The authors would like to thank the generous donors of eye tissues and the team from the New Zealand National Eye Bank for their support. This work was financially supported by project grants from the Maurice and Phyllis Paykel Trust and the Auckland Medical Research Foundation (1117015). IDR&#39;s directorship is supported by the Buchanan Charitable Foundation. CK&#39;s scholarship is provided by the New Zealand Association of Optometrists Education and Research Fund (CC36812) and HHL&#39;s scholarship is provided by the Buchanan Charitable Foundation.

Human donor eye cups were obtained from the New Zealand National Eye Bank following corneal excision for transplantation and as approved by the Northern B Health and Disability Ethics Committee (NTX/06/19/CPD/AM07).
NOTE: The culture should be done in a Class II biosafety cabinet to ensure sterile tissue culture conditions. The tissues must be cultured within 24 h post-mortem to avoid significant retinal integrity loss.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62046/62046fig02.jpg" /> 
Figure 2:&#160;Images showing the procedure for collecting the sandwich retinal explants.&#160;For explant preparation, use dissecting scissors with one sharp tip and one blunt end, with the blunt end facing the inside of the globe to reduce tissue damage during incision. Also use forceps with blunt ends to avoid scratching&#160;the intraocular tissues during handling. Using surgical scissors, with the blunt end facing the inside, cut along the limbus to remove the iris and lens (A-C). The ONH can be located if looking straight into the opened eyecup (D). Using forceps with blunt tips, hold the sclera to stabilize the globe (E). Divide the globe vertically into two halves by making two deep incisions towards the ONH but do not cut through the ONH. Repeat this along the horizontal meridian (G). Apply blunt forceps at the sclera and gently open the globe to a clover shape (H-K). Use forceps to carefully manipulate the vitreous to smoothen the folded retina, as the vitreous tugs onto the retina, but do not touch the retina directly (I). Remove the vitreous if it is causing retinal detachment or folding (not shown in&#160;Figure 2&#160;as the transparent vitreous is not well captured on photos). Locate areas where the retina is flat and extract retinal explants using a surgical trephine (L). Applying forceps at the sclera, carefully transfer the retinal explants into the pre-prepared culture medium (M,N). The entire sandwich explant should sink to the bottom of the well due to its weight, therefore each explant is submersed in the medium (O).&#160;<a href="https://www.jove.com/files/ftp_upload/62046/62046fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Extraction of the sandwich retinal explants from the eyecup
<ol>
	<li>Remove the iris and lens.
	<ol>
		<li>Place the eye cup inside a Petri dish, with the iris and lens facing upwards and the optic nerve head (ONH) contacting the Petri dish (Figure 2A).</li>
		<li>Hold the eye cup steady at the limbus using forceps (Figure 2B).</li>
		<li>Detach the iris and lens by making small cuts circumferentially along the outer edge of the limbus (Figure 2A).</li>
		<li>Remove the iris and lens carefully. Avoid disturbing the retina during manipulation (Figure 2C). 
		NOTE: The lens will not fall into the eye cup as it is attached, via zonule fibers, to the ciliary body.</li>
	</ol>
	</li>
	<li>Flatten the eye cup.
	<ol>
		<li>With the eye cup still sitting upright, identify the ONH. This is easier with a bright white light source (Figure 2D).</li>
		<li>Incise at the four quadrants towards the ONH (Figure 2E). The Petri dish can be rotated for easier handling. Do NOT cut the ONH.</li>
		<li>Spread and flatten the eye cup carefully (Figure 2E).</li>
		<li>Apply the forceps on the sclera instead of the retina to avoid disrupting retinal integrity. 
		NOTE: Peripheral retinal detachment and folding is unavoidable, as the weight of the overflowing vitreous will pull on&#160;the retina. In these areas, remove the vitreous to prevent further retinal folding. Remember to leave residual vitreous to stabilize the retina on top of the RPE-choroid and sclera.</li>
	</ol>
	</li>
	<li>Collect sandwich retinal explants.
	<ol>
		<li>Place a surgical trephine on the retina in a region without retinal folds (Figure 2F).</li>
		<li>Press hard to penetrate the sclera, which should generate a cracking sound.</li>
		<li>Twist the trephine by 180&#176; to ensure the sclera has been penetrated fully such that the retinal explant is now separated from the remaining tissue.</li>
		<li>Apply the forceps at the sclera and transfer the sandwich retinal explant to the culture medium (Figure 2G-I).</li>
		<li>Obtain 2-3 sandwich retinal explants from the peripheral retina of each quadrant. 
		NOTE: The sandwich retinal explant can sometimes be trapped in the opening of the trephine. Using forceps, gently tease out a small section of the base of the sclera. This occurs more often when the blade is blunt and can cause loss of the retina (Figure 3A-C).</li>
