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Structural Analysis of Cerebral Organoids Using Confocal Microscopy and Transmission/Scanning Electron Microscopy

Authors
Noh, SeulgiPark, YurimKim, BeomsueMun, Ji Young
Issue Date
Feb-2025
Publisher
OXFORD UNIV PRESS
Keywords
air-liquid interface; cerebral organoid; large-area scanning electron microscopy; transmission electron microscopy; ultrastructure
Citation
MICROSCOPY AND MICROANALYSIS, v.31, no.1
Journal Title
MICROSCOPY AND MICROANALYSIS
Volume
31
Number
1
URI
http://scholarworks.bwise.kr/kbri/handle/2023.sw.kbri/1254
DOI
10.1093/mam/ozae119
ISSN
1431-9276
1435-8115
Abstract
Cerebral organoid cultures from human-induced pluripotent stem cells are widely used to study complex human brain development; however, there is still limited ultrastructural information regarding the development. In this study, we examined the structural details of cerebral organoids using various microscopy techniques. Two protocols were chosen as representative methods for the development of brain organoids: the classic whole-cerebral organoid (Whole-CO) culture technique, and the air-liquid interface-cerebral organoid (ALI-CO) culture technique. Immunostained confocal laser scanning microscopy (CLSM) revealed the formation of the CTIP2- and TBR1-positive cortical deep layer on days 90 and 150, depending on the developmental progress of both methods. Furthermore, the presence of astrocytes and oligodendrocytes was verified through immunostained CLSM utilizing two-dimensional and three-dimensional reconstruction images after a 150-day period. Transmission electron microscopy analysis revealed nanometer-resolution details of the cellular organelles and neuron-specific structures including synapses and myelin. Large-area scanning electron microscopy confirmed the well-developed neuronal connectivity from each culture method on day 150. Using those microscopy techniques, we clearly showed significant details within two representative culture protocols, the Whole-CO and ALI-CO culture methods. These multi-level images provide ultrastructural insight into the features of cerebral organoids depending on the developmental stage.
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