The Authors would like to acknowledge Dr. Isaac Zambrano and the staff at the Manchester Eye Hospital Eye Bank for the supply of human donor eye tissue, and Mr. Pete Walker of the Faculty of Life Sciences Histology facility for the H&#38;E stained images. Special thanks goes to Mr. Roger Meadows for his help with the microscopy. SJC is a recipient of a Medical Research Council (MRC) Career Development Fellowship (MR/K024418/1) and the authors also acknowledge other recent research funding from MRC (G0900538 and&#160;K004441), Fight for Sight (1866) and The Macular Society.

Human eye tissue consented for research purposes was obtained from the Manchester Royal Eye Hospital Eye Bank following the removal of the cornea for transplantation. The use of human tissue for research was in accordance with the Human Tissue Act (2004) guidelines, and with University Research Ethics Committee approval (reference 11305).
1. Enrichment of Whole Bruch&#8217;s Membrane
<ol>
	<li>Clean the workspace around and underneath the dissecting microscope with 1% disinfectant (v/v) and then wipe down with 70% (v/v) ethanol.</li>
	<li>Ensure the following are to hand: one new disposable scalpel, two sterile cell scrapers (commonly used in tissue culture) and three pairs of tweezers (listed here as A, B, and C, see materials list).</li>
	<li>Place the eye globe into a disposable petri-dish, retaining the lid for later use. Using a new disposable scalpel, remove any residual optic nerve, which may interfere with creating a flat mount of the tissue. Next, make four incisions equally spaced apart to create four quadrants, if proceeding to part 3 (enrichment of macular Bruch&#8217;s membrane) do not cut through the macula region.
	<ol>
		<li>Ensure that the incisions extend through the entire depth of the eye tissue &#8211; from the innermost neurosensory retina (NSR), through the choroid and sclera (white of the eye). Continue the incision all the way round the eye from the margin of the eyecup to the optic nerve. This will allow the eyecup to be flattened out (see Figure 1B).</li>
	</ol>
	</li>
	<li>Holding one quadrant of flattened eyecup using tweezer A (to prevent choroid detachment), use the larger tweezer B to grip the vitreous and underlying NSR and pull this tissue away from the retinal pigment epithelium (RPE) starting at the periphery, moving towards the center of the opened eyecup and then to the opposing side. Usually, but not always both vitreous and NSR detach as one. Use the scalpel to excise any residual NSR anchored at the optic disc.</li>
	<li>Again, whilst holding one quadrant of tissue in place using tweezer A, use the sterile cell scraper to gently dislodge the RPE monolayer from the entire inside surface of the eyecup. These cells readily detach and can be seen as dark brown-pigmented clumps. Rinse with PBS to gently wash away dislodged RPE cells from the eyecup.</li>
	<li>Next, use tweezer A to gently peel off the overlying dark red tissue (comprising both Bruch&#8217;s membrane and the choroid) from all 4 eye quadrants. Place the excised tissue into a clean dish.</li>
	<li>Using the flattened edge of one cell scraper to hold the excised tissue in place, use the second scraper to gently scrape the tissue. Turn the tissue over and repeat, removing as much excess material as possible. Eventually, a translucent membrane will remain: this is the crudely enriched Bruch&#8217;s membrane. The use of the dissecting microscope at a minimum of 20X magnification is essential in aiding the removal of choroid and enrichment of Bruch&#8217;s membrane.</li>
	<li>Using a Pasteur pipette, briefly rinse the Bruch&#8217;s membrane with ultrapure water to remove residual choroid and blood before placing the membrane into a 1.5 ml microcentrifuge tube.</li>
	<li>Suspend the enriched Bruch&#8217;s membrane in approximately 1 ml ultrapure water and vortex briefly (approximately 15 sec) to help lyse any contaminating cellular matter (see Figure 2). Centrifuge the sample at 500 x g for 30 sec&#160;to pellet the membrane and aspirate off the supernatant. 
	Note: The remaining enriched Bruch&#8217;s membrane is now ready for solubilization (as described in section 2 below) or storage at -80 &#176;C for use later.</li>
