Involvement of Mitochondria in the Selective Response to Microsecond Pulsed Electric Fields on Healthy and Cancer Stem Cells in the Brain.

In the last few years, pulsed electric fields have emerged as promising clinical tools for tumor treatments. This study highlights the distinct impact of a specific pulsed electric field protocol, PEF-5 (0.3 MV/m, 40 µs, 5 pulses), on astrocytes (NHA) and medulloblastoma (D283) and glioblastoma (U87 NS) cancer stem-like cells (CSCs). We pursued this goal by performing ultrastructural analyses corroborated by molecular/omics approaches to understand the vulnerability or resistance mechanisms triggered by PEF-5 exposure in the different cell types. Electron microscopic analyses showed that, independently of exposed cells, the main targets of PEF-5 were the cell membrane and the cytoskeleton, causing membrane filopodium-like protrusion disappearance on the cell surface, here observed for the first time, accompanied by rapid cell swelling. PEF-5 induced different modifications in cell mitochondria. A complete mitochondrial dysfunction was demonstrated in D283, while a mild or negligible perturbation was observed in mitochondria of U87 NS cells and NHAs, respectively, not sufficient to impair their cell functions. Altogether, these results suggest the possibility of using PEF-based technology as a novel strategy to target selectively mitochondria of brain CSCs, preserving healthy cells.

In Vitro Imaging and Molecular Characterization of Ca2+ Flux Modulation by Nanosecond Pulsed Electric Fields.

In recent years, the application of pulsed electric fields with very short durations (nanoseconds) and extremely high amplitudes (MV/m) has been investigated for novel medical purposes. Various electric protocols have been explored for different objectives, including the utilization of fractionated pulse doses to enhance cell electrosensitization to the uptake of different markers or an increase in apoptosis. This study focused on the use of fluorescence imaging to examine molecular calcium fluxes induced by different fractionated protocols of short electric pulses in neuroblastoma (SH-SY5Y) and mesenchymal stem cells (HaMSCs) that were electroporated using nanosecond pulsed electric fields. In our experimental setup, we did not observe cell electrosensitization in terms of an increase in calcium flux following the administration of fractionated doses of nanosecond pulsed electric fields with respect to the non-fractionated dose. However, we observed the targeted activation of calcium-dependent genes (c-FOS, c-JUN, EGR1, NURR-1, ß3-TUBULIN) based on the duration of calcium flux, independent of the instantaneous levels achieved but solely dependent on the final plateau reached. This level of control may have potential applications in various medical and biological treatments that rely on calcium and the delivery of nanosecond pulsed electric fields.

Effects of Ultra-Short Pulsed Electric Field Exposure on Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most common brain cancer in adults. GBM starts from a small fraction of poorly differentiated and aggressive cancer stem cells (CSCs) responsible for aberrant proliferation and invasion. Due to extreme tumor heterogeneity, actual therapies provide poor positive outcomes, and cancers usually recur. Therefore, alternative approaches, possibly targeting CSCs, are necessary against GBM. Among emerging therapies, high intensity ultra-short pulsed electric fields (PEFs) are considered extremely promising and our previous results demonstrated the ability of a specific electric pulse protocol to selectively affect medulloblastoma CSCs preserving normal cells. Here, we tested the same exposure protocol to investigate the response of U87 GBM cells and U87-derived neurospheres. By analyzing different in vitro biological endpoints and taking advantage of transcriptomic and bioinformatics analyses, we found that, independent of CSC content, PEF exposure affected cell proliferation and differentially regulated hypoxia, inflammation and P53/cell cycle checkpoints. PEF exposure also significantly reduced the ability to form new neurospheres and inhibited the invasion potential. Importantly, exclusively in U87 neurospheres, PEF exposure changed the expression of stem-ness/differentiation genes. Our results confirm this physical stimulus as a promising treatment to destabilize GBM, opening up the possibility of developing effective PEF-mediated therapies.

Micro-RNA and Proteomic Profiles of Plasma-Derived Exosomes from Irradiated Mice Reveal Molecular Changes Preventing Apoptosis in Neonatal Cerebellum.

