This research is supported by Research to Prevent Blindness, New York, www.rpbusa.org, and the Greater New York Center for Retinal Degenerative Disease Foundation Fighting Blindness, www.blindness.org. The authors would like to thank Luanna Bartholomew, Ph.D., for her critical review of this manuscript.

The following protocol is performed in adherence to the tenets of the Declaration of Helsinki and the Yale University Human Research Ethics Committee guidelines.
1. Sourcing Human Donor Eyes
<ol>
	<li>Obtain human donor globes from the National Disease Research Interchange (NDRI, Philadelphia, PA). Depending on the project, prepare no less than three pairs of donor eyes for each experimental group.</li>
	<li>Enucleate donor eyes within 10 h and ship within 48 h post mortem. Then transport the eyes to the laboratory in sterile Dulbecco&#39;s modified Eagle medium (DMEM) culture medium on ice. Previously reported studies have demonstrated that donor eyes harvested and transported in this fashion preserve the biological features and activities of Bruch&#39;s membrane25,26,29.</li>
</ol>
2. Harvesting of Human Bruch&#39;s Membrane Explants
<ol>
	<li>Place each eye on a 1.5 x&#160;1.5 inch gauze soaked with carbon dioxide-independent DMEM media in a 100 mm culture dish, and use fine blade scissors to make an incision through the sclera 3 mm posterior to the limbus and circumferentially extending in either direction.</li>
	<li>Make a full-thickness circumferential incision (penetrating to vitreous) 1 mm posterior to the ora serrata. Remove and discard the anterior segment (cornea, lens, and iris), the vitreous and retina.</li>
	<li>Use fine surgical scissors to make four radial incisions in between the sclera and choroid, and then peel the sclera away from the periphery to the optic nerve, while taking care to avoid tearing the choroid/Bruch&#39;s membrane complex.</li>
	<li>Next, make a circumferential incision through the suprachoroidal space along the ora serrata and peel the RPE-Bruch&#8217;s membrane-choroid complex posteriorly from the sclera.</li>
	<li>Remove native RPE cells by bathing the explant with 0.02 M ammonium hydroxide in Dulbecco&#39;s phosphate-buffered saline (DPBS) in a 60 x&#160;15 mm polystyrene Petri dish for 20 min at room temperature, followed by washing it three-times in DPBS (Figure 1).</li>
	<li>Float the Bruch&#39;s membrane complex explant in carbon dioxide-free media over an unlaminated, hydrophobic 65 &#181;m-thick polytetrafluoroethylene membrane with 0.2 &#181;m pores, with the basal lamina layer of each explant facing the membrane.</li>
	<li>Place a polytetrafluoroethylene membrane with Bruch&#39;s membrane explant on a fritted glass support in a glass microanalysis vacuum filter-holder system.</li>
	<li>Flatten the curled edges from the choroidal side with fine forceps coated with Teflon, taking care not to touch the Bruch&#39;s membrane basal laminin side.</li>
	<li>Heat 15 mL of agarose (4% in DPBS) and then cool it to 37 &#176;C. Then pour 15 mL of the liquidized gel onto the Bruch&#39;s membrane-choroid complex from the choroidal-side while applying gentle suction to guarantee that the explant will remain flat. Keep the tissue at 4 &#176;C for 2 - 3 min to solidify the agarose and then peel the polytetrafluoroethylene membrane away.</li>
	<li>Place the Bruch&#39;s membrane explant (1.5 inches in diameter, within solidified agarose gel) in a 60 x&#160;15 mm polystyrene cell culture dish (with Bruch&#39;s membrane facing up) that has been lined with warm liquid agarose on the bottom of the dish to fix the Bruch&#39;s membrane explant on the bottom of the well with the explant basal laminin layer facing up.</li>
	<li>The agarose will solidify within 2 - 3 min at room temperature to stabilize the Bruch&#39;s membrane explants in the culture dish. Cover the Bruch&#39;s membrane explants in the culture dish with DPBS and store the dish at 4 &#176;C for future use. Figure 2A demonstrates the isolated Bruch&#39;s membrane explants in a 60 x&#160;15 mm culture dish.</li>
	<li>If the desired culture system is in a 96-well plate, place the Bruch&#39;s membrane explant (1.5 inches in diameter, with solidified agarose gel) on a flat Teflon sheet with the laminin-side up.</li>
	<li>Using a trephine, cut five or six, 6 mm-circular buttons from the Bruch&#39;s membrane complex explant and place each on 4% agarose at 37 &#176;C in a non-treated polystyrene well of a 96-well plate. The agarose will solidify within 2-3 min at room temperature to fix the Bruch&#39;s membrane explants (buttons) on the bottom of each well. 
	NOTE: Figure 2B demonstrates trephined Bruch&#39;s membrane buttons in a 96-well plate. Typically, 9 -&#160;11 buttons or explants can be harvested from each eye.</li>
</ol>
3. Culturing of Retinal Pigment Epithelial (RPE) Cells on Human Bruch&#39;s Membrane Explants
<ol>
	<li>Upon receipt of the donor eyes, clean the extraocular tissue with DPBS. Make a circumferential incision 5 mm below the iris-sclera touch point (pars plana) into the subretinal space. Discard the anterior segment, vitreous humor, and retina.</li>
	<li>Wash the eyecup, which contains the Bruch&#39;s membrane and RPE sheet, with cold DPBS. Dissociate the RPE cells with 5 mL of 0.25% trypsin for no longer than 10 min for each eye sample. Quench the trypsin reaction with 25 mL of DMEM complete medium with 15% fetal bovine serum (FBS) warmed at 37 &#176;C in a 50 mL tube.</li>
	<li>Centrifuge the cells at 200 &#215; g for 10 min at 24 &#176;C, and resuspend the pellet in 5 mL of DMEM complete medium (with 15% FBS).</li>
	<li>Count the cells and seed 15,000 viable RPE cells onto the basal lamina of each various aged Bruch&#39;s membrane explants (buttons) in a 96-well plate in 200 &#181;L of serum-free minimum essential media (MEM) containing antibiotics (100 IU/mL penicillin G, 100 mg/mL streptomycin, 5 mg/mL gentamicin, and 2.5 mg/mL amphotericin B). Assuming a cell diameter of 20 &#181;m, plating at this density will allow RPE cells to cover approximately 15% of the plating area.</li>
	<li>Plate the cells onto the button, whereby the cell density can then be increased stepwise up to 100% confluence depending on the effect of each parameter on cell behavior.</li>
	<li>Allow the cells to attach to the explant for 24 h in a humidified atmosphere of 95% air/5% CO2 at 37 &#176;C. Gently change medium with warm complete DMEM.</li>
</ol>

