We thank Lab members Jeffrey Adijanto, Tina Banzon, Rong Li, Qin Wan, Congxiao Zhang, Jing Zhao, Connie Zhi, Awais Zia, Natalia Strunnikova for help in characterizing these cell cultures. Special thanks to Jing Zhao, Connie Zhi, and Tina Banzon for their help in maintaining large stocks of cell cultures.
This work was supported by the National Institutes of Health Intramural Research Program.

1. Human Fetal Tissue
All human tissue related research follows the tenets of the Declaration of Helsinki and the NIH institutional review board. Fetal eyes are obtained by an independent procurer, Advanced Bioscience Resources (ABR, Alameda, CA), from random donors at 16 to 22 weeks of gestation, placed in RPMI-1640 media containing tubes (provided by ABR), packed on ice, and delivered by an overnight priority delivery service. Tissues remain viable up to 48 hours after enucleation.
2. Cell Culture Medium
MEM-alpha modified medium (Sigma-Aldrich) is used as the base medium to prepare 5% and 15% serum-containing media for culturing of RPE cells (RPE medium; Table 1 below).
<table border="1">
<tbody>
 <tr>
 <td>Name</td>
 <td>Sigma</td>
 <td>Gibco</td>
 <td>Amount</td>
 <td>Storage</td>
 </tr>
 <tr>
 <td>MEM, alpha modification</td>
 <td>M-4526</td>
 <td>&nbsp;</td>
 <td>500 mL</td>
 <td>+4&deg;C</td>
 </tr>
 <tr>
 <td>N1 supplement</td>
 <td>N-6530</td>
 <td>&nbsp;</td>
 <td>5 mL</td>
 <td>+4&deg;C</td>
 </tr>
 <tr>
 <td>Penicillin-streptomycin</td>
 <td>&nbsp;</td>
 <td>15140-148</td>
 <td>5 mL</td>
 <td>-20&deg;C</td>
 </tr>
 <tr>
 <td>GlutaMax - I </td>
 <td>&nbsp;</td>
 <td>35050</td>
 <td>5 mL</td>
 <td>-20&deg;C</td>
 </tr>
 <tr>
 <td>Non essential amino acids</td>
 <td>M-7145</td>
 <td>&nbsp;</td>
 <td>5 mL</td>
 <td>+4&deg;C</td>
 </tr>
 <tr>
 <td>THT*</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>-80&deg;C</td>
 </tr>
 <tr>
 <td>Taurine</td>
 <td>T-0625</td>
 <td>&nbsp;</td>
 <td>125 mg</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hydrocortisone</td>
 <td>H-0396 10</td>
 <td>&nbsp;</td>
 <td>10 &mu;g</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Triiodo-thyronin</td>
 <td>T-5516</td>
 <td>&nbsp;</td>
 <td>0.0065 &mu;g</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal bovine serum**</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>5% or 15%</td>
 <td>-80&deg;C</td>
 </tr>
</tbody>
</table>
Table 1. Human Fetal RPE Medium Components for Preparation of 500 mL Medium
* THT is made by dissolving taurine-hydrocortisone-triiodo-thyronin in 1 1.5 mL PBS before making the medium. Multiple aliquots are made and stored at -80&deg;C to simplify culturing preparation of the culture medium.
** Fetal bovine serum is not obtained from Sigma-Aldrich or Gibco.
Fetal bovine serum used in media preparation is obtained from Atlanta Biologicals (Norcross, GA). Each bottle of the serum is heat inactivated (56&deg;C for 1hr) prior to use. To ensure cell culture consistency large amounts of serum from the same lot are purchased and stored in -20&deg;C until they are used for media preparation. Sparsely seeded cells from a specific batch of hfRPE cells are grown in 24- or 12- well plates for a few weeks in media containing 20% FBS from different lots. The fastest growing melanated cells with the most classical RPE morphology (cobblestone) indicate an appropriate serum lot that can be used for cell culture.
Cell culture medium also contains: N1 supplement (Sigma-Aldrich) 1:100 mL/mL, GlutaMax/ penicillin-streptomycin 1:100 mL/mL (Gibco), and nonessential amino acid solution (Sigma-Aldrich) 1:100 mL/mL. In addition, hydrocortisone (20 &mu;g/L), taurine (250 mg/L), and triiodo-thyronin (0.013 &mu;g/L) (THT) is prepared ahead by dissolving these three components in PBS to a final concentration of 1:500 (mL/mL). Aliquots of THT are stored at 80&deg;C until added to the RPE medium.
