The authors would like to thank J&#233;r&#244;me Larghero and Val&#233;rie Vanneaux (H&#244;pital Saint Louis, Paris, France) for their input during the setting-up of the method described here.
This work was supported by grants from the ANR [GPiPS: ANR-2010-RFCS005; SightREPAIR: ANR-16-CE17-008-02], the Fondation pour la Recherche M&#233;dicale [Bio-engineering program - DBS20140930777] and from LABEX REVIVE [ANR-10-LABX-73] to Olivier Goureau and Christelle Monville. It was supported by NeurATRIS, a translational research infrastructure (Investissements d&#39;Avenir) for biotherapies in Neurosciences [ANR-11-INBS-0011] and INGESTEM, the national infrastructure (Investissements d&#39;Avenir) engineering for pluripotent and differentiated stem cells [ANR-11-INBS-000] to Christelle Monville. Karim Ben M&#39;Barek was supported by fellowships from DIM Stempole and LABEX REVIVE [ANR-10-LABX-73]. I-Stem is part of the Biotherapies Institute for Rare Diseases supported by the Association Fran&#231;aise contre les Myopathies (AFM)-T&#233;l&#233;thon.

All human materials used in this protocol were used in accordance with European Union regulations. The human ES cell line used in this study was derived from a unique embryo. The couple who had donated the embryo was fully informed and gave their consent for an anonymous donation. A clinical-grade human ES cell line was derived from this embryo, banked, qualified, and properly documented by Roslin Cells (UK). hAMs were procured under sterile conditions during a cesarean section in mothers who signed an informed consent for placenta donation according to hospital guidelines (APHP, H&#244;pital Saint Louis).
1. Preparation of Culture Media and Reagents
<ol>
	<li>Preparation of the RPE cell culture medium
	<ol>
		<li>To prepare the RPE cell culture medium, add 4% serum substitute (20 mL for a final volume of 500 mL), 1,000x diluted 2-mercaptoethanol (500 &#181;L for a final volume of 500 mL), and 1% Eagle&#8242;s minimum essential medium (MEM) non-essential amino acids solution (5 mL for a final volume of 500 mL) to Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) high glucose (475 mL for a final volume of 500 mL).</li>
	</ol>
	</li>
	<li>Preparation of the transport/conservation medium
	<ol>
		<li>To prepare the transport/conservation medium, add 1% of penicillin-streptomycin (5 mL for a final volume of 500 mL) to CO2-independent medium (495 mL for a final volume of 500 mL) and keep it at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Resuspension of the thermolysin enzyme
	<ol>
		<li>Preparation of a stock solution of thermolysin
		<ol>
			<li>To prepare a stock solution of thermolysin, thaw the thermolysin powder, which was stored at -20 &#176;C, at room temperature.</li>
			<li>As the enzymatic activity may be variable from batch to batch, calculate this activity based on the certificate of analysis provided with the thermolysin powder of the batch to be used. Divide the &#34;Activity Neutral Protease calculated = ANPC&#34; (indicated in the Certificate of Analysis) by 181 (which is tyrosine&#39;s molecular weight). The result obtained corresponds to the total enzymatic activity of the supplied thermolysin powder (U/vial).</li>
			<li>Prepare a stock solution at 200 U/mL by adding the volume of water corresponding to the total enzymatic activity (U/vial) divided by 200 (U/mL).</li>
			<li>Dissolve the enzyme by pipetting the water that was added up and down. Vortex the solution for 30 s and check whether the powder has completely been suspended. More pipetting may be required for a full dissolution.</li>
			<li>Aliquot the stock solution and store it at -20 &#176;C.</li>
		</ol>
		</li>
		<li>Preparation of a working solution of thermolysin 
		NOTE: The thermolysin solution must be prepared on the day of the hAM treatment.
		<ol>
			<li>Thaw the stock aliquots of thermolysin at 200 U/mL stored at -20 &#176;C. Dilute the stock solution 200x in phosphate-buffered saline (PBS) in order to obtain a final enzymatic activity of 1 U/mL. Prepare 40 mL to treat 1&#8211;4 hAM patches.</li>
			<li>Filter the thermolysin solution through a 0.2 &#181;m filter prior to use.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Resuspension of gelatin
	<ol>
		<li>Preparation of 20% gelatin solution and block 
		NOTE: The 20% gelatin solution and block may be prepared up to 1 week before use.
		<ol>
			<li>Warm the CO2-independent medium to 42 &#176;C in a water bath for 30 min (up to 1 h). In a 50 mL tube, add 10 g of gelatin to 40 mL of warmed CO2-independent medium and vortex the solution.</li>
			<li>Dissolve the gelatin for 30&#8211;60 min at 42 &#176;C. Vortex each 10 min to homogenize the solution.</li>
			<li>Once the solution is homogeneous, add 4 mL of the 20% gelatin solution to four 6 cm culture dishes. Avoid bubbles. Add a plastic paraffin film to protect the dishes, and allow the solution to solidify at 4 &#176;C.</li>
		</ol>
		</li>
		<li>Preparation of 8% gelatin solution 
		NOTE: The 8% gelatin solution may be prepared the day before use and stored at 4 &#176;C.
		<ol>
			<li>Warm the CO2-independent medium to 42 &#176;C for 30 min (up to 1 h). In a 50 mL tube, add 4 g of gelatin to 46 mL of warmed CO2-independent medium and vortex the solution.</li>
			<li>Dissolve the gelatin for 30&#8211;60 min at 42 &#176;C. Add a plastic paraffin film to protect the tube and keep it in a water bath at 37 &#176;C if it is to be used the same day, or store it at 4 &#176;C if it is to be used later.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Thermolysin Treatment of Human Amniotic Membranes
<ol>
	<li>Human amniotic membrane wash
	<ol>
		<li>Have hAMs supplied as small pieces (approximately 30 mm x 30 mm) fixed in a nylon scaffold, either already at 4 &#176;C in PBS or frozen. If provided frozen, thaw the membranes at 37 &#176;C in an incubator for 30 min.</li>
		<li>Wash the membranes:
		<ol>
			<li>Place 1&#8211;4 membranes in a 250 mL bottle containing 80 mL of PBS. Use as many bottles as required.</li>
			<li>Shake each bottle in a plate shaker at a high speed for 5 min, then discard the PBS. Add 80 mL of PBS and repeat.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Thermolysin treatment
	<ol>
		<li>Add 40 mL of the working solution of thermolysin (1 U/mL) per bottle containing the membranes (1&#8211;4 membranes per bottles; up to 3 bottles at a time).</li>
		<li>Shake the bottles for 5 min at 450 rpm and then vortex them for 30 s. Repeat 1x.</li>
		<li>Discard the thermolysin solution and add 80 mL of PBS.</li>
		<li>Shake the bottles for 5 min at 450 rpm. Discard the PBS and add 80 mL of PBS. Repeat 3x.</li>
	</ol>
	</li>
</ol>
3. Fixation of Human Amniotic Membranes on a Culture Insert
<ol>
	<li>Use long sterile forceps (that can reach the bottom of the bottle) to remove the hAMs one by one, and transfer each hAM to a 10 cm culture dish containing 10 mL of PBS.</li>
	<li>In a new 10 cm culture dish containing 10 mL of PBS, place one of the membranes with the nylon facing down. Detach two of the four clips that are used to fix the membrane to nylon.</li>
	<li>Insert the smaller ring of the culture insert between the nylon and the membrane.</li>
	<li>Make sure that the membrane covers the smaller ring completely. Clip the second part of the culture insert on top of the membrane. The basement membrane of the hAM is facing up inside the culture insert. Detach the last two clips.</li>
	<li>Cut, with sterile scissors, the excess of membrane outside the culture insert if necessary. Using forceps, transfer the membrane fixed in the culture insert to a 12-well plate, add PBS, and store the plate in an incubator at 37 &#176;C and 5% CO2.</li>
	<li>Repeat these operations (from step 3.2 to 3.5) with the other membranes.</li>
</ol>
4. Thawing and Seeding of Retinal Pigment Epithelium Cells on Human Amniotic Membranes
<ol>
	<li>Thawing of retinal pigment epithelium cells
	<ol>
		<li>The RPE cell bank is frozen at 1 million cells per cryogenic vial in liquid nitrogen. Thaw as many cryogenic vials as required (350,000 cells are seeded per membrane).</li>
		<li>Prepare a 15 mL tube containing 3 mL of RPE medium per cryogenic vial. Once the vial is thawed, transfer the cells to each tube. Repeat this operation for the other vials.</li>
		<li>Homogenize (pipette up and down a few times to resuspend the cells in the RPE medium) and take 10 &#181;L of the cell solution and transfer it to a 1.5 mL tube. Add 10 &#181;L of trypan blue. Place 15 &#181;L of this solution in a counting chamber, then count the number of viable cells (not colored in blue) under a microscope with a 4X objective. Repeat this operation for the other vials.</li>
		<li>In the meantime, centrifuge the 15 mL tubes at 110 x g for 5 min. Discard the supernatant and resuspend the cells at 700,000 cells/mL.</li>
	</ol>
	</li>
	<li>Seeding of retinal pigment epithelium cells into human amniotic membranes
	<ol>
		<li>Remove the 12-well plate containing the membranes from the incubator.</li>
		<li>Aspirate the PBS. Add 500 &#181;L of the cell suspension prepared in step 4.1.4 in the center of the culture insert. Add 1 mL of RPE medium outside the culture insert (inside the well). Repeat for the other membranes.</li>
		<li>Place the 12-well plate containing the membranes in the incubator.</li>
	</ol>
	</li>
</ol>
5. Maintenance of Retinal Pigment Epithelium Cell Cultures on Human Amniotic Membranes
<ol>
	<li>Renew the medium 2x per week, usually on Monday and Friday, until at least day 30 of the culture.</li>
	<li>Aspirate the medium inside and outside the culture insert for each well and renew it with fresh medium (1 mL outside the culture insert and 500 &#181;L inside). Place the plate in the incubator at 37 &#176;C and 5% CO2.</li>
</ol>
6. Preparation of the Retinal Pigment Epithelium Patch for Transplantation
NOTE: Starting at day 30 of the culture, the tissue is ready for transplantation.
<ol>
	<li>Warm the 8% gelatin solution in a water bath at 37 &#176;C for 30 min. Fill the internal chamber of the vibratome with cold CO2-independent medium (at 4 &#176;C).</li>
	<li>Place the 20% gelatin block into the vibratome.
	<ol>
		<li>Take the 6 cm culture dish containing the block of 20% gelatin from the fridge. Using a scalpel, cut out a 5 cm x 5 cm block. Paste the top side of the block to the support with cyanoacrylate glue or its equivalent.</li>
		<li>Place a new blade in the vibratome.</li>
		<li>Adjust the level of the block until it is at the same level of the razor blade.</li>
		<li>Fill the bath around the support with fresh CO2-independent medium at 4 &#176;C. Cut the block with the vibratome using a medium velocity until the block is cut uniformly, leading to a smooth surface. Set the position 0.</li>
		<li>Aspirate the medium around the block of gelatin until it is completely dry, and then take the 12-well culture plate containing the tissues from the incubator at 37 &#176;C.</li>
		<li>Using forceps, carefully open the culture insert on top of the gelatin block. Cut, with the scissors, all parts of the membranes that do not contain cells (outside the ring where cells were not cultured). Aspirate the entire residual medium.</li>
		<li>Add 1 mL of the liquid 8% gelatin solution at 37 &#176;C in order to cover the membranes. Carefully remove the excess. Wait 5&#8211;8 min to allow the gelatin to solidify.</li>
		<li>Add fresh CO2-independent medium (at 4 &#176;C) to the bath until the membrane is covered.</li>
		<li>Cut, with the vibratome at a position of -100 &#181;m, with a medium velocity, remaining aware of how the block behaves. At the end of the section, be careful to maintain the orientation of the tissue.</li>
		<li>With a scalpel, cut a corner to identify the orientation of the section.</li>
		<li>Collect the membrane embedded in gelatin with a spatula and place it in a 6-well plate filled with the conservation medium at 4 &#176;C, until grafting it into the recipient eye.</li>
		<li>At the time of the transplantation, adjust the size of the implant to the size of the recipient eye under a surgical microscope (1&#8211;3 for rats, 10&#8211;15 mm2 for non-human primates).</li>
	</ol>
	</li>
</ol>

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U., Heimann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descemet's+membrane+as+membranous+support+in+RPE/IPE+transplantation.">Descemet's membrane as membranous support in RPE/IPE transplantation.</a> Current Eye Research. 16 (12), 1236-1238 (1997).</li><li>Kiilgaard, J. F., Scherfig, E., Prause, J. U., la Cour, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+amniotic+membrane+to+the+subretinal+space+in+pigs.">Transplantation of amniotic membrane to the subretinal space in pigs.</a> Stem Cells International. 2012, 716968 (2012).</li><li>Capeans, C., 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=Amniotic+membrane+as+support+for+human+retinal+pigment+epithelium+(RPE)+cell+growth.">Amniotic membrane as support for human retinal pigment epithelium (RPE) cell growth.</a> Acta Ophthalmologica Scandinavica. 81 (3), 271-277 (2003).</li><li>Ohno-Matsui, 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=The+effects+of+amniotic+membrane+on+retinal+pigment+epithelial+cell+differentiation.">The effects of amniotic membrane on retinal pigment epithelial cell differentiation.</a> Molecular Vision. 11, 1-10 (2005).</li><li>Paolin, A., 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=Amniotic+membranes+in+ophthalmology:+long+term+data+on+transplantation+outcomes.">Amniotic membranes in ophthalmology: long term data on transplantation outcomes.</a> Cell and Tissue Banking. 17 (1), 51-58 (2016).</li><li>Hu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+for+subretinal+implantation+of+ultrathin+substrates+containing+stem+cell-derived+retinal+pigment+epithelium+monolayer.">A novel approach for subretinal implantation of ultrathin substrates containing stem cell-derived retinal pigment epithelium monolayer.</a> Ophthalmic Research. 48 (4), 186-191 (2012).</li><li>Pennington, B. O., Clegg, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+stem+cell-based+therapies+in+combination+with+substrate+for+the+treatment+of+age-related+macular+degeneration.">Pluripotent stem cell-based therapies in combination with substrate for the treatment of age-related macular degeneration.</a> Journal of Ocular Pharmacology and Therapeutics: The Official Journal of the Association. 32 (5), 261-271 (2016).</li><li>Song, M. J., Bharti, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+into+the+future:+Using+induced+pluripotent+stem+cells+to+build+two+and+three+dimensional+ocular+tissue+for+cell+therapy+and+disease+modeling.">Looking into the future: Using induced pluripotent stem cells to build two and three dimensional ocular tissue for cell therapy and disease modeling.</a> Brain Research. 1638 (Pt A), 2-14 (2016).</li><li>Ramsden, C. M., 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=Stem+cells+in+retinal+regeneration:+Past,+present+and+future).">Stem cells in retinal regeneration: Past, present and future).</a> Development. 140 (12), 2576-2585 (2013).</li><li>da Cruz, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+1+clinical+study+of+an+embryonic+stem+cell-derived+retinal+pigment+epithelium+patch+in+age-related+macular+degeneration.">Phase 1 clinical study of an embryonic stem cell-derived retinal pigment epithelium patch in age-related macular degeneration.</a> Nature Biotechnology. 36 (4), 328-337 (2018).</li><li>Kashani, A. H., 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=A+bioengineered+retinal+pigment+epithelial+monolayer+for+advanced,+dry+age-related+macular+degeneration.">A bioengineered retinal pigment epithelial monolayer for advanced, dry age-related macular degeneration.</a> Science Translational Medicine. 10 (435), (2018).</li><li>Binder, S., Stanzel, B. V., Krebs, I., Glittenberg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+the+RPE+in+AMD.">Transplantation of the RPE in AMD.</a> Progress in Retinal and Eye Research. 26 (5), 516-554 (2007).</li><li>Dunn, K. C., Aotaki-Keen, A. E., Putkey, F. R., Hjelmeland, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ARPE-19,+a+human+retinal+pigment+epithelial+cell+line+with+differentiated+properties.">ARPE-19, a human retinal pigment epithelial cell line with differentiated properties.</a> Experimental Eye Research. 62 (2), 155-169 (1996).</li><li>Salero, E., 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=Adult+human+RPE+can+be+activated+into+a+multipotent+stem+cell+that+produces+mesenchymal+derivatives.">Adult human RPE can be activated into a multipotent stem cell that produces mesenchymal derivatives.</a> Cell Stem Cell. 10 (1), 88-95 (2012).</li></ol>

