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Preparing Retinal Organoid Samples for Transmission Electron Microscopy
In This Article
Summary
This protocol provides an optimized and elaborate preparation procedure for retinal organoid samples for transmission electron microscopy. It is suitable for applications that involve the analysis of synapses in mature retinal organoids.
Abstract
Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem cells (iPSCs) under specific conditions. Synapse development and maturation in ROs have been studied immunocytochemically and functionally. However, the direct evidence of the synaptic contact ultrastructure is limited, containing both special ribbon synapses and conventional chemical synapses. Transmission electron microscopy (TEM) is characterized by high resolution and a respectable history elucidating retinal development and synapse maturation in humans and various species. It is a powerful tool to explore synaptic structure in ROs and is widely used in the research field of ROs. Therefore, to better explore the structure of RO synaptic contacts at the nanoscale and obtain high-quality microscopic evidence, we developed a simple and repeatable method of RO TEM sample preparation. This paper describes the protocol, reagents used, and detailed steps, including RO fixation preparation, post fixation, embedding, and visualization.
Introduction
The retina, a vital visual sensory organ in humans and mammals, exhibits a distinct laminated structure characterized by three nuclear layers housing neuron somas and two plexiform layers formed by synaptic connections1, including conventional synapses and the specialized ribbon synapse2,3. The ribbon synapse plays a crucial role in transmitting vesicle impulses in a graded manner2,3. The vision process involves electro-optical signal transmission across various levels of neurons and synapses, ultimately reaching the visual cort....
Protocol
1. Obtaining ROs from iPSCs25
NOTE: ROs were derived from iPSCs by modifying the previously reported procedure.
- Dissociate iPSCs at ~90% confluence using a bacterial protease (see Table of Materials). Chop up the colonies into pieces and scrape them with a cell lifter.
- After collection, resuspend the clusters of cells in 0.25 mL of ice-cold Matrigel. Following 20 min of incubation at 37 °C, sc.......
Representative Results
The establishment of 3D ROs through iPSC differentiation provides a powerful tool for studying retinal disease mechanisms and stem cell replacement therapy. Although others have demonstrated the synaptic connections in ROs functionally and immunocytochemically, direct evidence of conventional and ribbon synapses is very limited. Here we present a method for investigating the ultrastructure of two types of synapses in ROs by TEM. After 180 days of culture, ROs were fixed, stained, embedded, and ultrathin sliced. TEM obser.......
Discussion
In this article, we presented a detailed protocol for observing conventional and ribbon synaptic ultrastructure in ROs by TEM. This protocol is based on the previously described retinal preparation methods with some modifications20. To improve the success rate of sample treatment and the quality of TEM micrographs, consider the following key points. First, it is important to acknowledge that ROs develop from iPSCs, forming a cell mass lacking vasculature6,
Acknowledgements
This work was supported in part by grants from the National Key Research and Development Program of China (2022YFA1105503), the State Key Laboratory of Neuroscience (SKLN-202103), and the Zhejiang Natural Science Foundation of China (Y21H120019), the Natural Science Foundation of China (82070981).
....Materials
| Name | Company | Catalog Number | Comments |
| 100 mm Petri dish | Corning | 430167 | |
| Acetone | Electron Microscopy Science | 10000 | |
| B27 supplement | Gibco | A3582801 | |
| Cell lifter | Santa Cruz | sc-395251 | |
| Copper grids | Beijing Zhongjingkeyi Technology Co., Ltd. | AZH400HH | |
| DigitalMicrograph Software | Gatan, Inc. | Software | |
| Dispase | StemCell Technologies | #07913 | Bacterial protease |
| DMEM/F12 medium | Gibco | #11320033 | |
| Embedding mold | Beijing Zhongjingkeyi Technology Co., Ltd. | GZ10592 | |
| Epon-812 resin | Electron Microscopy Science | #14900 | |
| Fetal Bovine Serum (FBS) | Biological Industries | #04-0021A | |
| Glutaraldehyde | Electron Microscopy Science | 16020 | |
| hiPSC | Shownin Biotechnology Co. Ltd. | RC01001-A | |
| Lead citrate | Beijing Zhongjingkeyi Technology Co., Ltd. | GZ02618 | |
| L-GlutaMax | Life Technologies | #35050061 | L-glutamine substitute |
| Matrigel | Corning | 356234 | |
| Microscope slide | CITOTEST | 80312-3161 | |
| N2 supplement | Gibco | 17502048 | |
| Na2HPO4· 12H2O | Sigma | 71650 | A component of PB/PBS |
| NaH2PO4· H2O | Sigma | 71507 | A component of PB/PBS |
| Non-essential amino acids | Sigma | #M7145 | |
| Optical microscope | Lab Binocular Biological Microscope | Xsz-107bnii | |
| OsO4 | TED PELLA | 4008-160501 | |
| Oven | Bluepard | BPG9040A | |
| Paraformaldehyde | Electron Microscopy Science | 157-8 | |
| Penicillin-Streptomycin | Gibco | #15140-122 | |
| Semi/ultrathin microtome | Reichert-Jung | 396649 | |
| Taurine | Sigma | #T0625 | |
| Toluidine blue | Sangon Biotech | E670105-0100 | |
| Transmission Electron Microscopes | HITACHI | H-7500 | |
| Uranyl acetate | TED PELLA | CA96049 | |
| β-mercaptoethanol | Sigma | 444203 |
References
- Andreazzoli, M., Barravecchia, I., De Cesari, C., Angeloni, D., Demontis, G. C. Inducible pluripotent stem cells to model and treat inherited degenerative diseases of the outer retina: 3d-organoids limitations and bioengineering solutions. Cells. 10 (9), 2489 (2021).
- Moser, T., Grabner, C. P., Schmitz, F.
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