Acharya Prabha, Shrestha Sunil, Joshi Pranav, Choi Na Young, Lekkala Vinod Kumar Reddy, Kang Soo-Yeon, Ni Gabriel, Lee Moo-Yeal
Department of Biomedical Engineering, University of North Texas, Denton, Texas.
Bioprinting Laboratories Inc., Dallas, Texas.
bioRxiv. 2024 Mar 13:2024.03.11.584506. doi: 10.1101/2024.03.11.584506.
Despite the potential toxicity of commercial chemicals to the development of the nervous system (known as developmental neurotoxicity or DNT), conventional 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 didn't 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.
尽管商业化学品对神经系统发育具有潜在毒性(即发育性神经毒性,简称DNT),但传统细胞模型主要用于评估急性神经元毒性。另一方面,由于人类与动物之间存在异质性差异,用于评估DNT的动物模型与生理情况不相关。此外,动物模型通量低、耗时、成本高且存在伦理问题。最近,人类脑类器官已成为评估化学品对发育中大脑有害影响的一种有前景的替代方法。然而,传统类器官培养系统存在若干技术限制,包括通量低、缺乏可重复性、类器官成熟度不足以及由于营养物质和氧气扩散有限而形成坏死核心。为了解决这些问题并建立预测性DNT模型,在独特的柱/灌注板中于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/灌注板通过数字摇杆产生的快速双向流动促进了脑类器官的均匀、动态培养,提高了增殖和成熟度。将柱/灌注板中第9天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,测量类器官形态和神经基因表达的变化以确定DNT潜力。正如预期的那样,抗坏血酸未引起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发生显著变化。有趣的是,甲基汞在静态条件下未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。