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).

1. Obtaining ROs from iPSCs25
NOTE: ROs were derived from iPSCs by modifying the previously reported procedure.
<ol>
	<li>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.</li>
	<li>After collection, resuspend the clusters of cells in 0.25 mL of ice-cold Matrigel. Following 20 min of incubation at 37 &#176;C, scatter the solidified gel with the DMEM/F12 medium (0.5% N2 supplement, 1% B27 supplement, 0.1 mM &#946;-mercaptoethanol, 0.2 mM L-glutamine substitute, 100 U/mL penicillin and 100 mg/mL streptomycin). Define this time as day 0 of differentiation.</li>
	<li>After 5 days of floating culture, cells group to form cell cysts. Transfer these cell cysts into a 100 mm Petri dish where they will now adhere and grow.</li>
	<li>On day 15, use bacterial protease to dissociate the adhering cells and culture them in DMEM/F12 medium (2% B27 supplement, 0.1 mM non-essential amino acids).</li>
	<li>On day 21, change the medium with 20 mL of serum-containing medium (DMEM/F12 medium, 8% FBS, 2% B27 supplement, 100 mM Taurine, and 2 mM L-glutamine substitute), and replace the medium every week.</li>
</ol>
2. Anterior fixation of ROs
<ol>
	<li>Gently move one of the cultured ROs (~D180, a size of about 1~2 mm in diameter is optimal) from the Petri dish to 1.5 mL microcentrifuge tubes with a pipette and mark the relevant information about the ROs. 
	NOTE: Cut ROs in half or cut off the poorly structured tissue with a razor under the microscope in the dish containing fixative if the ROs are too large.</li>
	<li>Remove the culture medium from 1.5 mL microcentrifuge tubes with a pipette. Add 1.5 mL of 2% paraformaldehyde-2% glutaraldehyde fixative diluted in 0.1 M phosphate buffer (PB), pH 7.4, and fix at room temperature (RT) for 4 h. Store the microcentrifuge tubes at 4 &#176;C overnight. 
	NOTE: This step can be paused for 1 week but restarting as early as possible is recommended. If ROs are left for longer, it may cause autolysis of cells leading to destruction of the ultrastructure of ROs. Due to the fragility of the ROs, avoid rotating, oscillating, flipping, or harsh operations.</li>
</ol>
3. Post fixation of ROs
<ol>
	<li>Remove the 2% paraformaldehyde-2% glutaraldehyde fixative and wash with 1 mL of 0.01 M phosphate buffered saline (PBS) at pH 7.4 for 3 x 10 min. Gently suspend the ROs in the 1.5 mL microcentrifuge tubes during the washing process for fully rinsing. For every additional day of fixation, increase the number of washes by two to remove&#160;the residual fixative as much as possible.</li>
	<li>Remove any remaining PBS using a pipette and replace it with ~150 &#181;L of 1% osmic acid (OsO4) until the tissue blocks are submerged. Place the microcentrifuge tubes in a dark box to protect them from light and infiltrate at RT for 1 h. 
	NOTE: OsO4 is a strong oxidant and must be handled in a fume hood.</li>
</ol>
4. Staining and dehydration
NOTE: The dehumidifier must be turned on to dry the environment from this step.
<ol>
	<li>Remove OsO4 using a pipette, wash with 0.01 M PBS (pH 7.4) 3 x 10 min, and then wash 3 x 10 min with deionized water. 
	NOTE: To ensure complete rinsing of ROs, we recommend suspending the ROs. Aspirate OsO4 into a dedicated disposal bottle.</li>
	<li>Remove the last deionized water wash and replace with ~150 &#181;L per tube (ensure full infiltration of tissue blocks) of uranium acetate and stain for 1-2 h at RT. Protect the microcentrifuge tubes from light.</li>
	<li>Remove the uranium acetate and replace it with 1 mL of 50% acetone in the fume hood, dehydrating for 10 min. Then, use 70%, 80%, 90%, 100%, and 100% acetone for gradient dehydration of the ROs successively. Each dehydration lasts 10 min. 
