Name: characterization of a novel human organotypic retinal culture technique
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Description: Watch this Scientific Journal Video about Characterization of a Novel Human Organotypic Retinal Culture Technique at JoVE.com

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="/file...

<ol>
	<li>Kolb, H., Fernandez, E., Nelson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facts+and+Figures+Concerning+the+Human+Retina.">Facts and Figures Concerning the Human Retina.</a> Webvision-The Organization of the Retina and Visual System. , (2005).</li><li>Gemenetzi, M., De Salvo, G., Lotery, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+serous+chorioretinopathy:+an+update+on+pathogenesis+and+treatment.">Central serous chorioretinopathy: an update on pathogenesis and treatment.</a> Eye. 24 (12), 1743-1756 (2010).</li><li>Buschini, E., Piras, A., Nuzzi, R., Vercelli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age+related+macular+degeneration+and+drusen:+neuroinflammation+in+the+retina.">Age related macular degeneration and drusen: neuroinflammation in the retina.</a> Progress in Neurobiology. 95 (1), 14-25 (2011).</li><li>Chen, S. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+concepts+regarding+developmental+mechanisms+in+diabetic+retinopathy+in+Taiwan.">Current concepts regarding developmental mechanisms in diabetic retinopathy in Taiwan.</a> Biomedicine. 6 (2), (2016).</li><li>Kaaja, R., Loukovaara, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+retinopathy+in+type+1+diabetic+women+during+pregnancy.">Progression of retinopathy in type 1 diabetic women during pregnancy.</a> Current Diabetes Reviews. 3 (2), 85-93 (2007).</li><li>Azizzadeh Pormehr, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+organotypic+retinal+flat-mount+culture+(HORFC)+as+a+model+for+retinitis+pigmentosa11.">Human organotypic retinal flat-mount culture (HORFC) as a model for retinitis pigmentosa11.</a> Journal of Cellular Biochemistry. 119 (8), 6775-6783 (2018).</li><li>Fernandez-Bueno, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+modifications+in+organotypic+culture+of+human+neuroretina.">Time course modifications in organotypic culture of human neuroretina.</a> Experimental Eye Research. 104, 26-38 (2012).</li><li>Niyadurupola, N., Sidaway, P., Osborne, A., Broadway, D. 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R&amp;D Systems, I Available from: <a target="_blank" href="https://www.rndsystems.com/protocol-types/luminex">https://www.rndsystems.com/protocol-types/luminex</a> (2020)</li><li>Nakazawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuated+glial+reactions+and+photoreceptor+degeneration+after+retinal+detachment+in+mice+deficient+in+glial+fibrillary+acidic+protein+and+vimentin.">Attenuated glial reactions and photoreceptor degeneration after retinal detachment in mice deficient in glial fibrillary acidic protein and vimentin.</a> Investigative Ophthalmology, Visual Science. 48 (6), 2760-2768 (2007).</li><li>Okada, M., Matsumura, M., Ogino, N., Honda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M&#252;ller+cells+in+detached+human+retina+express+glial+fibrillary+acidic+protein+and+vimentin.">M&#252;ller cells in detached human retina express glial fibrillary acidic protein and vimentin.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 228 (5), 467-474 (1990).</li><li>Murali, A., Ramlogan-Steel, C. A., Andrzejewski, S., Steel, J. C., Layton, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+explant+culture:+A+platform+to+investigate+human+neuro-retina.">Retinal explant culture: A platform to investigate human neuro-retina.</a> Clinical &amp; Experimental Ophthalmology. 47 (2), 274-285 (2019).</li><li>Koleva-Georgieva, D. N., Sivkova, N. P., Terzieva, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+inflammatory+cytokines+IL-1beta,+IL-6,+TNF-alpha+and+VEGF+have+influence+on+the+development+of+diabetic+retinopathy.">Serum inflammatory cytokines IL-1beta, IL-6, TNF-alpha and VEGF have influence on the development of diabetic retinopathy.</a> Folia Medica (Plovdiv). 53 (2), 44-50 (2011).</li><li>Lee, J. -. 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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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 
			in this article</td>
		</tr>
		<tr>
			<td>Human Recombinant IL-1&#946;</td>
			<td>Peprotech, USA</td>
			<td>200-01B</td>
			<td>Working concentration: 10 ng/mL</td>
		</tr>
		<tr>
			<td>Human Recombinant&#160; TNF-a</td>
			<td>Peprotech, USA</td>
			<td>300-01A</td>
			<td>Working concentration: 10 ng/mL</td>
		</tr>
		<tr>
			<td>DAPI (1 &#181;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&#8217;s Modified Eagle Medium nutrient mixture F-12 containing a 1&#215; antibiotics and antimycotics mixture (AA, 100&#215; 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 (F...

