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.

  • Protocol
  • Published:

On-chip 3D neuromuscular model for drug screening and precision medicine in neuromuscular disease

Nature Protocols volume 15, pages 421–449 (2020)Cite this article

Subjects

Abstract

This protocol describes the design, fabrication and use of a 3D physiological and pathophysiological motor unit model consisting of motor neurons coupled to skeletal muscles interacting via the neuromuscular junction (NMJ) within a microfluidic device. This model facilitates imaging and quantitative functional assessment. The ‘NMJ chip’ enables real-time, live imaging of axonal outgrowth, NMJ formation and muscle maturation, as well as synchronization of motor neuron activity and muscle contraction under optogenetic control for the study of normal physiological events. The proposed protocol takes ~2–3 months to be implemented. Pathological behaviors associated with various neuromuscular diseases, such as regression of motor neuron axons, motor neuron death, and muscle degradation and atrophy can also be recapitulated in this system. Disease models can be created by the use of patient-derived induced pluripotent stem cells to generate both the motor neurons and skeletal muscle cells used. This is demonstrated by the use of cells from a patient with sporadic amyotrophic lateral sclerosis but can be applied more generally to models of neuromuscular disease, such as spinal muscular atrophy, NMJ disorder and muscular dystrophy. Models such as this hold considerable potential for applications in precision medicine, drug screening and disease risk assessment.

Access through your institution
Buy or subscribe

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

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Fig. 1
Fig. 2: Fabrication of an NMJ chip.
Fig. 3: Schematic illustration of the differentiation and co-culture of the MN spheroid and muscle cells in an NMJ chip.
Fig. 4: Schematic illustrating device coating and injection of skeletal muscle cells and the MN spheroid into an NMJ chip.
Fig. 5: Setup for optical stimulation.
Fig. 6: Calculation of muscle contractile force from pillar deflection.
Fig. 7: NMJ formation and its failure.

Similar content being viewed by others

Data availability

The data that support the findings of this study are available from the corresponding author on reasonable request.

References

  1. Nageshwaran, S., Davies, L. M., Rafi, I. & Radunović, A. Motor neurone disease. BMJ 349, g4052 (2014).

  2. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Metzker, M. L. Sequencing technologies—the next generation. Nat. Rev. Genet. 11, 31–46 (2010).

    Article  CAS  PubMed  Google Scholar 

  4. Andersen, J. K. Oxidative stress in neurodegeneration: cause or consequence? Nat. Med. 10, S18–S25 (2004).

    Article  PubMed  CAS  Google Scholar 

  5. Sreedharan, J. et al. TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science 319, 1668–1672 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Williams, K. L. et al. Pathophysiological insights into ALS with C9ORF72 expansions. J. Neurol. Neurosurg. Psychiatry 84, 931–935 (2013).

    Article  PubMed  Google Scholar 

  7. DeJesus-Hernandez, M. et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72, 245–256 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Giuliano, K. A., Haskins, J. R. & Taylor, D. L. Advances in high content screening for drug discovery. Assay. Drug Dev. Technol. 1, 565–577 (2003).

    Article  CAS  PubMed  Google Scholar 

  9. Egawa, N. et al. Drug screening for ALS using patient-specific induced pluripotent stem cells. Sci. Transl. Med. 4, 145ra104 (2012).

    Article  PubMed  CAS  Google Scholar 

  10. Fitzsimonds, R. M. & Poo, M.-M. Retrograde signaling in the development and modification of synapses. Physiol. Rev. 78, 143–170 (1998).

    Article  CAS  PubMed  Google Scholar 

  11. Wyatt, R. M. & Balice-Gordon, R. J. Activity-dependent elimination of neuromuscular synapses. J. Neurocytol. 32, 777–794 (2003).

    Article  CAS  PubMed  Google Scholar 

  12. Uzel, S. G. et al. Microfluidic device for the formation of optically excitable, three-dimensional, compartmentalized motor units. Sci. Adv. 2, e1501429 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Osaki, T., Uzel, S. G. M. & Kamm, R. D. Microphysiological 3D model of amyotrophic lateral sclerosis (ALS) from human iPS-derived muscle cells and optogenetic motor neurons. Sci. Adv. 4, eaat5847 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Park, J. W., Vahidi, B., Taylor, A. M., Rhee, S. W. & Jeon, N. L. Microfluidic culture platform for neuroscience research. Nat. Protoc. 1, 2128–2136 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. Qin, D., Xia, Y. & Whitesides, G. M. Soft lithography for micro- and nanoscale patterning. Nat. Protoc. 5, 491–502 (2010).

