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Retinal degenerative diseases are a leading cause of vision loss and blindness globally, impacting millions. These diseases result from progressive damage to retinal pigment epithelial (RPE) cells for which no curative or palliative treatments exist. Cell therapy, particularly RPE transplantation, has emerged as a promising strategy for vision restoration. This review provides a comprehensive overview of the recent advancements in clinical trials related to RPE transplantation. We discuss scaffold-free and scaffold-based approaches, including RPE cell suspensions and pre-organized RPE monolayers on biomaterial scaffolds. Key considerations, such as the form and preparation of RPE implants, delivery devices, strategies, and biodegradability of scaffolds, are examined. The article also explores the challenges and opportunities in RPE scaffold development, emphasising the crucial need for functional integration, immunomodulation, and long-term biocompatibility to ensure therapeutic efficacy. We also highlight ongoing efforts to optimise RPE transplantation methods and their potential to address retinal degenerative diseases., (© 2024. The Author(s).)

Excessive hydrogen peroxide (H 2 O 2 ) generated during retinal cell metabolic activity could lead to oxidative degeneration of retinal pigment epithelium (RPE) tissue, a specific pathological process implicated in various retinal diseases resulting in blindness, which can be mitigated by taking dietary antioxidants to prevent inflammation and impaired cellular dysfunction. This study tested the hypothesis that damages induced by oxidative stresses can be mitigated by lutein in a H 2 O 2 -challenged model, which was based on an ARPE-19 cell monolayer cultured on three-dimensional (3D)-printed fibrous scaffolds. Pretreating these models with lutein (0.5 μM) for 24 h can significantly lower the oxidative stress and maintain phagocytosis and barrier function. Moreover, lutein can modulate the NLRP3 inflammasome, leading to a ∼40% decrease in the pro-inflammatory cytokine (IL-1β and IL-18) levels. Collectively, this study suggests that the 3D RPE model is an effective tool to examine the capability of lutein to modulate cellular functionalities and regulate NLRP3 inflammation.

Retinal pigment epithelial (RPE) cell dysfunction and death are characteristics of age-related macular degeneration. A promising therapeutic option is RPE cell transplantation. Development of clinical grade stem-cell derived RPE requires efficient in vitro differentiation and purification methods. Enzymatic purification of RPE relies on the relative adherence of RPE and non-RPE cells to the culture plate. However, morphology and adherence of non-RPE cells differ for different stem cell sources. In cases whereby the non-RPE adhered as strongly as RPE cells to the culture plate, enzymatic method of purification is unsuitable. Thus, we hypothesized the need to customize purification strategies for RPE derived from different stem cell sources. We systematically compared five different RPE purification methods, including manual, enzymatic, flow cytometry-based sorting or combinations thereof for parameters including cell throughput, yield, purity and functionality. Flow cytometry-based approach was suitable for RPE isolation from heterogeneous cultures with highly adherent non-RPE cells, albeit with lower yield. Although all five purification methods generated pure and functional RPE, there were significant differences in yield and processing times. Based on the high purity of the resulting RPE and relatively short processing time, we conclude that a combination of enzymatic and manual purification is ideal for clinical applications., (© 2022. The Author(s).)

One common cause of vision loss after retinal detachment surgery is the formation of proliferative and contractile fibrocellular membranes. This aberrant wound healing process is mediated by epithelial-mesenchymal transition (EMT) and hyper-proliferation of retinal pigment epithelial (RPE) cells. Current treatment relies primarily on surgical removal of these membranes. Here, we demonstrate that a bio-functional polymer by itself is able to prevent retinal scarring in an experimental rabbit model of proliferative vitreoretinopathy. This is mediated primarily via clathrin-dependent internalisation of polymeric micelles, downstream suppression of canonical EMT transcription factors, reduction of RPE cell hyper-proliferation and migration. Nuclear factor erythroid 2-related factor 2 signalling pathway was identified in a genome-wide transcriptomic profiling as a key sensor and effector. This study highlights the potential of using synthetic bio-functional polymer to modulate RPE cellular behaviour and offers a potential therapy for retinal scarring prevention., (© 2022. The Author(s).)