</ol>
2. Solubilization of Whole Enriched Bruch&#8217;s Membrane and Western Blot Analysis
<ol>
	<li>Extract protein from enriched Bruch&#8217;s membrane by immersing the tissue in 500 &#181;l 8 M urea for 6 hr&#160;at RT&#160;in 1.5 ml microcentrifuge tubes ( Figure 3A).</li>
	<li>Centrifuge solubilized samples at 10,000 x g for 10 min to pellet any remaining extracellular matter. Remove the supernatant and transfer it to 3,500 Da MWCO dialysis membrane, then place the sample in 1 L of PBS to dialyse for 16 hr&#160;at 4 &#176;C.</li>
	<li>For each sample, add 40 &#181;l of protein isolation beads, and rotate sample/bead mix (20 rpm on a rotating mixer) at RT for 30 min.</li>
	<li>Centrifuge samples at 10,000 x g for 5 min to pellet the beads. Remove supernatant and solubilize protein bound to beads in 30 &#181;l 2x sample loading buffer (65.8 mM Tris-HCl, pH 6.8, 2.1% SDS, 26.3% (w/v) glycerol, 0.01% bromophenol blue, 3% &#946;-mercaptoethanol).</li>
	<li>Incubate the samples at 100 &#176;C for 10 min, and then centrifuge at 10,000 x g for 5 min to pellet the remaining beads.</li>
	<li>Load the supernatant that contains the released proteins on to a pre-cast SDS-page gradient gel (see materials list) for protein separation.</li>
	<li>Visualize separated protein bands using a common staining protocol, e.g., Coomassie blue.
	<ol>
		<li>In brief, add 20 ml of the brilliant blue stain (0.1% brilliant blue (w/v), 50% methanol, 10% glacial acetic acid, 40% water) to the gel, and incubate for 30 min at RT&#160;with gentle agitation.</li>
		<li>Discard the staining solution and wash the stained gel with deionized water until background staining is sufficiently reduced to allow stained protein band visualisation.</li>
		<li>Alternatively, transfer separated protein bands to nitrocellulose for western analysis, as described below.</li>
	</ol>
	</li>
	<li>Transfer separated protein bands onto nitrocellulose membrane at 80 mA for 2.5 hr using semidry transfer apparatus in transfer buffer (25 mM Tris, 192 mM glycine, 10% [v/v] methanol).</li>
	<li>Block the membrane in 10 ml PBS, 10% (w/v) milk, and 0.02% (w/v) BSA for 16 hr at 4 &#176;C before the addition of the desired primary antibodies for protein detection. 
	Note: As an example here, Figure 3C shows a representative immunoblot of solubilised Bruch&#8217;s membrane from three separate donors probed for a regulator of the complement cascade, factor H-like protein 1 (FHL-1).</li>
</ol>
3. Enrichment of Macular Bruch&#8217;s Membrane
<ol>
	<li>Follow steps 1.1 - 1.3 as previously described.</li>
	<li>Whilst the NSR is still in place, locate the yellow foveal colouration that represents the central macula region (see Figure 4). Using a sterile 6 mm biopsy punch, place it above the foveal region and firmly cut in to the macula.
	<ol>
		<li>Keeping the biopsy punch in place, make a separate incision with a scalpel from the edge of the eyecup to the edge of biopsy punch blade, and then using tweezer B, peel away the surrounding eye material to achieve a clean biopsy.</li>
	</ol>
	</li>
	<li>Using tweezer C, remove the 6 mm disc of tissue from the punch and place on the clean petri dish lid. Place the petri dish lid and macular punch under the dissecting microscope.</li>
	<li>Remove the thin NSR disc (check for yellow foveal staining to infer accuracy of biopsy). Use a sterile cell scraper to gently remove the RPE cells.</li>
	<li>Secure the macular disc via the sclera using the tips of tweezer A, and slowly peel off the choroid and Bruch&#8217;s membrane complex using tweezer C. Again, use the cell scraper to remove unwanted choroid and blood matter from Bruch&#8217;s membrane.</li>
	<li>Place the macular Bruch&#8217;s membrane into a 1.5 ml microcentrifuge tube and suspend in 1 ml ultrapure water. Vortex briefly (approximately 15 sec) to help lyse any contaminating cellular matter. Centrifuge the sample at 500 x g for 30 sec to pellet the membrane and aspirate off the supernatant. 
	Note: The enriched Bruch&#8217;s membrane isolated from the macula can now be used for proteomic analysis using the user&#8217;s preferred digestion protocols.</li>
</ol>

