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 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.
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'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'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'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'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 ("Total Consumptive Capacity"), helping eliminate this ambiguity. It is anticipated that insights about lipofuscin toxicity utilizing these protocols, including effects on OS phagocytosis rates utilizing the "Total Consumptive Capacity" method, will be used to shed light on the toxicity of lipofuscin in vivo.
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.
2. Building lipofuscin-like granules (UAM) in RPE cultures
3. Assessing effects of lipofuscin-like granules on RPE phagocytosis: Total Consumptive Capacity
NOTE: The rationale for measuring OS phagocytosis via the OS pulse only protocol below is detailed in the representative results section. The method, which is termed "Total Consumptive Capacity," avoids ambiguities about phagocytosis efficiency that can emerge with traditional OS pulse-chase phagocytosis assays. Assays are done on 24-well Transwell plates using 50 µL of media containing 4 x 106 OS/mL.
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 Figure 1Aiii. After photo-oxidation, OS autofluorescence increases significantly, as assessed by both microscopy and flow cytometry (Figure 1B). The degree of protein cross-linking after photo-oxidation can be assessed by SDS-PAGE with a Coomassie stain, shown as a conversion of the dominant protein band (monomeric rhodopsin) into higher order aggregates and smear (Figure 1C). Comparing photo-oxidation with a hand-held UV lamp (Figure 1Aii) versus the UV Crosslinker device (Figure 1Aiii) demonstrates that the hand-held UV lamp produces OxOS with as much autofluorescence as a 3 J/cm2 treatment from the UV Crosslinker device (Figure 1B), and as much protein cross-linking as a 6 J/cm2 treatment from the UV Crosslinker device (Figure 1C). If the UV lamp available to a researcher differs from the one used in this protocol, we suggest titrating the exposure time to achieve a level of cross-linking seen in the UV lamp lane in Figure 1C. The autofluorescence spectrum of OxOS is mildly blue-shifted compared to the spectrum of untreated OS, in both the protein-isolated fraction of the OS and the lipid-isolated fraction of OS16.
The amount of UAM buildup in RPE cultures depends on the number of OxOS feedings (Figure 2A), and 20 feedings over the course of approximately 4 weeks provides a robust amount of UAM accumulation. To distinguish UAM autofluorescence from autofluorescence of residual OxOS that remain stuck to the RPE apical surface even days to weeks after wash-off, we stain with a rhodopsin antibody, detected with a far-red dye. By using this rhodopsin fluorescence channel as a mask, we can subtract out OxOS autofluorescence from the UAM channel, ensuring we are truly quantifying autofluorescence from UAM only16. Utilizing staining protocols outlined above, accumulated UAM in this model has abundant neutral lipids, as assessed by Nile Red staining16, similar to native lipofuscin. However, we find little to no esterified or unesterified cholesterol accumulation (Figure 2B), which is consistent with the lack of cholesterol accumulation we see in native lipofuscin from healthy eyes (data not shown).
Following fluorescent markers of interest concurrent with lipofuscin autofluorescence is difficult, given the very broad fluorescence emission spectrum of lipofuscin. In the methods section above, several methods are detailed to overcome this problem. The method outlined in step 2.3.2.1 above takes advantage of the low autofluorescence emission intensity for lipofuscin in the far-red. In Figure 2Ci, the colocalization of UAM and the autophagy marker LC3 is assessed by staining LC3 with an Alexa 647 dye. Despite some bleedthrough of UAM into the LC3 channel, it is apparent where lipofuscin and LC3 are located in these images. In the method outlined in step 2.3.2.2 above, the very long fluorescence emission spectra of lipofuscin allows design of an emission channel that contains fluorescence from lipofuscin only. In Figure 2Cii, UAM is excited with a 488 nm laser, while emission is detected at both 500-535 nm and at 600-645 nm. The sample is co-stained with LC3, detected with Alexa 488 dye. The Alexa 488 channel (excitation: 488 nm, emission: 500-535 nm) contains both LC3 and UAM signal. However, the second channel, with 488 nm excitation and 600-645 nm emission, contains only UAM. This second channel can be used as a mask, applied to the Alexa 488/LC3 channel, to subtract out unwanted UAM signal from the LC3 signal. In the method outlined in step 2.3.2.4 above, the shorter fluorescence lifetime of autofluorescent lipofuscin allows one to distinguish the lipofuscin from co-staining fluorescence. In Figure 2Ciii, all fluorescence signals arriving to a photon-counting detector before 2 ns are eliminated, which gates out much of the UAM autofluorescence signal while largely preserving the co-staining signal from LC3. A combination of methods may be necessary to distinguish lipofuscin from co-staining fluorescence if there is strong co-localization of the lipofuscin and co-staining fluorophore.
As multiple studies have postulated an impact of RPE lipofuscin accumulation on OS phagocytosis rates5,36,37,38,39, OS phagocytosis capacity in UAM-laden cultures was assessed. Typical phagocytosis assays involve a brief incubation of cells with OS ("pulse") followed by wash-off of unbound OS and replacement of media without OS for a "chase" period. During the chase, as OS are degraded, there is a loss of rhodopsin, the most abundant protein in OS, within the RPE. At various timepoints during the chase, the OS-free media is removed, followed by cell lysis and SDS-PAGE for rhodopsin remaining within the RPE lysates. However, as the OS pulse period consists of both uptake and degradation of OS, RPE with high uptake and degradation ("phagocytosis efficient" cells) may have the same rhodopsin levels early in the chase period as RPE with low uptake and degradation ("phagocytosis inefficient" cells). This is schematically represented in the study by Zhang et al23. To overcome this ambiguity, a "pulse-only" assay was employed here. In this method, OS are pulsed onto RPE but not washed off. At various times after OS addition, the media and the cell lysate are collected together. The media contains undigested OS and the cell lysate contains a combination of intact OS on the cell surface, partly digested OS that have been internalized, and fully digested OS that have successfully made it through the lysosome. As a control, cells are exposed to OS but then the cell lysate and OS-containing supernatant are immediately collected. This control reveals how much total rhodopsin was introduced to the cells. As the lysate plus supernatant is collected at various points after OS introduction, one can assess for what fraction of the total rhodopsin signal has disappeared, using this as a marker for amount of all introduced/starting OS that are now completely degraded. The readout for this assay is termed, "Total Consumptive Capacity", since it accurately measures all OS degradation and is not subject to the confounding interpretations of a pulse-chase experiment described above.
Figure 3A and Supplementary Figure 1 demonstrate a Western blot of remaining rhodopsin using the Total Consumptive Capacity assay. Amount of OS fed to cells is measured by the rhodopsin band at 0 h. The main rhodopsin band is degraded with equal efficiency between the control and UAM-laden RPE samples (single arrow at 4 h and 24 h). However, there is a difference between the groups when looking at partly degraded fragments of rhodopsin, which appear below the main rhodopsin band. The differences in fragment abundance imply reduced lysosomal capacity in the UAM group, since proteolytically cleaved fragments of rhodopsin should be rapidly degraded with normal lysosomal function. Increased abundance of these fragments without increased abundance of the main rhodopsin fragment suggests more subtle defects in lysosomal degradative capacity in the UAM-laden cultures. Prior studies have suggested that lipofuscin can indeed induce defects in OS phagocytosis44, but no prior study has examined the effects of lipofuscin specifically on rhodopsin fragments, which allows a more precise pinpointing of the dysfunction to the end-stages of phagolysosomal degradation. Figure 3B demonstrates Western blotting of the main rhodopsin band plus rhodopsin fragments using two different anti-rhodopsin antibodies. Antibody 4D2 recognizes the N-terminus of rhodopsin, and N-terminal fragments are the last part of rhodopsin to be degraded in the lysosome. In contrast, antibody 1D4 recognizes the C-terminus of rhodopsin, which is degraded even prior to phagosome-lysosome fusion. Thus, understanding which fragments stain with which antibody provides a sense of rhodopsin processing defects that are pre-lysosomal vs. lysosomal32,40,41.
