Evaluation of Neurotoxicity With Human Pluripotent Stem Cell–Derived Cerebral Organoids

Abstract

The recent development of human cerebral organoids provides an invaluable in vitro model of human brain development to assess the toxicity of natural or man-made toxic substances. By recapitulating key aspects of early human neurodevelopment, investigators can evaluate with this three-dimensional (3D) model the effect of certain compounds on the formation of neuronal networks and their electrophysiological properties with more physiological relevance than neurons grown in monolayers and in cultures composed of a unique cell type. This promising potential has contributed to the development of a large number of diverse protocols to generate human cerebral organoids, making interlaboratory comparisons of results difficult. Based on a previously published protocol to generate human cortical organoids (herein called cerebral organoids), we detail several approaches to evaluate the effect of chemicals on neurogenesis, apoptosis, and neuronal function when exogenously applied to cultured specimens. Here, we take as an example 4-aminopyridine, a potassium channel blocker that modulates the activity of neurons and neurogenesis, and describe a simple and cost-effective way to test the impact of this agent on cerebral organoids derived from human induced pluripotent stem cells. We also provide tested protocols to evaluate neurogenesis in cerebral organoids with ethynyl deoxyuridine labeling and neuronal activity with live calcium imaging and microelectrode arrays. Together, these protocols should facilitate the implementation of cerebral organoid technologies in laboratories wishing to evaluate the effects of specific compounds or conditions on the development and function of human neurons with only basic cell culture equipment.

Integrating distribution kinetics and toxicodynamics to assess repeat dose neurotoxicity in vitro using human BrainSpheres: a case study on amiodarone

“…To overcome the difficulty of testing such a large number of chemicals and drugs, there is a general consensus that animal testing needs to be replaced by a combination of in vitro and in silico approaches ( Bal-Price et al, 2010 ; Anadón et al, 2014 ; Fischer et al, 2020 ; Caloni et al, 2022 ). Recent advances in human test models, including 3D models, on-a-chip technology and analytical techniques ( Pamies et al, 2017 ; Harrill et al, 2021 ; Olesti et al, 2021 ; Castiglione et al, 2022 ; Parmentier et al, 2023 ), in combination with adverse outcome pathways (AOPs) ( Tollefsen et al, 2014 ; Villeneuve et al, 2014 ), in vitro distribution kinetics modeling ( Pomponio et al, 2015a ), and physiologically based kinetic (PBK) modeling for quantitative in vitro to in vivo extrapolation (QIVIVE) ( Kasteel et al, 2021 ; Noorlander et al, 2022 ), provide us with a promising new toolbox of new approach methodologies (NAMs) for the 3Rs-based neurotoxicity testing approach or reducing, refining, and replacing (3Rs) animal-based toxicity tests ( Anson et al, 2011 ; Steimberg et al, 2020 ).…”