RT Journal Article T1 Enabling 3D electrical stimulation of adipose-derived decellularized extracellular matrix and reduced graphene oxide scaffolds in vitro using graphene electrodes A1 Martins, Patrícia Alexandra A1 Barroca, Nathalie A1 Vieira, Sandra I. A1 de Sousa, Bárbara M. A1 Gil, Guilherme A1 Cicuéndez Maroto, Mónica A1 Casarrubios Molina, Laura A1 Feito Castellano, María José A1 Díez Orejas, Rosalía María A1 Portolés Pérez, María Teresa A1 Figueiredo, Bruno A1 Silva, Rui A1 Garcia Lizarribar, Andrea A1 Fonseca, Pedro A1 Alves, Luís Nero A1 Marques, Paula A. A. P. AB Notwithstanding the demonstrated benefits of electrical stimulation in enhancing tissue functionality, existing state-of-the-art electrostimulation systems often depend on invasive electrodes or planar designs. This work exploits the versatility of graphene to fabricate biocompatible electrodes for the three-dimensional in vitro electrical stimulation of neural stem cells. A conductive green graphene-based ink was formulated and screen-printed as the bottom and top electrodes in a bottom-less standard culture well plate. Upon exposure to macrophages, although some oxidative stress was observed, this graphene-based ink did not elicit an increase in the pro-inflammatory cytokine IL-6. An analysis of the electrode impedance as a function of time and frequency was performed to optimize the 3D electrical stimulation. The efficacy of these graphene electrodes for electrically stimulating cells across 3D environments was investigated in scaffolds composed of a decellularized extracellular matrix and reduced graphene oxide, which had previously shown the capability to facilitate neuronal differentiation in vitro and to create a pro-regenerative microenvironment in vivo. Neural stem cells were seeded on these scaffolds and electrically stimulated with a 10 Hz bidirectional current signal of 200 μA for 1 hour daily. At the target frequency of 10 Hz, deemed advantageous for neural regeneration, a scaffold impedance below 800 Ω was ensured. The low-frequency 3D stimulation proved to enhance cellular mechanisms essential for the development of neuronal networks, including neuronal differentiation, neuritogenesis and neurite growth. PB Royal Society of Chemistry SN 2046-2069 YR 2025 FD 2025-09-01 LK https://hdl.handle.net/20.500.14352/138495 UL https://hdl.handle.net/20.500.14352/138495 LA eng NO Martins, P. A., Barroca, N., Vieira, S. I., de Sousa, B. M., Gil, G., Cicuéndez, M., Casarrubios, L., Feito, M. J., Diez-Orejas, R., Portolés, M. T., Figueiredo, B., Silva, R., Garcia-Lizarribar, A., Fonseca, P., Alves, L. N., & Marques, P. A. A. P. (2025). Enabling 3D electrical stimulation of adipose-derived decellularized extracellular matrix and reduced graphene oxide scaffolds in vitro using graphene electrodes. RSC Advances, 15(38), 31257-31271. https://doi.org/10.1039/D5RA02570B NO Patrícia Alexandra Martins is thankful to FCT for her Ph.D. grant 2022.13072.BD. This work was supported by the European Union's Horizon 2020 research and innovation programme under grant agreement no. 829060 (FETOPEN project: NeuroStimSpinal), and the Portuguese following funding: UIDB/00481/2020 and UIDP/00481/2020, iBiMED (UIDB/4501/2020 and UIDP/4501/2020), the LiM Bioimaging Facility (a PPBI node, POCI-01-0145-FEDER-022122) – Fundação para a Ciência e a Tecnologia (FCT) and CENTRO-01-0145-FEDER-022083 – Centro Portugal Regional Operational Programme (Centro2020) under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund. The authors wish to thank the staff of the Centro de Citometría y Microscopía de Fluorescencia of the Universidad Complutense de Madrid (Spain) for assistance in flow cytometry and confocal microscopy studies. NO European Commission NO Universidade de Aveiro (Portugal) NO Fundação para a Ciência e a Tecnologia(Portugal) NO Comissão de Coordenação e Desenvolvimento Regional do Centro (Portugal) DS Docta Complutense RD 20 ago 2026