Three‐dimensional spheroid cell culture of human MSC‐derived neuron‐like cells: New in vitro model to assess magnetite nanoparticle‐induced neurotoxicity effects

Abstract

As nanoparticles (NPs) can access the brain and impact on CNS function, novel in vitro models for the evaluation of NPs-induced neurotoxicity are advocated.Three-dimensional spheroids of primary neuron-like cells (hNLCs) of human origin have been generated, from differentiation of human umbilical cord mesenchymal stem cells (MSCs). The study evaluated Fe 3 O 4 NP impact on the differentiation process by applying the challenge at complete 3D hNLC spheroid formation (after 4 days, T4) or at beginning of neurogenic induction/simultaneously 3D forming (T0). Different endpoints were monitored over time (up to 10 days): spheroid growth, size, morphology, ATP, cell death, neuronal markers (β-Tub III, MAP-2, and NSE), NP uptake.At T0 application, a marked concentration-and time-dependent cell mortality occurred: effect started early (day 2) and low concentration (1 μg/ml) and exacerbated (80% mortality) after prolonged time (day 6) and increased concentrations (50 μg/ml). ATP was strikingly affected. All neuronal markers were downregulated, and spheroid morphology altered in a concentration-dependent manner (from ≥5 μg/ml) after day 2. Fe 3 O 4 NPs applied at complete 3D formation (T4) still induced adverse effects although less severe: cell mortality (20-60%) and ATP content decrease (10-40%) were observed in a concentration-dependent manner (from ≥ 5 μg/ml). A neuronal-specific marker effect and spheroid size reduction from 25 μg/ml without morphology alteration were evidenced. This finding provides additional information on neurotoxic effects of Fe 3 O 4 NPs in a new 3D hNLC spheroid model derived from MSCs that could find a consistent application as in a testing strategy serving in first step hazard identification for correct risk assessment. K E Y W O R D S in vitro alternative methods, mesenchymal stem cells, occupational and environmental health, predictive nanotoxicology, risk assessment 1 | INTRODUCTION With the development of nanotechnologies, even if towards a safe and sustainable innovation (Gottardo et al., 2021), uncertainties associated with the health and environmental impacts of free and manufactured nanoparticles (NPs) are growing. Regardless of the exposure route, once NPs are in the body, they are distributed by the bloodstream and translocated to the brain

Toxicity Factors of Magnetite Nanoparticles and Methods of Their Research

“…Blood cells (erythrocytes, leukocytes, thrombocytes) [35] Human monocytes and macrophages [37] Human erythrocytes [34,36] Rat erythrocytes [60] PC12 (rat pheochromocytoma) ReNcell VM (human neuronal stem cells) [38,56] NRK-52E (rat renal epithelium) [39] HFF2 (human embryonic stem cells) [40] NCTC 1469 (murine non-parenchimal hepatocytes) [41] Mesenchimal stem cells of the cord and bone marrow [42] 3D spheroids of the primary neuron-like cells [43] Caco-2 (human colorectal adenocarcinoma), HepG2 (human hepatocellular carcinoma), MDCK (Madin-Darby canine kidney cells), Calu-3 (human lung adenocarcinoma), Raw 264.7 (murine macrophage cell line) [47] BeWo b30 (fetal choriocarcinoma cells) [55] TK6 (human spleen lymphoblasts) [62] SK-Hep-1, Hep3B (human hepatoma cells) [63] Fibroblasts of the human periodontal ligament, murine dermal fibroblasts [67] D384 (human meduloblastoma cells), SH-SY5Y (human neuroblastoma cells) [70] HaCaT (human skin keratinocytes), HepG2 (human hepatocellular carciunoma) [75] SH-SY5Y (human neuroblastoma cells), A172 (human glioblastoma cells) [76] MCF-7 (human breast cancer cells) [77] human monocytes and macrophages at a concentration of 1 mg/ml for more than 72 h [37]. At the same time, according to other data, magnetite NPs stabilized by citric acid (11.44 nm) promoted the release of significantly more hemoglobin from erythrocytes than in the control, which indicated a hemolytic effect that was dose-dependent [34].…”

“…New in vitro models are proposed for evaluating NP-induced neurotoxicity based on the differentiation of human umbilical cord mesenchymal stem cells to three-dimensional (3D) spheroids of primary neuron-like cells [43]. When applied at the beginning of neurogenic induction and simultaneous formation of 3D structure, a nociceable concentration-and time-dependent cell death was observed: the effect started early (day 2) and at a low concentration (1 μg/ml).…”

Progress, Opportunities, and Challenges of Magneto-Plasmonic Nanoparticles under Remote Magnetic and Light Stimulation for Brain-Tissue and Cellular Regeneration

“…To evaluate the impact of iron oxide-NP-induced neurotoxicity, De Simone et al developed multiple brain organoids derived from human D384 astrocytes, SH-SY5Y neuronallike cells, and human-umbilical-cord mesenchymal stem cells [143,144]. The short-term exposure to iron oxide NPs resulted in differential cytotoxicity in astrocytes and neurons at concentrations of 10 µg/mL and 25 µg/mL, respectively.…”