Event Abstract

Assessing electrical activity of human neuronal cell culture networks grown on 3D fibrous scaffolds using different techniques: A comparative study

  • 1 Lund University, Institute of Clinical Sciences in Lund, Division of Ophthalmology, Sweden
  • 2 Lund University, Department of Biology, Unit of Functional Zoology, Sweden

Three-dimensional (3D) neural cell cultures will most likely be the future routine experimental platform in neuroscience research, judged by them being more in-vivo resembling than today’s widely used 2D neural cultures. The assessment of the functionality of neuronal cells in culture, i.e, the intrinsic property that neurons can initiate and propagate electrical signals, is essential. Hence, we here want to assess the functionality of 2D and 3D human neuronal cell cultures by using three different widely employed electrophysiological techniques, including single sharp microelectrodes, calcium imaging and multielectrode arrays (MEAs). A human neural progenitor cell (HNPCs) line, mitogen-expandable and multipotent, was cultured for up to 23 days in vitro (DIV) in differentiation medium (containing 1% fetal bovine serum) on 3D electrospun biodegradable poly-caprolactone (PCL) nanofibrous substrates, on 2D (flat) glass surfaces (control substrate), or directly seeded into a multielectrode device (MEA, Multichannel Systems). In selected cultures, induced pluripotent stem cell (iPSC, iNeuron, Cellular Dynamics)-derived neurons were utilized and cultured in a specific media on MEAs. The culture surfaces were either laminin-, polyethylene (PEI) coated, or non-coated. For sharp microelectrode recordings, a sharp electrode glass capillary filled with 1M KCl was used as the recording electrode, and to optically detect the calcium flux we used the Fluo-4 dye, a molecule that exhibit an increase in fluorescence upon binding to calcium. Calcium flux detection is an “indirect” measurement of electrical activity, as intracellular calcium concentration is correlated to neuronal physiological activity. Three different MEAs with titanium nitride (TiN) electrodes were tested. The electrodes were of either 10 µm or 30 µm in diameter, and the contact pads and tracks of the MEAs were of either titanium (Ti) or indium tin oxide (ITO). Cell cultures were also analyzed by using immunocytochemistry with glial-, neuronal and synaptic markers. Overall, we verified that the hNPCs differentiated into glial and neuronal cell lineages, and that cells matured into electrically active neurons. Calcium imaging provided some evidence of functional activity (spontaneous and induced with KCl)) on both 2D and 3D cultures, yet the experimental procedure takes a long period of time (over 3 hours), the recording time was over 10 min, and neuronal connectivity analysis is not sufficient. Recording of electrical activity using sharp microelectrodes was successfully achieved on 3D cultures, whereas no successful recordings could be made using 2D cultures as damage to the electrode frequently occurred. Although recording of electrical activity was achieved, this method only picks up local field potentials (small area). To assess functional neuronal connectivity and firing patterns, the MEA systems are advantageous comparative to sharp microelectrodes. Recording of 2D HNPC-derived neuronal cultures activity over time (0-23 days) was achieved using MEA when they were seeded directly on them, although sometimes, activity was not detected, most probably due to the insulation the glial cells create. Nevertheless, using iPSC-derived neuronal cultures electrical activity could be detected from 11 days onwards, and initial studies show that exposure to selected cultures to a toxic substance change the spiking pattern. Taken together, our early results show that electrical active 3D neuronal networks can be cultured on nanofibrous scaffolds, and their activity be recorded using sharp microelectrodes and calcium imaging. We believe that development of a system that allows direct, high-throughput and simultaneous recording of brain cells cultured on 3D artificial substrates will not only offer an attractive alternative for future research in the field of neuroscience, but also to pharmaceutical companies, as it could also be used as direct high-content screening tool during the pre-clinical drug developmental phase.


Cellevate AB is thanked for help regarding electrospinning. For financial support we gratefully thank Faculty of Medicine, Lund University, KMA, the fund for vision-disables in southern Sweden, O. Engqvist Foundation, The Royal Physiographic Society in Lund, E&O Johansson FOundation, C. Groschinsky Foundation, and NanoLund

Keywords: neural culture, 3D nanofibrous scaffolds, huma progenitor cells, Electrophysiological Techniques, calcium imaging, Sharp electrode, Multielectrode array (MEA)

Conference: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018.

Presentation Type: Poster Presentation

Topic: Neural Networks

Citation: Zalis MC, Nylander M, Enjin A, Stensmyr MC, Johansson P, O'Carroll D and Englund Johansson U (2019). Assessing electrical activity of human neuronal cell culture networks grown on 3D fibrous scaffolds using different techniques: A comparative study. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00048

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Received: 26 Mar 2018; Published Online: 17 Jan 2019.

* Correspondence: PhD. Marina C Zalis, Lund University, Institute of Clinical Sciences in Lund, Division of Ophthalmology, Lund, Sweden, marina.castro_zalis@med.lu.se