Australian Institute for Bioengineering and Nanotechnology, The University of Queensland;
Australian Institute for Bioengineering and Nanotechnology, The University of Queensland.
J Vis Exp. 2022 May 5(183). doi: 10.3791/63714.
Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.
脑类器官是三维的人类大脑发育模型，为疾病建模和大规模基因组学和药物筛选提供了引人注目的前沿平台。由于脑类器官中细胞的自组织特性以及越来越多的生成其的可用方案，类器官之间的异质性和可变性问题已经得到确认。在本方案论文中，我们描述了一种稳健且可重复的方案，该方案在很大程度上克服了这些问题，并在 1 个月内从神经外胚层祖细胞中生成皮质类器官，并且可以维持超过 1 年。该高度可重复的方案可以在标准的组织培养室中轻松进行，并且产生的类器官具有丰富多样的细胞类型，这些细胞类型通常存在于发育中的人类皮质中。尽管具有早期的发育结构，但神经元和其他人类脑细胞类型在体外培养延长后，将开始表现出神经元细胞中典型的衰老迹象，使它们成为研究与衰老相关的神经元过程的有价值和有用的平台。该方案还概述了使用衰老相关β-半乳糖苷酶染色检测皮质脑类器官中这些衰老细胞的方法。