Cell communication via exosomes is capable of influencing cell fate in stress situations such as exposure to ionizing radiation. In vitro and in vivo studies have shown that exosomes might play a role in out-of-target radiation effects by carrying molecular signaling mediators of radiation damage, as well as opposite protective functions resulting in resistance to radiotherapy. However, a global understanding of exosomes and their radiation-induced regulation, especially within the context of an intact mammalian organism, has been lacking. In this in vivo study, we demonstrate that, compared to sham-irradiated (SI) mice, a distinct pattern of proteins and miRNAs is found packaged into circulating plasma exosomes after whole-body and partial-body irradiation (WBI and PBI) with 2 Gy X-rays. A high number of deregulated proteins (59% of WBI and 67% of PBI) was found in the exosomes of irradiated mice. In total, 57 and 13 miRNAs were deregulated in WBI and PBI groups, respectively, suggesting that the miRNA cargo is influenced by the tissue volume exposed to radiation. In addition, five miRNAs (miR-99b-3p, miR-200a-3p, miR-200a, miR-182-5p, miR-182) were commonly overexpressed in the exosomes from the WBI and PBI groups. In this study, particular emphasis was also given to the determination of the in vivo effect of exosome transfer by intracranial injection in the highly radiosensitive neonatal cerebellum at postnatal day 3. In accordance with a major overall anti-apoptotic function of the commonly deregulated miRNAs, here, we report that exosomes from the plasma of irradiated mice, especially in the case of WBI, prevent radiation-induced apoptosis, thus holding promise for exosome-based future therapeutic applications against radiation injury.

50-Hz MF does not affect global DNA methylation of SH-SY5Y cells treated with the neurotoxin MPP.

Exposure to extremely low frequency magnetic fields (ELF-MFs) has been associated with an increased risk of neurodegenerative disorders. The underlying mechanisms, however, are still debated. Since epigenetics play a key role in the neurodegenerative process, we investigated whether exposure to ELF-MF (50 Hz, 1 mT) might affect global DNA methylation of SH-SY5Y dopaminergic-like neuroblastoma cells. We assessed the percentage of 5-methylcytosine (5-mC) of three repetitive interspersed sequences (ALU, LINE-1, or SATα), through pyrosequencing analysis. We demonstrated that ELF exposure (up to 72 h) does not induce any change in the methylation pattern of ALU, LINE-1, and SATα in both proliferating and differentiated SH-SY5Y cells. Furthermore, when administered in combination with 1-methyl-4-phenylpyridinium (MPP+ ), a neurotoxin mimicking the Parkinson's Disease (PD) phenotype, ELF-MF exposure does not trigger any modulation in the percentage of 5-mC of the repetitive elements. Our findings demonstrate that exposure to 50-Hz MF does not affect global DNA methylation in proliferating and dopaminergic differentiated SH-SY5Y cells, either under basal culture conditions or under neurotoxic stress. Bioelectromagnetics. 40:33-41, 2019. © 2018 Bioelectromagnetics Society.

Sine wave electropermeabilization reveals the frequency-dependent response of the biological membranes.

The permeabilization of biological membranes by electric fields, known as electroporation, has been traditionally performed with square electric pulses. These signals distribute the energy applied to cells in a wide frequency band. This paper investigates the use of sine waves, which are narrow band signals, to provoke electropermeabilization and the frequency dependence of this phenomenon. Single bursts of sine waves at different frequencies in the range from 8 kHz-130 kHz were applied to cells in vitro. Electroporation was studied in the plasma membrane and the internal organelles membrane using calcium as a permeabilization marker. Additionally, a double-shell electrical model was simulated to give a theoretical framework to our results. The electroporation efficiency shows a low pass filter frequency dependence for both the plasma membrane and the internal organelles membrane. The mismatch between the theoretical response and the observed behavior for the internal organelles membrane is explained by a two-step permeabilization process: first the permeabilization of the external membrane and afterwards that of the internal membranes. The simulations in the model confirm this two-step hypothesis when a variable plasma membrane conductivity is considered in the analysis. This study demonstrates how the use of narrow-band signals as sine waves is a suitable method to perform electroporation in a controlled manner. We suggest that the use of this type of signals could bring a simplification in the investigations of the very complex phenomenon of electroporation, thus representing an interesting option in future fundamental studies.

The epigenetic component of the brain response to electromagnetic stimulation in Parkinson's Disease patients: A literature overview.