	NOTE: Leave some liquid to soak the ROs for each exchange to prevent exposure to air and moisture. Additionally, operate quickly as soon as possible because acetone is volatile. Aspirate uranium acetate into a dedicated disposal bottle.</li>
</ol>
5. Infiltration
<ol>
	<li>Discard the acetone and add ~150 &#181;L of the mixture consisting of acetone and Epon-812 resin at a ratio of 1:1. Place it in a 37 &#176;C oven for 1 h.</li>
	<li>Remove the above mixture and replace it with a mixture consisting of acetone and Epon-812 resin at a ratio of 1:4. Place it in a 37 &#176;C oven overnight.</li>
	<li>Transfer the ROs into the new tubes containing ~500 &#181;L of pure epoxy resin carefully and place them in a 45 &#176;C oven for 1 h.</li>
</ol>
6. Embedding
<ol>
	<li>Prepare the 30-50 mL of pure epoxy resin in a 50 mL centrifuge tube by mixing the reagent kit according to the manufacturer&#39;s instructions in advance. Rotate the pure epoxy resin upside down slowly for 30 min to reduce air bubbles introduced by the mixer. Then, leave it in a 37 &#176;C oven for 20 min. Add 2/3 volume of pure epoxy resin to the groove of the embedding mold.</li>
	<li>Use a toothpick to pick ROs into the embedding mold equipped with pure epoxy resin.</li>
	<li>Fill the groove with pure epoxy resin until it slightly protrudes or bulges.</li>
	<li>Adjust the position of ROs at both ends of the embedding mold for directional embedding. Place the embedding mold in a 45 &#176;C oven for 1-2 h and then put it in a 65 &#176;C oven for 48-72 h.</li>
</ol>
7. Semi-thin positioning
<ol>
	<li>Sand the excess epoxy resin around the tissue from the ends of the longer four sides of the embedding block to form a trapezoidal face. Install the embedding block into the sample mounting stand and screw down the bolt with an L screwdriver. Then, install glass knives on the knife stand and tighten the nut manually.</li>
	<li>Trim away the excess resin until the desired area is exposed.</li>
	<li>Fill the trough of the glass knife with water. Cut semi-thin slices to a thickness of 1 &#181;m using a semi-thin microtome and lay them on a microscope slide with a needle.</li>
	<li>Add 50 &#181;L of 1% toluidine blue to dye semi-thin slices and incubate for 1 min at 95 &#176;C on the heating plate. Rinse with distilled water for 2 min. Use a 20x ordinary optical microscope to observe the structure of the sections.</li>
</ol>
8.&#160;Ultra-thin sectioning
<ol>
	<li>Install the embedding blocks and glass knives as described in step 7.1. Adjust the distance between the knife and the tissue block, the liquid level of the sink, and the slicing speed. Cut the ultrathin sections with a thickness of ~70-100 nm with an ultrathin microtome and scoop the slices onto the copper mesh (200 mesh) with the supporting film.</li>
	<li>Stain ultrathin sections with 3% lead citrate for 15 min to enhance contrast in a dual staining procedure.</li>
</ol>
9. Imaging ROs by TEM
<ol>
	<li>Launch the software (see the Table of Materials).</li>
	<li>Click on the Memory mode button and mark each ultrathin section roughly.</li>
	<li>Search for synaptic structures.
	<ol>
		<li>Distinguish the laminated structure of ROs at low magnification.</li>
		<li>Locate the outer nuclear layer (ONL), which shows a nuclear structure with a deeper electron density. Subsequently, navigate to the terminal of the photoreceptor where numerous vesicles accumulate. Search for the rod terminals with a single synaptic ribbon and the cone terminals with multiple synaptic ribbons.</li>
		<li>Move to the inner plexiform layer (IPL) and seek out a similar structure with a small ribbon.</li>
	</ol>
	</li>
	<li>Capture the microscopic image.
	<ol>
		<li>Click Start and input the corresponding magnifying power.</li>
		<li>Save the images.</li>
	</ol>
	</li>
</ol>