		<li>Use a new trephine after cutting out 1-2 retinal explants as the blade becomes blunt easily (Figure 3C<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62046/62046fig03.jpg" /> 
		Figure 3: Troubleshooting.&#160;During the extraction process, the retinal explant may be trapped at the opening of the surgical trephine (A). Gently tease out the tissue from the base of the sclera without touching the retina (B). This can happen more often if the surgical trephine is used for extracting more than two retinal explants, as the blade can easily become blunt (C).&#160;<a href="https://www.jove.com/files/ftp_upload/62046/62046fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a></li>
	</ol>
	</li>
	<li>Culture the retinal explants.
	<ol>
		<li>Prepare the culture medium containing Dulbecco&#39;s Modified Eagle Medium nutrient mixture F-12 (DMEM-F12) and a 1x antibiotics and antimycotics mixture (AA, 100x stock).</li>
		<li>Prior to explant extraction, place 500 &#181;L of medium in the wells of a 24-well plate and equilibrate in the incubator. This is important as adding the medium afterwards can dislodge the retina.</li>
		<li>Culture the sandwich retinal explants at 37 &#176;C for up to 72 h in a humidified 5% CO2 incubator in medium prepared in step 1.4.2.</li>
		<li>To induce DR-like changes, culture retinal explants in medium containing DMEM-F12 with a combination of 32.5 mM HG with Cyt, TNF-&#945; (10 ng/mL) and IL-1&#946; (10 ng/mL).</li>
	</ol>
	</li>
</ol>
2. Paraffin-embedding of the sandwich retinal explants
<ol>
	<li>Fix the sandwich retinal explants.
	<ol>
		<li>Immerse the sandwich retinal explants in 10% formalin for at least 24 h.</li>
		<li>Remove the formalin solution and transfer the sandwich retinal explants gently into tissue pads and cassettes.</li>
		<li>Soak the sandwich retinal explants in 70% ethanol for at least 24 h.</li>
		<li>Paraffin-embed retinal explants into blocks.</li>
		<li>Cut paraffin-embedded retinal tissues into 5 &#181;m thick sections using a microtome and mount onto glass slides. Store at room temperature until imaging.</li>
	</ol>
	</li>
	<li>Deparaffinize sections.
	<ol>
		<li>Immerse the sections in 70% xylene for 5 min.</li>
		<li>Immerse the sections in 100% xylene for 5 min.</li>
		<li>Rehydrate the sections in 70% ethanol for 5 min.</li>
		<li>Rehydrate the sections in 100% ethanol for 5 min.</li>
		<li>Wash the sections under running tap water for 10 min.</li>
	</ol>
	</li>
</ol>
3. Characterization using H&#38;E, IHC and a Magnetic Luminex&#160;assay
<ol>
	<li>H&#38;E staining protocol
	<ol>
		<li>Deparaffinize the sections using step 2.2.</li>
		<li>Hydrate the sections in tap water for 5 min.</li>
		<li>Stain the sections in Gill&#39;s 2 hematoxylin solution for 5 min.</li>
		<li>Wash the sections thoroughly under running tap water to remove excess stain.</li>
		<li>Differentiate by dipping the sections twice in 1% acid alcohol.</li>
		<li>Wash the sections quickly under running tap water.</li>
		<li>Stain the sections blue by dipping six times in 1% lithium carbonate (10 mg/mL).</li>
		<li>Wash the sections thoroughly under running tap water for 5 min to remove excess blue stain.</li>
		<li>Dip the sections in 1% eosin 10 times.</li>
		<li>Wash the sections quickly under&#160;tap water to remove excess stain.</li>
		<li>Dehydrate the sections by dipping them 10 times in 100% ethanol. Do this twice.</li>
		<li>Dip the sections in 70% xylene 10 times.</li>
		<li>Dip the sections in 100% xylene 10 times.</li>
		<li>Mount with a coverslip using dibutylphthalate polystyrene xylene (DPX) mounting medium.</li>
		<li>Take images using a light microscope.</li>
	</ol>
	</li>
	<li>IHC labeling protocol&#39;
	<ol>
		<li>Deparaffinize the sections using step 2.2.</li>
		<li>Place the slides into a solution containing 10 mM sodium citrate buffer with 0.05% Tween 20 at pH 6.0 and run an antigen retrieval in a pressure cooker automated at 121 &#176;C for 2 min.</li>
		<li>Wash sections in phosphate buffered saline (PBS) for 5 min. Do this 3 times.</li>
		<li>Block the sections with PBS containing 0.1% Triton X-100 and 10% normal goat serum for 1 h at room temperature.</li>
		<li>Incubate sections overnight at 4 &#176;C with primary antibodies conjugated to secondary antibodies (Table of Materials).</li>
		<li>Wash sections in PBS for 5 min. Do this 3 times.</li>
		<li>Stain nuclei using 4&#8242;,6-diamidino-2-phenylindole (DAPI) for 2 min.</li>
		<li>Wash and mount sections using an anti-fade reagent.</li>
		<li>Seal coverslips with nail polish.</li>
		<li>Take images using a confocal laser scanning microscope.</li>
	</ol>
	</li>
	<li>Magnetic Luminex assay
	<ol>
		<li>Transfer 75 &#181;L of media from each well to a 96-well-u-bottom plate at 24 and 72 h.</li>
		<li>Analyze the cell supernatant for IL-18, IL-6, IL-8 and vascular endothelial growth factor (VEGF) after 24 and 72 h using Luminex cytokine assay. Follow the manufacturer&#39;s instructions to conduct the assay16.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