3. Cell Culture
On receipt, intact globes are rinsed in antibiotic antimycotic solution (diluted to 10X; cat. no. 15240-096; Invitrogen) plus gentamicin (1 mg/mL) for 3 to 5 minutes (Figure 1 below). 
 <img src="/files/ftp_upload/2032/2032fig1.jpg" alt="Figure 1" /> 
 Figure 1. After incubation antibiotics are rinsed off twice with medium such as HBSS or PBS. One eye globe at the time is transferred to 10 cm Petri dish with coated with Sylgard-184 (WPI) and secured with 27G needles. Using ClearCut sideport knife (Alcon) an incision is made through the sclera below the ciliary body (1/3 of the distance from eye equator to the anterior surface). This incision is used to start a circular cut for removal of the anterior eye portion. This cut is made using a tungsten- carbide coated curved iris scissors with one blade serrated (FST). Prior to the removal of the anterior part of the eye, a cut is made through the vitreous body to avoid detaching the retina from the RPE at the posterior pole. After the anterior portion of the eye is removed, the posterior pole is incubated with dispase-I solution (2 U/mL, cat. no. 04942086001; Roche Diagnostics, Indianapolis, IN) in 5% serum containing medium for 40-60 minutes in 37&deg;C-5% CO2. After dispase treatment, the posterior poles are transferred to a HBSS in Petri dishes with silicon padding (Sylgard 184; Dow Corning, Midland, MI) and dissected into quadrants or larger pieces to sufficiently flatten tissues. Then the retina is gently removed with forceps. Single-cell RPE layers were peeled off in sheets and collected directly into cold trypsin-EDTA (Gibco, #25200-056) solution. After the RPE are collected, tubes with tissues in trypsin-EDTA are sealed and transferred into water bath for 10-15 mins at 37&deg;C. After 10 mins of incubation, the tubes are vigorously shaken to separate RPE into small clusters. If the separation is not complete, the tubes are placed back into water bath for another 5 mins. After trypsin-EDTA incubation, the tubes are inspected for possible un-dissolved mixed cell clusters. Any observed clusters are removed using fine tipped glass Pasteur pipette. After spinning down (1.4 rpm on clinical centrifuge for 4 mins), hfRPE cells are re-suspended in 15% RPE media and then put into Primaria flasks (example: cat. no. 08-772-45; Fisher Scientific, Pittsburgh, PA). This medium is replaced after 1 day with 5% serum-containing RPE medium, and subsequent changes were made every 2 to 3 days. After 3 to 4 weeks, the cells became confluent and uniformly pigmented. They are then trypsinized in 0.25% trypsin-EDTA for 10 to 15 minutes, re-suspended in 15% serum-containing RPE cell culture medium, and seeded onto clear cell culture inserts at 150 to 200K cells per well (Transwell; Corning Costar, Corning, NY), using 12-mm diameter inserts, 0.4-um pores, polyester membranes (example: cat. no. 07-200-161, Fisher Scientific). Before seeding, the wells were coated with human extracellular matrix (10 &mu;g in 150 &mu;L HBSS per well, cat. no. 354237; BD Biosciences, Franklin Lakes, NJ) and cured with UV light in the hood for 2 hours. In some cases, the trypsinization procedure was repeated for a second time, to collect the cells that did not detach after the first trypsinization. The same protocol (excluding coating with ECM) was used to culture cells on the flasks to generate the P1 population of cells. These cells were used in experiments when they had a total tissue resistance of &ge; 200 &Omega;&#8226;cm2 and were uniformly pigmented.
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/2032/2032fig1full.jpg">Please click here for a larger figure 1</a>.
4. Step by Step Procedures
<ol>
 <li> Prepare 12-well plate with multiple solutions (for each eye 4 wells needed):
 <ol class="lalpha">
 <li> add 10x antibiotic antimycotic solution - 1 well/eye</li>