Modulations of epigenetic machinery, namely DNA methylation pattern, histone modification, and non-coding RNAs expression, have been recently included among the key determinants contributing to Parkinson's Disease (PD) aetiopathogenesis and response to therapy. Along this line of reasoning, a set of experimental findings are highlighting the epigenetic-based response to electromagnetic (EM) therapies used to alleviate PD symptomatology, mainly Deep Brain Stimulation (DBS) and Transcranial Magnetic Stimulation (TMS). Notwithstanding the proven efficacy of EM therapies, the precise molecular mechanisms underlying the brain response to these types of stimulations are still far from being elucidated. In this review we provide an overview of the epigenetic changes triggered by DBS and TMS in both PD patients and neurons from different experimental animal models. Furthermore, we also propose a critical overview of the exposure modalities currently applied, in order to evaluate the technical robustness and dosimetric control of the stimulation, which are key issues to be carefully assessed when new molecular findings emerge from experimental studies. Bioelectromagnetics. 39:3-14, 2018. © 2017 Wiley Periodicals, Inc.

Frequency spectrum of induced transmembrane potential and permeabilization efficacy of bipolar electric pulses.

In this paper a simple prediction method for the bipolar pulse cancellation effect is proposed, based on the frequency analysis of the TMP spectra of a single cell and the computed relative global spectral content up to a defined frequency threshold. We present a spectral analysis of pulses applied in experiments, and we extract the induced TMP from a microdosimetric model of the cell. The induced TMP computation is carried out on a hemispherical multi-layered cell model in the time domain. The analysis is presented for a variety of unipolar and bipolar input signals in the nanosecond and the microsecond time scales. Our evaluations are in good agreement with experimental results for bipolar pulse cancellation of electropermeabilization-induced Ca2+ influx using 300ns, 750kV/m pulses and with other results reported in recent literature.

Comprehensive Characterization of the Interaction between Pulsed Electric Fields and Live Cells by Confocal Raman Microspectroscopy.

This study reports a comprehensive analysis of the effect of 100 µs electric pulses on the biochemical composition of live cells using a label-free approach, confocal Raman microspectroscopy. We investigated different regions of interest around the nucleus of the cells and the dose-effect relationship related to different electric pulse parameters. We also extended the study to another cell type. Membrane resealing was monitored by pulsing the cells in reversible or irreversible electropermeabilization condition at different temperatures. Our results confirmed a previous publication showing that proteins and lipids were highly impacted by the delivery of electric pulses. These chemical changes were similar in different locations around the cell nucleus. By sweeping the field magnitude, the number of electric pulses, or their repetition rate, the Raman signatures of live cells appeared to be related to the electropermeabilization state, verified by Yo-Pro-1 uptake. We also demonstrated that the chemical changes in the Raman signatures were cell-dependent even if common features were noticed between the two cell types used.

A Microdosimetric Study of Electropulsation on Multiple Realistically Shaped Cells: Effect of Neighbours.

Over the past decades, the effects of ultrashort-pulsed electric fields have been used to investigate their action in many medical applications (e.g. cancer, gene electrotransfer, drug delivery, electrofusion). Promising aspects of these pulses has led to several in vitro and in vivo experiments to clarify their action. Since the basic mechanisms of these pulses have not yet been fully clarified, scientific interest has focused on the development of numerical models at different levels of complexity: atomic (molecular dynamic simulations), microscopic (microdosimetry) and macroscopic (dosimetry). The aim of this work is to demonstrate that, in order to predict results at the cellular level, an accurate microdosimetry model is needed using a realistic cell shape, and with their position and packaging (cell density) characterised inside the medium.

Effects of exposure to gradient magnetic fields emitted by nuclear magnetic resonance devices on clonogenic potential and proliferation of human hematopoietic stem cells.

This study investigates effects of gradient magnetic fields (GMFs) emitted by magnetic resonance imaging (MRI) devices on hematopoietic stem cells. Field measurements were performed to assess exposure to GMFs of staff working at 1.5 T and 3 T MRI units. Then an exposure system reproducing measured signals was realized to expose in vitro CD34+ cells to GMFs (1.5 T-protocol and 3 T-protocol). CD34+ cells were obtained by Fluorescence Activated Cell Sorting from six blood donors and three MRI-exposed workers. Blood donor CD34+ cells were exposed in vitro for 72 h to 1.5 T or 3 T-protocol and to sham procedure. Cells were then cultured and evaluated in colony forming unit (CFU)-assay up to 4 weeks after exposure. Results showed that in vitro GMF exposure did not affect cell proliferation but instead induced expansion of erythroid and monocytes progenitors soon after exposure and for the subsequent 3 weeks. No decrease of other clonogenic cell output (i.e., CFU-granulocyte/erythroid/macrophage/megakaryocyte and CFU-granulocyte/macrophage) was noticed, nor exposed CD34+ cells underwent the premature exhaustion of their clonogenic potential compared to sham-exposed controls. On the other hand, pilot experiments showed that CD34+ cells exposed in vivo to GMFs (i.e., samples from MRI workers) behaved in culture similarly to sham-exposed CD34+ cells, suggesting that other cells and/or microenvironment factors might prevent GMF effects on hematopoietic stem cells in vivo. Accordingly, GMFs did not affect the clonogenic potential of umbilical cord blood CD34+ cells exposed in vitro together with the whole mononuclear cell fraction.

