<meta charset="utf8"></head><body>TEM用のレチナールオルガノイドサンプル調製のための最適化されたプロトコール

<ol>
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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducibility+and+staging+of+3d+human+retinal+organoids+across+multiple+pluripotent+stem+cell+lines.">Reproducibility and staging of 3d human retinal organoids across multiple pluripotent stem cell lines.</a> Development. 146 (1), 171686 (2019).</li><li>Cowan, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+types+of+the+human+retina+and+its+organoids+at+single-cell+resolution.">Cell types of the human retina and its organoids at single-cell resolution.</a> Cell. 182 (6), 1623-1640 (2020).</li><li>Cora, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cleared+view+on+retinal+organoids.">A cleared view on retinal organoids.</a> Cells. 8 (5), 391 (2019).</li><li>&#379;ak, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+in+situ+transmission+electron+microscopy&#9472;development,+challenges,+and+perspectives.">Light-induced in situ transmission electron microscopy&#9472;development, challenges, and perspectives.</a> Nano Lett. 22 (23), 9219-9226 (2022).</li><li>Tschulakow, A. V., Oltrup, T., Bende, T., Schmelzle, S., Schraermeyer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anatomy+of+the+foveola+reinvestigated.">The anatomy of the foveola reinvestigated.</a> PeerJ. 6, e4482 (2018).</li><li>Syrbe, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M&#252;ller+glial+cells+of+the+primate+foveola:+An+electron+microscopical+study.">M&#252;ller glial cells of the primate foveola: An electron microscopical study.</a> Exp Eye Res. 167, 110-117 (2018).</li><li>Xiao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rod+bipolar+cells+receive+cone+photoreceptor+inputs+through+both+invaginating+synapses+and+flat+contacts+in+the+mouse+and+guinea+pig+retinas.">Rod bipolar cells receive cone photoreceptor inputs through both invaginating synapses and flat contacts in the mouse and guinea pig retinas.</a> J Comp Neurol. 531 (11), 1184-1197 (2023).</li><li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+degeneration+in+rpgra+mutant+zebrafish.">Retinal degeneration in rpgra mutant zebrafish.</a> Front Cell Dev Biol. 11, 1169941 (2023).</li><li>Yang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+&#945;-synuclein+in+the+mouse+retina+is+confined+to+inhibitory+presynaptic+elements.">Expression of &#945;-synuclein in the mouse retina is confined to inhibitory presynaptic elements.</a> J Comp Neurol. 531 (10), 1057-1079 (2023).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+degeneration+of+photoreceptor+synapse+in+ccl2/cx3cr1-deficient+mice+on+crb1(rd8)+background.">Early degeneration of photoreceptor synapse in ccl2/cx3cr1-deficient mice on crb1(rd8) background.</a> Synapse. 67 (8), 515-531 (2013).</li><li>De Robertis, E., Franchi, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscope+observations+on+synaptic+vesicles+in+synapses+of+the+retinal+rods+and+cones.">Electron microscope observations on synaptic vesicles in synapses of the retinal rods and cones.</a> J Biophys Biochem Cytol. 2 (3), 307-318 (1956).</li><li>Frey, T. G., Mannella, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+internal+structure+of+mitochondria.">The internal structure of mitochondria.</a> Trends Biochem Sci. 25 (7), 319-324 (2000).</li><li>Gonzalez-Cordero, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recapitulation+of+human+retinal+development+from+human+pluripotent+stem+cells+generates+transplantable+populations+of+cone+photoreceptors.">Recapitulation of human retinal development from human pluripotent stem cells generates transplantable populations of cone photoreceptors.</a> Stem Cell Reports. 9 (3), 820-837 (2017).</li><li>Gao, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-specific+retinal+organoids+recapitulate+disease+features+of+late-onset+retinitis+pigmentosa.">Patient-specific retinal organoids recapitulate disease features of late-onset retinitis pigmentosa.</a> Front Cell Dev Biol. 8, 128 (2020).</li><li>Afanasyeva, T. a. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+look+into+retinal+organoids:+Methods,+analytical+techniques,+and+applications.">A look into retinal organoids: Methods, analytical techniques, and applications.</a> Cell Mol Life Sci. 78 (19-20), 6505-6532 (2021).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mm Petri dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Electron Microscopy Science</td>
			<td>10000</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>A3582801</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lifter</td>
			<td>Santa Cruz</td>
			<td>sc-395251</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper grids</td>
			<td>Beijing Zhongjingkeyi Technology Co., Ltd.</td>
			<td>AZH400HH</td>
			<td></td>
		</tr>
		<tr>
			<td>DigitalMicrograph Software</td>
			<td>Gatan, Inc.</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>StemCell Technologies</td>
			<td>#07913</td>
			<td>Bacterial protease</td>
		</tr>
		<tr>
			<td>DMEM/F12 medium</td>
			<td>Gibco</td>
			<td>#11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding mold</td>
			<td>Beijing Zhongjingkeyi Technology Co., Ltd.</td>
			<td>GZ10592</td>
			<td></td>
		</tr>
		<tr>
			<td>Epon-812 resin</td>
			<td>Electron Microscopy Science</td>
			<td>#14900</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Biological Industries</td>
			<td>#04-0021A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Electron Microscopy Science</td>
			<td>16020</td>
			<td></td>
		</tr>
		<tr>
			<td>hiPSC</td>
			<td>Shownin Biotechnology Co. Ltd.</td>
			<td>RC01001-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Lead citrate</td>
			<td>Beijing Zhongjingkeyi Technology Co., Ltd.</td>
			<td>GZ02618</td>
			<td></td>
		</tr>
		<tr>
			<td>L-GlutaMax</td>
			<td>Life Technologies</td>
			<td>#35050061</td>
			<td>L-glutamine substitute</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356234</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>CITOTEST</td>
			<td>80312-3161</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4&#183; 12H2O</td>
			<td>Sigma</td>
			<td>71650</td>
			<td>A component of PB/PBS</td>
		</tr>
		<tr>
			<td>NaH2PO4&#183; H2O</td>
			<td>Sigma</td>
			<td>71507</td>
			<td>A component of PB/PBS</td>
		</tr>
		<tr>
			<td>Non-essential amino acids</td>
			<td>Sigma</td>
			<td>#M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical microscope</td>
			<td>Lab Binocular Biological Microscope</td>
			<td>Xsz-107bnii</td>
			<td></td>
		</tr>
		<tr>
			<td>OsO4</td>
			<td>TED PELLA</td>
			<td>4008-160501</td>
			<td></td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Bluepard</td>
			<td>BPG9040A</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Science</td>
			<td>157-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>#15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Semi/ultrathin microtome</td>
			<td>Reichert-Jung</td>
			<td>396649</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma</td>
			<td>#T0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine blue</td>
			<td>Sangon Biotech</td>
			<td>E670105-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscopes</td>
			<td>HITACHI</td>
			<td>H-7500</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>TED PELLA</td>
			<td>CA96049</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Sigma</td>
			<td>444203</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparing retinal organoid samples for transmission electron microscopy

