Dynamic culture of cerebral organoids using a pillar/perfusion plate for the assessment of developmental neurotoxicity.
Dynamic culture of cerebral organoids using a pillar/perfusion plate for the assessment of developmental neurotoxicity.
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使用柱/灌注板动态培养脑类器官以评估发育神经毒性。
DOI：
10.1101/2024.03.11.584506
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发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Lee,Moo-Yeal
中科院分区：
文献类型：
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作者：
Acharya,Prabha;Shrestha,Sunil;Joshi,Pranav;Choi,NaYoung;Lekkala,VinodKumarReddy;Kang,Soo-Yeon;Ni,Gabriel;Lee,Moo-Yeal
尽管商业化学品对神经系统发育具有潜在毒性（称为发育神经毒性或DNT），但传统的体外细胞模型主要用于评估急性神经元毒性。另一方面，由于人和动物之间的异质性差异，用于评估DNT的动物模型不具有生理学相关性。此外，动物模型是低通量的，耗时的，昂贵的，并且在伦理上是有问题的。最近，人脑类器官已成为评估化学物质对发育中大脑的有害影响的一种有前途的替代方法。然而，常规的类器官培养系统具有若干技术限制，包括低通量、缺乏再现性、类器官的不充分成熟以及由于营养物和氧气的有限扩散而形成坏死核心。为了解决这些问题并建立预测性DNT模型，在独特的柱/灌注板中在动态条件下分化脑类器官，将其暴露于测试化合物以评估DNT潜力。柱/灌注板促进了脑类器官的均匀、动态培养，通过在数字摇杆上产生的快速、双向流动改善了增殖和成熟。第9天，将柱/灌注板中的脑类器官在动态条件下暴露于抗坏血酸（DNT阴性）和甲基汞（DNT阳性）1周和3周，并测量类器官形态和神经基因表达的变化以确定DNT电位。正如预期的那样，抗坏血酸没有诱导类器官形态和神经基因表达的任何变化。然而，第9天的脑类器官暴露于甲基汞导致类器官形态和神经基因表达的显着变化。有趣的是，甲基汞在静态条件下不会引起脑类器官的不良变化，从而突出了动态类器官培养在DNT评估中的重要性。
Despite the potential toxicity of commercial chemicals to the development of the nervous system (known as developmental neurotoxicity or DNT), conventional in vitro cell models have primarily been employed for the assessment of acute neuronal toxicity. On the other hand, animal models used for the assessment of DNT are not physiologically relevant due to the heterogenic difference between humans and animals. In addition, animal models are low-throughput, time-consuming, expensive, and ethically questionable. Recently, human brain organoids have emerged as a promising alternative to assess the detrimental effects of chemicals on the developing brain. However, conventional organoid culture systems have several technical limitations including low throughput, lack of reproducibility, insufficient maturity of organoids, and the formation of the necrotic core due to limited diffusion of nutrients and oxygen. To address these issues and establish predictive DNT models, cerebral organoids were differentiated in a dynamic condition in a unique pillar/perfusion plate, which were exposed to test compounds to evaluate DNT potential. The pillar/perfusion plate facilitated uniform, dynamic culture of cerebral organoids with improved proliferation and maturity by rapid, bidirectional flow generated on a digital rocker. Day 9 cerebral organoids in the pillar/perfusion plate were exposed to ascorbic acid (DNT negative) and methylmercury (DNT positive) in a dynamic condition for 1 and 3 weeks, and changes in organoid morphology and neural gene expression were measured to determine DNT potential. As expected, ascorbic acid did not induce any changes in organoid morphology and neural gene expression. However, exposure of day 9 cerebral organoids to methylmercury resulted in significant changes in organoid morphology and neural gene expression. Interestingly, methylmercury did not induce adverse changes in cerebral organoids in a static condition, thus highlighting the importance of dynamic organoid culture in DNT assessment.