    Article  CAS  PubMed  Google Scholar 

  16. Sakar, M. S. et al. Formation and optogenetic control of engineered 3D skeletal muscle bioactuators. Lab Chip 12, 4976–4985 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Lunn, M. R. & Wang, C. H. Spinal muscular atrophy. Lancet 371, 2120–2133 (2008).

    Article  PubMed  Google Scholar 

  18. Jaretzki, A. et al. Myasthenia gravis: recommendations for clinical research standards. Task Force of the Medical Scientific Advisory Board of the Myasthenia Gravis Foundation of America. Neurology 55, 16–23 (2000).

    Article  PubMed  Google Scholar 

  19. Vila, O. F. et al. Quantification of human neuromuscular function through optogenetics. Theranostics 9, 1232–1246 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Emery, A. E. H. The muscular dystrophies. Lancet 359, 687–695 (2002).

    Article  CAS  PubMed  Google Scholar 

  21. Bleicher, K. H., Böhm, H.-J., Müller, K. & Alanine, A. I. Hit and lead generation: beyond high-throughput screening. Nat. Rev. Drug Discov. 2, 369–378 (2003).

    Article  CAS  PubMed  Google Scholar 

  22. Mahmood, T. & Yang, P.-C. Western blot: technique, theory, and trouble shooting. N. Am. J. Med. Sci. 4, 429–434 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Nolan, T., Hands, R. E. & Bustin, S. A. Quantification of mRNA using real-time RT-PCR. Nat. Protoc. 1, 1559–1582 (2006).

    Article  CAS  PubMed  Google Scholar 

  24. Ran, F. A. et al. Genome engineering using the CRISPR–Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Brooks, B. R. Natural history of ALS: symptoms, strength, pulmonary function, and disability. Neurology 47, 71S–82S (1996).

    Article  Google Scholar 

  26. Chen, Y. et al. The relationship between four GWAS-identified loci in Alzheimer’s disease and the risk of Parkinson’s disease, amyotrophic lateral sclerosis, and multiple system atrophy. Neurosci. Lett. 686, 205–210 (2018).

    Article  CAS  PubMed  Google Scholar 

  27. Laing, N. G. Genetics of neuromuscular disorders. Crit. Rev. Clin. Lab Sci. 49, 33–48 (2012).

    Article  CAS  PubMed  Google Scholar 

  28. Rudnick, N. D. et al. Distinct roles for motor neuron autophagy early and late in the SOD1G93A mouse model of ALS. Proc. Natl Acad. Sci. USA 114, E8294–E8303 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Lutz, C. Mouse models of ALS: past, present and future. Brain Res. 1693, 1–10 (2018).

    Article  CAS  PubMed  Google Scholar 

  30. Hsieh-Li, H. M. et al. A mouse model for spinal muscular atrophy. Nat. Genet. 24, 66–70 (2000).

    Article  CAS  PubMed  Google Scholar 

  31. Daub, A., Sharma, P. & Finkbeiner, S. High-content screening of primary neurons: ready for prime time. Curr. Opin. Neurobiol. 19, 537–543 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Ribas, J., Pawlikowska, J. & Rouwkema, J. Microphysiological systems: analysis of the current status, challenges and commercial future. Microphysiol. Syst. 2, 10 (2018).

  33. Southam, K. A., King, A. E., Blizzard, C. A., McCormack, G. H. & Dickson, T. C. Microfluidic primary culture model of the lower motor neuron-neuromuscular junction circuit. J. Neurosci. Methods 218, 164–169 (2013).

    Article  PubMed  Google Scholar 

  34. Ionescu, A., Zahavi, E. E., Gradus, T., Ben-Yaakov, K. & Perlson, E. Compartmental microfluidic system for studying muscle-neuron communication and neuromuscular junction maintenance. Eur. J. Cell Biol. 95, 69–88 (2016).