HORC is currently the most clinically translatable model in preclinical retinal research. Compared to in vitro cell culture models, HORC can better represent the anatomy of the human retina in situ, through retaining the dynamic retinal cell types and their connections with neurons, vasculatures and the extracellular environment19. Compared to animal models, HORC are more advantageous in studying the pathophysiology of and designing pharmaceutical treatments for human retinal diseases due to inter-species variations, such as different retinal cell types as well as varying photoreceptor density and proportions, which could contribute to unsuccessful clinical trials despite positive results in animal models19. However, in previous HORC protocols, the retina was intentionally detached from the RPE-choroid and sclera. Without the tough sclera, the flimsy retina is difficult to handle and retinal integrity can be compromised if directly handled by forceps6,7,8,9. This is evidenced by the fact that previous studies using isolated retinas have reported increased M&#252;ller cell activation and TUNEL-positive nuclei by 72 h of culture. This is in contrast to our findings, suggesting that the sandwich method better preserves retinal integrity. Secondly, increased neuronal degeneration and glial proliferation were found in animal ORC models in retina cultured without RPE, compared to retina cultured with RPE10,11,12.
To minimize the chances of compromising retinal integrity, sandwich retinal explants were utilized in this novel HORC protocol. In contrast to previous HORC protocols, the trephine cut through not only the retina, but also the RPE-vitreous and the sclera, such that the resultant explant is &#34;sandwiched&#34; and stabilized between the residual vitreous and the underlying structures. The residual vitreous weighs the retina down to prevent folding and detachment from the underlying structures while the sclera acts as a scaffold to support the retinal architecture to protect the retina from forceps-induced injury. Furthermore, culturing retina with RPE can prevent retinal degeneration and glial proliferation in culture10,11,12.
Remember the following points when extracting sandwich retinal explants. When cutting along the limbus, the lens will weigh down the iris but will not fall inside the eye cup as it is attached, via zonule fibers, to the ciliary body. Apply the forceps on the sclera and avoid touching the retina to prevent disrupting retinal integrity. Fill the culturing wells with medium before placing the retinal explant inside the wells. Adding the medium in after placing the retinal explants inside the wells increases the risk of dislodging the retina from&#160;the RPE-choroid and sclera or&#160;flipping the entire explant upside down. When making deep incisions at the four quadrants, be mindful that the peripheral retina may detach due to the vitreous leaking out of the eye cup. To prevent detachment or folding of the retina, remove the vitreous in the area that is pulling on the retina. Although the previous point advises to remove the vitreous pulling on the retina, leave sufficient residual vitreous to weigh the retina on top of the RPE-choroid and sclera in order to increase the stability of the sandwich retinal explants. When cutting through the tough, fibrous sclera, the trephine can easily become blunt. As a result, excessive force may be required or the sclera may not be fully penetrated. Use a new trephine for every 1-2 extracted retinal explants to avoid incomplete penetration&#160;through the sclera. Experience in careful handling is crucial to protect the retina; therefore, training with porcine eyes, which are similar in size and structure to the human eye, is advisable before using the limited donor scleral eyecups available.
Retinal integrity was preserved in the explants sandwiched between residual vitreous and the underlying RPE-choroid and sclera. H&#38;E staining showed better structural integrity when the cultured retinal explant was attached to RPE-choroid and sclera, compared to culturing the retina alone. Supporting this finding, the lack of TUNEL-positive cell nuclei suggested&#160;an absence of apoptosis within all retinal layers, demonstrating preserved cellular viability even after 72 h. Furthermore, vimentin labelling confirmed M&#252;ller cell integrity and the restricted GFAP expression in GCL and INL, found in normal retina, again verified the lack of inflammatory or stress signals in the retina.
The HORC model characterized in this study resulted in preserved retinal structure and M&#252;ller cell integrity while preventing retinal inflammation and cellular apoptosis. Furthermore, it can be used to model retinal diseases, which is particularly useful to identify disease pathways and pharmaceutical mechanisms. Previous animal and in vitro studies have shown that DR inflammation can be induced when animal retina or cells are exposed to HG and Cyt13,14,15. In this&#160;HORC study, by applying HG and Cyt to the sandwich explants for 24 h in culture, secretion of IL-18, VEGF, IL-6 and IL-8 was significantly elevated above baseline. These cytokines are well known to increase in the vitreous and serum of patients with DR compared to non-diabetic controls, validating that this DR model is clinically translatable20,21,22,23,24,25. Although this study has only characterized DR using Magnetic Luminex&#160;assay, other techniques such as Western Blotting, IHC and polymerized chain reaction (PCR) can also be performed26.
Despite the clear advantages outlined above, this novel HORC model also has some limitations. A major limitation of this protocol is the low supply of tissue. Donor eye cups, which have all ocular structures but the cornea generally removed for transplantation, are rarely available for research due to the large demand for&#160;the sclera in surgical procedures such as glaucoma shunts, orbital implants, damage repair in retinal detachment and coverage of exposed sutures. In order to minimize this limitation, we suggest that all experimental groups are collected from the same donor. This removes the need to account for inter-donor-variabilities, such as age, duration of disease, baseline cytokine levels, and other factors that can contribute to confounding variables if comparing between different patients. Also, most donor tissues are from elderly people as it is harder to obtain samples from younger age groups, and even when available, the cause of death of young donors is often traumatic in nature leading to damaged tissues that are unsuitable for culture. Nonetheless, given that most chronic retinal diseases occur in aged patients, this model still retains usefulness in studying age-related chronic retinal diseases such as DR and age-related macular degeneration.