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None of the authors have any commercial disclosures to declare.

Aged human Bruch&#39;s membrane (extracellular matrix) contributes to the disease progression of AMD and thus there is a need to understand how this altered matrix contributes to RPE cell dysfunction. Given the lack of sufficient animal models to study age-related changes in AMD, ex vivo model systems that mimic the effects of disease can serve as a valuable tool to understand the pathophysiology. The methodology described in this manuscript can be used to consistently isolate human Bruch&#39;s membrane explants and culture RPE cells, including primary, immortalized, or stem cell-derived RPE cell lines.
As mentioned previously, human Bruch&#39;s membrane explants are sourced from the NDRI. A protocol is established with NDRI which specifies the criteria set for donor eyes to be acceptable. For example, donors must have no known retinal eye diseases, eyes/globes are retrieved within 10 h after death and globes arrive in the laboratory within 48 h after death (via overnight package delivery on ice). Specify the appropriate age range and number of eyes needed for statistical analysis of the study, e.g., five pairs of globes from donors between the ages of 20 - 49 years and five globe pairs from donors between the ages of 50 - 89 years.
The availability of eye globes varies, generally averaging ten pairs of globes available each month. The eye package arrives with basic, de-identified donor information: age, race, time of death, cause of death, and brief past medical history (disease listing or comorbidities).
Most importantly, harvesting human Bruch&#39;s membrane explants requires the removal of the anterior segment of the eye and vitreous, allowing for the isolation of the RPE-Bruch&#39;s membrane-choroid complex. Once the Bruch&#39;s membrane is isolated, RPE cells can be seeded onto the acellular explants at varying concentrations and cultured for downstream experiments. The preparation and procedural handling as described herein is critical, paying attention to the orientation of the isolated Bruch&#39;s membrane to make sure the laminal surface is facing-up while not touching the surface mechanically to avoid damaging the extracellular matrix surface structure.
It should also be noted that when using the Bruch&#39;s membrane explant systems, there are some variables that could affect the results of downstream experiments such as individual donor genomic background, age of the Bruch&#39;s membrane, or postmortem time of the Bruch&#39;s membrane, and the location of the Bruch&#39;s membrane, i.e., central or peripheral part of Bruch&#39;s membrane explants. As with any other human tissue study, one must recruit the appropriate donor eye sample number to have the necessary statistical power and match donor eye age if appropriate. Establishing strict criteria for accepting eyes from eye banks is a critical parameter. Only accept eyes that are enucleated less than 10 h postmortem and which the Bruch&#39;s membrane preparation can be harvested within 24 - 48 h after death. The optic nerve site may be used as a marker for orientation, and use the same locations for experimental and control study groups. By taking these measures, one can minimize experimental variability.
In summary, understanding of the RPE-Bruch&#39;s membrane-choroid complex and how it is affected by age and disease is critical to understanding its contribution to the pathophysiology of AMD. Model systems that employ ex vivo techniques are a valuable tool to investigate these effects using human tissue. Herein, we describe a methodology that employs human donor explants as a relevant ex vivo model of aged Bruch&#39;s membrane. This technique allows one to use aged and/or diseased human Bruch&#39;s membrane as a research tool to investigate how structural changes within the matrix can affect overlaying RPE cell behavior including attachment, proliferation, and gene expression25,27. Successful development of similar ex vivo model systems will further our understanding of AMD and facilitate the development of novel therapeutic options.

