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/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/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.

<ol>
	<li>Bharti, K., Miller, S. S., Arnheiter, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+new+paradigm:+Retinal+pigment+epithelium+cells+generated+from+embryonic+stem+cells+or+induced+pluripotent+cells.">The new paradigm: Retinal pigment epithelium cells generated from embryonic stem cells or induced pluripotent cells.</a> Pigment Cell &#38; Melanoma Research. , .</li><li>Strunnikova, N. V., Maminishkis, A., Barb, J. J., Wang, F., Zhi, C., Sergeev, Y., Chen, W., Edwards, A. O., Stambolian, D., Abecasis, G., Swaroop, A., Munson, P. J., Miller, S. S., S, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+analysis+and+molecular+signature+of+human+retinal+pigment+epithelium.">Transcriptome analysis and molecular signature of human retinal pigment epithelium.</a> Hum Mol Genet. 19 (12), 2468-2486 (2010).</li><li>Miller, S. S., Maminishkis, A., Li, R., Adijanto, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+184:+Phototransduction:+RPE+transport+Retina+Phototransduction:+RPE+transport.">Chapter 184: Phototransduction: RPE transport Retina Phototransduction: RPE transport.</a> Encyclopedia of the Eye. , 2540-2540 (2010).</li><li>Bryant, D. M., Mostov, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+cells+to+organs:+Building+polarized+tissue.">From cells to organs: Building polarized tissue.</a> Nature Reviews. Molecular Cell Biology. 9 (11), 887-901 (2008).</li><li>Maminishkis, A., Chen, S., Jalickee, S., Banzon, T., Shi, G., Wang, F. E., Ehalt, T., Hammer, J. A., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confluent+monolayers+of+cultured+human+fetal+retinal+pigment+epithelium+exhibit+morphology+and+physiology+of+native+tissue.">Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue.</a> Invest Ophthalmol Vis Sci. 47, 3612-3624 (2006).</li><li>Shi, G., Maminishkis, A., Banzon, T., Jalickee, S., Li, R., Hammer, J., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+chemokine+gradients+by+the+retinal+pigment+epithelium.">Control of chemokine gradients by the retinal pigment epithelium.</a> Invest Ophthalmol Vis Sci. 49, 4620-4630 (2008).</li><li>Economopoulou, M., Hammer, J., Wang, F. E., Fariss, R., Maminishkis, A., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression,+localization,+and+function+of+junctional+adhesion+molecule-C+(JAM-C)+in+human+retinal+pigment+epithelium.">Expression, localization, and function of junctional adhesion molecule-C (JAM-C) in human retinal pigment epithelium.</a> Invest Ophthalmol Vis Sci. 50 (3), 1454-1463 (2008).</li><li>Li, R., Maminishkis, A., Banzon, T., Wan, Q., Jalickee, S., Chen, S., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFN{gamma}+Regulates+Retinal+Pigment+Epithelial+Fluid+Transport.">IFN{gamma} Regulates Retinal Pigment Epithelial Fluid Transport.</a> Am J Physiol Cell Physiol. 297, C1452-C1465 (2009).</li><li>Li, R., Maminishkis, A., Wang, F. E., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDGF-C+and+-D+induced+proliferation/migration+of+human+RPE+is+abolished+by+inflammatory+cytokines.">PDGF-C and -D induced proliferation/migration of human RPE is abolished by inflammatory cytokines.</a> Invest Ophthalmol Vis Sci. 48, 5722-5732 (2007).</li><li>Maminishkis, A., Jalickee, S., Blaug, S. A., Rymer, J., Yerxa, B. R., Peterson, W. M., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+P2Y(2)+receptor+agonist+INS37217+stimulates+RPE+fluid+transport+in+vitro+and+retinal+reattachment+in+rat.">The P2Y(2) receptor agonist INS37217 stimulates RPE fluid transport in vitro and retinal reattachment in rat.</a> Invest Ophthalmol Vis Sci. 43, 3555-3566 (2002).</li><li>Wang, F. E., Zhang, C., Maminishkis, A., Dong, L., Zhi, C., Li, R., Zhao, J., Majerciak, V., Gaur, A. B., Chen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-204/211+alters+epithelial+physiology.">MicroRNA-204/211 alters epithelial physiology.</a> FASEB J. 24, (2010).</li><li>Adijanto, J., Banzon, T., Jalickee, S., Wang, N. S., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CO2-induced+ion+and+fluid+transport+in+human+retinal+pigment+epithelium.">CO2-induced ion and fluid transport in human retinal pigment epithelium.</a> J Gen Physiol. 133, 603-622 (2009).</li><li>Li, R., Maminishkis, A., Zahn, G., Vossmeyer, D., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+alpha5beta1+mediates+attachment,+migration,+and+proliferation+in+human+retinal+pigment+epithelium:+relevance+for+proliferative+retinal+disease.">Integrin alpha5beta1 mediates attachment, migration, and proliferation in human retinal pigment epithelium: relevance for proliferative retinal disease.</a> Invest Ophthalmol Vis Sci. 50, 5988-5996 (2009).</li><li>Peterson, W. M., Meggyesy, C., Yu, K., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+ATP+activates+calcium+signaling,+ion,+and+fluid+transport+in+retinal+pigment+epithelium.">Extracellular ATP activates calcium signaling, ion, and fluid transport in retinal pigment epithelium.</a> J Neurosci. 17, 2324-2337 (1997).</li><li>Voloboueva, L. A., Liu, J., Suh, J. H., Ames, B. N., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R)-alpha-lipoic+acid+protects+retinal+pigment+epithelial+cells+from+oxidative+damage.">R)-alpha-lipoic acid protects retinal pigment epithelial cells from oxidative damage.</a> Invest Ophthalmol Vis Sci. 46, 4302-4310 (2005).</li></ol>

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.