The retina is a highly specialized ocular structure responsible for transforming incoming light energy to electric signals, which are then processed by the brain for visual perception. The human retina contains a dynamic range of cell types, highly organized in a unique lamellar structure consisting of two synaptic and three nuclei layers1 (Figure 1). Retinal homeostasis is sustained by the intricate connections between neuroretinal cells, blood vessels, nerves, connective tissues and the RPE1. Due to the sophisticated retinal anatomy and physiology, mechanisms of many retinal diseases still remain poorly understood2,3,4,5. To better study retinal diseases, HORC models have been developed6,7,8,9. Compared to animal studies and in vitro cultures, HORC models are advantageous because they retain the dynamic cellular environment and complex neurovascular interactions in situ, providing a good model for clinical translation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62046/62046fig01.jpg" /> 
Figure 1: Posterior ocular structures of the human eye.&#160;Anterior to posterior, the retinal layers are: nerve fiber layer (NFL), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), photoreceptor inner segment (IS), and photoreceptor outer layer (OS). Cells within the retina include ganglion cells (blue), amacrine cells (yellow), bipolar cells (red), horizontal cells (purple), rod photoreceptors (pink) and cone photoreceptors (green). The vitreous is located anterior to the retina. The RPE, Bruch&#39;s membrane, choroid and sclera are located posterior to the retina. Note that the&#160;image shown is only a schematic representation of the retina and the ratio of cells/retinal connectivity within each layer may not be indicative of the in vivo setting.&#160;<a href="https://www.jove.com/files/ftp_upload/62046/62046fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Previously characterized HORC protocols6,7,8,9 have involved separating the retina from the underlying RPE-choroid and sclera using a surgical trephine. However, without the support provided by these underlying structures, the translucent retina becomes flimsy, difficult to handle and tools such as forceps can easily disrupt its integrity. Furthermore, isolating retina in culture without the RPE has been shown to cause ganglion cell apoptosis and photoreceptor degeneration10,11,12. Thus, an alternative HORC protocol that minimizes the loss of retinal integrity and better mimics the in vivo environment would be useful. This is particularly important when studying retinal disease mechanisms, as physical injury during explant handling could introduce artifacts. Therefore, the aim of this study was to develop a novel HORC model that includes the RPE-choroid and sclera in order to protect retinal integrity during explant handling and culture.
In order to achieve this aim, retinal explants &#34;sandwiched&#34; between the residual vitreous and the underlying RPE-choroid and sclera were extracted. In the sandwich explants, the vitreous weighs down the retina to prevent retinal detachment and folding, whereas the tough, fibrous sclera acts as both a scaffold for structural support and a contact point for forceps. Moreover, animal models have shown that retaining the RPE in culture can prevent retinal degeneration and glial proliferation, a response of M&#252;ller cells to danger signals such as hypoxia and inflammation10,11,12.
To characterize the model, sandwich retinal explants were stained with Hematoxylin and Eosin (H&#38;E) to assess anatomical structures and immunohistochemistry (IHC) was performed, labeling explants with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL, an apoptotic cell marker), glial fibrillary acidic protein (GFAP, a retinal inflammation and M&#252;ller cell activation marker), and vimentin, a marker of M&#252;ller cell integrity. To determine whether this model can be induced to develop molecular disease signs, the explants were exposed to high glucose (HG) with pro-inflammatory cytokines (Cyt), interleukin-1&#946; (IL-1&#946;) and tumor necrosis factor-&#945; (TNF-&#945;), a culturing environment that has been shown to mimic diabetic retinopathy (DR) in both cell and animal disease models13,14,15. Luminex&#160;assays were used in the DR model to measure cytokines&#160;released into the culture medium.