    Article  CAS  PubMed  Google Scholar 

  35. Fujimori, K. et al. Modeling sporadic ALS in iPSC-derived motor neurons identifies a potential therapeutic agent. Nat. Med. 24, 1579–1589 (2018).

    Article  CAS  PubMed  Google Scholar 

  36. Smith, A. S. T., Long, C. J., Pirozzi, K. & Hickman, J. J. A functional system for high-content screening of neuromuscular junctions in vitro. Technology 1, 37–48 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Yizhar, O., Fenno, L. E., Davidson, T. J., Mogri, M. & Deisseroth, K. Optogenetics in neural systems. Neuron 71, 9–34 (2011).

    Article  CAS  PubMed  Google Scholar 

  38. Maffioletti, S. M. et al. Three-dimensional human iPSC-derived artificial skeletal muscles model muscular dystrophies and enable multilineage tissue engineering. Cell Rep. 23, 899–908 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Afshar Bakooshli, M. et al. A 3D culture model of innervated human skeletal muscle enables studies of the adult neuromuscular junction. eLife 8, e44530 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Wilson, M. H. & Deschenes, M. R. The neuromuscular junction: anatomical features and adaptations to various forms of increased, or decreased neuromuscular activity. Int. J. Neurosci. 115, 803–828 (2005).

    Article  PubMed  Google Scholar 

  41. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  PubMed  Google Scholar 

  42. Gilestro, G. F. Video tracking and analysis of sleep in Drosophila melanogaster. Nat. Protoc. 7, 995–1007 (2012).

    Article  CAS  PubMed  Google Scholar 

  43. Afshar, M. E. et al. A 96-well culture platform enables longitudinal analyses of engineered human skeletal muscle microtissue strength. Preprint at https://www.biorxiv.org/content/10.1101/562819v1 (2019).

  44. Davie, J. T. et al. Dendritic patch-clamp recording. Nat. Protoc. 1, 1235–1247 (2006).

    Article  CAS  PubMed  Google Scholar 

  45. Spira, M. E. & Hai, A. Multi-electrode array technologies for neuroscience and cardiology. Nat. Nanotechnol. 8, 83–94 (2013).

    Article  CAS  PubMed  Google Scholar 

  46. Pearce, T. M., Wilson, J. A., Oakes, S. G., Chiu, S.-Y. & Williams, J. C. Integrated microelectrode array and microfluidics for temperature clamp of sensory neurons in culture. Lab Chip 5, 97–101 (2005).

    Article  CAS  PubMed  Google Scholar 

  47. Hochbaum, D. R. et al. All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins. Nat. Methods 11, 825–833 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Scanziani, M. & Häusser, M. Electrophysiology in the age of light. Nature 461, 930–939 (2009).

    Article  CAS  PubMed  Google Scholar 

  49. Jeong, J.-W. et al. Wireless optofluidic systems for programmable in vivo pharmacology and optogenetics. Cell 162, 662–674 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Psaltis, D., Quake, S. R. & Yang, C. Developing optofluidic technology through the fusion of microfluidics and optics. Nature 442, 381–386 (2006).

    Article  CAS  PubMed  Google Scholar 

  51. Albert-Smet, I. et al. Applications of light-sheet microscopy in microdevices. Front. Neuroanat. 13, 1 (2019).

  52. Compston, A. & Coles, A. Multiple sclerosis. Lancet 372, 1502–1517 (2008).

    Article  CAS  PubMed  Google Scholar 

  53. Montanez-Sauri, S. I., Sung, K. E., Puccinelli, J. P., Pehlke, C. & Beebe, D. J. Automation of three-dimensional cell culture in arrayed microfluidic devices. J. Lab. Autom. 16, 171-185 (2011).

  54. Chu, L. & Robinson, D. K. Industrial choices for protein production by large-scale cell culture. Curr. Opin. Biotechnol. 12, 180–187 (2001).

