<meta charset="utf8"></head><body>Protocollo ottimizzato per la preparazione di campioni di organoidi retinici per TEM

<ol>
	<li>Andreazzoli, M., Barravecchia, I., De Cesari, C., Angeloni, D., Demontis, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+pluripotent+stem+cells+to+model+and+treat+inherited+degenerative+diseases+of+the+outer+retina:+3d-organoids+limitations+and+bioengineering+solutions.">Inducible pluripotent stem cells to model and treat inherited degenerative diseases of the outer retina: 3d-organoids limitations and bioengineering solutions.</a> Cells. 10 (9), 2489 (2021).</li><li>Moser, T., Grabner, C. P., Schmitz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+processing+at+ribbon+synapses+in+the+retina+and+the+cochlea.">Sensory processing at ribbon synapses in the retina and the cochlea.</a> Physiol Rev. 100 (1), 103-144 (2020).</li><li>Tom Dieck, S., Brandst&#228;tter, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribbon+synapses+of+the+retina.">Ribbon synapses of the retina.</a> Cell Tissue Res. 326 (2), 339-346 (2006).</li><li>Kolb, H., Fernandez, E., Nelson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Webvision: The organization of the retina and visual. , (1995).</li><li>Soto, F., Zhao, L., Kerschensteiner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapse+maintenance+and+restoration+in+the+retina+by+ngl2.">Synapse maintenance and restoration in the retina by ngl2.</a> Elife. 7, e30388 (2018).</li><li>Kruczek, K., Swaroop, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+stem+cell-derived+retinal+organoids+for+disease+modeling+and+development+of+therapies.">Pluripotent stem cell-derived retinal organoids for disease modeling and development of therapies.</a> Stem Cells. 38 (10), 1206-1215 (2020).</li><li>O'Hara-Wright, M., Gonzalez-Cordero, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+organoids:+A+window+into+human+retinal+development.">Retinal organoids: A window into human retinal development.</a> Development. 147 (24), (2020).</li><li>Norrie, J. 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=Retinoblastoma+from+human+stem+cell-derived+retinal+organoids.">Retinoblastoma from human stem cell-derived retinal organoids.</a> Nat Commun. 12 (1), 4535 (2021).</li><li>Luni, C., Serena, E., Elvassore, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human-on-chip+for+therapy+development+and+fundamental+science.">Human-on-chip for therapy development and fundamental science.</a> Curr Opin Biotechnol. 25, 45-50 (2014).</li><li>Chahine Karam, F., 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=Human+ipsc-derived+retinal+organoids+and+retinal+pigment+epithelium+for+novel+intronic+rpgr+variant+assessment+for+therapy+suitability.">Human ipsc-derived retinal organoids and retinal pigment epithelium for novel intronic rpgr variant assessment for therapy suitability.</a> J Pers Med. 12 (3), 502 (2022).</li><li>Lo, 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=Therapeutic+strategies+for+glaucoma+and+optic+neuropathies.">Therapeutic strategies for glaucoma and optic neuropathies.</a> Mol Aspects Med. 94, 101219 (2023).</li><li>Capowski, E. 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>Autore in primo piano: Analisi dell'ultrastruttura sinaptica in organoidi retinici maturi utilizzando la TEM

Preparazione di campioni di organoidi retinici per la microscopia elettronica a trasmissione

Our research primarily focuses on analyzing the synaptic ultrastructure in mature retinal organoids. We aim to develop a reproducible and straightforward transmission electron microscope, or TEM, sample preparation method for retinal organoids, which could greatly benefit the field of retinal organoid research. Compared to other techniques such as optical microscope, transmission electron microscope offers the ability to characterize the ultrastructure of integral synapse and the subcellular organelles in retinal organoids at a nanoscale level.Our protocol offers optimized conditions for transmission electron microscope sample preparation of retinal organoids, returning simple and user-friendly stats. Our findings clearly show the ultrastructure morphology of synapse within retinal organoids, including both conventional synapse and ribbon synapse. This establishes a solid foundations and presents a reproducible methods for subsequent functional investigations of retinal organoids, along with the evaluation of their clinical applications such as drug screening.

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

455 Views.  Wenzhou Medical University. La nostra ricerca si concentra principalmente sull'analisi dell'ultrastruttura sinaptica in organoidi retinici maturi. Il nostro obiettivo è quello di sviluppare un metodo di preparazione dei campioni per organoidi retinici riproducibile e semplice, o TEM, che potrebbe essere di grande beneficio nel campo della ricerca sugli organoidi retinici. Rispetto ad altre tecniche come il microscopio ottico, il microscopio elettronico a trasmissione offre la capacità...