<table><tbody><tr><td>Disposable Biopsy Punches (5 mm)</td><td>Integra York PA Inc., USA</td><td>21909-142</td><td>Referred as surgical trephines&lt;br/&gt; in this article</td></tr><tr><td>Human Recombinant IL-1&amp;beta;</td><td>Peprotech, USA</td><td>200-01B</td><td>Working concentration: 10 ng/mL</td></tr><tr><td>Human Recombinant&amp;nbsp; TNF-a</td><td>Peprotech, USA</td><td>300-01A</td><td>Working concentration: 10 ng/mL</td></tr><tr><td>DAPI (1 &amp;micro;g/mL)</td><td>Sigma Aldrich, Germany</td><td>#D9542</td><td>Nuclear stain. Working dilution 1:1000</td></tr><tr><td>DMEM/F-12, GlutaMAX supplement</td><td>Gibco, Thermofisher, Scientific Inc., USA</td><td>10565018</td><td>Dulbecco&amp;rsquo;s Modified Eagle Medium nutrient mixture F-12 containing a 1&amp;times; antibiotics and antimycotics mixture (AA, 100&amp;times; stock)</td></tr><tr><td>Fine Scissors - Sharp-Blunt</td><td>Fine Science Tools (F.S.T)</td><td>14028-10</td><td>Tips: Sharp-Blunt, Cutting Edge: 27mm, Length: 10cm, Alloy/Material: Stainless Steel, Serrated: No, Tip Shape: Straight</td></tr><tr><td>Graefe Forceps</td><td>Fine Science Tools (F.S.T)</td><td>11050-10</td><td>Length:10cm, Tip shape: Straight, Tips: Serrated, Tip Width: 0.8mm, Tip Dimensions:0.8 x 0.7mm, Alloy/Materials: Stainless Steel</td></tr><tr><td>Mouse monoclonal GFAP-Cy3</td><td>Sigma Aldrich, Germany</td><td>#C9205</td><td>Primary antibody conjugated to Cy3. Working dilution 1:1000.</td></tr><tr><td>Mouse monoclonal Vimentin-Cy3</td><td>Sigma Aldrich, Germany</td><td>#C9080</td><td>Primary antibody conjugated to Cy3. Working dilution 1:50.</td></tr><tr><td>Rabbit polyclonal TUNEL (In Situ Cell Death Detection Kit, Fluorescein)</td><td>Sigma Aldrich, Germany</td><td>#11684795910</td><td>Primary antibody conjugated to Fluorescein-dUTP. Working dilution 1:10 with enzyme-buffer solution.</td></tr></tbody></table>

