This work is supported, in part, by grants from the Vitreo-Retinal Surgery Foundation (VRSF), Fight for Sight (FFS), and the International Retinal Research Foundation (IRRF). J.M.L.M. is currently supported by a K08 grant from the National Eye Institute (EY033420). No federal funds were used for HFT research. Further support comes from the James Grosfeld Initiative for Dry AMD and the following private donors: Barbara Dunn and Dee &#38; Dickson Brown.

The present protocol involving the acquisition and use of human tissue was reviewed and approved by the University of Michigan Institutional Review Board (HUM00105486).
1. Preparation of photo-oxidized outer segment tips and fragments
NOTE: Dark-adapted bovine retinas were purchased and shipped on ice (see Table of Materials). From these retinas, OS were purified following a previously published protocol23.
<ol>
	<li>Outer ...

Quantifying Retinal Pigment Epithelial Phagocytosis and Lipofuscin Accumulation

While RPE lipofuscin has been studied for decades, its toxicity is debated2,9,16,42. Given ambiguity about the toxicity of lipofuscin from animal models11, in vitro models using human RPE are valuable. A range of in vitro lipofuscin accumulation models have been described, but none have utilized both OS feeding and highly mature and differentiated human...

The retinal pigment epithelium (RPE) provides critical support for overlying photoreceptors, including the daily uptake and degradation of photoreceptor outer segment tips or fragments (throughout this protocol, the abbreviation OS stands for OS tips or fragments rather than whole outer segments). This daily uptake in the post-mitotic RPE eventually overloads phagolysosomal capacity and leads to the buildup of undigestible, autofluorescent intracellular material, termed lipofuscin. Interestingly, several studies have also demonstrated that RPE lipofuscin can accumulate without OS phagocytosis1,2. Lipofuscin has many components, including cross-linked adducts derived from visual cycle retinoids, and can occupy nearly 20% of RPE cell volume for those over the age of 803.
Whether lipofuscin is toxic has been hotly debated. Stargardt&#39;s disease is an autosomal recessive degeneration of the photoreceptors and RPE in which a mutation in ABCA4 triggers improper processing of visual cycle retinoids contained within photoreceptor outer segments. Improper retinoid processing leads to aberrant cross-linking and formation of bis-retinoid species, including the bis-retinoid N-retinylidene-N-retinylethanolamine (A2E). Studies have demonstrated multiple mechanisms for A2E toxicity4,5. Lipofuscin contributes to fundus autofluorescence signals during clinical imaging, and both Stargardt&#39;s patients and animal models display increased fundus autofluorescence prior to retinal degeneration, suggesting a correlation between lipofuscin levels and toxicity6,7. However, with age, lipofuscin accumulates in all humans without triggering an RPE degeneration. Further, in age-related macular degeneration (AMD), where RPE degeneration occurs only in elderly patients, those with early and intermediate forms of the disease have less fundus autofluorescence signals than age-matched non-diseased humans8. These clinical findings have been verified at the histologic level as well9,10.
Animal models of RPE lipofuscin accumulation have also left some ambiguity about lipofuscin toxicity. The ABCA4 knockout mouse does not display retinal degeneration on a pigmented background, whereas it does on an albino background or when exposed to blue light11,12. Further, the toxicity of lipofuscin derived via ABCA4 knockout likely differs from the more slowly accumulating lipofuscin that occurs with natural aging, as seen in AMD13.
In vitro models of lipofuscin accumulation provide an alternative to studying the effects of lipofuscin accumulation on RPE health. Such models allow for manipulating lipofuscin components, from feeding single retinoid components to feeding OS, and allow study in human rather than animal RPE. In the last couple of decades, multiple methods have been developed to model RPE lipofuscin in culture. Along with other groups, Dr. Boulton&#39;s group fed bovine OS daily for up to three months on passage 4 to 7 human primary RPE cells from donors aged 4 to 85 years old14. Alternatively, inhibition of autophagy has also led to lipofuscin accumulation in passages 3 to 7 primary human RPE cultures15. However, sub-lethal lysosomal inhibition in highly differentiated, passage 1, primary human pre-natal RPE (hfRPE) cultures failed to induce lipofuscin, even with the repeated addition of OS on a daily basis16.
As a more reductionist approach, others have fed single lipofuscin components to cultures, especially the bis-retinoid A2E4,17. Such studies are valuable in that they define potential direct mechanisms of toxicity for individual lipofuscin components, implicating, for example, lysosomal cholesterol and ceramide homeostasis18. At the same time, there is debate about the toxicity of A2E19, and feeding it directly to cells circumvents the typical pathway for lipofuscin accumulation, which involves phagocytosis of photoreceptor OS. In an attempt to deliver all components of lipofuscin to RPE cultures, Boulton and Marshall purified lipofuscin from human eyes and fed this to passage 4 to 7 human primary RPE cultures derived from both fetal and elderly human donors20. While innovative, this method represents a limited lipofuscin source for repeated experiments.
