The Neurosphere Assay as an In Vitro Method for Developmental Neurotoxicity (DNT) Evaluation
The human developing central nervous system is more vulnerable to the adverse effects of chemical agents than the adult brain. At present, due to the lack of available data on human neurodevelopmental toxicants, there is an urgent need for testing and sub
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ion Within the last two decades, there has been considerable concern that exposure toward chemicals might be a contributing factor to the increasing incidence of neurodevelopmental disorders in children [1–4]. For few compounds the evidence for causing developmental neurotoxicity (DNT) is clear, whereas for the majority of chemicals this concern hampers a solid scientific basis because they have not been evaluated for their neurodevelopmental toxicity [5, 6]. Laura Nimtz, Jördis Klose, and Stefan Masjosthusmann are contributed equally to this manuscript. Michael Aschner and Lucio Costa (eds.), Cell Culture Techniques, Neuromethods, vol. 145, https://doi.org/10.1007/978-1-4939-9228-7_8, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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The main reason for this data gap lies in the resource intensity of the current guideline studies: EPA 870.6300 developmental neurotoxicity (DNT) guideline [7] and the draft OECD 426 guideline [8]. These guidelines are highly demanding with regard to time, money, and animals [5, 9] and are therefore not suited for testing large number of chemicals. Therefore, international researchers have been establishing alternative methods for faster and cheaper DNT evaluation based on in vitro methods. In addition, concepts on how to use and interpret such methods with the final goal of regulatory application have been developed [9–16]. For alternative DNT evaluation, the complex procedure of brain development is disassembled into spatiotemporal neurodevelopmental processes that are necessary for forming a functional brain and can be tested for adverse effects of compounds in in vitro assays [10, 11, 16, 17]. Here, human-based systems are preferred because species differences in toxicokinetics, e.g., due to developmental timing, and/or toxicodynamics might affect responses to compounds [18–24]. In this chapter we describe one of the methods suitable for DNT evaluation, the “neurosphere assay.” This assay consists of six individual test methods (NPC1–6) measuring different endpoints, some of which can be multiplexed (Fig. 1) [11]. In the following
Fig. 1 Experimental setup of the “neurosphere assay”. (Adapted from Masjosthusmann et al. [23]). NPC were generated from fetal human brain (Lonza, Verviers, Belgium) or postnatal day (PND)1 mouse and rat brains and cultivated as free-floating neurospheres. The “neurosphere assay” consists of six individual assays: proliferation assay (NPC1), migration assay (NPC2), as well as neuronal differentiation assay (NPC3), neuronal morphology assay (NPC4), oligodendrocyte differentiation assay (NPC5), and thyroid hormone (TH)-dependent oligodendrocyte maturation assay (NPC6) for measuring different endpoints under chemical exposure. For saving time and resources, the assays NPC2-5 can be multiplexed. Gene expression analyses in the TH disruption assay (NPC6) quantify the oligodendrocyte-specific maturation genes MBP, MOBP, and MOG [18]
The ‘Neurosphere Assay’ for DNT Evaluation
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