Recent trials of retinal pigment epithelium (RPE) transplantation for the treatment of disorders such as age-related macular degeneration have been promising. However, limitations of existing strategies include the uncertain survival of RPE cells delivered by cell suspension and the inherent risk of uncontrolled cell proliferation in the vitreous cavity. Human RPE stem cell-derived RPE (hRPESC-RPE) transplantation can rescue vision in a rat model of retinal dystrophy and survive in the rabbit retina for at least 1 month. The present study placed hRPESC-RPE monolayers under the macula of a non-human primate model for 3 months. The transplant was able to recover in vivo and maintained healthy photoreceptors. Importantly, there was no evidence that subretinally transplanted monolayers underwent an epithelial-mesenchymal transition. Neither gliosis in adjacent retina nor epiretinal membranes were observed. These findings suggest that hRPESC-RPE monolayers are safe and may be a useful source for RPE cell replacement therapy., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Developing successful surgical strategies to deliver cell therapeutics to the back of the eye is an essential pillar to success for stem cell-based applications in blinding retinal diseases. Within this chapter, we have attempted to gather all key considerations during preclinical animal trials.Guidance is provided for choices on animal models, options for immunosuppression, as well as anesthesia. Subsequently we cover surgical strategies for RPE graft delivery, both as suspension as well as in monolayers in small rodents, rabbits, pigs, and nonhuman primate. A detailed account is given in particular on animal variations in vitrectomy and subretinal surgery, which requires a considerable learning curve, when transiting from human to animal. In turn, however, many essential subretinal implantation techniques in large-eyed animals are directly transferrable to human clinical trial protocols.A dedicated subchapter on photoreceptor replacement provides insights on preparation of suspension as well as sheet grafts, to subsequently outline the basics of subretinal delivery via both the transscleral and transvitreal route. In closing, a future outlook on vision restoration through retinal cell-based therapeutics is presented.

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision. Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology. The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Tissue engineered retinal pigment epithelial (RPE) transplantation is a promising cell-based therapy for age-related macular degeneration. The aim of this work is to develop a supportive scaffold with a favorable topography to aid functional RPE monolayer maintenance while being tolerated underneath the retina. To this end, films and electrospun substrates with fiber diameters ranging from 200 to 1000 nm were made of polyethylene terephthalate or poly(L-lactide-co-ε-caprolactone), and then tested using human fetal RPE cells in vitro and transplanted subretinally in rabbits. The results indicated that RPE on both 200 nm fiber variants showed the highest cell densities, adherent monolayers achieved deeper pigmentation, and more uniform hexagonal tight junctions. Facile subretinal implantation of flat 200 nm fiber membranes was achieved by electrospinning them onto a porous rigid-elastic carrier. Spectral-domain optical coherence tomography showed a reattached, slightly thinned retina overlying the implants over 2 weeks observation. Histology demonstrated native RPE variably migrated onto the nanofibers, and a reactive gliosis with some photoreceptor degeneration. In conclusion, scaffolds with 200 nm fiber topography enhanced RPE culture, showed subretinal biocompatibility, and should thus be considered for future cell-based therapies in blinding retinal diseases., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Purpose: To assess tight junction integrity in cultured human foetal retinal pigment epithelium (RPE) after exposure to clinically relevant indocyanine green (ICG) concentrations., Methods: Human foetal RPE was cultured with the Hu & Bok method. The apical compartments of well-differentiated cultures were exposed to 0.125, 0.05 and 0.025 mg/ml ICG with or without 10-min illumination. Vehicle and trypsin/EDTA or EDTA alone served as controls. Three minutes was chosen to mimic surgical exposure time, while 3 h was used for toxicity assays, with subsequent wash out. Cell-cell junctions were studied before and after exposure by phase contrast microscopy and immunofluorescence (ZO-1). Blood-retinal barrier function was measured through transepithelial electrical resistance (TER)., Results: At 6-8 weeks postconfluence, RPE had grown into pigmented hexagonal monolayers with stable TER (435-1227 Ω*cm(2) ). After 3 min ICG exposure, cell morphology remained unchanged, with patchy cell-cell dissociation in positive controls. A continuous ZO-1 signal was detected in ICG groups, whereas trypsin controls showed patchy loss of the tight junction stain. TER had dropped at 1.5 h after 3 min exposure to 22.8 ± 3.1%, compared with 10.2 ± 3.9% in positive controls. Surgical light illumination did not affect TER. After 3 h exposure to 0.05 mg/ml ICG, TER decreased to 58.1 ± 8.3%, while vehicle controls maintained similar levels as prior to exposure (92.7 ± 2.4%). TER recovered in all ICG groups to prior levels within 3 days., Conclusion: Indocyanine green (ICG) exposure induced a transient decrease in transepithelial electrical resistance, despite unaltered tight junction structure., (© 2013 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Transplantation of the retinal pigment epithelium (RPE) is being developed as a cell-replacement therapy for age-related macular degeneration. Human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC)-derived RPE are currently translating toward clinic. We introduce the adult human RPE stem cell (hRPESC) as an alternative RPE source. Polarized monolayers of adult hRPESC-derived RPE grown on polyester (PET) membranes had near-native characteristics. Trephined pieces of RPE monolayers on PET were transplanted subretinally in the rabbit, a large-eyed animal model. After 4 days, retinal edema was observed above the implant, detected by spectral domain optical coherence tomography (SD-OCT) and fundoscopy. At 1 week, retinal atrophy overlying the fetal or adult transplant was observed, remaining stable thereafter. Histology obtained 4 weeks after implantation confirmed a continuous polarized human RPE monolayer on PET. Taken together, the xeno-RPE survived with retained characteristics in the subretinal space. These experiments support that adult hRPESC-derived RPE are a potential source for transplantation therapies.