Watch this Scientific Journal Video about Characterization of a Novel Human Organotypic Retinal Culture Technique at JoVE.com

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 ...

This technique preserves all retinal layers and cell types in situ, making it more clinically relevant compared to animal and in vitro models. The main advantage of this protocol is that it minimizes retinal integrity disruption during tissue handling, which is essential when comparing healthy and diseased retina. Retinal diseases can be mimicked by culturing the tissue under specific conditions.This allows for testing of new drug therapies. The dissection and sample collection procedures are complex. Therefore, visual demonstration is critical to help the readers understand the written protocol and conduct the procedure successfully.Demonstrating the procedure will be Charisse Kuo, a second year PhD student from my laboratory. Prepare the culture medium containing Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 and a one times antibiotics and antimycotics mixture. First, place 500 microliters of medium in a 24-well plate and equilibrate it in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide.Having the media ready prior to explant extraction avoids dislodging of the retina upon subsequent addition. For extracting retinal explants, begin by placing the eyecup on a Petri dish with the iris and lens facing upwards and the ONH contacting the Petri dish. Hold the eyecup steady at the limbus using forceps and detach the iris and lens by making small cuts circumferentially along the outer edge of the limbus.Remove the iris and lens carefully, making sure to avoid disturbing the retina. Identify the ONH using a bright white light source and incise at the four quadrants towards the ONH rotating the Petri dish for easier handling. Spread and flatten the eyecup carefully.Apply the forceps to the sclera instead of the retina to avoid disrupting the retinal integrity. Remove the plate containing the prepared media from the incubator. Place a surgical trephine on the retina in a region without retinal folds and press hard to penetrate the sclera, which should generate a cracking sound as you break through the Petri dish.Rotate the trephine to ensure the sclera has been penetrated fully such that the retinal explant is now separated from the rest of the sample. Apply the forceps at the sclera and transfer the sandwich retinal explant to the culture medium. Culture the collected sandwich retinal explants at 37 degrees Celsius for up to 72 hours in a humidified 5%carbon dioxide incubator.Hematoxylin and eosin staining showed that the sandwich retinal explants preserved integrity and a distinct lamellae structure from the GCL to the ONL with compact nuclei in the inner and outer nuclear layers. 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 loss of nuclei in the INL and ONL. No TUNEL-positive cell nuclei, which marks cellular apoptosis, were found in retinal explants cultured in basal conditions, demonstrating sustained cellular vitality even after 72 hours.GFAP labeling was restricted to the GCL and the IPL without glial fibrosis, a sign of pathology which would be identified as extended expression of GFAP into the ONL. Vimentin was highly expressed from the ganglion cell layer to the outer nuclear layer, showing preserved Mueller cell integrity as seen in normal retinal tissues. Luminex magnetic assay showed that the IL-18, VEGF, IL-6, and IL-8 levels increased significantly above baseline in the sandwich retinal explant cultures in high glucose and pro-inflammatory cytokines after 24 hours.After 72 hours, IL-18 and VEGF levels remained significantly increased, but no significant difference was found in IL-6 and IL-8 levels. When performing this procedure, leave sufficient residual vitreous, which will weigh the retina down so that it does not detach from the RP choroid and float in the culture media. The cultured retina can be further characterized using immunohistochemistry and histology, while the inflammatory cytokines released into the cultured medium can be measured using a Luminex magnetic assay.This model is more clinically translatable compared to animal and in vitro models, which renders more suitable for testing the efficacy of novel therapeutic drugs for retinal diseases.

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A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

A Detailed Protocol for Perspiration Monitoring Using a Novel, Small, Wireless Device

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the ...

Minimally Invasive Muscle Embedding (MIME) - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

Minimally Invasive Muscle Embedding MIME - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new ...