While repeated feedings of OS to RPE cultures produce lipofuscin in many systems, it fails to do so in highly differentiated primary RPE cultures16. Photo-oxidizing OS induces cross-linking reactions like bis-retinoid formation that naturally occurs during lipofuscin formation in vivo. This can accelerate lipofuscin-like granule formation in RPE culture systems, even those that are highly differentiated and resistant to lipofuscin accumulation16. Here, a method to induce lipofuscin-like granule accumulation in highly differentiated hfRPE and human iPSC-RPE is introduced, modified from Wihlmark&#39;s published protocol21. This method has the advantage of inducing lipofuscin-like granules employing the same source (photoreceptor OS) and pathway (phagolysosomal OS uptake) as occurs for lipofuscinogenesis in vivo. Further, it is done on human RPE cultures that are highly differentiated and validated in multiple studies to replicate human RPE in vivo22,23,24. These lipofuscin-like granules are termed undigestible autofluorescent material (UAM), and provide data and discussion in this protocol comparing UAM to in vivo lipofuscin. Along with methods for building and evaluating UAM-laden cultures in highly differentiated human RPE, an updated method to assess RPE OS phagocytosis is also introduced. Multiple excellent pulse-chase methods for quantifying OS phagocytosis have been introduced, including Western blotting, immunocytochemistry, and FACS25,26,27. However, early in the OS pulse-chase, conditions that lead to poor OS uptake can be conflated with conditions that promote rapid degradation of internalized OS. The method presented here measures the total amount of introduced OS that is fully consumed/degraded by the RPE (&#34;Total Consumptive Capacity&#34;), helping eliminate this ambiguity. It is anticipated that insights about lipofuscin toxicity utilizing these protocols, including effects on OS phagocytosis rates utilizing the &#34;Total Consumptive Capacity&#34; method, will be used to shed light on the toxicity of lipofuscin in vivo.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mm cell culture dish</td>
			<td>Corning</td>
			<td>#353003</td>
			<td>Others also work</td>
		</tr>
		<tr>
			<td>24-well Transwells</td>
			<td>Corning</td>
			<td>#3470</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-LC3 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>#4801S</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Anti-rhodopsin antibody 1D4</td>
			<td>Abcam</td>
			<td>#5417</td>
			<td>1:1000 dilution. Epitope is C-terminal.</td>
		</tr>
		<tr>
			<td>Anti-rhodopsin antibody 4D2</td>
			<td>EnCor Biotech</td>
			<td>MCA-B630</td>
			<td>1:5000 dilution for western blot, 1:1000 dilution for immunostaining. Epitope is N-terminal.</td>
		</tr>
		<tr>
			<td>Autofluorescence quencher</td>
			<td>Biotium</td>
			<td>#23007</td>
			<td>TrueBlack Lipofuscin Autofluorescence Quencher</td>
		</tr>
		<tr>
			<td>Autofluorescence quencher</td>
			<td>Vector Laboratories</td>
			<td>SP-8400</td>
			<td>Vector TrueVIEW Autofluorescence Quenching Kit</td>
		</tr>
		<tr>
			<td>Bodipy 493/503</td>
			<td>Life Technologies</td>
			<td>D3922</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol esterase&#160;</td>
			<td>Life Technologies</td>
			<td>From A12216 kit</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica</td>
			<td>Leica Stellaris SP8 with FALCON module</td>
			<td></td>
		</tr>
		<tr>
			<td>Dark-adapted bovine retinas</td>
			<td>W. L. Lawson Company</td>
			<td>Dark-adapted bovine retinas (pre-dissected)</td>
			<td>Contact information: 
			https://wllawsoncompany.com/ 
			(402) 499-3161 
			stacy@wllawsoncompany.com</td>
		</tr>
		<tr>
			<td>Filipin</td>
			<td>Sigma-Aldrich</td>
			<td>F4767</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>Thermo Fisher</td>
			<td>Attune NxT</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer analysis software&#160;</td>
			<td>BD</td>
			<td>FlowJo</td>
			<td></td>
		</tr>
		<tr>
			<td>Handheld UV light&#160;</td>
			<td>Analytik Jena US</td>
			<td>UVGL-55</td>
			<td></td>
		</tr>
		<tr>
			<td>Human MFG-E8</td>
			<td>Sino Biological</td>
			<td>10853-H08B</td>
			<td></td>
		</tr>
		<tr>
			<td>Human purified Protein S</td>
			<td>Enzyme Research Laboratories</td>
			<td>HPS</td>
			<td></td>
		</tr>
		<tr>
			<td>Laemmli sample buffer</td>
			<td>Thermo Fisher</td>
			<td>J60015-AD</td>
			<td></td>
		</tr>
		<tr>
			<td>LDH assay</td>
			<td>Promega</td>
			<td>J2380</td>
			<td>LDH-Glo Cytotoxicity Assay</td>
		</tr>
		<tr>
			<td>Mounting media</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td>Prolong Gold antifade reagent</td>
		</tr>
		<tr>
			<td>Nile red</td>
			<td>Sigma-Aldrich</td>
			<td>#72485</td>
			<td></td>
		</tr>
		<tr>
			<td>Polytetrafluoroethylene-coated slides</td>
			<td>Tekdon</td>
			<td>Customized</td>
			<td>Customized specifications: PTFE mask with the following &#34;cut-outs&#34; -&#160; 3 glass rectangles, each measuring 17 mm x 9 mm, oriented so that the 17 mm side is 4 mm from the top of the slide and 4 mm from the bottom of the slide, assuming a standard microscope slide of 25 mm x 75 mm. Each rectangle is spaced at least 6 mm away from other rectangles and the edges of the slide. Print PTFE mask on a slide with frosted glass on one side to allow for labeling of the slide.</td>
		</tr>
		<tr>
			<td>Protease inhibitors&#160;</td>
			<td>Cell Signaling Technology</td>
			<td>#5872</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein assay</td>
			<td>Bio-Rad</td>
			<td>#5000122 RC DC protein assay</td>
			<td></td>
		</tr>
		<tr>
			<td>TEER electrode</td>
			<td>World Precision Instruments</td>
			<td>STX3</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-epithelial electrical resistance (TEER) meter</td>
			<td>World Precision Instruments</td>
			<td>EVOM3</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraviolet crosslinker device</td>
			<td>Analytik Jena US</td>
			<td>UVP CL-1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

