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Mass Generation, Neuron Labelling and 3D Imaging of Minibrains

View ORCID ProfileSubashika Govindan, Laura Batti, Samira F Osterop, Luc Stoppini, View ORCID ProfileAdrien Roux
doi: https://doi.org/10.1101/2020.07.11.198531
Subashika Govindan
1Tissue Engineering Laboratory, HEPIA HES-SO, University of Applied Sciences and Arts Western Switzerland, Geneva, Switzerland
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Laura Batti
2Wyss Center for Bio and Neuroengineering, Geneva, Switzerland
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Samira F Osterop
2Wyss Center for Bio and Neuroengineering, Geneva, Switzerland
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Luc Stoppini
1Tissue Engineering Laboratory, HEPIA HES-SO, University of Applied Sciences and Arts Western Switzerland, Geneva, Switzerland
3Swiss Center for Applied Human Toxicology (SCAHT), Bern, Switzerland
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Adrien Roux
1Tissue Engineering Laboratory, HEPIA HES-SO, University of Applied Sciences and Arts Western Switzerland, Geneva, Switzerland
3Swiss Center for Applied Human Toxicology (SCAHT), Bern, Switzerland
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  • For correspondence: adrien.roux@hesge.ch
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Abstract

Minibrain is a spherical in vitro 3D brain organoid model, composed of a mixed population of neurons and glial cells, generated from human iPSC derived neural stem cells. Despite the advances in human brain organoid models, there is a lack of labelling and imaging methodologies to characterize these models. In this study, we present a step-by-step methodology to generate human minibrain nurseries and novel strategies to subsequently label projection neurons, perform immunohistochemistry and 3D imaging of the minibrains at large multiplexable scales. To visualize projection neurons, we adapt viral transduction and to visualize the organization of cell types we implement immunohistochemistry. To facilitate 3D imaging of minibrains, we present here pipelines and accessories for one step mounting and clearing suitable for confocal microscopy. The pipelines are specifically designed in such a way that the assays can be multiplexed with ease for large-scale screenings using minibrains. Using the pipeline, we present i. dendrite morphometric properties obtained from 3D neuron morphology reconstructions and ii. distribution and quantification of cell types in 3D across whole mount organoids.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted July 17, 2020.
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Mass Generation, Neuron Labelling and 3D Imaging of Minibrains
Subashika Govindan, Laura Batti, Samira F Osterop, Luc Stoppini, Adrien Roux
bioRxiv 2020.07.11.198531; doi: https://doi.org/10.1101/2020.07.11.198531
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Mass Generation, Neuron Labelling and 3D Imaging of Minibrains
Subashika Govindan, Laura Batti, Samira F Osterop, Luc Stoppini, Adrien Roux
bioRxiv 2020.07.11.198531; doi: https://doi.org/10.1101/2020.07.11.198531

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