<ol>
	<li>Coleman, H. R., Chan, C. -. C., Ferris, F. L., Chew, E. Y. <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+macular+degeneration.">Age-related macular degeneration.</a> Lancet. 372, 1835-1845 (2008).</li><li>Wong, W. 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=Global+prevalence+of+age-related+macular+degeneration+and+disease+burden+projection+for+2020+and+2040:+A+systematic+review+and+meta-analysis.">Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: A systematic review and meta-analysis.</a> Lancet Glob Health. 2 (2), e106-e116 (2014).</li><li>Coleman, A. 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=Impact+of+age-related+macular+degeneration+on+vision-specific+quality+of+life:+Follow-up+from+the+10-year+and+15-year+visits+of+the+study+of+osteoporotic+fractures.">Impact of age-related macular degeneration on vision-specific quality of life: Follow-up from the 10-year and 15-year visits of the study of osteoporotic fractures.</a> Am J Ophthalmol. 150 (5), 683-691 (2010).</li><li>Hageman, G. S., 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+common+haplotype+in+the+complement+regulatory+gene+factor+H+(HF1/CFH)+predisposes+individuals+to+age-related+macular+degeneration.">A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration.</a> Proc Natl Acad Sci U S A. 102 (20), 7227-7232 (2005).</li><li>Klein, R. J., 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=Complement+factor+H+polymorphism+in+age-related+macular+degeneration.">Complement factor H polymorphism in age-related macular degeneration.</a> Science. 308 (5720), 385-389 (2005).</li><li>Haines, J. 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=Complement+factor+H+variant+increases+the+risk+of+age-related+macular+degeneration.">Complement factor H variant increases the risk of age-related macular degeneration.</a> Science. 308 (5720), 419-421 (2005).</li><li>Edwards, A. O., Ritter, R., Abel, K. J., Manning, A., Panhuysen, C., Farrer, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement+factor+H+polymorphism+and+age-related+macular+degeneration.">Complement factor H polymorphism and age-related macular degeneration.</a> Science. 308 (5720), 421-424 (2005).</li><li>Fritsche, L. G., 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=Seven+new+loci+associated+with+age-related+macular+degeneration.">Seven new loci associated with age-related macular degeneration.</a> Nat Genet. 45 (4), 433-439 (2013).</li><li>Clark, S., Bishop, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Factor+H+and+Related+Proteins+in+Regulating+Complement+Activation+in+the+Macula,+and+Relevance+to+Age-Related+Macular+Degeneration.">Role of Factor H and Related Proteins in Regulating Complement Activation in the Macula, and Relevance to Age-Related Macular Degeneration.</a> J Clin Med. 4 (1), 18-31 (2014).</li><li>Booij, J. C., Baas, D. C., Beisekeeva, J., Gorgels, T. G. M. F., Bergen, A. A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+nature+of+Bruch&#8217;s+membrane.">The dynamic nature of Bruch&#8217;s membrane.</a> Prog Retin Eye Res. 29 (1), 1-18 (2010).</li><li>Clark, S. J., 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=Identification+of+factor+H-like+protein+1+as+the+predominant+complement+regulator+in+Bruch&#8217;s+membrane:+implications+for+age-related+macular+degeneration.">Identification of factor H-like protein 1 as the predominant complement regulator in Bruch&#8217;s membrane: implications for age-related macular degeneration.</a> J Immunol. 193 (10), 4962-4970 (2014).</li><li>Sim, E., Palmer, M. S., Puklavec, M., Sim, 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=Monoclonal+antibodies+against+the+complement+control+protein+factor+H+(beta+1+H).">Monoclonal antibodies against the complement control protein factor H (beta 1 H).</a> Biosci Rep. 3 (12), 1119-1131 (1983).</li><li>Ripoche, J., Day, A. J., Harris, T. J., Sim, 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=The+complete+amino+acid+sequence+of+human+complement+factor+H.">The complete amino acid sequence of human complement factor H.</a> Biochem J. 249 (2), 593-602 (1988).</li><li>Moore, D. J., Clover, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+age+on+the+macromolecular+permeability+of+human+Bruch&#8217;s+membrane.">The effect of age on the macromolecular permeability of human Bruch&#8217;s membrane.</a> Invest Ophthalmol Vis Sci. I. 42 (12), 2970-2975 (2001).</li><li>McHarg, S., Clark, S. J., Day, A. J., Bishop, P. N. <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+macular+degeneration+and+the+role+of+the+complement+system.">Age-related macular degeneration and the role of the complement system.</a> Mol Immunol. 67 (1), 43-50 (2015).</li></ol>