Optimized Protocol for Retinal Organoid Sample Preparation for TEM

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</ol>

The authors have no conflicts of interest to disclose.

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 vasculature6,26&#160;and glial cells26. This absence may lead to a less compact organization compared to the in vivo retina. Thus, it is crucial to handle Ros gently for maintaining their structure and integrity. Add liquids along the tube wall to minimize disturbance.
Importantly, ensure thorough dehydration of the tissue in protocol step 4 and switch on the dehumidifier to reduce room humidity before starting the dehydration step. When replacing acetone, operate quickly and leave some liquid to submerge the tissue, preventing severe destruction of the RO tissue structure. Undehydrated tissue contracts sharply at high EM altitudes, and water in tissue&#160;hampers the penetration of the embedding agent.
Since ROs present as transparent spheres, ensure the correct direction when cutting semi-thin slices to obtain intact lamellar structures. Lastly, ROs in stage 3 (around after D100-D150) are considered mature and possess integral synapses12, as a result of which, the investigation of synaptic contacts, including ribbon synapses and chemical synapses, is recommended to be conducted in those ROs.
Although limited ultrastructural proof of RO synapses has been provided in previous studies, there is no detail about the complete procedure of RO sample preparation as well as the identification of various types of synaptic structures12,13,24. Capowski et al. used different techniques to investigate the characteristics of ROs induced by different hPSC lines at different stages, including electron micrographs showing the existence of vesicle-laden ribbon synapses in the cone pedicle, which revealed that ROs in stage 3 induced by various hPSC lines are mature and functional12. Using TEM and two-photon imaging, Cowan et al. confirmed the formation of functional ribbon synaptic structures in mature ROs13, but the type of synapse was not indicated (cone or rod synapses). However, our several batches of micrograph results showed the membrane structure of the ribbon synapses with an ideal clarity and contrast, which illustrates that our method is feasible and reproducible. In addition, we identified different types of synapses according to their different morphological characteristics: cone synapses are usually larger and have several ribbons while rod synapses are usually smaller, showing only one ribbon; In chemical synapses, vesicles accumulate in the presynaptic structures, and the electron density of the synaptic cleft is enhanced.
The ultrastructural integrity of the synaptic contacts in ROs is vital for their function. Employing TEM to investigate these contacts offers broad and significant advantages in various fields, such as studying the pathogenesis of the retina in vitro, conducting drug screening in ROs, and evaluating differentiation techniques of ROs, among other applications. Limited by 2D TEM imaging, only a specific aspect of the ROs ultrastructure is observable, making it challenging to analyze the synapse&#39;s entire structure stereoscopically. In addition, the boundedness of differentiation contributes to misplaced cells13,26, further complicating the observation process. Thus, combining volume EM with 3D reconstruction may overcome these shortcomings in the future.

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.

<table><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>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot; 12H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>71650</td><td>A component of PB/PBS</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;middot; H&lt;sub&gt;2&lt;/sub&gt;O</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>OsO&lt;sub&gt;4&lt;/sub&gt;</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>&amp;beta;-mercaptoethanol</td><td>Sigma</td><td>444203</td><td></td></tr></tbody></table>

preparing retinal organoid samples for transmission electron microscopy

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.