Controlled release from magnetoliposomes aqueous suspensions exposed to a low intensity magnetic field.

Recently, the use of liposomes loaded with magnetic nanoparticles (magnetoliposomes, (MLs)) has been intensely growing as a new drug delivery system. With the use of alternating magnetic fields, it is possible to remotely control the delivery of a drug or any other macromolecule loaded inside the MLs. In this experiment, the release of a fluorescent dye from MLs is achieved through an alternating magnetic field of 20 kHz and amplitude below 100 A/m, and without a macroscopic temperature increase.

On the effects of 30.5 GHz sinusoidal wave exposure on glioblastoma organoids.

Introduction: Glioblastoma (grade IV) is the most aggressive primary brain tumor in adults, representing one of the biggest therapeutic challenges due to its highly aggressive nature. In this study, we investigated the impact of millimeter waves on tridimensional glioblastoma organoids derived directly from patient tumors. Our goal was to explore novel therapeutic possibilities in the fight against this challenging disease. Methods: The exposure setup was meticulously developed in-house, and we employed a comprehensive dosimetry approach, combining numerical and experimental methods. Biological endpoints included a global transcriptional profiling analysis to highlight possible deregulated pathways, analysis of cell morphological changes, and cell phenotypic characterization which are all important players in the control of glioblastoma progression. Results and discussion: Our results revealed a significant effect of continuous millimeter waves at 30.5 GHz on cell proliferation and apoptosis, although without affecting the differentiation status of glioblastoma cells composing the organoids. Excitingly, when applying a power level of 0.1 W (Root Mean Square), we discovered a remarkable (statistically significant) therapeutic effect when combined with the chemotherapeutic agent Temozolomide, leading to increased glioblastoma cell death. These findings present a promising interventional window for treating glioblastoma cells, harnessing the potential therapeutic benefits of 30.5 GHz CW exposure. Temperature increase during treatments was carefully monitored and simulated with a good agreement, demonstrating a negligible involvement of the temperature elevation for the observed effects. By exploring this innovative approach, we pave the way for improved future treatments of glioblastoma that has remained exceptionally challenging until now.

Microdosimetric study for nanosecond pulsed electric fields on a cell circuit model with nucleus.

Recently, scientific interest in electric pulses, always more intense and shorter and able to induce biological effects on both plasma and nuclear membranes, has greatly increased. Hence, microdosimetric models that include internal organelles like the nucleus have assumed increasing importance. In this work, a circuit model of the cell including the nucleus is proposed, which accounts for the dielectric dispersion of all cell compartments. The setup of the dielectric model of the nucleus is of fundamental importance in determining the transmembrane potential (TMP) induced on the nuclear membrane; here, this is demonstrated by comparing results for three different sets of nuclear dielectric properties present in the literature. The results have been compared, even including or disregarding the dielectric dispersion of the nucleus. The main differences have been found when using pulses shorter than 10 ns. This is due to the fact that the high spectral components of the shortest pulses are differently taken into account by the nuclear membrane transfer functions computed with and without nuclear dielectric dispersion. The shortest pulses are also the most effective in porating the intracellular structures, as confirmed by the time courses of the TMP calculated across the plasma and nuclear membranes. We show how dispersive nucleus models are unavoidable when dealing with pulses shorter than 10 ns because of the large spectral contents arriving above 100 MHz, i.e., over the typical relaxation frequencies of the dipolar mechanism of the molecules constituting the nuclear membrane and the subcellular cell compartments.

Complex magnetic field exposure system for in vitro experiments at intermediate frequencies.

