Skip to main content

Design and Fabrication of Miniaturized Neuronal Circuits on Microelectrode Arrays Using Agarose Hydrogel Micro-molding Technique

BioChip Journal volume 12, pages 193–201 (2018)Cite this article

Abstract

Dissociated neuronal cultures combined with planar-type microelectrode arrays (MEAs) have been used as a promising read-out platform for the application of cell-based biosensors. There are increasing interests in engineering neuronal cultures to form the desired network topology by surface micropatterning technology. Here, we report a long-term cultivation of primary hippocampal neurons on microelectrode arrays using soft-lithography. Ordered hippocampal neuronal networks were formed by seeding neurons in agarose-microwells and inducing neurite outgrowth through microgrooves. Unlike previous approaches, our technique allowed us to design networks with various microwells on microelectrode arrays with high repeatability. These hippocampal network chips were cultivated for 30 days with excellent pattern fidelity, and neural spikes were successfully measured. We also found that spontaneous activity of the networks could be enhanced by acute disinhibition of inhibitory synapses. The proposed patterning method for neuronal network chips will be a potentially powerful tool for cell-based drug-screening applications.

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

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price includes VAT (USA)
Tax calculation will be finalised during checkout.

References

  1. Bakkum, D.J., Chao, Z.C. & Potter, S.M. Spatio-temporal electrical stimuli shape behavior of an embodied cortical network in a goal-directed learning task. J. Neural Eng. 5, 310–323 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Droge, M.H., Gross, G.W., Hightower, M.H. & Czisny, L.E. Multielectrode analysis of coordinated, multisite, rhythmic bursting in cultured CNS monolayer networks. J. Neurosci. 6, 1583–1592 (1986).

    Article  CAS  PubMed  Google Scholar 

  3. Eytan, D. & Marom, S. Dynamics and effective topology underlying synchronization in networks of cortical neurons. J. Neurosci. 26, 8465–8476 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. Gross, G.W., Rhoades, B.K., Azzazy, H.M. & Wu, M.C. The use of neuronal networks on multielectrode arrays as biosensors. Biosens. Bioelectron. 10, 553–567 (1995).

    Article  CAS  PubMed  Google Scholar 

  5. Jimbo, Y., Robinson, H.P. & Kawana, A. Strengthening of synchronized activity by tetanic stimulation in cortical cultures: application of planar electrode arrays. IEEE Trans. Biomed. Eng. 45, 1297–1304 (1998).

    Article  CAS  PubMed  Google Scholar 

  6. Jimbo, Y., Tateno, T. & Robinson, H.P. Simultaneous induction of pathway-specific potentiation and depression in networks of cortical neurons. Biophys. J. 76, 670–678 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Marom, S. & Shahaf, G. Development, learning and memory in large random networks of cortical neurons: lessons beyond anatomy. Q. Rev. Biophys. 35, 63–87 (2002).

    Article  PubMed  Google Scholar 

  8. Shahaf, G. & Marom, S. Learning in networks of cortical neurons. J. Neurosci. 21, 8782–8788 (2001).

    Article  CAS  PubMed  Google Scholar 

  9. Wagenaar, D.A., Madhavan, R., Pine, J. & Potter, S.M. Controlling bursting in cortical cultures with closedloop multi-electrode stimulation. J. Neurosci. 25, 680–688 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Berdondini, L. et al. Active pixel sensor array for high spatio-temporal resolution electrophysiological recordings from single cell to large scale neuronal networks. Lab Chip 9, 2644–2651 (2009).

    Article  CAS  PubMed  Google Scholar 

  11. Eversmann, B. et al. A 128×128 CMOS biosensor array for extracellular recording of neural activity. IEEE J. Solid-State Circuits 38, 2306–2317 (2003).

    Article  Google Scholar 

  12. Frey, U., Egert, U., Heer, F., Hafizovic, S. & Hierlemann, A. Microelectronic system for high-resolution mapping of extracellular electric fields applied to brain slices. Biosens. Bioelectron. 24, 2191–2198 (2009).

    Article  CAS  PubMed  Google Scholar 

  13. Chang, J.C., Brewer, G.J. & Wheeler, B.C. Modulation of neural network activity by patterning. Biosens. Bioelectron. 16, 527–533 (2001).

