Advances in 3D neural cell culture by means of microsieve electrode arrays
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1
Eindhoven University of Technology, Netherlands
Single cell analysis is of paramount importance in cell biology. Cell populations are heterogeneous and the detection of subpopulations of cells and cellular events at the single cell level are challenging in every day cell culture. In particular, the contribution of a single neuron within a network is hard to elucidate and can have dramatic impact on network function resulting in diseases [1]. We have contributed to this exciting field by the development of a fabrication process for electrically functionalized micro sieves using silicon micromachining that contains highly uniform 3D-micropores (Figure 1) that allow us to capture hundreds of individual neurons in parallel and directing and pairing of these neurons onto arrays of single electrodes [2]. This new silicon-based technology platform for multi-site electrophysiology recordings was termed µSieve Electrode Array (µSEA) and it facilitates the 3D morphology of a neuron being paired to an electrode. We demonstrate that capillary pumping across a microsieve generates gentle cell trapping velocities (< 13.3μm/s), enabling reproducible neuron (SH-SY5Y neuroblastomas cells differentiated into neuron by retinoic acid exposure) trapping efficiencies of 80% with high cell survival rates (90% over 1 week of culture) and allowing the formation of spatially standardized neuronal networks [3] (Figure 2). Growing neurons in a spatially standardized fashion (i.e., arrays) can also ease the analysis of neurite connectivity (which is a hallmark neurodevelopmental end point indicator) and will lead to an easier method for relating changes in connectivity to electrophysiology and biological function. Although the electrical component of the µSEA device is not yet optimal for electrical recordings of neurons, the communication between adjacent single neurons trapped within the sieves we can also study network behavior through calcium imaging by loading neurons with a Ca-sensitive fluorescent dye [4]. Events relating to the release of calcium-like inter-neuronal signaling can be subsequently tracked by fluorescent microscopy. The fluorescent neuron activity can be recorded into peaks for each neuron and a spatiotemporal map can be generated of active neurons within a network. We demonstrate here as a proof of principle that single neuron activity can be dynamically analysed within an arrayed network and cell to cell networking can be established based on calcium imaging footage (Figure 3). This is obtained by comparing the neuron blinking patterns of single neurons trapped inside the microsieve in time and identifying which neurons blink in the same time frame, therefore linking them together. In this way, neurons blinking in quick succession and in close proximity can be mapped paving the road for dynamic single neuron interrogation within an arrayed neuronal network in vitro as a model for distinguished brain functions.
Figure 1: The microsieve electrode array (µSEA). Overall image of the µSEA with patterned and boron doped poly-silicon (A). The poly-silicon pattern forms the electrode layer consisting of contact electrodes and lead wires (scale bar 200 µm). A cross section of a 3-D micropores with a thickness of approximately 16 µm, top micropore opening of 20 μm and bottom apertures of 3.2 μm (B) (scale bar 5 µm) [2].
Figure 2: Result of the trapping of neurons by capillary pumping on microsieves. Following 7 days in culture, neurons connected the micropores. An example with 100% filling efficiency is shown (A) as well as a close up of 16 micropores connected by neuron outgrowths.
Figure 3: Calcium imaging of neurons trapped by capillary pumping on the silicon µSEA. Neurons fluorescently labelled with calcium dye and location of calcium measurements (A). The resulting spatio-temporal map and neuronal network structure construction (B). Neuronal network structure construction. Fluorescent peaks from the detected cells in (A) are correlated between all the neurons over time for network structure detection. Synchronized peak detection will be interconnected and networked as shown by red lines (B).
References
[1] Silva da F.H.L., Blanes W., Klitzin S. N., Parra J., Suffczynski P., Velis D.N. (2003). Dynamical diseases of brain systems: different routes to epileptic seizures, IEEE Trans. Biomed. Eng. 50 (5), 540-8. DOI:10.1109/TBME.2003.810703.
[2] Schurink B., Tiggelaar R.M., Gardeniers J.G.E., Luttge R. (2017). Fabrication and characterization of microsieve electrode array (μSEA) enabling cell positioning on 3D electrodes. J. Micromech. Microeng. 27 (015017). doi:10.1088/0960-1317/27/1/015017.
[3] Frimat J.P., Schurink B., Luttge R. (2017). Passive pumping for the parallel trapping of single neurons onto microsieve electrode array. J. Vac. Sci. & Tech. B, 35 (6), 06GA01. doi: 10.1116/1.4991827.
[4] Romano S.A., Pérez-Schuster V., Jouary A., Boulanger-Weill J., Candeo A., Pietri T., Sumbre G. (2017). An integrated calcium imaging processing toolbox for the analysis of neuronal population dynamics. PLoS Comput Biol 13(6): e1005526. https://doi.org/10.1371/journal.pcbi.1005526.
Keywords:
neuronal networks,
Single neuron analysis,
microsieve electrode array,
calcium imaging,
Lab on-a-Chip
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:
Frimat
J and
Luttge
R
(2019). Advances in 3D neural cell culture by means of microsieve electrode arrays.
Conference Abstract:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays.
doi: 10.3389/conf.fncel.2018.38.00075
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Received:
18 Mar 2018;
Published Online:
17 Jan 2019.
*
Correspondence:
Dr. Jean-Philippe Frimat, Eindhoven University of Technology, Eindhoven, Netherlands, j.m.s.frimat@tue.nl