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. 2025 Aug 13;15:29738. doi: 10.1038/s41598-025-97186-x

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© The Author(s) 2025

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

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Fig. 2 — Characterization of co-culture of human Glutamatergic and GABAergic neurons in microfluidic device. (a) Timeline of cell culture maintenance with milestones during culture. (b) Brightfield contrast pictures of human glutamatergic and GABAergic neurons seeded in Dualink MEA at day 16. (c) Fluorescent picture of Live/Dead assay in the co-culture. Live cells were stained in green, and dead cells in red. (d, e) Immunofluorescent pictures of respectively, pluripotency markers (d), and specific neuronal markers (e) for each cell type. Sox2 was marked in red and Nestin in green. Β-III-tubulin was marked in green, vGlut1 in yellow, and GABA in red. In each immunofluorescent picture, DAPI was in blue. Scale bar represent 500 µm. (f, g, h) Graph bars showing the quantification in channel 1 and channel 3 of the Live/dead assay, pluripotency markers and neuronal specific markers, respectively. (i) Raster plots from electrophysiological recordings at day 7, day 14 and day 21, showing spikes in time per electrode, in each DuaLink compartment. (j, k) Bar graphs showing, respectively mean firing rate (MFR), weighted mean firing rate (WMFR) and Synchrony index at day 7, day 14, and day 21 measured in channels and microchannels. The data were analyzed using a two-way ANOVA to evaluate the effects of Ch1, Ch3, 1µ2 and 2µ3 and their interaction in time. Fisher’s Least Significant Difference (LSD) test was used as a post-hoc analysis to compare specific group means. * p-value < 0.05, *** p-value < 0.001 and **** p-value < 0.001. Error bar = SD.