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dc.contributor.author
Tran, Hung Tri
dc.contributor.author
Lucas, Miriam S.
dc.contributor.author
Ishikawa, Takashi
dc.contributor.author
Shahmoradian, Sarah H.
dc.contributor.author
Padeste, Celestino
dc.date.accessioned
2021-10-08T11:45:08Z
dc.date.available
2021-09-27T02:55:34Z
dc.date.available
2021-10-08T11:45:08Z
dc.date.issued
2021-09
dc.identifier.issn
1662-453X
dc.identifier.issn
1662-4548
dc.identifier.other
10.3389/fnins.2021.726763
en_US
dc.identifier.uri
http://hdl.handle.net/20.500.11850/507049
dc.identifier.doi
10.3929/ethz-b-000507049
dc.description.abstract
The human brain contains a wide array of billions of neurons and interconnections, which are often simplified for analysis in vitro using compartmentalized microfluidic devices for neuronal cell culturing, to better understand neuronal development and disease. However, such devices are traditionally incompatible for high-pressure freezing and high-resolution nanoscale imaging and analysis of their sub-cellular processes by methods including electron microscopy. Here we develop a novel compartmentalized neuronal co-culture platform allowing reconstruction of neuronal networks with high variable spatial control, which is uniquely compatible for high-pressure freezing. This cryo-fixation method is well-established to enable high-fidelity preservation of the reconstructed neuronal networks and their sub-cellular processes in a near-native vitreous state without requiring chemical fixatives. To direct the outgrowth of neurites originating from two distinct groups of neurons growing in the two different compartments, polymer microstructures akin to microchannels are fabricated atop of sapphire disks. Two populations of neurons expressing either enhanced green fluorescent protein (EGFP) or mCherry were grown in either compartment, facilitating the analysis of the specific interactions between the two separate groups of cells. Neuronally differentiated PC12 cells, murine hippocampal and striatal neurons were successfully used in this context. The design of this device permits direct observation of entire neuritic processes within microchannels by optical microscopy with high spatial and temporal resolution, prior to processing for high-pressure freezing and electron microscopy. Following freeze substitution, we demonstrate that it is possible to process the neuronal networks for ultrastructural imaging by electron microscopy. Several key features of the embedded neuronal networks, including mitochondria, synaptic vesicles, axonal terminals, microtubules, with well-preserved ultrastructures were observed at high resolution using focused ion beam – scanning electron microscopy (FIB-SEM) and serial sectioning – transmission electron microscopy (TEM). These results demonstrate the compatibility of the platform with optical microscopy, high-pressure freezing and electron microscopy. The platform can be extended to neuronal models of brain disease or development in future studies, enabling the investigation of subcellular processes at the nanoscale within two distinct groups of neurons in a functional neuronal pathway, as well as pharmacological testing and drug screening.
en_US
dc.format
application/pdf
en_US
dc.language.iso
en
en_US
dc.publisher
Frontiers
en_US
dc.rights.uri
http://creativecommons.org/licenses/by/4.0/
dc.subject
high pressure freezing
en_US
dc.subject
neuronal co-culture
en_US
dc.subject
focused ion beam
en_US
dc.subject
scanning electron microscopy
en_US
dc.subject
serial sectioning and imaging
en_US
dc.subject
photolithography
en_US
dc.subject
transmission electron microscopy
en_US
dc.subject
microfluidics
en_US
dc.subject
neuronal networks
en_US
dc.title
A Compartmentalized Neuronal Cell-Culture Platform Compatible With Cryo-Fixation by High-Pressure Freezing for Ultrastructural Imaging
en_US
dc.type
Journal Article
dc.rights.license
Creative Commons Attribution 4.0 International
dc.date.published
2021-09-08
ethz.journal.title
Frontiers in Neuroscience
ethz.journal.volume
15
en_US
ethz.journal.abbreviated
Front Neurosci
ethz.pages.start
726763
en_US
ethz.size
15 p.
en_US
ethz.version.deposit
publishedVersion
en_US
ethz.identifier.wos
ethz.identifier.scopus
ethz.publication.place
Lausanne
en_US
ethz.publication.status
published
en_US
ethz.date.deposited
2021-09-27T02:55:40Z
ethz.source
WOS
ethz.eth
yes
en_US
ethz.availability
Open access
en_US
ethz.rosetta.exportRequired
true
ethz.COinS
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