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Open-Chamber Co-Culture Microdevices for Single-Cell Analysis of Skeletal Muscle Myotubes and Motor Neurons with Neuromuscular Junctions

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Abstract

Degeneration of motor neurons and skeletal muscles or the collapse of neuromuscular junctions (NMJs) causes progressive motility disturbances in many neuromuscular diseases. Although various microdevices for the co-culture of skeletal muscle myotubes and motor neurons have been developed to investigate neuromuscular diseases in vitro, it remains difficult to isolate single myotubes and motor neurons from the device for single-cell analyses, such as gene expression analysis. Here, we developed open chamber-coculture microdevices that contain cell culture chambers with narrow widths. Given the small chamber width (0.2 mm), the device significantly prevented the overlap among myotubes within the chamber. The percentage of non-overlapping was 95.6 ± 7.7% for the 0.2-mmwidth chamber and 11.8 ± 6.4% for the 7-mm-width chamber as a control. In addition, the device with the 0.2-mm chamber promoted myotube maturation, as indicated by the longer widths and lengths of the myotubes relative to those in the control chamber. Single C2C12 myotubes and human induced pluripotent stem cell (hiPSC)-derived motor neurons were successfully collected from the device with the 0.2-mm chamber using a micromanipulator equipped with a glass capillary. Furthermore, myotubes and hiPSC-derived motor neurons were co-cultured in the device with the 0.2- mm chamber, and the formation of NMJs were observed. Thus, the developed device is a useful tool for performing single-cell analysis for studying neuromuscular diseases in vitro.

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References

  1. Witzemann, V. Development of the neuromuscular junction. Cell Tissue Res. 326, 263–271 (2006).

    Article  Google Scholar 

  2. Darabid, H., Perez-Gonzalez, A.P. and Robitaille, R. Neuromuscular synaptogenesis: coordinating partners with multiple functions. Nat. Rev. Neurosci. 15, 703–718 (2014).

    Article  CAS  Google Scholar 

  3. Fischer, L.R., Culver, D.G., Tennant, P., Davis, A. A., Wang, M.S., Castellano-Sanchez, A., Khan, J., Polak, M.A. and Glass, J.D. Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and man. Exp. Neurol. 185, 232–240 (2004).

    Article  Google Scholar 

  4. Wong, M. and Martin, L.J. Skeletal muscle-restricted expression of human SOD1 causes motor neuron degeneration in transgenic mice. Hum. Mol. Genet. 19, 2284–2302 (2010).

    Article  CAS  Google Scholar 

  5. Robitaille, R., Garcia, M.L., Kaczorowski, G.J. and Charlton, M.P. Functional colocalization of calcium and calcium-gated potassium channels in control of transmitter release. Neuron 11, 645–655 (1993).

    Article  CAS  Google Scholar 

  6. Gurney, M., Pu, H., Chiu, A., Dal Canto, M., Polchow, C., Alexander, D., Caliendo, J., Hentati, A., Kwon, Y., Deng, H. and et al. Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. Sciences (N. Y.) 264, 1772–1775 (1994).

    Article  CAS  Google Scholar 

  7. He, X.P., Yang, F., Xie, Z.P. and Lu, B. Intracellular Ca2+ and Ca2+/calmodulin-dependent kinase II mediate acute potentiation of neurotransmitter release by neurotrophin-3. J. Cell Biol. 149, 783–791 (2000).

    Article  CAS  Google Scholar 

  8. Guo, X.F., Gonzalez, M., Stancescu, M., Vandenburgh, H.H. and Hickman, J.J. Neuromuscular junction formation between human stem cell-derived motoneurons and human skeletal muscle in a defined system. Biomaterials 32, 9602–9611 (2011).

    Article  CAS  Google Scholar 

  9. Das, M., Rumsey, J.W., Bhargava, N., Stancescu, M. and Hickman, J.J. A defined long-term in vitro tissue engineered model of neuromuscular junctions. Biomaterials 31, 4880–4888 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Tong, Z., Seira, O., Casas, C., Reginensi, D., Homs-Corbera, A., Samitier, J. and Antonio Del Rio, J. Engineering a functional neuro-muscular junction model in a chip. RSC Adv. 4, 54788–54797 (2014).