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 observation revealed ribbon synapses at the axonal terminals of photoreceptors (Figure 1 and Figure 2), which are distributed in the OPL. Under TEM, ribbons of rods spherule around the synaptic invagination, forming a horseshoe-shaped profile with horizontal cells (Figure 1). Rods typically have only a single ribbon, but cones have multiple ribbons (Figure 2). Additionally, classical chemical synapses between amacrine cells in the IPL were observed (Figure 3). Under TEM, the chemical synapse consists of three parts: the presynaptic, the synaptic cleft, and the postsynaptic. The cell membranes corresponding to the presynaptic and postsynaptic parts are slightly thicker than the rest of the parts, and the narrow gap between the two membranes is called the synaptic cleft. There are many synaptic vesicles in the cytoplasm of the presynaptic membrane site. This study confirms the presence of complete synaptic connections in 3D ROs derived from iPSC. However, it should be noted that incomplete rinsing between steps would lead to the precipitation of black particles during the process of sample preparation (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66590/66590fig01.jpg" /> 
Figure 1: TEM images of rod ribbon synapses in ROs. (A,B) Rod and horizontal cells form a horseshoe-shaped profile. A single ribbon (arrowheads) was observed at each rod spherule in the OPL of ROs, surrounded by numerous vesicles. Scale bars = 0.2 &#181;m. Abbreviations: ROs = retinal organoids; HC = horizontal cell; OPL = outer plexiform layer. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66590/66590fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66590/66590fig02.jpg" /> 
Figure 2: TEM images of cone ribbon synapses in ROs. Three ribbon synapses (arrowheads) were obvious at a cone pedicle in the OPL of ROs. Scale bar = 0.2 &#181;m. Abbreviations: TEM = transmission electron microscopy; ROs = retinal organoids; OPL = outer plexiform layer. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66590/66590fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66590/66590fig03.jpg" /> 
Figure 3: TEM image of chemical synapses in ROs. A conventional chemical synapse was observed between amacrine cells in the IPL layer of ROs. The chemical synapse consists of three parts: the presynaptic, the synaptic cleft, and the postsynaptic. There are many synaptic vesicles in the cytoplasm of the presynaptic membrane site. An arrow denotes the direction of synaptic transmission, indicating the transfer of numerous vesicles from one AC1 process to another AC2 profile. Scale bar = 0.2 &#181;m. Abbreviations: TEM = transmission electron microscopy; ROs = retinal organoids; IPL = inner plexiform layer; ACs = amacrine cells. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66590/66590fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66590/66590fig04.jpg" /> 
Figure 4: An example of a problem that can occur during the process of sample preparation. There are a lot of black particles (circles) in the cone pedicle because of incomplete rinsing between steps. Arrows: ribbon synapse. Scale bar = 0.2 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66590/66590fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Author Spotlight: Analyzing the Synaptic Ultrastructure in Mature Retinal Organoids Using TEM

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.

Preparing Retinal Organoid Samples for Transmission Electron Microscopy

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

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

859 Views.  Wenzhou Medical University. 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.

Video: Author Spotlight: Analyzing the Synaptic Ultrastructure in Mature Retinal Organoids Using TEM

The Gateway to the Brain: Dissecting the Primate Eye

The visual system in humans is considered the gateway to the world and plays a principal role in the plethora of sensory, perceptual and cognitive processes. It is therefore not surprising that quality of vision is tied to quality of life . Despite widespread clinical and basic research surrounding the causes of visual disorders, many forms of visual impairments, such as retinitis pigmentosa and macular degeneration, lack effective treatments. Non-human primates have the closest general features of eye development to that of humans. Not only do they have a similar vascular anatomy, but amongst other mammals, primates have the unique characteristic of having a region in the temporal retina specialized for high visual acuity, the fovea1. Here we describe a general technique for dissecting the primate retina to provide tissue for retinal histology, immunohistochemistry, laser capture microdissection, as well as light and electron microscopy. With the extended use of the non-human primate as a translational model, our hope is that improved understanding of the retina will provide insights into effective approaches towards attenuating or reversing the negative impact of visual disorders on the quality of life of affected individuals.

The non-human primate is an important translational species for our understanding of development and aging. The anatomical organization of the primate retina may provide important insights into normal and pathological conditions in humans.

The visual system in humans is considered the  gateway  to the world and plays a principal role in the plethora of sensory, perceptual and cognitive ...

Biology

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 &#181;m thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 &#181;m is discarded before 200 &#181;m of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 &#176;C. Pieces of outer layer are incubated in 500 &#181;l of Ringer&#39;s solution with 2 units of activated papain for 20 min at 37 &#176;C. The reaction is stopped by adding 500 &#181;l 10% fetal calf serum (FCS) in Dulbecco&#39;s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 &#181;l of DMEM and seeded at 1 x 105 cells/cm2. The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 &#181;m) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

