TY - JOUR
T1 - A Compartmentalized Neuronal Cell-Culture Platform Compatible With Cryo-Fixation by High-Pressure Freezing for Ultrastructural Imaging
AU - Tran, Hung Tri
AU - Lucas, Miriam S.
AU - Ishikawa, Takashi
AU - Shahmoradian, Sarah H.
AU - Padeste, Celestino
N1 - Funding Information:
SS devised the project and obtained project grant funding. SS, CP, and HT planned the experiments and interpreted the results. HT designed and manufactured the device, performed cell culturing, lentivirus preparation and transfection, chemical fixation, SEM characterizations, and high-pressure freezing with advice by SS, and made all figures and schematic models, and wrote the manuscript together with CP and SS, with input from all other authors. HT, TI, and ML built the strategy of freeze substitution and resin embedment of frozen cell cultures, ultramicrotomy, and TEM imaging of ultrathin sections. ML performed the strategy. SS, CP, and TI supervised HT. TI further supported HT in project logistical needs. All authors contributed to the article and approved the submitted version.
Funding Information:
We thank Philipp Berger (Laboratory for Nanoscale Biology, Paul Scherrer Institute) for his expert help with the lentivirus experiments, Bibhas Roy (Laboratory for Nanoscale Biology, Paul Scherrer Institute) for assistance in collecting images by confocal microscopy, and Vitaliy Guzenko (Laboratory for Micro and Nanotechnology, Paul Scherrer Institute) for the fabrication of the photomasks by e-beam lithography. We also thank Gebhard Schertler, Marco Stampanoni, G. V. Shivashankar, and Gregor Cicchetti for scientific discussions and suggestions, Konrad Vogelsang, Christopher Dennis Wild, and Dario Theodor Marty (Laboratory for Micro and Nanotechnology, Paul Scherrer Institute) for technical assistance with sample preparation and maintenance of equipment in cleanroom facility, and Eline Pecho-Vrieseling and Margarita Dinamarca Ceballos (University of Basel, Switzerland) for providing the primary striatal neurons.
Publisher Copyright:
© Copyright © 2021 Tran, Lucas, Ishikawa, Shahmoradian and Padeste.
PY - 2021/9/8
Y1 - 2021/9/8
N2 - 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.
AB - 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.
KW - focused ion beam – scanning electron microscopy
KW - high pressure freezing
KW - microfluidics
KW - neuronal co-culture
KW - neuronal networks
KW - photolithography
KW - serial sectioning and imaging
KW - transmission electron microscopy
UR - http://www.scopus.com/inward/record.url?scp=85115754561&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85115754561&partnerID=8YFLogxK
U2 - 10.3389/fnins.2021.726763
DO - 10.3389/fnins.2021.726763
M3 - Article
C2 - 34566569
AN - SCOPUS:85115754561
SN - 1662-4548
VL - 15
JO - Frontiers in Neuroscience
JF - Frontiers in Neuroscience
M1 - 726763
ER -