The video outlines a detailed protocol for preparing and characterizing photo-oxidized outer segments (OS) from retinal cells. It covers the sterilization of slides, treatment with UV light, the resuspension and dilution of OS in culture medium, and assessing lipofuscin accumulation using confocal microscopy.

preparation of photo-oxidized outer segment tips and fragments

Quantifying Retinal Pigment Epithelial Phagocytosis and Lipofuscin Accumulation

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.

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

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

While lipofuscin universally accumulates in the RPE with aging, discerning its toxicity has been difficult. Here, we develop protocols that allow us to develop lipofuscin-like accumulation in highly polarized and mature RPE cultures to help discern lipofuscin's effects on RPE physiology. Studying lipofuscin toxicity in humans is confounded by the fact that lipofuscin decreases as the RPE dies.Animal models of lipofuscin toxicity have had widely disparate results. We have carefully created in vitro models of lipofuscin-like material accumulation to facilitate the study of lipofuscin toxicity. The key to the success of our models is maintaining highly differentiated cultures while inducing lipofuscin genesis via the same process that triggers lipofuscin in vivo, namely phagocytosis of photoreceptor outer segments.Our in vitro protocol is unique in that lipofuscin-like material accumulates in RPE cultures that are highly differentiated to faithfully mimic RPE in vivo. In these circumstances, we found that RPE culture are highly resistant to the lipofuscin accumulation and to toxic effect from lipofuscin. Additionally, in the process of assaying for phenotypes induced by lipofuscin-like accumulation in our RPE cultures, we created a new type of phagocytosis assay called total consumptive capacity.This assay allows us to determine the entire capacity of the RPE to phagocytose outer segments while avoiding some of the confounding interpretations that come with classical pulse-chase phagocytosis assays. We are excited to utilize this model of lipofuscin-like material accumulation to better understand what RPE stressors may take normally inert lipofuscin into a more pathological role in RPE cell deaths.

Watch this Scientific Journal Video about Preparation of Photo-Oxidized Outer Segment Tips and Fragments at JoVE.com

Preparation of Photo-Oxidized Outer Segment Tips and Fragments  

Begin by sterilizing polytetrafluoroethylene, or PTFE-coated slides by immersing them in 70%ethanol for 10 minutes in a biosafety cabinet. Allow the slides to air-dry in a sterile 100-millimeter cell culture dish. Place each slide in 1 new 100-millimeter cell culture dish and add 200 microliters of serum-containing cell culture medium into each rectangle.Then place a handheld 254-nanometer UV light on the 100-millimeter cell culture dish with the bulb directly facing the slide for 20 minutes. Thaw the frozen aliquots of the outer segments, or OS, in a 37-degree Celsius water bath. Then spin the OS at 2, 400G for five minutes at room temperature.Immediately aspirate the supernatant in the biosafety cabinet using a pipette and resuspend the OS in PBS. Now, aspirate the medium from the slides and place up to 500 microliters of the 2 x 10 to the 8th OS per milliliter PBS solution of the 2 x 10 to the 8th OS per milliliter PBS solution on each slide rectangle. Place a handheld 254-nanometer UV light over the cell culture dish with the bulb directly facing the OS for 40 minutes.Collect OS-PBS solution in a 1.5-milliliter tube. Wash the rectangle of the coated slide with 200 to 500 microliter of PBS and collect each wash in the microcentrifuge tube. Then spin down the OS-PBS solution at 2, 400G for five minutes at room temperature.Aspirate the PBS in the biosafety cabinet with a pipetter and resuspend the pellet in 500 microliters of standard medium to be used for the retinal pigment epithelium, or RPE cultures. Place 10 microliters of the suspension in a new microcentrifuge tube. Dilute 50 to 100 times with more cell culture medium, and count the OS with a hemocytometer.Dilute the photo-oxidized OS, or OxOS suspension, at a 5.6 x 10 to the 7th OS per milliliter concentration into a standard RPE medium. Add phagocytosis bridging ligands, which facilitate OS uptake and lipofuscin accumulation. Then aliquot OxOS into single-use stocks and snap-freeze in liquid nitrogen.Thaw OxOS aliquots by diluting the frozen aliquot in 45 microliters of cell culture media before adding it to RPE transwells to induce lipofuscin accumulation. Then characterize the oxidized OS by measuring the OxOS emission spectrum using Lambda scanning on a confocal microscope. Confocal imaging showed that treated OS had increased autofluorescence compared to untreated OS.

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142 visualizações.  University of Michigan, Ann Arbor. Comece esterilizando as lâminas de politetrafluoretileno ou PTFE, imergindo-as em etanol 70% por 10 minutos em um gabinete de biossegurança. Deixe as lâminas secarem ao ar em uma placa estéril de cultura de células de 100 milímetros. Coloque cada lâmina em 1 nova placa de cultura de células de 100 milímetros e adicione 200 microlitros de meio de cultura celular contendo soro em cada retângulo.Em seguida, coloque uma luz UV portátil de 254 nanômetros na placa de cultura de c...