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 cortex4,5.
Retinal organoids (ROs) represent a three-dimensional (3D) culture system derived from induced pluripotent stem cells (iPSCs), mimicking the physiological states of retinal tissue in vitro1,6,7. This approach holds promise for studying retinal diseases8, drug screening9, and serving as a potential therapy for irreversible retinal degenerative conditions such as retinitis pigmentosa10&#160;and glaucoma11. As a powerful in vitro optical conduction system, the synapse within ROs is a crucial structure facilitating effective signal transformation and transfer5.
RO development can be roughly divided into three stages according to their morphological traits and molecular expression profiles6,12. ROs in stage 1 (around D21-D60) comprise neural progenitor cells of the retina, many retinal ganglion cells (RGCs), and a few starburst amacrine cells (SACs), corresponding to the first epoch of human fetal development. In stage 2 (around D50-D150), ROs express some photoreceptor precursors, interneurons, and synaptogenesis-related genes, which represents a phase of transition. Photoreceptors develop maturity in stage 3 ROs (around after D100-D150), corresponding&#160;to the third stage of human fetal development6,12,13. Notably, compared with ROs in stage 1 and stage 2, ROs in stage 3 have a distinct lamellar structure whose synapses have matured12, including the presence of ribbon synapses14. Moreover, a recent study has confirmed that the mature synapses exist the transmission of light signals, indicating they are functional13. Thus, ROs in stage 3 are often selected to investigate synaptic structure.
Immunohistochemistry is widely applied to the study of the expression of various molecular proteins. However, the limitation of optical microscopy lies in its ability to observe only a restricted number of specific cells and molecules at a time, resulting in a lack of comprehensive analysis of the relationships between cells and their surrounding environment. Transmission Electron Microscopy (TEM) is characterized by high resolution, with a limited resolution of 0.1-0.2 nm, surpassing the light microscope by ~10-20 times15. It makes up for the defects of optical microscopy and is used to elucidate retinal development and synapse maturation in humans16,17 and various species18,19,20,21. TEM enables the direct distinction of presynaptic and postsynaptic components18,20, and even allows for comprehensive observation of subcellular structures such as ribbons2,3, vesicles22, and mitochondria23. Therefore, TEM is an essential tool for identifying types of synapses and exploring the ultrastructure of synaptic contacts in ROs at the nanoscale.
It is crucial to note that sample preparation is of great importance for acquiring high-quality electron micrographs. Although some studies have performed EM on ROs12,13,24, the detailed procedures are unclear. Since the quality of the electron microscopy image depends on the effect of RO fixation and reagent permeation to a large extent, various important factors need to be considered during preparation. Consequently, to better investigate synaptic contacts in ROs, we present a method with good reproducibility that shows the operation points of RO fixing, embedding, and the identification of observation sites.

<meta charset="utf8"></head><body>著者スポットライト:TEMを使用した成熟Retinalオルガノイドのシナプス超微細構造の解析

透過型電子顕微鏡法のためのレチナールオルガノイドサンプルの調製

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem ...

preparing-retinal-organoid-samples-for-transmission-electron

Research

JoVE Journal

Neuroscience

Preparing Retinal Organoid Samples for Transmission Electron Microscopy

553 ビュー.  Wenzhou Medical University. 私たちの研究は主に、成熟した網膜オルガノイドのシナプス超微細構造の解析に焦点を当てています。私たちは、網膜オルガノイドの研究分野に大きな利益をもたらす可能性のある、網膜オルガノイド用の再現性のある簡便な透過型電子顕微鏡(TEM)試料調製法の開発を目指しています。光学顕微鏡などの他の技術と比較して、透過型電子顕微鏡?...

ビデオ: 著者スポットライト:TEMを使用した成熟Retinalオルガノイドのシナプス超微細構造の解析

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.

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.