Generation of Human Patient iPSC-derived Retinal Organoids to Model Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority of RP patients have progressive photoreceptor degeneration leading to peripheral vision loss, night blindness, and finally, total blindness. To date, thousands of mutations in more than 90 genes have been reported to be associated with RP. Currently, there are few animal models available for all the affected genes and different types of mutations, which largely hampers the deciphering of the mechanisms underlying the gene/mutation pathology and limits treatment and drug development. Patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids (ROs) have provided a better system to model the human early-onset disease than cells and animals. In order to study RP, those patient-derived 3D retinal organoids were utilized to recapitulate the clinical phenotypes of RP. In the RP patient-derived ROs, Rhodopsin mislocalization was clearly displayed. Compared with other animal models, patient iPSC-derived retinal organoid models more closely recapitulated RP features and represent an ideal approach for investigating the disease pathogenesis and for drug development.

In this protocol, retinitis pigmentosa patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids were generated. Those organoids successfully recapitulated some clinical phenotypes of the retinitis pigmentosa disease.

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority ...

Developmental Biology

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M). These immature retinal organoids contain PAX6+ and ChX10+ proliferating, multipotent retinal precursors. The precursor cells are linearly self-assembled within the organoids and appear as distinct radial striations. At 4 weeks after suspension culture, the retinal progenitors undergo post-mitotic arrest and lineage differentiation to form mature retinal organoids (OC-2M). The photoreceptor lineage committed precursors develop within the outermost layers of retinal organoids. These CRX+ and RCVRN+ photoreceptor cells morphologically mature to display inner segment-like extensions. This method can be adopted for generating retinal organoids using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). All steps and procedures are clearly explained and demonstrated to ensure replicability and for wider applications in basic science and translational research.

This protocol describes an efficient method of differentiating hiPSCs into eye field clusters and generating neuro-retinal organoids using simplified culture conditions involving both adherent and suspension culture systems. Other ocular cell types, such as the RPE and corneal epithelium, can also be isolated from mature eye fields in retinal cultures.

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative ...

Retinal Pericytes Visualization Using Tissue-Clearing and Fluorescent Labeling

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining 

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal vasculature. To provide a detailed view of the morphological characteristics of pericytes, this study described a new approach combining the retro-orbital injection of a fluorescent agent, immunofluorescent-staining, and tissue-clearing treatment. Firstly, the fluorescent tomato lectin was injected into the retro-orbital sinus of the live mouse to label the retinal vasculature. After 5 min, the mouse was sacrificed, and its intact retina was carefully removed from the retinal cup and immunofluorescently stained with platelet-derived growth factor receptor &#946; to reveal the pericytes. Additionally, the stained retina was treated with tissue-clearing reagent and whole mounted on the microscope slide. Through these approaches, the retinal vasculature and pericytes were clearly observed in the transparent retina. Under a confocal microscope, we obtained more opportunities to take high-resolution images for further reconstructing and analyzing the morphological characteristics of pericytes along the retinal vascular tree in a three-dimensional view. Methodologically, this protocol offers an effective approach for visualizing pericytes within the retinal vasculature, providing valuable insights into their role under both physiological and pathological conditions.

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

The pericytes in retinal vasculature were examined by immunofluorescent staining with platelet-derived growth factor receptor &#946; after retro-orbital injection of fluorescent tomato lectin. The labeled retina was further treated with the tissue-clearing method and whole mounted for visualizing the three-dimensional views of pericytes surrounding retinal vasculature under a confocal microscope.

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal ...

Biochemistry

Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and throughput of cell culture. Mature human retinal organoids are utilized to screen potential pharmaceutical treatments for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).
We have recently shown that MacTel can be caused by elevated levels of an atypical lipid species, deoxysphingolipids (deoxySLs). These lipids are toxic to the retina and may drive the photoreceptor loss that occurs in MacTel patients. To screen drugs for their ability to prevent deoxySL photoreceptor toxicity, we generated human retinal organoids from a non-MacTel induced pluripotent stem cell (iPSC) line and matured them to a post-mitotic age where they develop all of the neuronal lineage-derived cells of the retina, including functionally mature photoreceptors. The retinal organoids were treated with a deoxySL metabolite and apoptosis was measured within the photoreceptor layer using immunohistochemistry. Using this toxicity model, pharmacological compounds that prevent deoxySL-induced photoreceptor death were screened. Using a targeted candidate approach, we determined that fenofibrate, a drug commonly prescribed for the treatment of high cholesterol and triglycerides, can also prevent deoxySL toxicity in the cells of the retina.
The toxicity screen successfully identified an FDA-approved drug that can prevent photoreceptor death. This is a directly actionable finding owing to the highly disease-relevant model tested. This platform can be easily modified to test any number of metabolic stressors and potential pharmacological interventions for future treatment discovery in retinal diseases.