 <li> add PBS/HBSS solution for rinse - 2 wells/eye</li>
 <li> add dispase solution - 1 well/eye</li>
 </ol>
 </li>
 <li> Prepare dissecting Petri dish by unpacking 5 fixation needles and filling it with HBSS</li>
 <li> Unpack eyes and place them into antibiotic-antimycotic solution for 3-5 mins (prepared in step 1)</li>
 <li> Rinse eyes in two wells (prepared in step 1) with PBS/HBSS, then transfer them to dissecting dish.</li>
 <li> Trim excessive muscle and connective tissues around the eye</li>
 <li> Using 27G needles secure (pin down to silicon base) eyes in dissecting dish aligning them in a way where cornea faces up.</li>
 <li> Using sideport knife make incision below cornea, where circular cut will begin</li>
 <li> Using iris scissors make cut around the eye, then using same scissors cut through vitreous and lift anterior portion of the eye away. 
 Note: in steps 3 to 8 avoid any excessive mechanical pressure to eyeball.</li>
 <li> Transfer open eye into dispase solution (prepared in step 1)</li>
 <li> Incubate eye cup for 40-60 minutes at 37&deg;C with 5 % CO2</li>
 <li> Replace HBSS solution in dissecting dish with fresh one.</li>
 <li> Transfer eye from dispase solution to dissecting dish.</li>
 <li> Position eye in Petri dish (eyeball cup facing up) and secure with two 27G needles.</li>
 <li> Gently lift partially separated retina and using retinal scissors cut retina away from the optic nerve. Discard retina.</li>
 <li> Using iris scissors make one incision from periphery of the eye towards optic nerve.</li>
 <li> Using all five 27G needles flatten eye making RPE layer nicely stretched.</li>
 <li> Using retinal scissors make circular cut around optic nerve separating RPE layer from attachment to optic nerve. 
 Note: steps 13-17 can be done with low magnification or without stereo microscope</li>
 <li> Adjust stereomicroscope to 250x magnification or more and find edge of RPE sheet close to optic nerve along the cut made by iris scissors.</li>
 <li> Using two forceps separate RPE-Bruch's membrane from the choroidal tissue layer. It may require a few attempts to find area along the edge where the connection of RPE and choroid is weakest.</li>
 <li> Place RPE sheets into cold trypsin-EDTA solution in 15 mL tube</li>
 <li> After the RPE are collected, cap and transfer tubes with tissues in trypsin-EDTA into water bath for 10-15mins at 37&deg;C.</li>
 <li> After 10 mins of incubation, vigorously shake 15 mL tubes to separate RPE into small clusters. If the separation is not complete, place tubes back into water bath for another 5 mins.</li>
 <li> Inspect the tubes for possible un-dissolved mixed cell clusters. Any observed clusters should be removed using fine tipped glass Pasteur pipette.</li>
 <li> Spin down (1.4 rpm on clinical centrifuge for 4 mins) hfRPE cells, remove supernatant and re-suspend cells in 15% RPE media (9 mL total)</li>
 <li> Put 3 mL of cell suspension into Primaria flasks add 2 mL of fresh 15% RPE media, place flask into incubator until next day (37&deg;C, 5% CO2)</li>
</ol>
5. Representative Results
Functional validation of hfRPE cell culture
In previous experiments we have used these primary cultures to determine the expression, polarization and function of plasma membrane transport proteins and identify specific signaling pathways that regulate RPE function 1-11 These characteristics as they are accumulated provide a validation set of properties that can be applied to each cell generation. The experiments summarized below identify the proteins that determine transepithelial fluid transport and the integrity of the paracellular pathway. These in vitro experiments have been validated in an animal model of retinal re-reattachment 6.