Purpose: To assess tight junction (TJ) integrity in cultured human fetal retinal pigment epithelium (HFRPE) after exposure to clinically relevant novel vital dyes., Methods: HFRPE floater cells were harvested from RPE primary cultures of 4 donor eyes and seeded on polyester Transwell® for 4-6 weeks. The apical compartments of well-differentiated cultures were exposed to 0.005 mg/ml Coomassie violet R200 (CVR), methyl 2B (M2B) or Orange II. Periods of 30-300 s were chosen to mimic surgical exposure times, while 3 h was used for toxicity assays, with subsequent washout. Cell-cell junctions were studied by immunofluorescence (zonula occludens-1, ZO-1). Transepithelial electrical resistance (TER) was measured regarding blood-retina barrier (BRB) function., Results: At 4-6 weeks after confluence, HFRPE had grown into pigmented hexagonal monolayers with stable TER values (451-1,520 Ω·cm(2)). After 300-second dye treatments, a continuous ZO-1 signal was detected in all vital dye-treated groups 1.5 h after exposure, whereas trypsin controls showed patchy loss of the TJ stain. TER of CVR-, M2B- and Orange-II-treated groups had dropped 1.5 h after exposure to 148 ± 58.4, 162 ± 23.7 and 164 ± 18.5 Ω·cm(2), respectively, compared to 73 ± 44.9 Ω·cm(2) in positive controls. After 3 h of exposure to 0.005 mg/ml vital dyes in thick drops, TER maintained similar levels to those prior to exposure (90.8 ± 4.7% of the original values, 93.8 ± 6.5 and 91.9 ± 3.6%, respectively), together with no difference from the vehicle controls (94.8 ± 6.6%). TER values recovered in all groups to prior levels within 3 days., Conclusion: Novel vital dyes (CVR, M2B and Orange II) caused no outer BRB function alteration., (© 2013 S. Karger AG, Basel.)

Purpose: To develop a surgical technique for the subretinal implantation of cell carriers suitable for the transplantation of cultured retinal pigment epithelium (RPE) in a preclinical animal model., Methods: Cell carriers were porous 10-μm-thick polyester membranes. A custom-made shooter instrument consisted of a 20-gauge metallic nozzle with a nonstick plunger. Fetal human RPE cultures were used for vitality assessment during instrument handling. Transvitreal subretinal implantation of carriers without RPE was performed in 31 rabbits after vitrectomy. Fourteen of 31 implants were encapsulated in gelatin. Fluid turbulence over the implantation site was minimized using a novel infusion cannula. Six rabbits had intravitreal plasmin injections before surgery. SD-OCT in vivo images were obtained after 3, 7, and 14 days, followed by perfusion-fixed histology., Results: Gelatin encapsulation of RPE/polyester implants made cell loss during handling reproducible, compared with 40% of controls showing random, large damage zones. Gelatin implants were ejected smoothly in 12 of 14 surgeries (86%), whereas "naked" implants frequently became trapped with the instrument, which reduced success to 9 of 17 cases (53%). Vitreous remnants after vitrectomy alone complicated subretinal placement of encapsulated and naked implants in 7 of 25 cases (28%). Plasmin-assisted vitrectomy resulted in implant ejection unperturbed by vitreous adhesions in six experiments. SD-OCT and histology demonstrated atraumatic subretinal implant delivery after uncomplicated surgery., Conclusions: A novel shooter instrument design allows for safe and atraumatic transvitreal delivery of hydrogel-encapsulated, ultrathin, rigid-elastic carriers into the subretinal space. The procedure may be used in the future to deliver cultured RPE.

Background: To evaluate the influence of surface topography on the proliferation of the retinal pigment epithelium (RPE) by comparing nanofibrillar and smooth substrates., Methods: Electrospun polyamide nanofibers (EPN) are an engineered surface mimicking native basement membranes. Commonly used plastic (polystyrene, PS) and glass substrates have a smooth topography. All were analyzed by scanning electron microscopy. RPE cultures were established from fetal and adult donors. Growth curves were established on the above substrates. Cell cycle and growth fractions were analyzed with 5-ethynyl-2'-deoxyuridine (EdU) and 4',6-diamidino-2-phenylindole (DAPI)., Results: At a magnification of ×5,000, EPN showed randomly overlapping fibers and pores. The surface of glass was slightly studded yet regular, in contrast to ideally smooth PS. Polygonal cells grew on nanofibers in a colony-like distribution, while randomly spread spindle-shaped cell morphologies were seen on smooth surfaces. This was observed at all donor ages. Initial proliferation rates were higher on EPN, and similar final cell densities were reached in all age groups, compared to an age-related decline on PS. EdU/DAPI revealed faster cell cycles on EPN. Growth fractions were higher and maintained longer on EPN. Observed substrate differences in growth behavior were statistically significant., Conclusion: Surface topography appears to induce distinct RPE proliferation characteristics., (Copyright © 2011 S. Karger AG, Basel.)

[Display omitted] • The first 3D RPE monolayer was used for studying dietary antioxidants. • Phone screen light reduced cell viability and increased oxidative stress levels. • Lutein promoted cell survival by reducing oxidative stress. • Lutein improved barrier and phagocytosis functions of light-damaged RPE monolayer. • The highest protective effects were observed with 0.5 µM lutein. Cumulative light exposure, including electronic device radiation, may induce retinal pigment epithelium (RPE)-related retinal diseases, potentially mitigated by lutein. However, there is limited 3D RPE monolayer model to study the effect of phytochemicals on their activity and action mechanisms in vitro. Here, we present a method using a monolayer RPE tissue model on a 3D-printed polycaprolactone scaffold. Six-hour of phone screen light increased reactive oxygen species (ROS) in RPE cells, leading to oxidative stress, cytotoxicity, and inhibited metabolism. Pre-treating RPE cells (24 h) with 0.5 µM lutein reduced ROS levels and maintained viability under the same light exposure. Additionally, lutein demonstrated protective effects on crucial RPE functions, including phagocytosis and the retinal-blood barrier. This study underscores the importance of optimal lutein content in shielding RPE from phone screen light damage, and our model holds promise for evaluating the efficacy of antioxidants against light radiation. [ABSTRACT FROM AUTHOR]