Protocol for the generation of cultured cortical brain organoid slices

• Published in: STAR protocols Vol. 5; no. 3; p. 103212
• Main Authors: Petersilie, Laura, Kafitz, Karl W., Neu, Louis A., Heiduschka, Sonja, Le, Stephanie, Prigione, Alessandro, Rose, Christine R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 20.09.2024; Elsevier
• Subjects: Brain - cytology; Brain - metabolism; Cell culture; Cell Culture Techniques - methods; Cerebral Cortex - cytology; Humans; Microscopy; Neuroscience; Organoids; Organoids - cytology; Organoids - metabolism; Pluripotent Stem Cells - cytology; Pluripotent Stem Cells - metabolism; Protocol; Cell culture; Neuroscience; Microscopy; Organoids
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2024.103212

Three-dimensional brain organoids from human pluripotent stem cells are a powerful tool for studying human neural networks. Here, we present a protocol for generating cortical brain organoid slices (cBOS) derived from regionalized cortical organoids and grown at the air-liquid interphase. We provide steps for slicing organoids and maintaining them in long-term culture. We then detail approaches for quality control including the evaluation of cell death and cellular identity. Finally, we describe procedures for the expression of a genetically encoded nanosensor for ATP. For complete details on the use and execution of this protocol, please refer to Petersilie et al.1 [Display omitted] •Protocol to prepare robust human cortical brain organoid slices (cBOS)•Detailed steps for maintenance of cBOS in long-term culture•Evaluation of cell death and identification of different cell types in cBOS•Functional characterization of cells using fluorescence-based nanosensors Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Three-dimensional brain organoids from human pluripotent stem cells are a powerful tool for studying human neural networks. Here, we present a protocol for generating cortical brain organoid slices (cBOS) derived from regionalized cortical organoids and grown at the air-liquid interphase. We provide steps for slicing organoids and maintaining them in long-term culture. We then detail approaches for quality control including the evaluation of cell death and cellular identity. Finally, we describe procedures for the expression of a genetically encoded nanosensor for ATP.