<img src="/files/ftp_upload/2032/2032fig2.jpg" alt="Figure 2" /> Figure 2. Localization of CFTR in hfRPE cells. Left upper panel: CFTR was detected in hfRPE cells using membrane-enriched extracts. M, molecular weight marker; lane 1, major mature (band C) and immature (bands A and B). Center lower panel: immunofluorescence localization of CFTR (green label). Top right panel: enlarged cross section view through the z plane showing maximum-intensity projection through the z-axis. ZO-1 (labeled as red) serves as a tight junction marker delineating apical and basolateral sides of the RPE. DAPI (blue) labels the nuclei located close to the basal membrane. ZO-1 appears as yellow/orange, since its red label overlaps green fluorescence from CFTR.
<img src="/files/ftp_upload/2032/2032fig3.jpg" alt="Figure 3" /> Figure 3. IFN&gamma;-induced physiological changes in hfRPE. A: addition of IFN&gamma; to the basal bath increased transepithelial fluid transport (Jv) across monolayer of primary, cultured hfRPE cells. Jv is plotted as a function of time in the top trace and net fluid absorption (apical to basal bath) is indicated by positive values; transepithelial potential (TEP) and total tissue resistance (RT) are plotted as a function of time in the bottom traces. B: Addition of CFTRinh-172(5 &mu;M) to the basal bath inhibited the IFN&gamma;-stimulated Jv increase.
Below is an example of animal model experiments confirming hfRPE in vitro findings. In this experiment, artificially created retinal detachment was significantly reduced after addition of IFNg to the exterior eye surface (Figure 4 A,B). This effect can be partially blocked by: (1) addition of cAMP blocker (Figure 4D); (2) completely blocked after addition of JAK+cAMP blockers. Images below are obtained using OCT scanner.
<img src="/files/ftp_upload/2032/2032fig4.jpg" alt="Figure 4" /> Figure 4. In vivo retinal detachment experiments. The procedures for these in vivo experiments have been previously described in detail 6. In these experiments, retinal detachments were created in rat eye by injection of 0.5-3 &mu;L of modified PBS solution into the subretinal space (SRS), alone or with a combination of JAK-STAT and PKA pathway inhibitors. Each experiment had an initial control period of 40-70 min after creation of the retinal detachment. During this time, the rate of change of bleb volume was measured to insure bleb stability. Optical coherence tomography imaging (Institute of Applied Physics, Russian Academy of Science, Nizhniy Novgorod, Russia) was used to measure the time course of the volume change in SRS.
The time course of re-attachment volume change was measured by optical coherence tomography (OCT). OCT images from 4 different experiments (panels on the left; A-D) that show a change in detachment sizes (arrows in A, B, and D) following the addition of IFN&gamma; to the anterior surface for 40-70 min. Panels A and B show that IFN&gamma; increased Jv from its control rate (&asymp; 2 &mu;l&#8226;cm2&#8226;hr-1) to 14 and 12 &mu;l&#8226;cm2&#8226;hr-1, respectively. Following addition of JAK-STAT pathway and PKA inhibitors (C and D), the IFN&gamma; - induced absorption rates were significantly reduced to 0.2 and 7.9 &mu;l&#8226;cm2&#8226;hr-1, respectively. Arrows indicate the boundary of the bleb for comparison with the area enclosed within the dashed line, (starting volume). Right side of figure: top panel- summary of measured Jv rates from several experiments; middle panel- movie (<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/2032/Bleb%205.wmv">click here</a>); lower panel- 3D sections of experiment summarized in B. Pseudocolor in blue indicates spatial extent of detachment at t = 0 and 40 min after addition of INF&gamma;.
All animal experiments were conducted in compliance with the Association for Research in Vision and Ophthalmology statement. The protocol was approved by the Animal Care and Use Committee of the National Institutes of Health.

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No conflicts of interest declared.

In the present experiments, we describe additional modifications of our previously published techniques 3 designed to streamline the multiple steps needed to produce consistent hfRPE primary cultures with a higher numbers of available cells per eye. Each change in the original procedure was rigorously tested in multiple physiology and molecular biology experiments to ensure that the changes did not introduce artifacts and being continually tested by many other labs using these cells. Finally, additional experiments were carried out to compare the in vitro results to similar perturbations in animal models.

<table><tbody><tr><td colspan="4">Instruments needed for dissection protocol</td></tr><tr><td>Stereo Microscope - any available with working magnification x250 needed for dissection</td><td></td><td></td><td></td></tr><tr><td>Dissecting dish - e.g. Kimble 100 x 20mm #23062 (one per dissection) any source</td><td></td><td></td><td></td></tr><tr><td>Sylgard 184 - WPI Cat. #SYLG184* </td><td></td><td></td><td></td></tr><tr><td>* To prepare dissecting dish follow Sylgard 184 included mixing instructions, pour mixed liquid elastomer into Petri dish to form 5-8mm layer. Allow elastomer to cure for at least 24hrs. Dish with cured elastomer can be sterilized using 70% ethanol and reused multiple times (~100 times)</td><td></td><td></td><td></td></tr><tr><td>27 G needles from B-D PrecissionGlide 1&amp;#188; length (5 per dissection) any source</td><td></td><td></td><td></td></tr><tr><td> HBSS 1X Solution containing Ca and Mg salts - GIBCO Cat.#14025 500ml (good for multiple dissections)</td><td></td><td></td><td></td></tr><tr><td>Iris scissors carbide serrated blade, curved - FST Cat.# 14559-11 (one)</td><td></td><td></td><td></td></tr><tr><td>Retinal scissors - Katena Cat.#K4-5300 (one)</td><td></td><td></td><td></td></tr><tr><td>Forceps - e.g. Dumont Tweezers #5 with polished tips from WPI Cat.#500085 (2 pieces)</td><td></td><td></td><td></td></tr><tr><td>Sideport knife - Alcon, ClearCut 1mm, Dual Bevel, angled Cat.#8065921540</td><td></td><td></td><td></td></tr><tr><td>Centrifuge tube 15ml - (one per eye) any source</td><td></td><td></td><td></td></tr><tr><td>Pasteur glass pipette - (one per dissection) any source</td><td></td><td></td><td></td></tr><tr><td>Plate 12 well (one per dissection) any source</td><td></td><td></td><td></td></tr><tr><td>Primaria 25cm&lt;sup&gt;2&lt;/sup&gt; flask - (2-3 per eye) any source</td><td></td><td></td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology at JoVE.com

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

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 ...

experimental-models-for-study-retinal-pigment-epithelial-physiology

Nanyang Technological University

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JoVE Journal

Neuroscience

19.2K Views.  National Institutes of Health. Wij bieden een reproduceerbare methode voor het kweken van samenvloeiing monolagen van de menselijke foetus retinale pigment epitheelcellen (hfRPE) cellen die vertonen morfologie, fysiologie, polariteit, en eiwit-en genexpressie-patronen van de volwassen autochtone weefsel. Dit werk is uitgebreid tot een diermodel van verschillende oogziekten.

Video: Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms remain elusive mainly due to the lack of a good in vivo model for studying BEST1 and its mutations under physiological conditions. BEST1 encodes an ion channel, namely BESTROPHIN1 (BEST1), which functions in retinal pigment epithelium (RPE); however, the extremely limited accessibility to native human RPE cells represents a major challenge for scientific research. This protocol describes how to generate human RPEs bearing BEST1 disease-causing mutations by induced differentiation from human pluripotent stem cells (hPSCs). As hPSCs are self-renewable, this approach allows researchers to have a steady source of hPSC-RPEs for various experimental analyses, such as immunoblotting, immunofluorescence, and patch clamp, and thus provides a very powerful disease-in-a-dish model for BEST1-associated retinal conditions. Notably, this strategy can be applied to study RPE (patho)physiology and other genes of interest natively expressed in RPE.

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Here we present a protocol to differentiate retinal pigment epithelium (RPE) cells from human pluripotent stem cells bearing patient-derived mutations. The mutant cell lines may be used for functional analyses including immunoblotting, immunofluorescence, and patch clamp. This disease-in-a-dish approach circumvents the difficulty of obtaining native human RPE cells.

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms ...

Genetics

A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal pigmented epithelium (RPE) is a key pathobiological event in AMD. To understand the mechanisms that lead to RPE dysfunction, mouse models can be utilized by researchers. It has been established by previous studies that mice can develop RPE pathologies, some of which are observed in the eyes of individuals diagnosed with AMD. Here, we describe a phenotyping protocol to assess RPE pathologies in mice. This protocol includes the preparation and evaluation of retinal cross-sections using light microscopy and transmission electron microscopy, as well as that of RPE flat mounts by confocal microscopy. We detail the common types of murine RPE pathologies observed by these techniques and ways to quantify them through unbiased methods for statistical testing. As proof of concept, we use this RPE phenotyping protocol to quantify the RPE pathologies observed in mice overexpressing transmembrane protein 135 (Tmem135) and aged wild-type C57BL/6J mice. The main goal of this protocol is to present standard RPE phenotyping methods with unbiased quantitative assessments for scientists using mouse models of AMD.

Mouse models can be useful tools for investigating the biology of the retinal pigmented epithelium (RPE). It has been established that mice can develop an array of RPE pathologies. Here, we describe a phenotyping protocol to elucidate and quantify RPE pathologies in mice using light, transmission electron, and confocal microscopy.

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal ...

Direct-Coupled Electroretinogram (DC-ERG) for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a specialized monolayer of cells strategically located between the retina and the choriocapillaris that maintain the overall health and structural integrity of the photoreceptors. The RPE is polarized, exhibiting apically and basally located receptors or channels, and performs vectoral transport of water, ions, metabolites, and secretes several cytokines.
In vivo noninvasive measurements of RPE function can be made using direct-coupled ERGs (DC-ERGs). The methodology behind the DC-ERG was pioneered by Marmorstein, Peachey, and colleagues using a custom-built stimulation recording system and later demonstrated using a commercially available system. The DC-ERG technique uses glass capillaries filled with Hank&#8217;s buffered salt solution (HBSS) to measure the slower electrical responses of the RPE elicited from light-evoked concentration changes in the subretinal space due to photoreceptor activity. The prolonged light stimulus and length of the DC-ERG recording make it vulnerable to drift and noise resulting in a low yield of useable recordings. Here, we present a fast, reliable method for improving the stability of the recordings while reducing noise by using vacuum pressure to reduce/eliminate bubbles that result from outgassing of the HBSS and electrode holder. Additionally, power line artifacts are attenuated using a voltage regulator/power conditioner. We include the necessary light stimulation protocols for a commercially available ERG system as well as scripts for analysis of the DC-ERG components: c-wave, fast oscillation, light peak, and off response. Due to the improved ease of recordings and rapid analysis workflow, this simplified protocol is particularly useful in measuring age-related changes in RPE function, disease progression, and in the assessment of pharmacological intervention.

Direct-Coupled Electroretinogram DC-ERG for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium

Here, we present a method for recording light-evoked electrical responses of the retinal pigment epithelium (RPE) in mice using a technique known as DC-ERGs first described by Marmorstein, Peachey, and colleagues in the early 2000s.

The retinal pigment epithelium (RPE) is a specialized monolayer of cells strategically located between the retina and the choriocapillaris that maintain ...

Chemistry

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 ...

Medicine

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 ...

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 ...

Biology

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. ...

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and vision. Proteins on the RPE apical surface are tightly associated with proteins on the photoreceptor outer segment surface, making it difficult to consistently separate the RPE from the photoreceptors/retina. We developed a method to efficiently separate the retina from the RPE of human eyes to generate complete RPE/choroid and retina flatmounts for separate cellular analysis of the photoreceptors and RPE cells. An intravitreal injection of a high-osmolarity solution of D-mannitol, a sugar not transported by the RPE, induced the separation of the RPE and retina across the entire posterior chamber without causing damage to the RPE cell junctions. No RPE patches were observed attached to the retina. Phalloidin labeling of actin showed RPE shape preservation and allowed morphometric analysis of the entire epithelium. An artificial intelligence (AI)-based software was developed to accurately recognize and segment the RPE cell borders and quantify 30 different shape metrics. This dissection method is highly reproducible and can be easily extended to other animal models.

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...

Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential application in molecular biology studies, including gene expression analyses. In the context of eye growth regulation and myopia, the RPE likely plays a role as a cellular relay for growth modulatory signals, as it is located between the retina and the two walls of the eye, such as the choroid and sclera. While protocols for isolating the RPE have been developed for both chicks and mice, these protocols have proven not to be directly translatable to the guinea pig, which has become an important and widely used mammalian myopia model. In this study, molecular biology tools were used to examine the expression of specific genes to confirm that the samples were free of contamination from the adjacent tissues. The value of this protocol has already been demonstrated in an RNA-Seq study of RPE from young pigmented guinea pigs exposed to myopia-inducing optical defocus. Beyond eye growth regulation, this protocol has other potential applications in studies of retinal diseases, including myopic maculopathy, one of the leading causes of blindness in myopes, in which the RPE has been implicated. The main advantage of this technique is that it is relatively simple and&#160;once perfected, yields high-quality RPE samples suitable for molecular biology studies, including RNA analysis.

We describe a simple and efficient method for isolating cells of the retinal pigment epithelium (RPE) cells from the eyes of young pigmented guinea pigs. This procedure allows for follow-up molecular biology studies on the isolated RPE, including gene expression analyses.

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential ...

Modeling and Analyzing Retinal Degeneration

LipidUNet-Machine Learning-Based Method of Characterization and Quantification of Lipid Deposits Using iPSC-Derived Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a monolayer of hexagonal cells located at the back of the eye. It provides nourishment and support to photoreceptors and choroidal capillaries, performs phagocytosis of photoreceptor outer segments (POS), and secretes cytokines in a polarized manner for maintaining the homeostasis of the outer retina. Dysfunctional RPE, caused by mutations, aging, and environmental factors, results in the degeneration of other retinal layers and causes vision loss. A hallmark phenotypic feature of degenerating RPE is intra and sub-cellular lipid-rich deposits. These deposits are a common phenotype across different retinal degenerative diseases. To reproduce the lipid deposit phenotype of monogenic retinal degenerations in vitro, induced pluripotent stem cell-derived RPE (iRPE) was generated from patients&#39; fibroblasts. Cell lines generated from patients with Stargardt and Late-onset retinal degeneration (L-ORD) disease were fed with POS for 7 days to replicate RPE physiological function, which caused POS phagocytosis-induced pathology in these diseases. To generate a model for age-related macular degeneration (AMD), a polygenic disease associated with alternate complement activation, iRPE was challenged with alternate complement anaphylatoxins. The intra and sub-cellular lipid deposits were characterized using Nile Red, boron-dipyrromethene (BODIPY), and apolipoprotein E (APOE). To quantify the density of lipid deposits, a machine learning-based software, LipidUNet, was developed. The software was trained on maximum-intensity projection images of iRPE on culture surfaces. In the future, it will be trained to analyze three-dimensional (3D) images and quantify the volume of lipid droplets. The LipidUNet software will be a valuable resource for discovering drugs that decrease lipid accumulation in disease models.

Author Spotlight: Unraveling the Pathogenesis of Age-Related Macular Degeneration and Discovering Potential Therapies 

Degenerative eye diseases that affect the retinal pigment epithelium layer of the eye have monogenic and polygenic origins. Several disease models and a software application, LipidUNet, have been developed to study mechanisms of disease, as well as potential therapeutic interventions.

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

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