The authors declare that they have no competing financial interests.

Here we describe the enrichment of Bruch&#8217;s membrane for further analysis allowing subsequent analyses including western blotting11, mass spectrometry and the diffusion properties of Bruch&#8217;s membrane utilising modified Ussing chambers11,14. Critical steps include a) the thorough scraping and washing of the inner surface of the eye to remove all RPE cells b) the repeated and careful scraping of the underlying choroid without causing the Bruch&#8217;s to disintegrate and c) washing of the excised Bruch&#8217;s in ultrapure water to ensure lysis of residual cells. Furthermore, if the enriched Bruch&#8217;s membrane is to be utilized for proteomic analysis, protein extraction by urea treatment is the most effective in breaking down this resilient tissue as compared to other methods such as mechanical homogenisation. Histology of enriched Bruch&#8217;s membrane indicates the ECM of the choriocapillaris is not removed. This is to be expected as the outer layer of the pentilaminar Bruch&#8217;s membrane is formed by choriocapillaris endothelial cell basement membranes. Indeed it may be advantageous that the ECM of the choriocapillaris remains as changes occur in this in AMD including the deposition of terminal complement complex/membrane attack complex (MAC)15. It should be noted that for detailed histological analysis of Bruch&#8217;s membrane, its structure would be better preserved if sectioned in the presence of sclera, choroid, and Bruch&#8217;s membrane. Indeed, the histology presented in Figure 2 is solely intended to demonstrate the degree of enrichment resulting from the technique described in this manuscript.
Enrichment of macular Bruch&#8217;s requires careful dissection of the eyecup, ensuring that the NSR and particularly the fovea are not damaged; otherwise identification and thus isolation of the macula are not possible. The integrity of the tissue is also important to this process and it is therefore recommended that samples under 48 hr post-mortem time be used. The degree of enrichment required varies between donors and can be affected by donor age, post-mortem time, and the presence of any eye pathology. The use of the term enriched is deliberate in acknowledging a limitation of the technique, this being that &#8216;purification&#8217; or &#8216;isolation&#8217; of Bruch&#8217;s membrane using this method simply is not possible. Indeed, given that the ECM of the choriocapillaris merges into Bruch&#8217;s membrane there complete separation is not feasible.
Understanding the biochemical changes of the respective ocular tissues is essential in understanding eye disease progression. This unique enrichment of Bruch&#8217;s membrane facilitates this understanding; ongoing studies are investigating the proteome of enriched macular Bruch&#8217;s membrane using mass spectrometry at different stages of AMD and in samples originating from subjects with different genotypes to improve understanding of the molecular pathology of AMD. Already the technique described here has been used to successfully identify FHL-1 as the predominant complement regulator in Bruch&#8217;s membrane 11 and further studies on enriched Bruch&#8217;s membrane are likely to result in more new insights into the molecular pathology of AMD.