Age-related structural changes to human Bruch&#39;s membrane, which is caused by many factors, including nitrosative and oxidative stress, exerts multiple deleterious effects on the function of retinal pigment epithelial (RPE) cells and contributes to the pathology of age-related macular degeneration (AMD)1,2,3,4,5,6,7,8,9. When considering cell replacement therapy for advanced atrophic AMD or geographic atrophy, treatment will likely require the transplantation of cells onto a bed of RPE cell atrophy. Age-related changes within human Bruch&#39;s membrane can adversely affect the success of transplanted RPE cell grafts, given the damage to the extracellular matrix6,9,10,11,12,13,14,15,16,17,18,19,20,21,22. Investigating human Bruch&#39;s membrane biology and how structural changes within the matrix contribute to the progression to AMD is vital to understanding disease pathology. Thus, there is a critical need for the investigators in the age-related eye field to develop protocols that describe the ex vivo harvesting of aged and/or diseased human Bruch&#39;s membrane.
Historically, it has been difficult to model age-related disorders such as geographic atrophy and an aged human Bruch&#39;s membrane in animals23. This difficulty arises from multiple factors including the longevity of humans compared to rodents and other species frequently used for disease modeling, as well as the lack of a macula in most vertebrates24. The advantage of the method described herein is that cells can be tested directly on Bruch&#39;s membrane extracted from post mortem eyes of aged and/or diseased individuals. The overall goal of this article is to provide a detailed methodology for an ex vivo model of aged and/or diseased human Bruch&#39;s membrane, including the isolation of human Bruch&#39;s membrane explants from human donors and the seeding of RPE cells for downstream experiments. This model can serve as a relevant model to investigate the contribution of extracellular matrix damage on RPE cell function and pathology20,25,26,27,28.

