The cerebellum plays an important role in maintaining balance and movement coordination. Some studies showed that the functional defects of different cerebellar neurons may result in cerebellar dysfunction. In the past decades, postmortem tissues are major source for research of disease-related neuronal phenotypes. Subsequently, immortalized cell lines, as well as rodent animal models, have been used in neurodegenerative diseases study. In recent years, human induced pluripotent stem cells (iPSCs) present great potentials for disease modeling, the cerebral organoids generated from patients' iPSCs are beneficial for the proposal of treatment strategies.
Studies have shown that the maturation of cerebellar neurons requires isolation of organoids, sorting of cerebellar progenitor cells, and co-culture with feeder cells in a monolayer culture system. Protein analysis can be used to detect different cerebellar cell populations, and immunostaining of organoid slices can further evaluate the spatial organization of specific cerebellar cell populations.
Fig.1 Efficient cerebellar differentiation and maturation in human iPSC-derived organoids. (Silva, 2020)
3D Cerebellar Organoid Culture System
Creative Biolabs has developed a dynamic 3D culture system to produce cerebellar organoids based on iPSCs. It is a single-use vertical wheel bioreactor equipped with a large vertical impeller to provide optimal culture conditions. During the maturation process, different types of cerebellar neurons can be observed, such as Purkinje cells, granule cells, Golgi cells, unipolar brush cells, as well as deep cerebellar nuclei projection neurons. After 35 days, these neurons can survive for almost 90 days for disease modeling use.
Protocols for Cerebellar Organoid Generation
According to the protocol for dynamic 3D culture system, high-quality cerebellar organoids can be obtained for a series of biomedical applications.
Passaging and Maintenance of Human iPSCs in Monolayer CultureSeeding of Human iPSCs in the BioreactorDifferentiation and Maturation of Human iPSC-derived Aggregates in Cerebellar OrganoidsPreparation of Organoids for Cryosectioning and Immunohistochemistry Fig.2 Schematics show the protocol for cerebellar organoid generation.
Features of Cerebellar Organoid Culture System
Repeatability to generate PSC-derived cerebellar-specific organoids
Applications of Cerebellar Organoid
High-throughput screening for drugs that restore cerebellar function
Gene function studies
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Silva, T.; et al. Scalable generation of mature cerebellar organoids from human pluripotent stem cells and characterization by immunostaining. JoVE (Journal of Visualized Experiments). 2020 (160): e61143.