The use of post mortem human eye tissue in ophthalmic research is an invaluable resource for the understanding of eye disease pathogenesis. Analysis of human donor eyes has made important contributions to discerning the mechanisms underpinning age-related macular degeneration (AMD), which is the leading cause of blindness in the western world1. Globally, AMD accounts for approximately 8.7% of all blindness and with an increasingly aging population, it is predicted to affect 196 million people by 20202. AMD results in the loss of central vision and has a profound impact on patient independence and quality of life3. Early AMD is characterised by the formation of drusen and it can progress to geographic atrophy (sometimes referred to as &#8216;dry&#8217; AMD), or choroidal neovascularization (also referred to as neovascular or &#8216;wet&#8217; AMD). Whilst anti-VEGF injections can stabilise or improve vision in neovascular AMD it is not curative, and at present, no treatment exists for geographic atrophy.
Whilst certain genetic variants have been shown to modify AMD risk 4&#8211;8, less is known about how these variants affect the macula at a biochemical level. An important site in AMD pathogenesis is Bruch&#8217;s membrane. Drusen form within this structure and various studies have provided evidence of complement activation in and around Bruch&#8217;s membrane in AMD9. Bruch&#8217;s membrane is a sheet of extracellular matrix that separates the retinal pigment epithelial (RPE) cells from the choroid, and is approximately 4 &#181;m thick. Bruch&#8217;s membrane merges into the choriocapillaris, a layer of the choroid that contains fenestrated capillaries and external to this are layers containing larger blood vessels10 (Figure 1A).
Bruch&#8217;s membrane is a pentilaminar sheet of extracellular matrix (ECM), and this methodology details how to isolate Bruch&#8217;s membrane, along with the ECM of the underlying choriocapillaris (henceforth referred to enriched Bruch&#8217;s membrane), which is largely decellularized and free from red blood cells. Enriched Bruch&#8217;s membrane was then used for western blotting and histology experiments. Furthermore, methodology for enriching Bruch&#8217;s membrane solely from the macula region of the eye is described, which will be of particular interest to those investigating the biochemical aspects of AMD.

<table><tbody><tr><td>Auxillary objective 0.5X</td><td>GT-Vision Ltd</td><td>3638</td><td>Useful (but not essential) for lower magnification imaging of whole eye flat-mount</td></tr><tr><td>Biopsy Punch 6 mm</td><td>Oncall Medical Supplies</td><td>SCH-33-36</td><td>6mm Biopsy Punches</td></tr><tr><td>Bovine serum albumin</td><td>Sigma - Aldrich</td><td>A3059</td><td></td></tr><tr><td>Bromophenol Blue</td><td>Sigma - Aldrich</td><td>B0126</td><td></td></tr><tr><td>Cell scrapers</td><td>Sarstedt</td><td>83.183</td><td>For membrane enrichment</td></tr><tr><td>CyroPure Cyrovials</td><td>Sarstedt</td><td>72.38</td><td></td></tr><tr><td>Disposable Scalpels</td><td>Fisher Scientific</td><td>12387999</td><td>Sterile, individually wrapped Swann-Morton scalpels.</td></tr><tr><td>Dual Gooseneck LED spot lights on 30 cm arms</td><td>GT-Vision Ltd</td><td>0153</td><td>Flexible light source essential for dissection and imaging</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Sigma - Aldrich</td><td>D8537</td><td>Used to prevent macula drying out and during membrane enrichment.</td></tr><tr><td>(A) Forceps</td><td>Scientific Laboratory Supplies</td><td>INS4280</td><td>(A) Fine (100 mm) and straight</td></tr><tr><td>(B) Forceps</td><td>Scientific Laboratory Supplies</td><td>INS4272</td><td>(B) Broad and blunt (150 mm). Useful for lifting large portions of sample</td></tr><tr><td>(C) Forceps</td><td>Scientific Laboratory Supplies</td><td>INS4323</td><td>(C) Straight fine points</td></tr><tr><td>Glycerol</td><td>Sigma - Aldrich</td><td>G1345</td><td></td></tr><tr><td>Glycine</td><td>Sigma - Aldrich</td><td>B0126</td><td></td></tr><tr><td>GXCAM 5 digital colour camera &amp;amp; GT-Vision software</td><td>GT-Vision Ltd</td><td>1122</td><td></td></tr><tr><td>Methanol</td><td>Sigma - Aldrich</td><td>M/4000/17</td><td></td></tr><tr><td>Nitrocellulose membrane</td><td>Life Technologies</td><td>NP0007</td><td></td></tr><tr><td>NuPAGE Novex 4 - 12% Bis-Tris Protein Gels</td><td>Life Technologies</td><td>NP0322BOX</td><td></td></tr><tr><td>Petri dish</td><td>Sterilin</td><td>101VR20</td><td>90 mm, used as a dish for samples</td></tr><tr><td>Protein isolation beads (Strataclean resin)</td><td>Agilent</td><td>400714-61</td><td></td></tr><tr><td>SDS</td><td>Bio-Rad</td><td>161-0301</td><td></td></tr><tr><td>Stereozoom microscope GXMXTL3T (magnification range 7X - 45X)</td><td>GT-Vision Ltd</td><td>5595</td><td>Stereozoom microscope attached to boom stand for ease of use during dissection</td></tr><tr><td>Tris-HCl</td><td>Sigma - Aldrich</td><td>T3253</td><td></td></tr></tbody></table>

enrichment of bruch's membrane from human donor eyes

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

The protocol described above to produce enriched Bruch&#8217;s membrane results in the isolation of Bruch&#8217;s membrane, the ECM of the choriocapillaris and removes most of the cells/cellular debris including all of the RPE (Figure 2). The washing of the enriched Bruch&#8217;s membrane in water is a key step in lysing any remaining choroidal cells. The pressure exerted during scrapping of the choroid appears to compact some of the few remaining cell nuclei into the region defined as Bruch&#8217;s membrane (Figure 2B).
The solubilisation of enriched Bruch&#8217;s membrane allows the downstream analysis of its protein content. The separation of protein bands from solubilised enriched Bruch&#8217;s membrane using a 4 - 12% gradient SDS-PAGE gel, while not useful in its own right to identify protein content, does demonstrate the ability of this technique to minimise contaminating blood proteins that are non-specifically bound (Figure 3B). In this regard, one notable absentee from the protein gel run is a sizeable band of human serum albumin (~66 kDa) that may otherwise make subsequent western blotting more difficult to interpret: the histological analysis of enriched Bruch&#8217;s membrane shown in Figure 2 also supports this. Indeed, as part of work described previously11, solubilised Bruch&#8217;s membrane from a series of individual donors were probed for a number of proteins using specific antibodies via western blotting (Figure 3C). In the case shown here, an antibody (termed OX23)12 directed against a 155 kDa regulator of innate immunity, called complement factor H (FH), was used and demonstrated the presence of a lower molecular weight band in Bruch&#8217;s membrane samples from three separate donors (Figure 3C). This subsequently turned out to be factor H-like protein 1 (FHL-1), a 49 kDa protein related to FH and arising from a splice variation of the same gene13. FHL-1 appears to be the main complement regulator present in Bruch&#8217;s membrane11.
<img alt="Figure 1" src="/files/ftp_upload/53382/53382fig1.jpg" /> 
Figure 1. Dissection of the Human Eye and Enrichment of Bruch&#8217;s Membrane.&#160;(A) Bruch&#8217;s membrane is an extracellular matrix barrier. The choroid contains a layer of fenestrated capillaries that continuously merge into Bruch&#8217;s membrane (called the choriocapillaris) and these separate the rest of the choroid (containing large blood vessels) from the retinal pigment epithelium (RPE) cell monolayer, which physiologically supports the photoreceptor cells of the neurosensory retina (NSR). (B) Four incisions into the human eyecup are made allowing it to be opened to create a flat mount. The vitreous can be removed with the use of tweezers. The NSR may come off along with the vitreous but can also be removed with tweezers. A cell scraper is used to remove the RPE cell layer from Bruch&#8217;s membrane while it is still anchored to the choroid and sclera. The Bruch&#8217;s membrane/choroid complex can be peeled away from the sclera and using a new cell scraper, the outer choroid can be removed. A final wash in ultrapure water lyses any remaining cellular matter. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53382/53382fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53382/53382fig2.jpg" /> 
Figure 2. Histology of Bruch&#8217;s Membrane and the Choroid. H&#38;E stained sections before (A) and after (B) scraping away the outer choroidal structures from Bruch&#8217;s membrane and washing in ultrapure water to generate enriched Bruch&#8217;s membrane. Scale bars represent 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53382/53382fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53382/53382fig3.jpg" /> 
Figure 3. Solubilisation of Enriched Bruch&#8217;s Membrane for Protein Content Analysis.&#160;(A) Protein can be extracted from enriched Bruch&#8217;s membrane by incubating in 8 M urea for 6 hr at RT, before being dialysed against PBS. For Western analysis, proteins from the whole Bruch&#8217;s membrane dialysate can be isolated using protein isolation beads and eluted by boiling with laemmli reducing sample loading buffer. (B) A representative Coomassie blue stained SDS-PAGE gel, showing both neat and diluted solubilised Bruch&#8217;s membrane (BM) using the method described in (A). (C) A representative western blot of solubilised Bruch&#8217;s membrane from 3 individual donors (lanes 1 - 3) probed for factor H-like protein 1 (FHL-1). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53382/53382fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53382/53382fig4.jpg" /> 
Figure 4. Isolation of Enriched Bruch&#8217;s Membrane from the Macula. The macula region of the retina is a 5 mm area that can be readily identified by the yellow colouration of the overlying fovea, and its proximity to the adjacent optic disc. Using the fovea as a guide, place a 6 mm biopsy punch on the flat mounted human eye to excise a 6 mm macula tissue block. Remove the overlying NSR and scrape off RPE cells from the excised macula. The remaining Bruch&#8217;s membrane/choroid complex can then be peeled away from the sclera and thoroughly scraped to remove choroid, lyse remaining cellular material by submersion in ultrapure water. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53382/53382fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Enrichment of Bruch's Membrane from Human Donor Eyes at JoVE.com

