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

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. © 2018 by John Wiley & Sons, Inc.

SUBMITTER: Janssens S

PROVIDER: S-EPMC6336506 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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

Janssens Sylvie S Schotsaert Michael M Manganaro Lara L Dejosez Marion M Simon Viviana V García-Sastre Adolfo A Zwaka Thomas P TP

Current protocols in stem cell biology 20181024 1

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 cu ...[more]

PMID: 30358129

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Project description:In vitro brain-on-a-chip platforms hold promise in many areas including: drug discovery, evaluating effects of toxicants and pathogens, and disease modelling. A more accurate recapitulation of the intricate organization of the brain in vivo may require a complex in vitro system including organization of multiple neuronal cell types in an anatomically-relevant manner. Most approaches for compartmentalizing or segregating multiple cell types on microfabricated substrates use either permanent physical surface features or chemical surface functionalization. This study describes a removable insert that successfully deposits neurons from different brain areas onto discrete regions of a microelectrode array (MEA) surface, achieving a separation distance of 100 μm. The regional seeding area on the substrate is significantly smaller than current platforms using comparable placement methods. The non-permanent barrier between cell populations allows the cells to remain localized and attach to the substrate while the insert is in place and interact with neighboring regions after removal. The insert was used to simultaneously seed primary rodent hippocampal and cortical neurons onto MEAs. These cells retained their morphology, viability, and function after seeding through the cell insert through 28 days in vitro (DIV). Co-cultures of the two neuron types developed processes and formed integrated networks between the different MEA regions. Electrophysiological data demonstrated characteristic bursting features and waveform shapes that were consistent for each neuron type in both mono- and co-culture. Additionally, hippocampal cells co-cultured with cortical neurons showed an increase in within-burst firing rate (p = 0.013) and percent spikes in bursts (p = 0.002), changes that imply communication exists between the two cell types in co-culture. The cell seeding insert described in this work is a simple but effective method of separating distinct neuronal populations on microfabricated devices, and offers a unique approach to developing the types of complex in vitro cellular environments required for anatomically-relevant brain-on-a-chip devices.

Dataset Information

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

Publications

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

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