improved lipofuscin models and quantification of outer segment phagocytosis capacity in highly polarized human retinal pigment epithelial cultures

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging pigment termed lipofuscin. The toxicity of lipofuscin is well established in Stargardt's disease, the most common inherited retinal degeneration, but is more controversial in age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world. Determining lipofuscin toxicity in humans has been difficult, and animal models of Stargardt's have limited toxicity. Thus, in vitro models that mimic human RPE in vivo are needed to better understand lipofuscin generation, clearance, and toxicity. The majority of cell culture lipofuscin models to date have been in cell lines or have involved feeding RPE a single component of the complex lipofuscin mixture rather than fragments/tips of the entire photoreceptor outer segment, which generates a more complete and physiologic lipofuscin model. Described here is a method to induce the accumulation of lipofuscin-like material (termed undigestible autofluorescence material, or UAM) in highly differentiated primary human pre-natal RPE (hfRPE) and induced pluripotent stem cell (iPSC) derived RPE. UAM accumulated in cultures by repeated feedings of ultraviolet light-treated OS fragments taken up by the RPE via phagocytosis. The key ways that UAM approximates and differs from lipofuscin in vivo are also discussed. Accompanying this model of lipofuscin-like accumulation, imaging methods to distinguish the broad autofluorescence spectrum of UAM granules from concurrent antibody staining are introduced. Finally, to assess the impact of UAM on RPE phagocytosis capacity, a new method for quantifying outer segment fragment/tips uptake and breakdown has been introduced. Termed "Total Consumptive Capacity", this method overcomes potential misinterpretations of RPE phagocytosis capacity inherent in classic outer segment "pulse-chase" assays. The models and techniques introduced here can be used to study lipofuscin generation and clearance pathways and putative toxicity.

Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures

The set-up for photo-oxidation of OS is demonstrated in Figure 1Ai. The polytetrafluoroethylene-coated slides allow for a large volume of OS in solution to be loaded per open rectangle without spreading across the rest of the slide. The slide with OS is contained within a sterile Petri dish with the lid off, and a UV lamp is placed over the slide as shown in Figure 1Aii. Alternatively, the slide can be placed in a UV Crosslinker device, as shown in <strong class...

Watch this Scientific Journal Video about Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures at JoVE.com

Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures (Video) | JoVE 

This protocol describes a lipofuscin accumulation model in highly differentiated and polarized human retinal pigment epithelial (RPE) cultures and an improved outer segment (OS) phagocytosis assay to detect the total OS consumption/degradation capacity of the RPE. These methods overcome the limitations of previous lipofuscin models and classical pulse-chase outer segment phagocytosis assays.

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging ...