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>

<ol>
	<li>Murdaugh, L. S., Wang, Z., Del Priore, L. V., Dillon, J., Gaillard, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+accumulation+of+3-nitrotyrosine+and+nitro-A2E+in+human+Bruch's+membrane.">Age-related accumulation of 3-nitrotyrosine and nitro-A2E in human Bruch's membrane.</a> Exp. Eye Res. 90 (5), 564-571 (2010).</li><li>Wiradjaja, F., DiTommaso, T., Smyth, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basement+membranes+in+development+and+disease.">Basement membranes in development and disease.</a> Birth Defects Res C Embryo Today. 90 (1), 8-31 (2010).</li><li>Nystrom, A., Bornert, O., Kuhl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+therapy+for+basement+membrane-linked+diseases.">Cell therapy for basement membrane-linked diseases.</a> Matrix Biol. , (2016).</li><li>Pauleikhoff, D., Harper, C. A., Marshall, J., Bird, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+changes+in+Bruch's+membrane.+A+histochemical+and+morphologic+study.">Aging changes in Bruch's membrane. A histochemical and morphologic study.</a> Ophthalmology. 97 (2), 171-178 (1990).</li><li>Marshall, G. E., Konstas, A. G., Reid, G. G., Edwards, J. G., Lee, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+IV+collagen+and+laminin+in+Bruch's+membrane+and+basal+linear+deposit+in+the+human+macula.">Type IV collagen and laminin in Bruch's membrane and basal linear deposit in the human macula.</a> Brit. J. Ophthalmol. 76 (10), 607-614 (1992).</li><li>Sarks, S. H., Arnold, J. J., Killingsworth, M. C., Sarks, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+drusen+formation+in+the+normal+and+aging+eye+and+their+relation+to+age+related+maculopathy:+a+clinicopathological+study.">Early drusen formation in the normal and aging eye and their relation to age related maculopathy: a clinicopathological study.</a> Brit. J. Ophthalmol. 83 (3), 358-368 (1999).</li><li>Spraul, C. W., Lang, G. E., Grossniklaus, H. E., Lang, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histologic+and+morphometric+analysis+of+the+choroid,+Bruch's+membrane,+and+retinal+pigment+epithelium+in+postmortem+eyes+with+age-related+macular+degeneration+and+histologic+examination+of+surgically+excised+choroidal+neovascular+membranes.">Histologic and morphometric analysis of the choroid, Bruch's membrane, and retinal pigment epithelium in postmortem eyes with age-related macular degeneration and histologic examination of surgically excised choroidal neovascular membranes.</a> Surv. Ophthalmol. 44 (Suppl 1), S10-S32 (1999).</li><li>Abdelsalam, A., Del Priore, L., Zarbin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drusen+in+age-related+macular+degeneration:+pathogenesis,+natural+course,+and+laser+photocoagulation-induced+regression.">Drusen in age-related macular degeneration: pathogenesis, natural course, and laser photocoagulation-induced regression.</a> Surv. Ophthalmol. 44 (1), 1-29 (1999).</li><li>Mullins, R. F., Aptsiauri, N., Hageman, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+composition+of+drusen+associated+with+glomerulonephritis:+implications+for+the+role+of+complement+activation+in+drusen+biogenesis.">Structure and composition of drusen associated with glomerulonephritis: implications for the role of complement activation in drusen biogenesis.</a> Eye. 15 (Pt 3), 390-395 (2001).</li><li>Caldwell, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+alterations+precede+vascularization+of+the+retinal+pigment+epithelium+in+dystrophic+rats.">Extracellular matrix alterations precede vascularization of the retinal pigment epithelium in dystrophic rats.</a> Curr. Eye Res. 8 (9), 907-921 (1989).</li><li>Pauleikhoff, D., Harper, C. A., Marshall, J., Bird, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+changes+in+Bruch's+membrane.+A+histochemical+and+morphologic+study.">Aging changes in Bruch's membrane. A histochemical and morphologic study.</a> Ophthalmology. 97 (2), 171-178 (1990).</li><li>Abdelsalam, A., Del Priore, L., Zarbin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drusen+in+age-related+macular+degeneration:+pathogenesis,+natural+course,+and+laser+photocoagulation-induced+regression.">Drusen in age-related macular degeneration: pathogenesis, natural course, and laser photocoagulation-induced regression.</a> Surv. Ophthalmol. 44 (1), 1-29 (1999).</li><li>Spraul, C. W., Lang, G. E., Grossniklaus, H. E., Lang, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histologic+and+morphometric+analysis+of+the+choroid,+Bruch's+membrane,+and+retinal+pigment+epithelium+in+postmortem+eyes+with+age-related+macular+degeneration+and+histologic+examination+of+surgically+excised+choroidal+neovascular+membranes.">Histologic and morphometric analysis of the choroid, Bruch's membrane, and retinal pigment epithelium in postmortem eyes with age-related macular degeneration and histologic examination of surgically excised choroidal neovascular membranes.</a> Surv Ophthalmol. 44, S10-S32 (1999).</li><li>Del Priore, L. V., Tezel, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reattachment+rate+of+human+retinal+pigment+epithelium+to+layers+of+human+Bruch's+membrane.">Reattachment rate of human retinal pigment epithelium to layers of human Bruch's membrane.</a> Arch. Ophthalmol. 116 (3), 335-341 (1998).</li><li>Ho, T. C., Del Priore, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reattachment+of+cultured+human+retinal+pigment+epithelium+to+extracellular+matrix+and+human+Bruch's+membrane.">Reattachment of cultured human retinal pigment epithelium to extracellular matrix and human Bruch's membrane.</a> Invest. Ophth. Vis. Sci. 38 (6), 1110-1118 (1997).</li><li>Tezel, T. H., Del Priore, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reattachment+to+a+substrate+prevents+apoptosis+of+human+retinal+pigment+epithelium.">Reattachment to a substrate prevents apoptosis of human retinal pigment epithelium.</a> Graefes Arch. Clin. Exp. Ophthalmol. 235 (1), 41-47 (1997).</li><li>Tezel, T. H., Del Priore, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF+beta+secretion+modulates+the+density-dependent+growth+of+pig+retinal+pigment+epithelium+in+vitro.">TGF beta secretion modulates the density-dependent growth of pig retinal pigment epithelium in vitro.</a> Ophthalmic Res. 31 (3), 192-202 (1999).</li><li>Gullapalli, V. K., Sugino, I. K., Van Patten, Y., Shah, S., Zarbin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+RPE+survival+on+aged+submacular+human+Bruch's+membrane.">Impaired RPE survival on aged submacular human Bruch's membrane.</a> Exp. Eye Res. 80 (2), 235-248 (2005).</li><li>Wang, H., Yagi, F., Cheewatrakoolpong, N., Sugino, I. K., Zarbin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+study+of+retinal+pigment+epithelium+sheet+transplants+onto+Bruch's+membrane.">Short-term study of retinal pigment epithelium sheet transplants onto Bruch's membrane.</a> Exp. Eye Res. 78 (1), 53-65 (2004).</li><li>Tezel, T. H., Del Priore, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repopulation+of+different+layers+of+host+human+Bruch's+membrane+by+retinal+pigment+epithelial+cell+grafts.">Repopulation of different layers of host human Bruch's membrane by retinal pigment epithelial cell grafts.</a> Invest. Ophth. Vis. Sci. 40 (3), 767-774 (1999).</li><li>Tezel, T. H., Del Priore, L. V., Kaplan, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reengineering+of+aged+Bruch's+membrane+to+enhance+retinal+pigment+epithelium+repopulation.">Reengineering of aged Bruch's membrane to enhance retinal pigment epithelium repopulation.</a> Invest. Ophth. Vis. Sci. 45 (9), 3337-3348 (2004).</li><li>Castellarin, A. A., Sugino, I. K., Vargas, J. A., Parolini, B., Lui, G. M., Zarbin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+transplantation+of+fetal+human+retinal+pigment+epithelial+cells+onto+human+cadaver+Bruch's+membrane.">In vitro transplantation of fetal human retinal pigment epithelial cells onto human cadaver Bruch's membrane.</a> Exp. Eye Res. 66 (1), 49-67 (1998).</li><li>Nguyen, H. V., Li, Y., Tsang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-specific+iPSC-derived+RPE+for+modeling+of+retinal+diseases.">Patient-specific iPSC-derived RPE for modeling of retinal diseases.</a> J. Clin. Med. 4 (4), 567-578 (2015).</li><li>Fletcher, E. L., Jobling, A. I., Greferath, U., Mills, S. A., Waugh, M., Ho, T., De Longh, R. U., Phipps, J. A., Vessey, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+age-related+macular+degeneration+using+animal+models.">Studying age-related macular degeneration using animal models.</a> Optometry Vision Sci. 91 (8), 878-886 (2014).</li><li>Del Priore, L. V., Tezel, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reattachment+rate+of+human+retinal+pigment+epithelium+to+layers+of+human+Bruch's+membrane.">Reattachment rate of human retinal pigment epithelium to layers of human Bruch's membrane.</a> Arch. Ophthalmol. 116 (3), 335-341 (1998).</li><li>Tezel, T. H., Kaplan, H. J., Del Priore, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+human+retinal+pigment+epithelial+cells+seeded+onto+layers+of+human+Bruch's+membrane.">Fate of human retinal pigment epithelial cells seeded onto layers of human Bruch's membrane.</a> Invest. Ophth. Vis. Sci. 40 (2), 467-476 (1999).</li><li>Cai, H., Del Priore, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bruch+membrane+aging+alters+the+gene+expression+profile+of+human+retinal+pigment+epithelium.">Bruch membrane aging alters the gene expression profile of human retinal pigment epithelium.</a> Curr. Eye Res. 31 (2), 181-189 (2006).</li><li>Moreira, E. F., Cai, H., Tezel, T. H., Fields, M. A., Del Priore, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reengineering+human+Bruch's+membrane+increases+rod+outer+segment+phagocytosis+by+human+retinal+pigment+epithelium.">Reengineering human Bruch's membrane increases rod outer segment phagocytosis by human retinal pigment epithelium.</a> Transl. Vis. Sci. Technol. 4 (5), 10 (2015).</li><li>Sun, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bruch's+membrane+aging+decreases+phagocytosis+of+outer+segments+by+retinal+pigment+epithelium.">Bruch's membrane aging decreases phagocytosis of outer segments by retinal pigment epithelium.</a> Mol. Vis. 13, 2310-2319 (2007).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="1219">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:599px;">Human donor globes</td>
			<td style="width:305px;">National Disease Research Interchange, NDRI</td>
			<td style="width:148px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s modified eagle medium (DMEM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carbon-dioxide independent media</td>
			<td>Thermo Fisher Scientific</td>
			<td>18045-088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60-mm polystyrene petri dish</td>
			<td>Corning Inc.</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s phosphate buffered saline, PBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrophobic 65 &#181;m-thick polytetrafluoroethylene membrane with 0.2 &#181;m pores</td>
			<td>Merck Millipore</td>
			<td>JGWP04700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisherbrand glass microanalysis vacuum filter holder system&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>09-753-102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A2576-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 &#215; 10mm culture dish</td>
			<td>VWR International</td>
			<td>25373-041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trephine</td>
			<td>Accutome</td>
			<td>AM0570 60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well plate</td>
			<td>Corning Inc.</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minimum essential media (MEM)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>11095-080&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin G</td>
			<td>Sigma-Aldrich</td>
			<td>P3032-1MU</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>S6501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamicin</td>
			<td>Sigma-Aldrich</td>
			<td>G1914</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amphotericin B</td>
			<td>Sigma-Aldrich</td>
			<td>1397-89-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium hydroxide solution</td>
			<td>Sigma-Aldrich</td>
			<td>1336-21-6 
			&#160;</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 ...

