3D Organotypic Spinal Cultures: Exploring Neuron and Neuroglia Responses Upon Prolonged Exposure to Graphene Oxide

Abstract

Graphene-based nanomaterials are increasingly engineered as components of biosensors, interfaces or drug delivery platforms in neuro-repair strategies. In these developments, the mostly used derivative of graphene is graphene oxide (GO). To tailor the safe development of GO nanosheets, we need to model in vitro tissue responses, and in particular the reactivity of microglia, a sub-population of neuroglia that acts as the first active immune response, when challenged by GO. Here, we investigated central nervous system (CNS) tissue reactivity upon long-term exposure to GO nanosheets in 3D culture models. We used the mouse organotypic spinal cord cultures, ideally suited for studying long-term interference with cues delivered at controlled times and concentrations. In cultured spinal segments, the normal presence, distribution and maturation of anatomically distinct classes of neurons and resident neuroglial cells are preserved. Organotypic explants were developed for 2 weeks embedded in fibrin glue alone or presenting GO nanosheets at 10, 25 and 50 μg/mL. We addressed the impact of such treatments on premotor synaptic activity monitored by patch clamp recordings of ventral interneurons. We investigated by immunofluorescence and confocal microscopy the accompanying glial responses to GO exposure, focusing on resident microglia, tested in organotypic spinal slices and in isolated neuroglia cultures. Our results suggest that microglia reactivity to accumulation of GO flakes, maybe due to active phagocytosis, may trim down synaptic activity, although in the absence of an effective activation of inflammatory response and in the absence of neuronal cell death.

Optimization of Organotypic Cultures of Mouse Spleen for Staining and Functional Assays

“…Organotypic culture technology is routinely used to perform organotypic culture of central nervous system sections (20,21). However, its application to lymphoid organs has lagged behind.…”

Tuning Neuronal Circuit Formation in 3D Polymeric Scaffolds by Introducing Graphene at the Bio/Material Interface

“…Nuclei were stained in all conditions with 4′,6‐diamidino‐2‐phenylindole (DAPI, 1:200, Invitrogen). Graphene was visualized by reflection mode during the confocal acquisition 57,58. To evaluate the density of neurons and astrocytes, immunolabeled 3D cultures were analyzed using an inverted confocal microscope (Nikon, Japan), acquiring serial confocal planes (z‐stack) every 1 µm across the entire 3D sections.…”

Advances in 3D neuronal microphysiological systems: towards a functional nervous system on a chip

“…While organotypic slice cultures are still used to study the spinal cord (Musto et al . 2019 ) and brain, recent advances have seen central nervous system neurons grown on mechanically controlled anisotropic scaffolds (Kim et al . 2017 ), porous microlattices (S. Li et al .…”