Olivier Goureau is the inventor on pending patents related to the generation of retinal cells from human pluripotent stem cells. The other authors have nothing to disclose.

We described a method for the culture of RPE cells on a biological scaffold and its preparation for implantation in animal models. One of the critical steps of the protocol is the maintenance of the orientation of the hAM all along the procedure until its inclusion into gelatin. Indeed, the native epithelium of the membrane is removed and its basement membrane becomes exposed9. The RPE cells have to be seeded on top of this basement membrane. Upon preparation for gelatin embedding, it is crucial t...

RPE is crucial for the survival and homeostasis of the photoreceptors with which it is tightly associated1. Several pathological conditions alter its functionality and/or survival, including RP and AMD.
RP is a group of inherited monogenic mutations that affect the functions of photoreceptors or RPE cells or both2,3. It is estimated that mutations that affect specifically the RPE cells account for 5% of RP2. AMD is another condition where the RPE layer is altered, leading ultimately to central vision loss. AMD is caused by the complex interactions of genetic and environmental factors and affects the elderly4,5,6. According to projections, AMD will be a concern for 196 million patients worldwide by 20207. For these disorders, no effective cure exists, and one of the strategies proposed is the transplantation of new RPE cells in order to compensate for dead/nonfunctional preexisting RPE cells8.
The mode of formulation of the final product to be grafted is essential to ensure the best functional outcomes. RPE cells injected as a cell suspension, despite being an easy and straightforward method of delivery, raise concerns regarding their survival, integration, and functionality9,10,11,12,13. Scientists are now developing more complex formulations to deliver engineered retinal tissue9,13,14,15,16. In this context, we developed an original method to generate in vitro RPE tissue that could be used for transplantation9.
RPE cell banks derived from human embryonic stem (ES) cells are used in this protocol. However, alternative RPE cell banks from different cell sources (human-induced pluripotent stem cells, primary RPE cells, etc.) and differentiated with a different method are also suitable for this protocol. It includes directed differentiation protocols using cytokines and/or small molecules17,18,19,20,21,22.
To be transplanted, the engineered tissue should be prepared on a scaffold. In the past few years, different scaffolds were developed based on a polymer or on a matrix of biological origin13,23,24. Here, the biological substrate used is the hAM, but other substrates, like denuded Bruch membranes, could be implemented. The method described herein has the advantage of using a biological scaffold that is more relevant to the RPE native environment.
Human ES cell-derived RPE cells are cultured for at least 4 weeks in order to be fully organized as a cobblestone monolayer. At that stage, the epithelium obtained is functional and polarized9. Finally, as this tissue wrinkles easily, it is embedded in a thin layer of a hydrogel carrier to give it more rigidity and elasticity and to protect it during the injection procedure. This product is then stored at 4 &#176;C until grafting.

<table><tbody><tr><td>Sterile biosafety cabinet</td><td>TechGen International</td><td>Not applicable</td><td></td></tr><tr><td>Liquid waste disposal system for aspiration</td><td>Vacuubrand</td><td>BVC 21</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt;-controlled +37&amp;nbsp;&amp;deg;C cell incubator</td><td>Thermo Electron Corporation</td><td>BVC 21 NT</td><td></td></tr><tr><td>200&amp;nbsp;&amp;micro;L pipette: P200</td><td>Gilson</td><td>F144565</td><td></td></tr><tr><td>1&amp;nbsp;mL pipette: P1000</td><td>Gilson</td><td>F144566</td><td></td></tr><tr><td>Pipet aid</td><td>Drummond</td><td>75001</td><td></td></tr><tr><td>+4&amp;nbsp;&amp;deg;C refrigerator</td><td>Liebherr</td><td>Not applicable</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000S</td><td></td></tr><tr><td>Fine scissors</td><td>WPI</td><td>501758</td><td></td></tr><tr><td>Forceps (x2)</td><td>WPI</td><td>555227F</td><td></td></tr><tr><td>Water bath</td><td>Grant subaqua pro</td><td>SUB6</td><td></td></tr><tr><td>Precision balance</td><td>Sartorius</td><td>CP225D</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorff</td><td>5804</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>SC30</td><td></td></tr><tr><td>Horizontal Rocking Shaker</td><td>IKA-WERKE</td><td>IKA MTS 214D</td><td></td></tr><tr><td>Vortex</td><td>VWR</td><td>LAB DANCER S40</td><td></td></tr><tr><td>Disposable Scalpel</td><td>WPI</td><td>500351</td><td></td></tr><tr><td>plastic paraffin film</td><td>VWR</td><td>PM992</td><td></td></tr><tr><td>0.200 &amp;micro;m single use syringe filter</td><td>SARTORIUS</td><td>16532</td><td></td></tr><tr><td>Syringe without needle 50 mL</td><td>Dutscher</td><td>50012</td><td></td></tr><tr><td>Bottles 250 mL</td><td>Dutscher</td><td>28024</td><td></td></tr><tr><td>15 mL sterile Falcon tubes</td><td>Dutscher</td><td>352097</td><td></td></tr><tr><td>50 mL sterile Falcon tubes</td><td>Dutscher</td><td>352098</td><td></td></tr><tr><td>culture insert</td><td>Scaffdex</td><td>C00001N</td><td></td></tr><tr><td>60&amp;nbsp;mm cell culture disches: B6</td><td>Dutscher</td><td>353004</td><td></td></tr><tr><td>12 well cell culture plate</td><td>Corning</td><td>3512</td><td></td></tr><tr><td>6-well culture plates</td><td>Corning</td><td>3506</td><td></td></tr><tr><td>Razor blades</td><td>Ted Pella, Inc</td><td>121-9</td><td></td></tr><tr><td>Cyanoacrylate glue</td><td>Castorama</td><td>3178040670105</td><td></td></tr><tr><td>PBS 1x (500&amp;nbsp;mL)</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>Thermolysine</td><td>Roche</td><td>5339880001</td><td></td></tr><tr><td>DMEM, high glucose, GlutaMAX</td><td>Invitrogen</td><td>61965-026</td><td></td></tr><tr><td>KSR CTS (KnockOut SR XenoFree CTS)</td><td>Invitrogen</td><td>12618-013</td><td></td></tr><tr><td>MEM-NEAA (100x)</td><td>Invitrogen</td><td>11140-035</td><td></td></tr><tr><td>b-mercaptoethanol (50&amp;nbsp;mM)</td><td>Invitrogen</td><td>31350-010</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140122</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt;-independent medium</td><td>GIBCO</td><td>18045-054</td><td></td></tr><tr><td>Gelatin</td><td>MERCK</td><td>104078</td><td></td></tr><tr><td>human amniotic membrane</td><td>Tissue bank St Louis hospital (Paris, France)</td><td>Not applicable</td><td></td></tr></tbody></table>

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.

hAMs contain an epithelial layer that should be removed before the seeding of RPE cells. An enzymatic treatment of the membrane is performed with the thermolysin under shaking. In order not to not lose the polarity of the membrane (the epithelium is on one side), it is fixed on a support which composition could be different depending on the provider (Figure 1A). Check the adhesion of the membrane to its support at this step and add clips if necessary. At the ...