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.

Retinal integrity was preserved in this HORC model. Retinal integrity was preserved in the cultured sandwich retinal explants but was lost in the retina cultured without adjacent structures. H&#38;E was conducted to examine the structural integrity of sectioned sandwich retinal explants after 72 h in culture. The sandwich retinal explants showed preserved integrity and a distinct lamellae structure from GCL to ONL with compact nuclei in INL and ONL (Figure 4A). However, disrupted retinal integrity was found at the edges of the same sample, where the retina had detached from the underlying RPE-choroid and sclera, showing reduced retinal thickness and sever loss of nuclei in INL and ONL (Figure 4B).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62046/62046fig04.jpg" /> 
Figure 4: H&#38;E images of retinal explants cultured in medium for 72 h. A) Structural integrity maintained in areas attached to the underlying RPE-choroid and sclera, depicted by the distinct separation of each retinal layer from GCL to ONL and compact nuclei in INL and ONL.&#160;B) Severe loss in retinal integrity in regions detached from the RPE-choroid and sclera,&#160;shown by the overall retinal thickness reduction and decreased numbers of nuclei in INL and ONL. Scale bar = 50 &#181;m.&#160;<a href="https://www.jove.com/files/ftp_upload/62046/62046fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Characterization of retinal explants using IHC demonstrated well-preserved retinal integrity even after 72 h in culture. No TUNEL-positive cell nuclei, which mark&#160;apoptosis, were found in retinal explants cultured in basal conditions, demonstrating sustained cell viability&#160;even after 72 h (Figure 5A-C). GFAP expression remained localized in GCL and IPL, as seen in normal retinal tissues17,18. There was no glial fibrosis, a sign of M&#252;ller cell activation and proliferation in response to retinal inflammation, which would otherwise be identified as upregulated GFAP expression that extends into the ONL10,11,12(Figure 5D-F). Vimentin labelling (red) was highly expressed from GCL to ONL, showing preserved M&#252;ller cell integrity, as seen in normal retinal tissues (Figure 5G-I).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62046/62046fig05.jpg" /> 
Figure 5: IHC images of retinal explants after culturing in basal media for 72 h.&#160;The lack of TUNEL-positive staining (green) suggests that no cellular apoptosis has occurred in the retinal layers from GCL to ONL (A-C). GFAP labelling (red) was restricted to GCL and IPL without glial fibrosis, which would be identified as extended expression of GFAP into the ONL (D-F). Vimentin (red) was highly expressed from GCL to ONL, locating the M&#252;ller cells that span through these retinal layers (G-I). Cell nuclei were stained with DAPI (blue). Scale bar = 50 &#181;m.&#160;<a href="https://www.jove.com/files/ftp_upload/62046/62046fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A DR model was developed by culturing&#160;sandwich retinal explants in HG and Cyt. HG and Cyt, IL-1&#946; and TNF-&#945;, increased the secretion of IL-18, VEGF, IL-6 and IL-8 relative to baseline levels. A Luminex assay was used to measure the cytokines&#160;IL-18, VEGF, IL-6 and IL-8 secreted by retinal explants after culturing for 24 and 72 h under HG + Cyt conditions (Figure 6). To minimize inter-donor differences, secreted cytokine levels were compared to baseline (indicated by the dotted red line), represented by cytokine levels secreted by the sandwich explants from the same donor but cultured in basal&#160;medium (prepared in step 1.4.1). After 24 h, secreted IL-18, VEGF, IL-6 and IL-8 levels increased significantly relative to baseline. After 72 h, only IL-18 and VEGF levels remained significantly elevated, whereas no significant difference was found in IL-6 and IL-8 levels compared to baseline.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62046/62046fig06.jpg" /> 
Figure 6: Cytokines release by the sandwich retinal explants cultured in HG and Cyt, IL-1&#946; and TNF-&#945;, after 24 and 72 h.&#160;The secreted IL-18, VEGF, IL-6 and IL-8 levels increased significantly above baseline after 24 h (p &#60; 0.05). After 72 h, IL-18 and VEGF levels remained significantly increased (p &#60; 0.05), but no significant difference was found in IL-6 and IL-8 levels, compared to baseline. The dotted red line indicates the baseline level, which represents cytokine levels secreted by the explants from the same donor but cultured in normal medium.&#160;<a href="https://www.jove.com/files/ftp_upload/62046/62046fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Characterization of a Novel Human Organotypic Retinal Culture Technique