<table><tbody><tr><td>Human donor globes</td><td>National Disease Research Interchange, NDRI</td><td></td><td></td></tr><tr><td>Dulbecco&#039;s modified eagle medium (DMEM)</td><td>Thermo Fisher Scientific</td><td>11995-065</td><td></td></tr><tr><td>Carbon-dioxide independent media</td><td>Thermo Fisher Scientific</td><td>18045-088</td><td></td></tr><tr><td>60-mm polystyrene petri dish</td><td>Corning Inc.</td><td>351007</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline, PBS</td><td>Thermo Fisher Scientific</td><td>14190144</td><td></td></tr><tr><td>Hydrophobic 65 &amp;micro;m-thick polytetrafluoroethylene membrane with 0.2 &amp;micro;m pores</td><td>Merck Millipore</td><td>JGWP04700</td><td></td></tr><tr><td>Fisherbrand glass microanalysis vacuum filter holder system&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>09-753-102</td><td></td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>A2576-5G</td><td></td></tr><tr><td>35 &amp;times; 10mm culture dish</td><td>VWR International</td><td>25373-041</td><td></td></tr><tr><td>Trephine</td><td>Accutome</td><td>AM0570 60</td><td></td></tr><tr><td>96 well plate</td><td>Corning Inc.</td><td>3595</td><td></td></tr><tr><td>Minimum essential media (MEM)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11095-080&amp;nbsp;</td><td></td></tr><tr><td>Penicillin G</td><td>Sigma-Aldrich</td><td>P3032-1MU</td><td></td></tr><tr><td>Streptomycin</td><td>Sigma-Aldrich</td><td>S6501</td><td></td></tr><tr><td>Gentamicin</td><td>Sigma-Aldrich</td><td>G1914</td><td></td></tr><tr><td>Amphotericin B</td><td>Sigma-Aldrich</td><td>1397-89-3</td><td></td></tr><tr><td>Ammonium hydroxide solution</td><td>Sigma-Aldrich</td><td>1336-21-6&lt;br/&gt; &amp;nbsp;</td><td></td></tr></tbody></table>

culturing of retinal pigment epithelial cells on an ex vivo model of aged human bruch's membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

When this protocol is performed properly, one can determine the effects of aged and/or diseased human Bruch&#39;s membrane on RPE cell function by the establishment of polarity and the outer blood retinal barrier, polarized secretion of growth factors and cytokines, and phagocytosis of photoreceptor rod outer segments.
We have generated data that demonstrate a disease phenotype on aged human Bruch&#39;s membrane. For example, culturing RPE cells on aged human Bruch&#39;s membrane explants decreases RPE cell reattachment (Figure 3). RPE cells cultured on aged human Bruch&#39;s membrane decreases the capacity of these cells to phagocytize rod outer segments (ROS), a critical RPE function (Figure 4). Moreover, apoptotic cells can be identified and compared on these button explants using a terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) stain as described in previous work26. The effects of an aged and/or diseased human Bruch&#39;s membrane on RPE cell gene expression profile can be determined using microarray technology. We have demonstrated that RPE cell gene expression is altered when cultured on human Bruch&#39;s membrane from older individuals compared to explants from younger individuals (Figure 5)27. For statistical analysis, explants from at least 3 - 5 human donors are used per group.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57084/57084fig1v2.jpg" /> 
Figure 1. Schematic representation of the isolation of human Bruch&#39;s membrane from donor eyes. Human Bruch&#39;s membranes are harvested by removing the sclera, anterior segment, retina, vitreous, and native retinal pigment epithelial (RPE) cells. <a href="https://cloudflare.jove.com/files/ftp_upload/57084/57084fig1v2large.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/57084/57084fig2.jpg" /> 
Figure 2. Isolated human Bruch&#39;s membrane (BM) in a culture dish. (A) Schematic of human Bruch&#39;s membrane explants (each 1.5 inches in diameter, with solidified agarose gel) placed in a 60 &#215; 15 mm polystyrene cell culture dish (BM face-up) filled with warm liquid agarose on the bottom of dish. (B) A human Bruch&#39;s membrane explant that has been trephined into a few 6 mm circular buttons (explants), each ow which was then placed on 4% agarose at 37 &#176;C in a non-treated polystyrene well of a 96-well plate. <a href="https://cloudflare.jove.com/files/ftp_upload/57084/57084fig2large.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/57084/57084fig3.jpg" /> 
Figure 3. Retinal pigment epithelial (RPE) cell reattachment rate on young and older human Bruch&#39;s membrane (BM). Primary RPE cells were seeded onto human Bruch&#39;s membrane explants from young (&#60;50-year-old, n = 3) or older (&#62;70-year-old, n = 5) donor eyes for three weeks. Cell attachment rate was measured by MTT cell viability assay. Older human Bruch&#39;s membranes reduced the RPE cell attachment rate by 24% (young BM 100 +/-8.54 S.E. vs. older BM 76.65 +/-1.44 S.E.). **p &#60;0.05, S.E. = standard error.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57084/57084fig4.jpg" /> 
Figure 4. Retinal pigment epithelial (RPE) cell phagocytosis is affected by the age of the human Bruch&#39;s membrane (BM). Primary RPE cells cultured on older human Bruch&#39;s membrane explants had a reduced ability to phagocytize rod outer segments (ROS) (young BM 873 +/-9.3 S.E., n = 5 vs. older BM 608 +/-25.4 S.E., n = 5). ***p &#60;0.01, S.E. = standard error.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57084/57084fig5.jpg" /> 
Figure 5. Number of retinal pigment epithelial (RPE) cell genes expressed in each sample cultured on young or older human Bruch&#39;s membrane explants. There was more scatter in the number of genes expressed in primary RPE cells seeded onto older versus younger human Bruch&#39;s membrane (6201 &#177; 388 vs. 6439 &#177; 80, respectively); five explants were tested for each age group. Presented with the full permission of all authors of Cai, H et al.27. <a href="https://cloudflare.jove.com/files/ftp_upload/57084/57084fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane at JoVE.com