Instruments needed for dissection protocol
<ol>
<li> Stereo Microscope - any available with working magnification x250 needed for dissection</li>
<li> Dissecting dish - e.g. Kimble 100 x 20mm #23062 (one per dissection) any source</li>
<li> Sylgard 184 - WPI Cat. #SYLG184* 
* 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)</li>
<li> 27 G needles from B-D PrecissionGlide 1&frac14; length (5 per dissection) any source</li>
<li> HBSS 1X Solution containing Ca and Mg salts - GIBCO Cat.#14025 500ml (good for multiple dissections)</li>
<li> Iris scissors carbide serrated blade, curved - FST Cat.# 14559-11 (one)</li>
<li> Retinal scissors - Katena Cat.#K4-5300 (one)</li>
<li> Forceps - e.g. Dumont Tweezers #5 with polished tips from WPI Cat.#500085 (2 pieces)</li>
<li> Sideport knife - Alcon, ClearCut 1mm, Dual Bevel, angled Cat.#8065921540</li>
<li> Centrifuge tube 15ml - (one per eye) any source</li>
<li> Pasteur glass pipette - (one per dissection) any source</li>
<li> Plate 12 well (one per dissection) any source</li>
<li> Primaria 25cm2 flask - (2-3 per eye) any source</li>
</ol>

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 ...

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19.2K Views.  National Institutes of Health. 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.

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

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Olfactory Assays for Mouse Models of Neurodegenerative Disease

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process and may be a useful behavioral marker for early detection. Earlier detection in neurodegenerative disease is a major goal in the field because this is when neuroprotective therapies have the best potential to be effective. Therefore, in preclinical studies testing novel neuroprotective strategies in rodent models of neurodegenerative disease, olfactory assessment could be highly useful in determining therapeutic potential of compounds and translation to the clinic. In the present study we describe a battery of olfactory assays that are useful in measuring olfactory function in mice. The tests presented in this study were chosen because they measure olfaction abilities in mice related to food odors, social odors, and non-social odors. These tests have proven useful in characterizing novel genetic mouse models of Parkinson&#8217;s disease as well as in testing potential disease-modifying therapies.

Impairment in olfactory function is a common feature in many neurodegenerative disorders including Parkinson, Alzheimer, and Huntington diseases. In the present article, we describe a set of tests for assessing olfaction discrimination and detection in mice that can be used to measure olfactory abilities in mouse models of neurodegenerative diseases.

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process ...

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell leukodystrophy (GLD) is unclear. This gap in knowledge has been hindered by the lack of an appropriate in vitro model for study. Herein, we describe a primary murine glial culture system in which treatment with psychosine results in multinucleation of microglia resembling the characteristic globoid cells found in GLD. Using this novel system, we defined the conditions and modes of analysis for study of globoid cells. The potential use of this model system was validated in our previous study, which identified a potential role for matrix metalloproteinase (MMP)-3 in GLD. This novel in vitro system may be a useful model in which to study the formation and function, but also the potential therapeutic manipulation, of these unique cells.

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

Globoid cells are a defining pathological feature of Krabbe disease, a leukodystrophy currently lacking an effective long-term therapy. We have developed a cell culture model to study the innate biology and pathogenic potential of activated microglia and their transformation into globoid cells.

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell ...

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for neuroprotection, postconditioning treatments have proven to be promising therapies against cerebral ischemia. However, complicated procedures and potential safety issues limit their clinical application. To overcome these disadvantages, we have developed acidic postconditioning (APC) as a therapy for experimental focal cerebral ischemia. APC refers to the mild acidosis treatment by inhaling CO2 during reperfusion following ischemia. Here we present two models to execute APC in vitro and in vivo, respectively. The oxygen-glucose deprivation (OGD) treatment of mice and the corticostriatal occlusion and middle cerebral artery occlusion (MCAO) of mice were employed to mimic cerebral ischemia. APC can be simply achieved by transferring brain slices to acidic buffer bubbled with 20% CO2, or by mice inhaling 20% CO2. APC showed significant protective effects against cerebral ischemia, as reflected by tissue viability and brain infarct volume.

Acidic postconditioning protects against cerebral ischemia. Here we present two models to execute APC. They are achieved respectively by transferring corticostriatal slices to acidic buffer after oxygen-glucose deprivation in vitro and by inhaling 20% CO2 after middle cerebral artery occlusion in vivo.

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

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 ...

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

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Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Olfactory Assays for Mouse Models of Neurodegenerative Disease

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

Low-Density Primary Hippocampal Neuron Culture

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

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