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 ...

characterization-novel-human-organotypic-retinal-culture

Nanyang Technological University

Research

JoVE Journal

Medicine

3.9K Views.  University of Auckland. 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 epithelium-choroid (RPE-choroid) and sclera.

Video: Characterization of a Novel Human Organotypic Retinal Culture Technique

Organotypic Culture of Adult Rabbit Retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with imaging techniques, genetic manipulation, and electrophysiological recording. Here we present a simple interphase tissue culture system for adult rabbit retina that requires no specialized equipment and very little maintenance. We demonstrate the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Rabbit retinas cultured this way can be kept alive for up to 6 days with very little changes of the overall anatomical structure or the morphology of individual ganglion- and amacrine cells.

This article demonstrates the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells.

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

Biology

Organotypic Culture of Full-thickness Adult Porcine Retina

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human retina and is therefore a valuable species for studying mechanisms of human retinal injury and degenerative disease. Here we describe a cost-effective technique for organotypic culture of adult porcine retina isolated from eyes obtained from an abattoir. After removing the anterior segment, a trephine blade was used to create multiple neural retina-Bruch's membrane-RPE-choroid-sclera explants from the posterior segment of adult porcine eyes. A piece of sterile filter paper was used to lift the neural retina off from each explant. The filter paper-retina complex was cultured (photoreceptor side up) atop an insert, which was held away from the bottom of the culture dish by a custom-made stand. The stand allows for good circulation of the culture medium to both sides of the retina. Overall, this procedure is simple, reproducible, and permits preservation of native retinal structure for at least seven days, making it a useful model for a variety of morphological, pharmacological, and biochemical studies on mammalian retina.

Here we describe a cost-effective technique for organotypic culture of adult porcine retina for seven days. Briefly, a sterile filter paper was used to lift the neural retina off from the RPE and place photoreceptor side up on an insert raised by a custom-made stand.

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human ...

Neuroscience

In vivo-like Organotypic Murine Retinal Wholemount Culture

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages.
Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used.
By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5.
In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.