    Article  CAS  PubMed  Google Scholar 

  55. Miller, J. D. et al. Human iPSC-based modeling of late-onset disease via progerin-induced aging. Cell Stem Cell 13, 691–705 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Ziff, O. J. & Patani, R. Harnessing cellular aging in human stem cell models of amyotrophic lateral sclerosis. Aging Cell 18, e12862 (2019).

    Article  PubMed  CAS  Google Scholar 

  57. Hargus, G. et al. Origin-dependent neural cell identities in differentiated human iPSCs in vitro and after transplantation into the mouse brain. Cell Rep. 8, 1697–1703 (2014).

    Article  CAS  PubMed  Google Scholar 

  58. Mao, T. et al. Long-range neuronal circuits underlying the interaction between sensory and motor cortex. Neuron 72, 111–123 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Lake, M. et al. Microfluidic device design, fabrication, and testing protocols. Protocol Exchange https://doi.org/10.1038/protex.2015.069 (2015).

Download references

Acknowledgements

AAV-CAG-hChR2H134R-tdTomato was a gift from K. Svoboda at Howard Hughes Medical Institute (Addgene viral prep no. 28017-AAVrg; http://n2t.net/addgene:28017; RRID: Addgene_28017). pMD2.G was a gift from D. Trono at EPLF (Addgene plasmid no. 12259; http://n2t.net/addgene:12259; RRID: Addgene_12259). psPAX2 was a gift from D. Trono (Addgene plasmid no. 12260; http://n2t.net/addgene:12260; RRID: Addgene_12260). The pLEX307-EF1a-ChR2[H134R]-mCherry-Puro-WPRE plasmid can be obtained from Addgene (no. 125256). T.O. was supported by an overseas research fellowship (from the Japan Society for the Promotion of Science). T.O., S.G.M.U. and R.D.K. also acknowledge support from the National Science Foundation for a Science and Technology Center on Emergent Behaviors of Integrated Cellular Systems grant (CBET-0939511) and partially from the Cancer Center Support (core) grant P30-CA14051 from the NCI.

Author information

Authors and Affiliations

  1. Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Tatsuya Osaki & Roger D. Kamm

  2. Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA, USA

    Sebastien G. M. Uzel

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

    Sebastien G. M. Uzel

  4. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Roger D. Kamm

  5. Koch Institute for Integrative Cancer Research, Cambridge, MA, USA

    Roger D. Kamm

Authors
  1. Tatsuya Osaki
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Sebastien G. M. Uzel
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Roger D. Kamm
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

T.O., S.G.M.U. and R.D.K. conceived and designed the study. T.O. conducted the experiment, and collected and analyzed the data. T.O. and S.G.M.U. designed the microfluidic devices for the human NMJ model. T.O. modified the microfluidic devices for the human NMJ model. T.O. prepared entire procedures and all of the figures. All authors wrote and revised the manuscript.

Corresponding author

Correspondence to Roger D. Kamm.

Ethics declarations

Competing interests

A part of this study was funded by Biogen Inc.

Additional information

Peer review information Nature Protocols thanks Eran Perlson, Francesco Saverio Tedesco and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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

Related links

Key references using this protocol

Osaki, T., Uzel, S. G. M. & Kamm, R. D. Sci. Adv. 4, eaat5847 (2018): https://doi.org/10.1126/sciadv.aat5847.

Uzel, S. G. et al. Sci. Adv. 2, e1501429 (2016): https://doi.org/10.1126/sciadv.1501429.

Vila, O. F. et al. Theranostics 9, 1232–1246 (2019): https://doi.org/10.7150/thno.25735.

Supplementary information

Supplementary Data 1

Photomask-1 for photolithography (Step 5)

Reporting summary

Supplementary Data 2

Photomask-2 for photolithography (Step 11)

Supplementary Data 3

Source code of TTL control for optogenetics in Arduino

Supplementary Data 4

Description: ImageJ macro for calculating pillar deflection by Method A (Steps 77B)

Supplementary Data 5

ImageJ macro for calculating pillar deflection by Method B (Steps 77B)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Osaki, T., Uzel, S.G.M. & Kamm, R.D. On-chip 3D neuromuscular model for drug screening and precision medicine in neuromuscular disease. Nat Protoc 15, 421–449 (2020). https://doi.org/10.1038/s41596-019-0248-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-019-0248-1

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research