Watch this Scientific Journal Video about Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells at JoVE.com

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

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 ...

engineering-transplantation-suitable-retinal-pigment-epithelium

Research

JoVE Journal

Developmental Biology

7.6K Views.  Institut National de la Santé et de la Recherche Médicale (INSERM). Beschrijven we een methode om ingenieur een retinale weefsel samengesteld van retinale pigment epitheliale cellen afgeleid van menselijke pluripotente stamcellen gekweekt op de top van menselijke vruchtwater membranen en de voorbereiding voor de transplantatie in diermodellen.

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

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 ...

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 ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Medicine

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 ...

Neuroscience

Subretinal Implantation of RPE on a Carrier in Minipigs: Guidelines for Preoperative Preparations, Surgical Techniques, and Postoperative Care

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial (RPE) layer, lead to a progressive disorganization of the retinal anatomy and the deterioration of visual function. The substitution of damaged RPE cells (RPEs) with in vitro cultured RPE cells using a subretinal cell carrier has shown potential for re-establishing the anatomical structure of the outer retinal layers and is, therefore, being further studied. Here, we present the principles of a surgical technique that allows for the effective subretinal transplantation of a cell carrier with cultivated RPEs into minipigs. The surgeries were performed under general anesthesia and included a standard lens-sparing three-port pars plana vitrectomy (PPV), subretinal application of a balanced salt solution (BSS), a 2.7 mm retinotomy, implantation of a nanofibrous cell carrier into the subretinal space through an additional 3.0 mm sclerotomy, fluid-air exchange (FAX), silicone oil tamponade, and closure of all the sclerotomies. This surgical approach was used in 29 surgeries (18 animals) over the past 8 years with a success rate of 93.1%. Anatomic verification of the surgical placement was carried out using in vivo fundus imaging (fundus photography and optical coherence tomography). The recommended surgical steps for the subretinal implantation of RPEs on a carrier in minipig eyes can be used in future preclinical studies using large-eye animal models.

The subretinal implantation of retinal pigmented epithelium (RPE) is one of the most promising approaches for the treatment of degenerative retinal diseases. However, the performance of preclinical studies on large-eye animal models remains challenging. This report presents guidelines for the subretinal transplantation of RPE on a cell carrier into minipigs.

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial ...

Bioengineering

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...

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

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

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

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

Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. Subretinal transplantation of therapeutic donor cells into mouse recipients is challenging due to the limited surgical space allowed by the small volume of the mouse eye. We developed a trans-scleral surgical transplantation platform with direct transpupillary vision guidance to facilitate the subretinal delivery of exogenous cells in mouse recipients. The platform was tested using retinal cell suspensions and three-dimensional retinal sheets collected from rod-rich Rho::EGFP mice and cone-rich OPN1LW-EGFP;NRL-/- mice, respectively. Live/dead assay showed low cell mortality for both forms of donor cells. Retinal grafts were successfully delivered into the subretinal space of a mouse model of retinal degeneration, Rd1/NS, with minimum surgical complications as detected by multimodal confocal scanning laser ophthalmoscope (cSLO) imaging. Two months post-transplantation, histological staining demonstrated evidence of advanced maturation of the retinal grafts into &#39;adult&#39; rods and cones (by robust Rho::EGFP, S-opsin, and OPN1LW:EGFP expression, respectively) in the subretinal space. Here, we provide a surgical platform that can enable highly accurate subretinal delivery with a low rate of complications in mouse recipients. This technique offers precision and relative ease of skill acquisition. Furthermore, the technique could be used not only for studies of subretinal cell transplantation but also for other intraocular therapeutic studies including gene therapies.

This protocol presents a transpupillary vision-guided trans-scleral approach to safely and precisely deliver subretinal cellular grafts, with a low rate of surgical complications, in mouse recipients with or without retinal degeneration.

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

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

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