In vivo-like Organotypic Murine Retinal Wholemount Culture

This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

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 ...

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina&#160;with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2&#8211;9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day.
The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

The protocol describes organotypic explants of mouse neuroretina, cultivated together with its retinal pigment epithelium (RPE), in R16 defined medium, free of serum and antibiotics. This method is relatively simple to perform, less expensive, and time-consuming when compared to in vivo experiments, and can be adapted to numerous experimental applications.

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of ...

Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual capabilities to the degenerated retina. Nevertheless, progress in cell replacement therapy presently faces the challenges of requiring an off-the-shelf source of high quality and standardized human retinas. Therefore, an easy and stable protocol is needed for the experiments. Here, we develop an optimized protocol, based on a self-organizing method with the use of exogenous molecules and reagent A as well as manual excision to generate the three-dimensional human retina organoids (RO). The human Pluripotent Stem Cells (PSCs)-derived RO expresses specific markers for photoreceptors. With the addition of COCO, a multifunctional antagonist, the differentiation efficiency of photoreceptor precursors and cones is significantly increased. The efficient use of this system, which has the benefits of cell lines and primary cells, and without the sourcing issues associated with the latter, could produce confluent retinal cells, especially photoreceptors. Thus, the differentiation of PSCs to RO provides an optimal and biorelevant platform for disease modelling, drug screening and cell transplantation.

Using a self-organizing method, we develop a protocol with the addition of COCO that could significantly increase the generation of photoreceptors.

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual ...

Organotypic Retinal Explant Cultures from Macaque Monkey

Hereditary retinal degeneration (RD) is characterized by progressive photoreceptor cell death. Overactivation of the cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) pathway in photoreceptor cells causes photoreceptor cell death, especially in models harboring phosphodiesterase 6b (PDE6b) mutations. Previous studies on RD have used mainly murine models such as rd1 or rd10 mice. Given the genetic and physiological differences between mice and humans, it is important to understand to which extent the retinas of primates and rodents are comparable. Macaques share a high level of genetic similarity with humans. Therefore, wild-type macaques (aged 1-3 years) were selected for the in vitro culture of retinal explants that included the retina-retinal pigment epithelium (RPE)-choroid complex. These explants were treated with different concentrations of the PDE6 inhibitor zaprinast to induce the cGMP-PKG signaling pathway and simulate RD pathogenesis. cGMP accumulation and cell death in primate retinal explants were subsequently verified using immunofluorescence and the TUNEL assay. The primate retinal model established in this study may serve for relevant and effective studies into the mechanisms of cGMP-PKG-dependent RD, as well as for the development of future treatment approaches.

Retinal explants obtained from wild-type macaques were cultured in vitro. Retinal degeneration and the cGMP-PKG signaling pathway was induced using the PDE6 inhibitor zaprinast. cGMP accumulation in the explants at different zaprinast concentrations was verified using immunofluorescence.

Hereditary retinal degeneration (RD) is characterized by progressive photoreceptor cell death. Overactivation of the cyclic guanosine monophosphate ...

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to differentiate into all types of retinal cells, even mini-retinal tissues, hold huge promises for patients with these diseases and many opportunities in disease modeling and drug screening. However, the induction process from hPSCs to retinal cells is complicated and time-consuming. Here, we describe an optimized retinal induction protocol to generate retinal tissues with high reproducibility and efficiency, suitable for various human pluripotent stem cells. This protocol is performed without the addition of retinoic acid, which benefits the enrichment of cone photoreceptors. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction. With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate downstream applications, such as disease modeling and cell therapy.

Here we describe an optimized retinal organoid induction system, which is suitable for various human pluripotent stem cell lines to generate retinal tissues with high reproducibility and efficiency.

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to ...

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Summary

Explore More Videos

Chapters in this video

Organotypic Culture of Adult Rabbit Retina

Organotypic Culture of Full-thickness Adult Porcine Retina

In vivo-like Organotypic Murine Retinal Wholemount Culture

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

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells

Organotypic Retinal Explant Cultures from Macaque Monkey

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases