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Image-Based Analysis of a Human Neurosphere Stem Cell Model for the Evaluation of Potential Neurotoxicants

Brad Larson1, Hilary Sherman2, Hannah Gitschier2, Alexandra Wolff3, Wini Luty3
1BioTek Instruments, Inc. Winooski, VT USA
2Corning Incorporated, Life Sciences Kennebunk, ME USA
3Enzo Life Sciences Farmingdale, NY USA

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Developmental neurotoxicity (DNT) of environmental chemicals has long been identified as a threat to the health of the human population, as the developing nervous system is particularly susceptible to toxicant exposure. The resulting neurological deficits can have long-term effects on families and society both financially and emotionally. Current DNT testing guidelines involve the use of animal models; primarily rodents. The testing strategy incorporates large numbers of animals, which can be extremely time- and cost-intensive; particularly due to the backlog of chemicals needing to be tested (Lein et al., 2005). This demand, in addition to current and future proposed regulations on the use of animals for testing makes it imperative that new models be found to reduce animal experimentation while providing a suitable method to test new chemicals.

Three-dimensional cell models, which incorporate human neural stem cells (hNSCs) aggregated into neurospheres, have been proposed as a viable alternative for DNT testing. The in vitro system has the ability to recapitulate the processes of brain development, including proliferation, migration, differentiation and apoptosis (Salma et al., 2015). Inclusion of human cells, as opposed to murine, also meets recommendations to circumvent the drawback of species differences between in vivo testing and actual exposure effects.

Here we demonstrate the use of a 3D neurosphere model, composed of hNSCs, to conduct toxicity testing of potential neurotoxicants. A spheroid microplate was used to create and maintain cells in the 3D model. 3D neurosphere proliferation, multipotency, along with the continued capacity to differentiate into neurons, astrocytes, and oligodendrocytes was initially validated. Neurotoxicity testing was then performed using neurospheres maintained in the 3D spheroid plate. Detection of induced levels of oxidative stress, apoptotic, and necrotic activity within treated neurospheres, compared to negative control spheres, was evaluated. Monitoring of cell proliferation, differentiation, multipotency and experimental testing was performed using a novel cell imaging multi-mode reader.

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