Here we present a step-by-step protocol to generate mature human retinal organoids and utilize them in a photoreceptor toxicity assay to identify pharmaceutical candidates for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and ...

Bioengineering

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...

Medicine

Visualizing Retinal Synaptic Structures in 3D with Volume Electron Microscopy

Preparation of Retinal Samples for Volume Electron Microscopy

Volume electron microscopy (Volume EM) has emerged as a powerful tool for visualizing the 3D structure of cells and tissues with nanometer-level precision. Within the retina, various types of neurons establish synaptic connections in the inner and outer plexiform layers. While conventional EM techniques have yielded valuable insights into retinal subcellular organelles, their limitation lies in providing 2D image data, which can hinder accurate measurements. For instance, quantifying the size of three distinct synaptic vesicle pools, crucial for synaptic transmission, is challenging in 2D. Volume EM offers a solution by providing large-scale, high-resolution 3D data. It is worth noting that sample preparation is a critical step in Volume EM, significantly impacting image clarity and contrast. In this context, we outline a sample preparation protocol for the 3D reconstruction of photoreceptor axon terminals in the retina. This protocol includes three key steps: retina dissection and fixation, sample embedding processes, and selection of the area of interest.

Author Spotlight: Retinal Neuroscience Studies with Volume Electron Microscopy

This protocol describes the detailed steps for preparing retinal samples for volume electron microscopy, focusing on the structural features of retinal photoreceptor terminals.

Volume electron microscopy (Volume EM) has emerged as a powerful tool for visualizing the 3D structure of cells and tissues with nanometer-level ...

Synaptic Circuits Analysis and Protein Localization in the Mouse Retina

Investigating Retinal Circuits and Molecular Localization by Pre&#45;Embedding Immunoelectron Microscopy

The retina comprises numerous cells forming diverse neuronal circuits, which constitute the first stage of the visual pathway. Each circuit is characterized by unique features and distinct neurotransmitters, determining its role and functional significance. Given the intricate cell types within its structure, the complexity of neuronal circuits in the retina poses challenges for exploration. To better investigate retinal circuits and cross-talk, such as the link between cone and rod pathways, and precise molecular localization (neurotransmitters or neuropeptides), such as the presence of substance P-like immunoreactivity in the mouse retina, we employed a pre-embedding immunoelectron microscopy (immuno-EM) method to explore synaptic connections and organization. This approach enables us to pinpoint specific intercellular synaptic connections and precise molecular localization and could play a guiding role in exploring its function. This article describes the protocol, reagents used, and detailed steps, including (1) retina fixation preparation, (2) pre-embedding immunostaining, and (3) post-fixation and embedding.

Author Spotlight: Understanding the Ultrastructural Basis of Retinal Synaptic Connectivity and Neurotransmitter Localization in Mice 

This protocol outlines the detailed steps of pre-embedding immunoelectron microscopy, with a focus on exploring synaptic circuits and protein localization in the retina.

Preparing Retinal Organoid Samples for Transmission Electron Microscopy

Podsumowanie

Przeglądaj więcej filmów

The Gateway to the Brain: Dissecting the Primate Eye

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

Generation of Human Patient iPSC-derived Retinal Organoids to Model Retinitis Pigmentosa

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Characterization of a Novel Human Organotypic Retinal Culture Technique

Preparation of Retinal Samples for Volume Electron Microscopy

Investigating Retinal Circuits and Molecular Localization by Pre-Embedding Immunoelectron Microscopy