Human Embryonic Stem Cell Derived Neurospheres – 2D and 3D Cell Culture in one sample
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1
Aschaffenburg University of Applied Sciences, BioMEMS Lab, Germany
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2
GSI Helmholtzzentrum für Schwerionenforschung, Biophysics division, Germany
Abstract
Various studies have shown that two dimensional (2D) neuronal cell cultures does not recapitulate structure and physiology of three-dimensional (3D) in vivo tissues. These findings are of paramount importance for drug screening, since the response to neurotoxicological substances may differ for 2D und 3D cell culture models. To address this topic, we present human embryonic stem cell (hESC) derived neurospheres (NS) coupled onto microelectrode array (MEA) chips as a model system that includes a 3D NS as well as an outgrowing 2D monolayer allowing direct comparison of functionality within one culture. Preliminary results revealed an enhanced functional reactions of 3D NS to GABA receptor antagonist bicuculline compared to the 2D domain. Thus we have the first evidence that hESC derived NS are a promising model system for neurotoxicity testing enabling a direct comparison between functionality of neurons grown in 2D or 3D.
Motivation
Cells in a living organism are embedded in a three-dimensional (3D) microenvironment that fulfills key functions in cell development, behavior and physiology not reflected in conventional 2D cell cultures [1]. So it is not surprising that functional properties of 3D cell cultures differ from two-dimensional (2D) networks with respect to spike and burst rates as well as network burst (NB) rates [2]. More important for drug screening application is the question, whether response to neurotoxicological substances may differ for 2D und 3D cell culture models [3]. This issue however is not systematically studied and if so, 2D and 3D models were prepared with different protocols which makes comparison difficult. To overcome this problem, we present a model based on human embryonic stem cell (hESC) derived neurospheres (NS), where NS represent the 3D and its flat outgrowth the 2D culture. NS are cultured on microelectrode arrays (MEA) and one day after plating neurons and neurites grew radially out of the NS as previously described [4]. Neuronal activity was measured directly from the intact 3D NS as well as from the 2D monolayer of outgrowing cells allowing for direct comparison of functionality within one sample.
Material and Methods
hESC derived NS were used as a model system as previously described [4]. Briefly, hESC (H9, WiCell, Madison, USA) were differentiated to neural stem cells (NSC) using an established differentiation protocol [5]. NSC were enzymatically dissociated using accutase and seeded in 6-well low attachment plates at a concentration of 100 cells / µl in a total volume of 5 ml to generate NS by reaggregation of NSC. After 18 days in suspension, individual NS were plated on the MEA chips (60MEA200/30iR-Ti, Multichannel Systems, Reutlingen). Before plating, MEA chips were coated with Polyethyleneimine (0,1 % in boric acid) and laminin (20 µg/ml in PBS). Pharmacological response to GABA receptor antagonist bicuculline (10 µM in PBS) was tested 19 days after the first signals were detected (n=1). Data analysis was performed using the custom made Matlab®-based software tool “DrCell” [6]. Spike detection was accomplished using simple threshold based spike detection in combination with the SWTTEO algorithm [4].
Results
One day after NS were placed on MEA chips, neurons and neurites migrated radially out of the NS (Fig. 1). Neuronal signals were detected from electrodes beneath the 3D NS (red electrodes in Fig 1) as well as from electrodes covered by the 2D monolayer of cells (black electrodes in Fig. 1). In order to compare the response of NS and outgrowing cells to neuroactive substances, cultures were treated with bicuculline. Immediately after application, a regular pattern of NB appeared that was detected by electrodes beneath 3D (9 electrodes) as well as 2D (21 electrodes) neuronal culture (Fig 2A). This supports the assumption that outgrowing neurons and NS form a functional and connected neuronal network. Yet, our preliminary results clearly revealed differently pronounced pharmacological responses of neurons grown as 2D or within a 3D structure. The number of spikes increased about twofold for neurons grown as monolayer and threefold for the NS after application of bicuculline (Fig. 2B). This effect was observed for about 10 minutes before network activity returned to baseline activity after washout (data not shown). The distinct increase in spiking activity of the NS could be due to a higher number of neurons affected by bicuculline within the detection range of an electrode or due to a higher structural and functional connectivity of neurons within the NS.
Conclusion
In this study, we present hESC-derived NS coupled onto MEA chips as a model system that includes a 3D neurosphere and an outgrowing 2D monolayer. We compare the functional reaction of both domains towards the application of bicuculline and find that the network answer is in principal very close for 2D and 3D while the strength of the effect is clearly enlarged for 3D. This finding suggests that 3D models may facilitate the detection of small drug effect and thus increase the sensitivity of the neuronal assay. However, to examine the underlying mechanisms responsible for the enlarged reaction of 3D structure, the cell distribution and the grade of self-organization within the NS will be analyzed in future studies.
Figure Legend
Figure 1: NS on a MEA chip 1 day after plating.
Figure 2: Electrophysiological response of NS (3D) and outgrowing cells (2D) to GABA receptor antagonist bicuculline. Immediately after bicuculline was applied, a regular pattern of NB appeared, detected by electrodes covered by 2D (black) and 3D culture (red) (A). Further, the number of spikes increased as expected with a clearly stronger effect for the 3D culture (B). Data are normalized to baseline activity (n = 1).
Acknowledgements
This work was supported by the Federal Ministry of Education and Research, Bonn, Germany (02NUK049, 02NUK034C and 202507).
References
[1] Huang, G. et al. (2017) Chem. Rev. 117: 12764-12850
[2] Frega, M. et al. (2014) Sci rep. 4: 5489
[3] Bosi, S. (2015) Sci. rep. 5: 9562
[4] Mayer, M. (2018) Biosens. Bioelectron. 100: 462-468
[5] Yan, Y. et al. (2013) StemCells Transl Med 2 (11): 862-870
[6] Nick, C. et al. (2013) SPIJ 7 (2): 96-1
Keywords:
MEA Chips,
neurospheres,
human embryonic stem cells,
3D,
Bicuculline
Conference:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018.
Presentation Type:
Poster Presentation
Topic:
Stem cell-derived applications
Citation:
Mayer
M,
Arrizabalaga
O,
Schröder
I,
Ritter
S and
Thielemann
C
(2019). Human Embryonic Stem Cell Derived Neurospheres – 2D and 3D Cell Culture in one sample.
Conference Abstract:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays.
doi: 10.3389/conf.fncel.2018.38.00078
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Received:
18 Mar 2018;
Published Online:
17 Jan 2019.
*
Correspondence:
Dr. Margot Mayer, Aschaffenburg University of Applied Sciences, BioMEMS Lab, Aschaffenburg, Germany, Margot.Mayer@h-ab.de