In occupational environments, an increasing number of electromagnetic sources emitting complex magnetic field waveforms in the range of intermediate frequencies is present, requiring an accurate exposure risk assessment with both in vitro and in vivo experiments. In this article, an in vitro exposure system able to generate complex magnetic flux density B-fields, reproducing signals from actual intermediate frequency sources such as magnetic resonance imaging (MRI) scanners, for instance, is developed and validated. The system consists of a magnetic field generation system and an exposure apparatus realized with a couple of square coils. A wide homogeneity (99.9%) volume of 210 × 210 × 110 mm(3) was obtained within the coils, with the possibility of simultaneous exposure of a large number of standard Petri dishes. The system is able to process any numerical input sequence through a filtering technique aimed at compensating the coils' impedance effect. The B-field, measured in proximity to a 1.5 T MRI bore during a typical examination, was excellently reproduced (cross-correlation index of 0.99). Thus, it confirms the ability of the proposed setup to accurately simulate complex waveforms in the intermediate frequency band. Suitable field levels were also attained. Moreover, a dosimetry index based on the weighted-peak method was evaluated considering the induced E-field on a Petri dish exposed to the reproduced complex B-field. The weighted-peak index was equal to 0.028 for the induced E-field, indicating an exposure level compliant with the basic restrictions of the International Commission on Non-Ionizing Radiation Protection. Bioelectromagnetics 34:211-219, 2013. © 2012 Wiley Periodicals, Inc.

Biological effects of ultrashort electric pulses in a neuroblastoma cell line: the energy density role.

BACKGROUND: Despite the numerous literature results about biological effects of electromagnetic field (EMF) exposure, the interaction mechanisms of these fields with organisms are still a matter of debate. Extremely low frequency (ELF) MFs can modulate redox homeostasis and we showed that 24 h exposure to 50 Hz-1 mT has a pro-oxidant effect and effects on the epigenome of SH-SY5Y cells, decreasing miR-34b/c expression through the hypermethylation of their promoter. METHODS: Here, we investigated the role of the electromagnetic deposited energy density (ED) during exposures lasting 24 h to 1 mT amplitude MFs at a frequency of 50 Hz in inducing the above mentioned effects. To this end, we delivered ultrashort electric pulses, in the range of microsecond and nanosecond duration, with the same ED of the previously performed magnetic exposure to SH-SY5Y cells. Furthermore, we explored the effect of higher deposited energy densities. Analysis of i) gene and microRNA expression, ii) cell morphology, iii) reactive oxygen species (ROS) generation, and iv) apoptosis were carried out. RESULTS: We observed significant changes in egr-1 and c-fos expression at very low deposited ED levels, but no change of the ROS production, miR-34b/c expression, nor the appearance of indicators of apoptosis. We thus sought investigating changes in egr-1 and c-fos expression caused by ultrashort electric pulses at increasing deposited ED levels. The pulses with the higher deposited ED caused cell electroporation and even other morphological changes such as cell fusion. The changes in egr-1 and c-fos expression were more intense, but, again, no change of the ROS production, miR-34b/c expression, nor apoptosis induction was observed. CONCLUSIONS: These results, showing that extremely low levels of electric stimulation (never investigated until now) can cause transcriptional changes, also reveal the safety of the electroporating pulses used in biomedical applications and open up the possibility to further therapeutic applications of this technology.

Changes in hydration of liposome membranes exposed to nanosecond electric pulses detected by wide-field Coherent anti-Stokes Raman microspectroscopy.

Electropulsation has become a powerful technological platform for electromanipulation of cells and tissues for various medical and biotechnological applications, but the molecular changes that underlay the very first initiation step of this process have not been experimentally observed. Here, we endowed a wide-field Coherent anti-Stokes Raman Scattering platform with an ad-hoc electromagnetic exposure device and we demonstrated, using artificial lipid vesicles (i.e. liposomes), that electropulsation is initiated by the increase of interstitial water content in liposome membranes. A pulse-dependent accumulation of the interstitial water molecules is observed in the membranes and a plausible mechanism supported by a computational electrochemical model is presented and discussed.

MiRNA-Mediated Fibrosis in the Out-of-Target Heart following Partial-Body Irradiation.

Recent reports have shown a link between radiation exposure and non-cancer diseases such as radiation-induced heart disease (RIHD). Radiation exposures are often inhomogeneous, and out-of-target effects have been studied in terms of cancer risk, but very few studies have been carried out for non-cancer diseases. Here, the role of miRNAs in the pathogenesis of RIHD was investigated. C57Bl/6J female mice were whole- (WBI) or partial-body-irradiated (PBI) with 2 Gy of X-rays or sham-irradiated (SI). In PBI exposure, the lower third of the mouse body was irradiated, while the upper two-thirds were shielded. From all groups, hearts were collected 15 days or 6 months post-irradiation. The MiRNome analysis at 15 days post-irradiation showed that miRNAs, belonging to the myomiR family, were highly differentially expressed in WBI and PBI mouse hearts compared with SI hearts. Raman spectral data collected 15 days and 6 months post-irradiation showed biochemical differences among SI, WBI and PBI mouse hearts. Fibrosis in WBI and PBI mouse hearts, indicated by the increased deposition of collagen and the overexpression of genes involved in myofibroblast activation, was found 6 months post-irradiation. Using an in vitro co-culture system, involving directly irradiated skeletal muscle and unirradiated ventricular cardiac human cells, we propose the role of miR-1/133a as mediators of the abscopal response, suggesting that miRNA-based strategies could be relevant for limiting tissue-dependent reactions in non-directly irradiated tissues.