    Article  CAS  PubMed  Google Scholar 

  14. Jun, S.B. et al. Low-density neuronal networks cultured using patterned poly-l-lysine on microelectrode arrays. J. Neurosci. Methods 160, 317–326 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Jungblut, M., Knoll, W., Thielemann, C. & Pottek, M. Triangular neuronal networks on microelectrode arrays: An approach to improve the properties of lowdensity networks for extracellular recording. Biomed. Microdevices 11, 1269–1278 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Marconi, E. et al. Emergent functional properties of neuronal networks with controlled topology. PLoS One 7, e34648 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Nam, Y., Branch, D.W. & Wheeler, B.C. Epoxy-silane linking of biomolecules is simple and effective for patterning neuronal cultures. Biosens. Bioelectron. 22, 589–597 (2006).

    Article  CAS  PubMed  Google Scholar 

  18. Vishwanathan, A., Bi, G.-Q. & Zeringue, H.C. Ringshaped neuronal networks: a platform to study persistent activity. Lab Chip 11, 1081–1088 (2011).

    Article  CAS  PubMed  Google Scholar 

  19. Suzuki, I., Sugio, Y., Jimbo, Y. & Yasuda, K. Stepwise pattern modification of neuronal network in photo-thermally-etched agarose architecture on multi-electrode array chip for individual-cell-based electrophysiological measurement. Lab Chip 5, 241–247 (2005).

    Article  CAS  PubMed  Google Scholar 

  20. Jimbo, Y., Robinson, H.P. & Kawana, A. Simultaneous measurement of intracellular calcium and electrical activity from patterned neural networks in culture. IEEE Trans. Biomed. Eng. 40, 804–810 (1993).

    Article  CAS  PubMed  Google Scholar 

  21. Kang, G., Lee, J.-H., Lee, C.-S. & Nam, Y. Agarose microwell based neuronal micro-circuit arrays on microelectrode arrays for high throughput drug testing. Lab Chip 9, 3236–3242 (2009).

    Article  CAS  PubMed  Google Scholar 

  22. Shein Idelson, M., Ben-Jacob, E. & Hanein, Y. Innate synchronous oscillations in freely-organized small neuronal circuits. PLoS One 5, e14443 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Shein, M. et al. Engineered neuronal circuits shaped and interfaced with carbon nanotube microelectrode arrays. Biomed. Microdevices 11, 495–501 (2009).

    Article  CAS  PubMed  Google Scholar 

  24. Sorkin, R. et al. Compact self-wiring in cultured neural networks. J. Neural Eng. 3, 95–101 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. Ferrarini, L. et al. Hierarchical functional modularity in the resting-state human brain. Hum. Brain Mapp. 30, 2220–2231 (2009).

    Article  PubMed  Google Scholar 

  26. Mountcastle, V.B. The columnar organization of the neocortex. Brain 120, 701–722 (1997).

    Article  PubMed  Google Scholar 

  27. Kato-Negishi, M., Tsuda, Y., Onoe, H. & Takeuchi, S. A neurospheroid network-stamping method for neural transplantation to the brain. Biomaterials 31, 8939–8945 (2010).

    Article  CAS  PubMed  Google Scholar 

  28. Pan, L., Alagapan, S., Franca, E., Brewer, G.J. & Wheeler, B.C. Propagation of action potential activity in a predefined microtunnel neural network. J. Neural Eng. 8, 46031 (2011).

    Article  Google Scholar 

  29. Kim, R., Hong, N. & Nam, Y. Gold nanograin microelectrodes for neuroelectronic interfaces. Biotechnol. J. 8, 206–214 (2013).

    Article  CAS  PubMed  Google Scholar 

  30. Joo, S., Kang, K. & Nam, Y. In vitro neurite guidance effects induced by polylysine pinstripe micropatterns with polylysine background. J. Biomed. Mater. Res. A 103, 2731–2739 (2015).

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Author notes

  1. These authors contributed equally to this work.

Authors and Affiliations

  1. Department Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea

    Sunghoon Joo, Jisoon Lim & Yoonkey Nam

Authors
  1. Sunghoon Joo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jisoon Lim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yoonkey Nam
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yoonkey Nam.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Joo, S., Lim, J. & Nam, Y. Design and Fabrication of Miniaturized Neuronal Circuits on Microelectrode Arrays Using Agarose Hydrogel Micro-molding Technique. BioChip J 12, 193–201 (2018). https://doi.org/10.1007/s13206-018-2308-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13206-018-2308-y

Keywords

  • Neuron patterning
  • Microelectrode arrays
  • Surface patterning
  • Micromolding in capillaries
  • Long-term culture