culturing-retinal-pigment-epithelial-cells-on-an-ex-vivo-model-aged

Research

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

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Deriving Retinal Pigment Epithelium (RPE) from Induced Pluripotent Stem (iPS) Cells by Different Sizes of Embryoid Bodies

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of techniques to reprogram somatic cells into induced pluripotent stem (iPS) cells has generated interest and excitement towards the possibility of customized personal regenerative medicine. However, the efficiency of stem cell differentiation towards a desired lineage remains low. The purpose of this study is to describe a protocol to derive retinal pigment epithelium (RPE) from iPS cells (iPS-RPE) by applying a tissue engineering approach to generate homogenous populations of embryoid bodies (EBs), a common intermediate during in vitro differentiation. The protocol applies the formation of specific size of EBs using microwell plate technology. The methods for identifying protein and gene markers of RPE by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) are also explained. Finally, the efficiency of differentiation in different sizes of EBs monitored by fluorescence-activated cell sorting (FACS) analysis of RPE markers is described. These techniques will facilitate the differentiation of iPS cells into RPE for future applications.

Deriving Retinal Pigment Epithelium RPE from Induced Pluripotent Stem iPS Cells by Different Sizes of Embryoid Bodies

The objective of this report is to describe the protocols to derive the retinal pigment epithelium (RPE) from induced pluripotent stem (iPS) cells using different sizes of embryoid bodies.

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of ...

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune cells, and the coordination with lymphatic vessels and nerves. The multi-cell, multi-system interactions necessitate the investigation of angiogenesis in a physiologically relevant environment. Thus, while the use of in vitro cell-culture models have provided mechanistic insights, a common critique is that they do not recapitulate the complexity associated with a microvascular network. The objective of this protocol is to demonstrate the ability to make time-lapse comparisons of intact microvascular networks before and after angiogenesis stimulation in cultured rat mesentery tissues. Cultured tissues contain microvascular networks that maintain their hierarchy. Immunohistochemical labeling confirms the presence of endothelial cells, smooth muscle cells, pericytes, blood vessels and lymphatic vessels. In addition, labeling tissues with BSI-lectin enables time-lapse comparison of local network regions before and after serum or growth factor stimulation characterized by increased capillary sprouting and vessel density. In comparison to common cell culture models, this method provides a tool for endothelial cell lineage studies and tissue specific angiogenic drug evaluation in physiologically relevant microvascular networks.

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis.

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune ...

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the efficacy of cell persistence and biodistribution of haMSCs in animal models. We demonstrated a method to label the cell membrane of haMSCs with lipophilic fluorescent dye. Subsequently, intra-articular injection of the labeled cells in rats with surgically induced KOA was monitored dynamically by an in vivo imaging system. We employed the lipophilic carbocyanines DiD (DilC18 (5)), a far-red fluorescent Dil (dialkylcarbocyanines) analog, which utilized a red laser to avoid excitation of the natural green autofluorescence from surrounding tissues. Furthermore, the red-shifted emission spectra of DiD allowed deep-tissue imaging in live animals and the labeling procedure caused no cytotoxic effects or functional damage to haMSCs. This approach has been shown to be an efficient tracking method for haMSCs in a rat KOA model. The application of this method could also be used to determine the optimal administration route and dosage of MSCs from other sources in pre-clinical studies.

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

This protocol describes an efficient way to monitor the cell persistence and biodistribution of human adipose-derived mesenchymal stem cells (haMSCs) by far-red fluorescence labeling in a rat knee osteoarthritis (KOA) model via intra-articular (IA) injection.

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms of retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Cell therapy is one of the most promising therapeutic strategies proposed to cure these diseases, with already encouraging preliminary results in humans. However, the method of preparation of the graft has a significant impact on its functional outcomes in vivo. Indeed, RPE cells grafted as a cell suspension are less functional than the same cells transplanted as a retinal tissue. Herein, we describe a simple and reproducible method to engineer RPE tissue and its preparation for an in vivo implantation. RPE cells derived from human pluripotent stem cells are seeded on a biological support, the human amniotic membrane (hAM). Compared to artificial scaffolds, this support has the advantage of having a basement membrane that is close to the Bruch&#39;s membrane where endogenous RPE cells are attached. However, its manipulation is not easy, and we developed several strategies for its proper culturing and preparation for grafting in vivo.

We describe a method to engineer a retinal tissue composed of retinal pigment epithelial cells derived from human pluripotent stem cells cultured on top of human amniotic membranes and its preparation for grafting in animal models.

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...