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

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.

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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.

Data availability

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

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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

Affiliations

Authors

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.

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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)

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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

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