Figure 1: Photo-oxidized outer segment (OxOS) treatment and characterization. (A) Depiction of OS on a polytetrafluoroethylene-coated slide (i) treated with either a 254 nm UV handheld lamp (ii) or a UV Crosslinker device (iii). (B) Compared to untreated (regular) OS (RegOS), treated OS (OxOS) had increased autofluorescence as shown by confocal imaging (left) (scale bar = 10 µm) and flow cytometry (right). Autofluorescent intensity of OxOS treated with the handheld UV lamp was similar to OS treated with the UV Crosslinker Device at a radiant exposure of 3 J/cm2 (error bar = S.E.M.). (C) SDS-PAGE demonstrating the crosslinking of OS protein induced by UV exposure. OxOS crosslinking via the handheld UV lamp was similar to OS treated with the UV Crosslinker Device at a radiant exposure of 6 J/cm2. Monomeric rhodopsin is highlighted with an arrow. Please click here to view a larger version of this figure.
Figure 2: Imaging OxOS-induced lipofuscin-like granules in hfRPE cultures. (A) Autofluorescent granules (green) induced by OxOS accumulated significantly more after 20 feedings compared to 5 feedings. Residual OxOS stain with rhodopsin antibody (magenta). Scale bar = 10 µm. (B) UAM induced by OxOS (green) was not enriched with free cholesterol or cholesterol ester, as assessed by filipin staining (red). Scele bar = 10 µm. (C) Imaging methods for fluorescence co-staining in the presence of lipofuscin. (i) Use of far-red fluorophore (Alexa 647) to stain autophagy marker LC3 (magenta), which minimizes UAM bleedthrough. Pure UAM (green) imaged with a standard green channel excitation and emission filter set-up. Scale bar = 2 µm. (ii) Use of ratiometric imaging helps decipher UAM autofluorescence from LC3 staining labeled with Alexa 488. Excitation is 488 nm, green channel emission bandpass is 500-535 nm while red channel emission bandpass is 600-645 nm. Red channel can be applied as a subtractive mask to isolate LC3 signal from the green channel. Scale bar = 5 µm. (iii) As fluorescence lifetime of lipofuscin/UAM is typically shorter than specific dyes, UAM autofluorescence can be reduced by gating out fluorescence signals arriving at a photon-counting detector in less than 2 ns. Top image is without gating. Bottom image is with gating, keeping only fluorescence signals with a lifetime longer than 2 ns. There is a greater relative reduction in UAM signal compared to specific LC3 signal (labeled with Alexa 488) between top and bottom images. Scale bar = 5 µm. Please click here to view a larger version of this figure.
Figure 3: "Total Consumptive Capacity" method for measuring OS phagocytosis. (A) Total remaining rhodopsin protein in both cell lysate and supernatant after introduction of OS to RPE culture, as assayed by western blot. After feeding regular OS in 50 µL media, both the conditioned media/supernatant and cells were lysed together at 0, 4, and 24 h. The 0 h timepoint serves as a control, indicating the total amount of OS fed to each Transwell. The intact rhodopsin band (single arrow) was not different between control and UAM-laden RPE cells, suggesting no large effect of UAM on RPE phagocytosis. However, the cleavage products of rhodopsin, indicated by the double arrows, were higher in the UAM group, suggesting some mild degradative dysfunction in the phagolysosomal system. Equal levels of GAPDH indicate that cell count between wells, which can affect phagocytosis rates, was equal. (B) Different rhodopsin antibodies recognize different degradation fragments. 4D2 recognizes the N-terminus of rhodopsin, which is intact until the very last steps of lysosomal degradation. The fragments it recognizes are therefore small (double arrows). In contrast, 1D4 recognizes the C-terminus of rhodopsin, which is degraded earlier in the phagolysosomal process. This antibody therefore recognizes rhodopsin fragments of a higher molecular weight (double arrows). Both antibodies recognize intact rhodopsin (single arrow). Please click here to view a larger version of this figure.
Supplementary Figure 1: Unedited blot corresponding to Figure 3A. Please click here to download this File.