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...

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JoVE Journal

Developmental Biology

10.1K Views.  Yale School of Medicine. The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch's membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

Video: Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

Neuroscience

Enrichment of Bruch's Membrane from Human Donor Eyes

Age-related macular degeneration (AMD) is a leading cause of visual impairment in the developed world. The disease manifests itself by the destruction of the center&#160;of the retina, called the macula, resulting in the loss of central vision. Early AMD is characterised by the presence of small, yellowish lesions called soft drusen that can progress onto late AMD such as geographic atrophy (dry AMD) or neovascularisation (wet AMD). Although the clinical changes are well described, and the understanding of genetic influences on conferring AMD risk are getting ever more detailed, one area lacking major progress is an understanding of the biochemical consequences of genetic risk. This is partly due to difficulties in understanding the biochemistry of Bruch&#8217;s membrane, a very thin extracellular matrix that acts as a biological filter of material from the blood supply and a scaffold on which the retinal pigment epithelial (RPE) cell monolayer resides. Drusen form within Bruch&#8217;s membrane and their presence disrupts nutrient flow to the RPE cells. Only by investigating the protein composition of Bruch&#8217;s membrane, and indeed how other proteins interact with it, can researchers hope to unravel the biochemical mechanisms underpinning drusen formation, development of AMD and subsequent vision loss. This paper details methodologies for enriching either whole Bruch&#8217;s membrane, or just from the macula region, so that it can be used for downstream biochemical analysis, and provide examples of how this is already changing the understanding of Bruch&#8217;s membrane biochemistry.

Identifying proteins specifically associated with Bruch&#8217;s membrane in human eyes is an important step in understanding the biochemical mechanisms behind eye diseases such as age-related macular degeneration. This protocol describes how to enrich this sheet of extracellular matrix for down-stream biochemical analysis.

Age-related macular degeneration (AMD) is a leading cause of visual impairment in the developed world. The disease manifests itself by the destruction of ...

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 ...

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

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

Characterization and Morphological Dynamics of Cultured Primary Murine RPE

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important retinal diseases, such as age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy. We present a streamlined approach for the isolation of RPE from murine adult eyes. In contrast to previously reported methods, this approach enables the isolation and culture of highly pure RPE from adult mice. This simple and fast method does not require extensive technical skill and is achievable with basic scientific tools and reagents. Primary RPE are isolated from C57BL/6 background mice aged 3- to 14-weeks by enucleation of the eye followed by the removal of the anterior segment. Enzymatic trypsinization and centrifugation are used to dissociate and isolate the RPE from the eyecup. In conclusion, this approach offers a quick and effective protocol for the utilization of RPE in the study of retinal function and disease.

Author Spotlight: Efficient Isolation of Primary Retinal Pigment Epithelial Cells from Adult Mice for Retinopathy Research

Retinal pigment epithelium (RPE) acts as a crucial barrier between the choroid and retina, promoting the health and function of retinal cell types, such as photoreceptors. Herein, we describe a simple and effective protocol for isolating and culturing adult murine RPE.

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Biology

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo&#160;analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo&#160;analyses and&#160;in vivo studies transferable to humans.

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

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Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

Enrichment of Bruch's Membrane from Human Donor Eyes

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

Characterization of a Novel Human Organotypic Retinal Culture Technique

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Primary Culture of Porcine Retinal Pigment Epithelial Cells