Enrichment of Bruch's Membrane from Human Donor Eyes

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

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

enrichment-of-bruch-s-membrane-from-human-donor-eyes

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.0K Views.  University of Manchester. Identifying proteins specifically associated with Bruch’s membrane in human eyes is an important step in understanding the biochemical mechanisms behind eye diseases such as age-related macular degeneration. This protocol describes how to enrich this sheet of extracellular matrix for down-stream biochemical analysis.

Video: Enrichment of Bruch's Membrane from Human Donor Eyes

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.

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.

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 ...

Developmental Biology

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 ...

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately understood, thus impeding the establishment of defined treatments. Cell-based additive gene therapies for the increased expression of a protective factor are considered as a promising option to medicate neurodegenerative diseases, such as age-related macular degeneration (AMD). We have developed a method for the stable expression of the gene encoding pigment epithelium-derived factor (PEDF), which is characterized as a neuroprotective and anti-angiogenic protein in the nervous system, into the genome of primary human pigment epithelial (PE) cells using the Sleeping Beauty (SB) transposon system. Primary PE cells were isolated from human donor eyes and maintained in culture. After reaching confluence, 1 x 104 cells were suspended in 11 &#181;L of resuspension buffer and combined with 2 &#181;L of a purified solution containing 30 ng of hyperactive SB (SB100X) transposase plasmid and 470 ng of PEDF transposon plasmid. Genetic modification was carried out with a capillary electroporation system using the following parameters: two pulses with a voltage of 1,100 V and a width of 20 ms. Transfected cells were transferred into culture plates containing medium supplemented with fetal bovine serum; antibiotics and antimycotics were added with the first medium exchange. Successful transfection was demonstrated in independently performed experiments. Quantitative polymerase chain reaction (qPCR) showed the increased expression of the PEDF transgene. PEDF secretion was significantly elevated and remained stable, as evaluated by immunoblotting, and quantified by enzyme-linked immunosorbent assay (ELISA). SB100X-mediated transfer allowed for a stable PEDF gene integration into the genome of PE cells and ensured the continuous secretion of PEDF, which is critical for the development of a cell-based gene addition therapy to treat AMD or other retinal degenerative diseases. Moreover, analysis of the integration profile of the PEDF transposon into human PE cells indicated an almost random genomic distribution.

We have developed a protocol to transfect primary human pigment epithelial cells by electroporation with the gene encoding pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB) transposon system. Successful transfection was demonstrated by quantitative polymerase chain reaction (qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA).

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately ...

Medicine

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...

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 ...