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 RPE cultures. This combination represents an ideal model, as OS feeding recapitulates the method for lipofuscin accumulation in vivo and highly mature human RPE cultures best replicate RPE behavior in vivo. Interestingly, when utilizing highly differentiated hfRPE cultures, it was discovered that routine OS exposure was insufficient to induce lipofuscin-like material, even after repeated feedings16. Thus, this protocol accelerates the lipofuscin accumulation process by photo-oxidizing OS (OxOS) in a controlled fashion. Feeding OxOS to both human iPSC-RPE and hfRPE cultures resulted in robust accumulation of lipofuscin-like granules, which is termed undigestible autofluorescent material (UAM). UAM accumulation allows for modeling of lipofuscin in culture systems that mimic healthy human RPE in vivo22,23,29,43,44. In both a prior publication and in this study, extensive characterization of UAM compared to native lipofuscin from healthy older adults demonstrates both similarities and differences. UAM mimics the ultrastructure of lipofuscin in vivo, including the tendency for lipofuscin granules to fuse with melanin granules to form melanolipofuscin16. Both UAM and lipofuscin contain substantial neutral lipids, no rhodopsin, and no significant enrichment in cholesterol (Figure 2A,B from our prior publication16, Figure 2A,B above, and unpublished data). We also compared native lipofuscin granule size and spectrum to those from UAM (Figure 3 from our prior publication16). UAM granules are initially slightly larger and blue-shifted compared to native lipofuscin, but over significant periods of time in culture, the UAM compact to a smaller size and red-shift in their emission spectrum, becoming much more similar to the size and spectra profile of native lipofuscin.
The OxOS UAM model has been utilized for a range of applications, including the effects of autophagy inducers on preventing and removing lipofuscin granules50 and the effects of lipofuscin on RPE polarity and metabolism16. Further, these granules have been shown to persist in cultured RPE for more than a year, allowing one to study the evolution of lipofuscin spectra, morphology, and behavior over prolonged periods of time16. Other applications for the model include the study of lipofuscin accumulation on complement activation51, lysosomal stability and inflammatory responses52, and mitochondrial compromise53.
There are a few critical steps to this protocol. First, RPE cultures have to be highly differentiated and mature. OxOS feedings should only start after RPE has been on Transwells for at least 8 weeks and has obtained all the qualities characteristic of highly mature RPE (the 5 'P's elaborated by Finnemann et al. – polygonal in morphology, postmitotic, pigmented, polarized, and phagocytic54). Second, OS are highly fragile and should be handled with care during pipetting. Excessive pipetting or freeze-thaws will break apart the OS. Lastly, the ratio of OS to total UV flux exposure is critical for generating OxOS that remain intact but can still induce UAM; this ratio has been refined in this protocol. Too much UV light for a given number of OS causes excessive cross-linking and destroys critical chemical structures in the OS. Too little UV light for a given number of OS will fail to induce UAM in culture.
Modifications to the protocol largely involve altering OxOS feeding duration or concentration. Empirically, 20 feedings over the course of approximately 4 weeks produces a robust amount of UAM accumulation. Feedings beyond this may incrementally add to UAM accumulation, while fewer feedings are likely to cause only sporadic UAM accumulation. The number of feedings can be adjusted based on the purpose, needs, and resources of the experiment and experimenter. In less differentiated RPE cultures (higher passage number, cell lines, or cultures demonstrating epithelial-mesenchymal transition properties), fewer feedings and lower OxOS concentrations are needed for robust UAM induction.
The protocol has several limitations. Utilizing a handheld UV lamp for photo-oxidation of OS prevents precise quantification of the total radiant exposure delivered to OS. However, photo-oxidation of OS using the hand-held lamp was correlated in this study to a much more expensive (but quantitative) UV Crosslinker Device in Figure 1 to help provide parameters for radiant exposure. It is recommended that experimenters alter the duration of UV exposure until the cross-linking/rhodopsin smearing pattern mimics that seen in the UV lamp column of the Western blot in Figure 1C.
