynamic osmotic loading of chondrocytes using a novel microfluidic device. J. Biomech 38, 1273–1281 (2005).

    Article  Google Scholar 

  6. 6

    Baudoin, R., Griscom, L., Monge, M., Legallais, C. & Leclerc, E. Development of a renal microchip for in vitro distal tubule models. Biotechnol. Prog. 23, 1245–1253 (2007).

    CAS  PubMed  Google Scholar 

  7. 7

    Leclerc, E., Sakai, Y. & Fujii, T. Microfluidic PDMS (polydimethylsiloxane) bioreactor for large-scale culture of hepatocytes. Biotechnol. Prog. 20, 750–755 (2004).

    CAS  Article  Google Scholar 

  8. 8

    Powers, M.J. et al. A microfabricated array bioreactor for perfused 3D liver culture. Biotechnol. Bioeng. 78, 257–269 (2002).

    CAS  Article  Google Scholar 

  9. 9

    Tilles, A.W., Baskaran, H., Roy, P., Yarmush, M.L. & Toner, M. Effects of oxygenation and flow on the viability and function of rat hepatocytes cocultured in a microchannel flat-plate bioreactor. Biotechnol. Bioeng. 73, 379–389 (2001).

    CAS  Article  Google Scholar 

  10. 10

    Shin, M. et al. Endothelialized networks with a vascular geometry in microfabricated poly(dimethyl siloxane). Biomed. Microdevices 6, 269–278 (2004).

    CAS  Article  Google Scholar 

  11. 11

    Huh, D., Torisawa, Y.S., Hamilton, G.A., Kim, H.J. & Ingber, D.E. Microengineered physiological biomimicry: organs-on-chips. Lab Chip 12, 2156–2164 (2012).

    CAS  Article  Google Scholar 

  12. 12

    van der Meer, A.D. & van den Berg, A. Organs-on-chips: breaking the in vitro impasse. Integr. Biol. 4, 461–470 (2012).

    CAS  Article  Google Scholar 

  13. 13

    Sung, J.H. et al. Microfabricated mammalian organ systems and their integration into models of whole animals and humans. Lab Chip 13, 1201–1212 (2013).

    CAS  Article  Google Scholar 

  14. 14

    Moraes, C., Mehta, G., Lesher-Perez, S.C. & Takayama, S. Organs-on-a-chip: a focus on compartmentalized microdevices. Ann. Biomed. Eng. 40, 1211–1227 (2012).

    Article  Google Scholar 

  15. 15

    Ghaemmaghami, A.M., Hancock, M.J., Harrington, H., Kaji, H. & Khademhosseini, A. Biomimetic tissues on a chip for drug discovery. Drug Discov. Today 17, 173–181 (2012).

    CAS  Article  Google Scholar 

  16. 16

    Esch, M.B., King, T.L. & Shuler, M.L. The role of body-on-a-chip devices in drug and toxicity studies. Annu. Rev. Biomed. Eng. 13, 55–72 (2011).

    CAS  Article  Google Scholar 

  17. 17

    Jang, K.J. et al. Fluid-shear-stress-induced translocation of aquaporin-2 and reorganization of actin cytoskeleton in renal tubular epithelial cells. Integr. Biol. 3, 134–141 (2011).

    CAS  Article  Google Scholar 

  18. 18

    Jang, K.J. & Suh, K.Y. A multi-layer microfluidic device for efficient culture and analysis of renal tubular cells. Lab Chip 10, 36–42 (2010).

    CAS  Article  Google Scholar 

  19. 19

    Jang, K.J. et al. Human kidney proximal tubule-on-a-chip for drug transport and nephrotoxicity assessment. Integr. Biol. 5, 1119–1129 (2013)

    CAS  Article  Google Scholar 

  20. 20

    Carraro, A. et al. In vitro analysis of a hepatic device with intrinsic microvascular-based channels. Biomed. Microdevices 10, 795–805 (2008).

    Article  Google Scholar 

  21. 21

    Kane, B.J., Zinner, M.J., Yarmush, M.L. & Toner, M. Liver-specific functional studies in a microfluidic array of primary mammalian hepatocytes. Anal. Chem. 78, 4291–4298 (2006).

    CAS  Article  Google Scholar 

  22. 22

    Khetani, S.R. & Bhatia, S.N. Microscale culture of human liver cells for drug development. Nat. Biotechnol. 26, 120–126 (2008).

    CAS  Article  Google Scholar 

  23. 23

    Lee, P.J., Hung, P.J. & Lee, L.P. An artificial liver sinusoid with a microfluidic endothelial-like barrier for primary hepatocyte culture. Biotechnol. Bioeng. 97, 1340–1346 (2007).

    CAS  Article  Google Scholar 

  24. 24

    Nakao, Y., Kimura, H., Sakai, Y. & Fujii, T. Bile canaliculi formation by aligning rat primary hepatocytes in a microfluidic device. Biomicrofluidics 5, 22212 (2011).

    Article  Google Scholar 

  25. 25

    Griep, L.M. et al. BBB ON CHIP: microfluidic platform to mechanically and biochemically modulate blood-brain barrier function. Biomed. Microdevices 15, 145–150 (2013).

    CAS  Article  Google Scholar 

  26. 26

    Khademhosseini, A. et al. Microfluidic patterning for fabrication of contractile cardiac organoids. Biomed. Microdevices 9, 149–157 (2007).

    Article  Google Scholar 

  27. 27

    Grosberg, A., Alford, P.W., McCain, M.L. & Parker, K.K. Ensembles of engineered cardiac tissues for physiological and pharmacological study: heart on a chip. Lab Chip 11, 4165–4173 (2011).

    CAS  Article  Google Scholar 

  28. 28

    Feinberg, A.W. et al. Muscular thin films for building actuators and powering devices. Science 317, 1366–1370 (2007).

    CAS  Article  Google Scholar 

  29. 29

    Nawroth, J.C. et al. A tissue-engineered jellyfish with biomimetic propulsion. Nat. Biotechnol. 30, 792–797 (2012).

    CAS  Article  Google Scholar 

  30. 30

    McCain, M.L., Sheehy, S.P., Grosberg, A., Goss, J.A. & Parker, K.K. Recapitulating maladaptive, multiscale remodeling of failing myocardium on a chip. Proc. Natl. Acad. Sci. USA 110, 9770–9775 (2013).

    CAS  Article  Google Scholar 

  31. 31

    Giridharan, G.A. et al. Microfluidic cardiac cell culture model (muCCCM). Anal. Chem. 82, 7581–7587 (2010).

    CAS  Article  Google Scholar 

  32. 32

    Wilson, K., Das, M., Wahl, K.J., Colton, R.J. & Hickman, J. Measurement of contractile stress generated by cultured rat muscle on silicon cantilevers for toxin detection and muscle performance enhancement. PLoS ONE 5, e11042 (2010).

    Article  Google Scholar 

  33. 33

    Esch, M.B. et al. On chip porous polymer membranes for integration of gastrointestinal tract epithelium with microfluidic 'body-on-a-chip' devices. Biomed. Microdevices 14, 895–906 (2012).

    CAS  Article  Google Scholar 

  34. 34

    Imura, Y., Asano, Y., Sato, K. & Yoshimura, E. A microfluidic system to evaluate intestinal absorption. Anal. Sci. 25, 1403–1407 (2009).

    CAS  Article  Google Scholar 

  35. 35

    Kimura, H., Yamamoto, T., Sakai, H., Sakai, Y. & Fujii, T. An integrated microfluidic system for long-term perfusion culture and on-line monitoring of intestinal tissue models. Lab Chip 8, 741–746 (2008).

    CAS  Article  Google Scholar 

  36. 36

    Mahler, G.J., Esch, M.B., Glahn, R.P. & Shuler, M.L. Characterization of a gastrointestinal tract microscale cell culture analog used to predict drug toxicity. Biotechnol. Bioeng. 104, 193–205 (2009).

    CAS  Article  Google Scholar 

  37. 37

    Sung, J.H., Yu, J., Luo, D., Shuler, M.L. & March, J.C. Microscale 3-D hydrogel scaffold for biomimetic gastrointestinal (GI) tract model. Lab Chip 11, 389–392 (2011).

    CAS  Article  Google Scholar 

  38. 38

    Wang, L., Murthy, S.K., Barabino, G.A. & Carrier, R.L. Synergic effects of crypt-like topography and ECM proteins on intestinal cell behavior in collagen based membranes. Biomaterials 31, 7586–7598 (2010).

    CAS  Article  Google Scholar 

  39. 39

    Puleo, C.M., McIntosh Ambrose, W., Takezawa, T., Elisseeff, J. & Wang, T.H. Integration and application of vitrified collagen in multilayered microfluidic devices for corneal microtissue culture. Lab Chip 9, 3221–3227 (2009).

    CAS  Article  Google Scholar 

  40. 40

    Chung, S. et al. Cell migration into scaffolds under co-culture conditions in a microfluidic platform. Lab Chip 9, 269–275 (2009).

    CAS  Article  Google Scholar 

  41. 41

    Shin, Y. et al. Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels. Nat. Protoc. 7, 1247–1259 (2012).

    CAS  Article  Google Scholar 

  42. 42

    Sung, K.E. et al. Control of 3-dimensional collagen matrix polymerization for reproducible human mammary fibroblast cell culture in microfluidic devices. Biomaterials 30, 4833–4841 (2009).

    CAS  Article  Google Scholar 

  43. 43

    Sung, K.E. et al. Transition to invasion in breast cancer: a microfluidic in vitro model enables examination of spatial and temporal effects. Integr. Biol. 3, 439–450 (2011).

    CAS  Article  Google Scholar 

  44. 44

    Zervantonakis, I.K., Kothapalli, C.R., Chung, S., Sudo, R. & Kamm, R.D. Microfluidic devices for studying heterotypic cell-cell interactions and tissue specimen cultures under controlled microenvironments. Biomicrofluidics 5, 13406 (2011).

    Article  Google Scholar 

  45. 45

    Achyuta, A.K. et al. A modular approach to create a neurovascular unit-on-a-chip. Lab Chip 13, 542–553 (2013).

    CAS  Article  Google Scholar 

  46. 46

    Park, J., Koito, H., Li, J. & Han, A. Multi-compartment neuron-glia co-culture platform for localized CNS axon-glia interaction study. Lab Chip 12, 3296–3304 (2012).

    CAS  Article  Google Scholar 

  47. 47

    Prabhakarpandian, B. et al. SyM-BBB: a microfluidic Blood Brain Barrier model. Lab Chip 13, 1093–1101 (2013).

    CAS  Article  Google Scholar 

  48. 48

    Ma, S.H., Lepak, L.A., Hussain, R.J., Shain, W. & Shuler, M.L. An endothelial and astrocyte co-culture model of the blood-brain barrier utilizing an ultra-thin, nanofabricated silicon nitride membrane. Lab Chip 5, 74–85 (2005).

    CAS  Article  Google Scholar 

  49. 49

    Allen, J.W. & Bhatia, S.N. Formation of steady-state oxygen gradients in vitro: application to liver zonation. Biotechnol. Bioeng. 82, 253–262 (2003).

    CAS  Article  Google Scholar 

  50. 50

    Kim, S.H., Kang, J.H., Chung, I.Y. & Chung, B.G. Mucin (MUC5AC) expression by lung epithelial cells cultured in a microfluidic gradient device. Electrophoresis 32, 254–260 (2011).

    Article  Google Scholar 

  51. 51

    Torisawa, Y.S. et al. Microfluidic platform for chemotaxis in gradients formed by CXCL12 source-sink cells. Integr. Biol. 2, 680–686 (2010).

    CAS  Article  Google Scholar 

  52. 52

    Douville, N.J. et al. Combination of fluid and solid mechanical stresses contribute to cell death and detachment in a microfluidic alveolar model. Lab Chip 11, 609–619 (2011).

    CAS  Article  Google Scholar 

  53. 53

    Huh, D. et al. Acoustically detectable cellular-level lung injury induced by fluid mechanical stresses in microfluidic airway systems. Proc. Natl. Acad. Sci. USA 104, 18886–18891 (2007).

    CAS  Article  Google Scholar 

  54. 54

    Huh, D. et al. Reconstituting organ-level lung functions on a chip. Science 328, 1662–1668 (2010).

    CAS  Article  Google Scholar 

  55. 55

    Huh, D. et al. A human disease model of drug toxicity-induced pulmonary edema in a lung-on-a-chip microdevice. Sci. Transl. Med. 4, 159ra147 (2012).

    Article  Google Scholar 

  56. 56

    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).

    CAS  Article  Google Scholar 

  57. 57

    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).

    CAS  Article  Google Scholar 

  58. 58

    Ochs, M. et al. The number of alveoli in the human lung. Am. J. Respir. Crit. Care Med. 169, 120–124 (2004).

    Article  Google Scholar 

  59. 59

    Toepke, M.W. & Beebe, D.J. PDMS absorption of small molecules and consequences in microfluidic applications. Lab Chip 6, 1484–1486 (2006).

    CAS  Article  Google Scholar 

  60. 60

    Wang, J.D., Douville, N.J., Takayama, S. & ElSayed, M. Quantitative analysis of molecular absorption into PDMS microfluidic channels. Ann. Biomed. Eng. 40, 1862–1873 (2012).

    Article  Google Scholar 

  61. 61

    Gomez-Sjoberg, R., Leyrat, A.A., Houseman, B.T., Shokat, K. & Quake, S.R. Biocompatibility and reduced drug absorption of sol-gel-treated poly(dimethyl siloxane) for microfluidic cell culture applications. Anal. Chem. 82, 8954–8960 (2010).

    CAS  Article  Google Scholar 

  62. 62

    Borysiak, M.D. et al. Simple replica micromolding of biocompatible styrenic elastomers. Lab Chip 13, 2773–2784 (2013).

    CAS  Article  Google Scholar 

  63. 63

    Domansky, K. et al. Clear castable polyurethane elastomer for fabrication of microfluidic devices. Lab Chip 13, 3956–3964 (2013).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank D. Levner and C. Hinojosa for their assistance in preparing the protocols for device fabrication. This work was supported by the Wyss Institute for Biologically Inspired Engineering at Harvard University and grants from US National Institutes of Health (NIH) NIEHS (ES016665-01A1) and the NIH Common Fund (U01 NS073474) through the Division of Program Coordination, Planning, and Strategic Initiatives (DPCPSI), Office of the Director, NIH and the US Food and Drug Administration (FDA). Additional funds were provided by the Defense Advanced Research Projects Agency (DARPA) under Cooperative Agreement Number W911NF-12-2-0036, and FDA contract HHSF223201310079C. The content of the information does not necessarily reflect the position or the policy of DARPA or the US Government, and no official endorsement should be inferred.

Author information

Affiliations

Authors

Contributions

D.H. led development of the lung-on-a-chip, performed experiments, analyzed data and prepared the manuscript. H.J.K. led development of the gut-on-a-chip, performed experiments, analyzed data and contributed to preparation of the manuscript. J.P.F., D.E.S., M.K., A.B. and G.A.H. provided assistance in experiments, data analysis and manuscript preparation. D.E.I. led the organ-on-chip effort, assisted in experimental design and analysis and helped in writing of the manuscript.

Corresponding authors

Correspondence to Dongeun Huh or Donald E Ingber.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Huh, D., Kim, H., Fraser, J. et al. Microfabrication of human organs-on-chips. Nat Protoc 8, 2135–2157 (2013). https://doi.org/10.1038/nprot.2013.137

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

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