OCT-Based Multimodal Imaging of Human Donor Eyes for AMD Research

Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging could add prognostic value to laboratory findings. In this work, ex vivo OCT and MMI were applied to human donor eyes prior to retinal tissue sectioning. The eyes were recovered from non-diabetic white donors aged &#8805;80 years old, with a death-to-preservation time (DtoP) of &#8804;6 h. The globes were recovered on-site, scored with an 18 mm trephine to facilitate cornea removal, and immersed in buffered 4% paraformaldehyde. Color fundus images were acquired after anterior segment removal with a dissecting scope and an SLR camera using trans-, epi-, and flash illumination at three magnifications. The globes were placed in a buffer within a custom-designed chamber with a 60 diopter lens. They were imaged with spectral domain OCT (30&#176; macula cube, 30 &#181;m spacing, averaging = 25), near-infrared reflectance, 488 nm autofluorescence, and 787 nm autofluorescence. The AMD eyes showed a change in the retinal pigment epithelium (RPE), with drusen or subretinal drusenoid deposits (SDDs), with or without neovascularization, and without evidence of other causes. Between June 2016 and September 2017, 94 right eyes and 90 left eyes were recovered (DtoP: 3.9 &plusmn; 1.0 h). Of the 184 eyes, 40.2% had AMD, including early intermediate (22.8%), atrophic (7.6%), and neovascular (9.8%) AMD, and 39.7% had unremarkable maculas. Drusen, SDDs, hyper-reflective foci, atrophy, and fibrovascular scars were identified using OCT. Artifacts included tissue opacification, detachments (bacillary, retinal, RPE, choroidal), foveal cystic change, an undulating RPE, and mechanical damage. To guide the cryo-sectioning, OCT volumes were used to find the fovea and optic nerve head landmarks and specific pathologies. The ex vivo volumes were registered with the in vivo volumes by selecting the reference function for eye tracking. The ex vivo visibility of the pathology seen in vivo depends on the preservation quality. Within 16 months, 75 rapid DtoP donor eyes at all stages of AMD were recovered and staged using clinical MMI methods.

Author Spotlight: Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration  

Laboratory assays can leverage prognostic value from the longitudinal optical coherence tomography (OCT)-based multimodal imaging of age-related macular degeneration (AMD). Human donor eyes with and without AMD are imaged using OCT, color, near-infrared reflectance scanning laser ophthalmoscopy, and autofluorescence at two excitation wavelengths prior to tissue sectioning.

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging ...

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, ...

Biology

A Protocol to Isolate, Culture, and Identify Human Limbal Niche Cells

Isolation and Identification of Limbal Niche Cells

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used for LNCs isolation. The tissue was divided into 12 pieces under aseptic conditions and digested for 18 h at 37 &#176;C in the cell culture incubator using collagenase A to obtain cell clusters with LNCs and limbal&#160;epithelial progenitor cells. The cell clusters were further digested for 15 min at 37 &#176;C using 0.25% trypsin-EDTA to obtain single cells and then cultured in modified embryonic stem cell medium (MESCM) on a plastic surface coated with 5% Matrigel. Cells were passaged upon 70% confluence, and LNCs were identified using immunofluorescence, real-time quantitative PCR (qPCR), and flow cytometry. Primary LNCs were isolated and passaged more than 12 times. The proliferation activity of LNCs from P4 to P6 was the highest. LNCs expressed higher stem cell markers than BMMSCs (SCF, Nestin, Rex1, SSEA4, CD73, CD90, MSX1, P75NTR, and PDGFR&#946;). Furthermore, results showed that P4 LNCs uniformly expressed VIM, CD90, CD105, and PDGFR&#946;, but not Pan-CK, which could be used as a marker for the identification of LNCs. Flow cytometric analysis showed that approximately 95%, 97%, 92%, and 11% of LNCs expressed CD73, CD90, CD105, and SCF respectively, while they were&#160;68%, 99%, 20%, and 3% in&#160;BMMSCs. The standard process for LNC&#160;isolation and identification could provide&#160;a reliable laboratory basis for the widespread use of LNCs.

Author Spotlight: Standardizing Limbal Niche Cell (LNC) Isolation and Characterization to Support Widespread LNC Research 

Here, we present a protocol to isolate and identify the human limbal niche cells.

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used ...

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

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

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

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

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

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

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

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

Enrichment of Bruch's Membrane from Human Donor Eyes

Summary

Explore More Videos

Chapters in this video

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

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

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

Characterization of a Novel Human Organotypic Retinal Culture Technique

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

Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration

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

Isolation and Identification of Limbal Niche Cells

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

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