A second limitation of the method is that it likely lacks many of the features of lipofuscin accumulation in vivo. Indeed, throughout this protocol, the lipofuscin-like granules are referred to as undigestible autofluroescent material (UAM) to make this distinction clear. Photo-oxidizing OS recapitulates some of the natural chemical cross-linking that happens in photoreceptor outer segments in disease states, but the cross-linking is greatly accelerated and likely more non-specific in the UAM model. Further, despite nearly a month of OxOS feedings, the UAM model still represents an "acute" exposure to lipofuscin-inducing OS, compared to the decades it takes for lipofuscin to accumulate in humans. With a more prolonged lipofuscin accumulation in culture, there may be fewer phenotypic effects. Nevertheless, because UAM granules persist for more than a year in the culture system presented here, there is opportunity to study their long-term effects on RPE health16.
A limitation of the "Total Consumptive Capacity" method presented here to assess for RPE phagocytosis capacity is that it fails to distinguish whether phagocytosis defects may be due to OS binding vs. uptake vs. degradation. Indeed, when the goal of the phagocytosis assay is mechanistic insight into dysfunction of specific steps of the phagocytosis process, traditional OS pulse-chase assays are more appropriate. Measurement of "Total Consumptive Capacity" should be employed when the goal is simply to unambiguously measure the OS phagocytosis competence of the RPE culture under control versus experimental conditions.
In conclusion, a protocol is presented for lipofuscin-like granule accumulation in highly differentiated human RPE cultures. This method merges the physiologic process for lipofuscin accumulation – repeated feedings of photo-oxidized OS – with RPE culture models that have been shown in repeated studies to strongly recapitulate human RPE in vivo. The resulting cultures laden with lipofuscin-like granules can be used for myriad applications, ranging from assessment of lipofuscin effects on RPE biology to tests of small molecules, genes, and signaling pathways important for modulating lipofuscin accumulation.
The authors have nothing to disclose.
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 & Dickson Brown.
100 mm cell culture dish | Corning | #353003 | Others also work |
24-well Transwells | Corning | #3470 | |
Anti-LC3 antibody | Cell Signaling Technology | #4801S | 1:1000 dilution |
Anti-rhodopsin antibody 1D4 | Abcam | #5417 | 1:1000 dilution. Epitope is C-terminal. |
Anti-rhodopsin antibody 4D2 | EnCor Biotech | MCA-B630 | 1:5000 dilution for western blot, 1:1000 dilution for immunostaining. Epitope is N-terminal. |
Autofluorescence quencher | Biotium | #23007 | TrueBlack Lipofuscin Autofluorescence Quencher |
Autofluorescence quencher | Vector Laboratories | SP-8400 | Vector TrueVIEW Autofluorescence Quenching Kit |
Bodipy 493/503 | Life Technologies | D3922 | |
Cholesterol esterase | Life Technologies | From A12216 kit | |
Confocal microscope | Leica | Leica Stellaris SP8 with FALCON module | |
Dark-adapted bovine retinas | W. L. Lawson Company | Dark-adapted bovine retinas (pre-dissected) | Contact information: https://wllawsoncompany.com/ (402) 499-3161 stacy@wllawsoncompany.com |
Filipin | Sigma-Aldrich | F4767 | |
Flow cytometer | Thermo Fisher | Attune NxT | |
Flow cytometer analysis software | BD | FlowJo | |
Handheld UV light | Analytik Jena US | UVGL-55 | |
Human MFG-E8 | Sino Biological | 10853-H08B | |
Human purified Protein S | Enzyme Research Laboratories | HPS | |
Laemmli sample buffer | Thermo Fisher | J60015-AD | |
LDH assay | Promega | J2380 | LDH-Glo Cytotoxicity Assay |
Mounting media | Invitrogen | P36930 | Prolong Gold antifade reagent |
Nile red | Sigma-Aldrich | #72485 | |
Polytetrafluoroethylene-coated slides | Tekdon | Customized | Customized specifications: PTFE mask with the following "cut-outs" - 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. |
Protease inhibitors | Cell Signaling Technology | #5872 | |
Protein assay | Bio-Rad | #5000122 RC DC protein assay | |
TEER electrode | World Precision Instruments | STX3 | |
Trans-epithelial electrical resistance (TEER) meter | World Precision Instruments | EVOM3 | |
Ultraviolet crosslinker device | Analytik Jena US | UVP CL-1000 |