Microsecond Pulsed Electric Fields: An Effective Way to Selectively Target and Radiosensitize Medulloblastoma Cancer Stem Cells.

PURPOSE: Cancer stem cells constitute an endless reserve for the maintenance and progression of tumors, and they could be the reason for conventional therapy failure. New therapeutic strategies are necessary to specifically target them. In this context, microsecond pulsed electric fields have been selected to expose D283Med cells, a human medulloblastoma cell line resulted to be rich in cancer stem cells, and normal human astrocytes. METHODS: We analyzed in vitro different endpoints at different times after microsecond pulsed electric field exposure, such as permeabilization, reactive oxygen species generation, cell viability/proliferation, cell cycle, and clonogenicity, as well as the expression of different genes involved in cell cycle, apoptosis, and senescence. Furthermore, the response of D283Med cells exposed to microsecond pulsed electric fields was validated in vivo in a heterotopic mouse xenograft model. RESULTS: Our in vitro results showed that a specific pulse protocol (ie, 0.3 MV/m, 40 µs, 5 pulses) was able to induce irreversible membrane permeabilization and apoptosis exclusively in medulloblastoma cancer stem cells. In the surviving cells, reactive oxygen species generation was observed, together with a transitory G2/M cell-cycle arrest with a senescence-associated phenotype via the upregulation of GADD45A. In vivo results, after pulsed electric field exposure, demonstrated a significant tumor volume reduction with no eradication of tumor mass. In conjunction, we verified the efficacy of electric pulse pre-exposure followed by ionizing irradiation in vivo to enable complete inhibition of tumor growth. CONCLUSIONS: Our data reveal novel therapeutic options for the targeting of medulloblastoma cancer stem cells, indicating nonionizing pulsed electric field pre-exposure as an effective means to overcome the radioresistance of cancer stem cells.

Exposure of the SH-SY5Y Human Neuroblastoma Cells to 50-Hz Magnetic Field: Comparison Between Two-Dimensional (2D) and Three-Dimensional (3D) In Vitro Cultures.

We here characterize the response to the extremely low-frequency (ELF) magnetic field (MF, 50 Hz, 1 mT) of SH-SY5Y human neuroblastoma cells, cultured in a three-dimensional (3D) Alvetex® scaffold compared to conventional two-dimensional (2D) monolayers. We proved that the growing phenotype of proliferating SH-SY5Y cells is not affected by the culturing conditions, as morphology, cell cycle distribution, proliferation/differentiation gene expression of 3D-cultures overlap what reported in 2D plates. In response to 72-h exposure to 50-Hz MF, we demonstrated that no proliferation change and apoptosis activation occur in both 2D and 3D cultures. Consistently, no modulation of Ki67, MYCN, CCDN1, and Nestin, of invasiveness and neo-angiogenesis-controlling genes (HIF-1α, VEGF, and PDGF) and of microRNA epigenetic signature (miR-21-5p, miR-222-3p and miR-133b) is driven by ELF exposure. Conversely, intracellular glutathione content and SOD1 expression are exclusively impaired in 3D-culture cells in response to the MF, whereas no change of such redox modulators is observed in SH-SY5Y cells if grown on 2D monolayers. Moreover, ELF-MF synergizes with the differentiating agents to stimulate neuroblastoma differentiation into a dopaminergic (DA) phenotype in the 3D-scaffold culture only, as growth arrest and induction of p21, TH, DAT, and GAP43 are reported in ELF-exposed SH-SY5Y cells exclusively if grown on 3D scaffolds. As overall, our findings prove that 3D culture is a more reliable experimental model for studying SH-SY5Y response to ELF-MF if compared to 2D conventional monolayer, and put the bases for promoting 3D systems in future studies addressing the interaction between electromagnetic fields and biological systems.