Organoids—or pluripotent stem cell–derived in vitro-grown simplified mini organs—have become a tremendously important model to study human organ development and disease. To restrict the noise inherent to the heterogeneous cell mixtures derived from organoid cultures, we developed a new technique of fluorescence-assisted cell sorting (FACS) of virus-infected cerebral organoid cultures. This method still includes the advantage of growing cells in a more natural environment than traditional cell culture, but now renders samples suitable for downstream cell type-specific multi-omics analyses. The protocol starts from stem cell-derived mature brain organoids and includes steps for: preparing the culture for viral infection, production of the viral stocks, FACS sample preparation, and gating and sorting implementation. The protocol has been developed for Zika virus infection, but can be extrapolated to other viruses or fluorescent marker expression as illustrated in an alternate protocol using a single-cycle lentivirus expressing a fluorescent reporter protein.

FACS-Mediated Isolation of Neuronal Cell Populations From Virus-Infected Human Embryonic Stem Cell–Derived Cerebral Organoid Cultures / S. Janssens, M. Schotsaert, L. Manganaro, M. Dejosez, V. Simon, A. Garcia-Sastre, T.P. Zwaka. - In: CURRENT PROTOCOLS IN STEM CELL BIOLOGY. - ISSN 1941-7322. - 48:1(2019), pp. e65.1-e65.15. [10.1002/cpsc.65]

FACS-Mediated Isolation of Neuronal Cell Populations From Virus-Infected Human Embryonic Stem Cell–Derived Cerebral Organoid Cultures

L. Manganaro;
2019

Abstract

Organoids—or pluripotent stem cell–derived in vitro-grown simplified mini organs—have become a tremendously important model to study human organ development and disease. To restrict the noise inherent to the heterogeneous cell mixtures derived from organoid cultures, we developed a new technique of fluorescence-assisted cell sorting (FACS) of virus-infected cerebral organoid cultures. This method still includes the advantage of growing cells in a more natural environment than traditional cell culture, but now renders samples suitable for downstream cell type-specific multi-omics analyses. The protocol starts from stem cell-derived mature brain organoids and includes steps for: preparing the culture for viral infection, production of the viral stocks, FACS sample preparation, and gating and sorting implementation. The protocol has been developed for Zika virus infection, but can be extrapolated to other viruses or fluorescent marker expression as illustrated in an alternate protocol using a single-cycle lentivirus expressing a fluorescent reporter protein.
cerebral organoids; FACS; HIV; human ESC; lentiviral vector; virus; Zika; Brain; Cell Culture Techniques; Cell Separation; Cells, Cultured; Flow Cytometry; Human Embryonic Stem Cells; Humans; Neurons; Organoids; Zika Virus Infection; Zika Virus
Settore BIO/19 - Microbiologia Generale
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/886578
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