Cerebral organoids, or brain organoids, can be generated from a wide array of emerging technologies for modeling brain development and disease. The fact that they are cultured in vitro makes them easily accessible both genetically and for live assays such as fluorescence imaging. In this Protocol Extension, we describe a modified version of our original protocol (published in 2014) that can be used to reliably generate cerebral organoids of a telencephalic identity and maintain long-term viability for later stages of neural development, including axon outgrowth and neuronal maturation. The method builds upon earlier cerebral organoid methodology, with modifications of embryoid body size and shape to increase surface area and slice culture to maintain nutrient and oxygen access to the interior regions of the organoid, enabling long-term culture. We also describe approaches for introducing exogenous plasmid constructs and for sparse cell labeling to image neuronal axon outgrowth and maturation over time. Together, these methods allow for modeling of later events in cortical development, which are important for neurodevelopmental disease modeling. The protocols described can be easily performed by an experimenter with stem cell culture experience and take 2–3 months to complete, with long-term maturation occurring over several months.

大脑类器官或大脑类器官可以从一系列用于模拟大脑发育和疾病的新兴技术中生成。它们在体外培养的事实使它们很容易从遗传和荧光成像等活体检测中获得。在本协议扩展中，我们描述了我们原始协议（2014 年发布）的修改版本，可用于可靠地生成端脑身份的大脑类器官，并为神经发育的后期阶段保持长期活力，包括轴突生长和神经元成熟。该方法建立在早期的大脑类器官方法的基础上，通过修改胚胎体的大小和形状来增加表面积和切片培养，以保持营养和氧气进入类器官的内部区域，从而实现长期培养。我们还描述了引入外源质粒构建体和稀疏细胞标记的方法，以随着时间的推移对神经元轴突的生长和成熟进行成像。总之，这些方法允许对皮质发育中的后期事件进行建模，这对于神经发育疾病建模很重要。所描述的方案可由具有干细胞培养经验的实验者轻松执行，需要 2-3 个月才能完成，长期成熟需要几个月的时间。