    Article  CAS  Google Scholar 

  12. Shimizu, K., Genma, R., Gotou, Y., Nagasaka, S. and Honda, H. Three-Dimensional Culture Model of Skeletal Muscle Tissue with Atrophy Induced by Dexamethasone. Bioengineering 4, (2017).

  13. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K. and Yamanaka, S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    Article  CAS  Google Scholar 

  14. Nagashima, T., Shimizu, K., Matsumoto, R. and Honda, H. Selective Elimination of Human Induced Pluripotent Stem Cells Using Medium with High Concentration of L-Alanine. Sci. Rep. 8, 12427 (2018).

    Article  Google Scholar 

  15. Shimojo, D., Onodera, K., Doi-Torii, Y., Ishihara, Y., Hattori, C., Miwa, Y., Tanaka, S., Okada, R., Ohyama, M., Shoji, M., Nakanishi, A., Doyu, M., Okano, H. and Okada, Y. Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells. Mol. Brain 8, 79 (2015).

    Article  Google Scholar 

  16. Arai, S., Okochi, M., Shimizu, K., Hanai, T. and Honda, H. A single cell culture system using lectin-conjugated magnetite nanoparticles and magnetic force to screen mutant cyanobacteria. Biotechnol. Bioeng. 113, 112–119 (2016).

    Article  CAS  Google Scholar 

  17. Shimizu, K., Fujita, H. and Nagamori, E. Micropatterning of single myotubes on a thermoresponsive culture surface using elastic stencil membranes for single-cell analysis. J. Biosci. Bioeng. 109, 174–178 (2010).

    Article  CAS  Google Scholar 

  18. Velleman, S.G. and McFarland, D.C. Myotube morphology, and expression and distribution of collagen type I during normal and low score normal avian satellite cell myogenesis. Dev., Growth Differ. 41, 153–161 (1999).

    Article  CAS  Google Scholar 

  19. Bettadapur, A., Suh, G.C., Geisse, N.A., Wang, E.R., Hua, C., Huber, H.A., Viscio, A.A., Kim, J.Y., Strickland, J.B. and McCain, M.L. Prolonged Culture of Aligned Skeletal Myotubes on Micromolded Gelatin Hydrogels. Sci. Rep. 6, 28855 (2016).

    Article  CAS  Google Scholar 

  20. Ostrovidov, S., Ahadian, S., Ramon-Azcon, J., Hosseini, V., Fujie, T., Parthiban, S.P., Shiku, H., Matsue, T., Kaji, H., Ramalingam, M., Bae, H. and Khademhosseini, A. Three-dimensional co-culture of C2C12/PC12 cells improves skeletal muscle tissue formation and function. J. Tissue Eng. Regener. Med. 11, 582–595 (2017).

    Article  CAS  Google Scholar 

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Acknowledgements

This research was supported in part by the Japan Society for the Promotion of Science (grant numbers 26630429 and 18H01796).

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Authors and Affiliations

  1. Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Nagoya, 464-8603, Japan

    Nao Yamaoka, Kazunori Shimizu, Yu Imaizumi & Hiroyuki Honda

  2. Department of Neurology, Aichi Medical University School of Medicine, Aichi, 480-1195, Japan

    Takuji Ito & Yohei Okada

  3. Innovative Research Center for Preventive Medical Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan

    Hiroyuki Honda

Authors
  1. Nao Yamaoka
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  2. Kazunori Shimizu
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  3. Yu Imaizumi
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  4. Takuji Ito
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  5. Yohei Okada
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  6. Hiroyuki Honda
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Correspondence to Kazunori Shimizu.

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Yamaoka, N., Shimizu, K., Imaizumi, Y. et al. Open-Chamber Co-Culture Microdevices for Single-Cell Analysis of Skeletal Muscle Myotubes and Motor Neurons with Neuromuscular Junctions. BioChip J 13, 127–132 (2019). https://doi.org/10.1007/s13206-018-3202-3

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  • DOI: https://doi.org/10.1007/s13206-018-3202-3

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