An Automated Visual Psychophysics Method to Measure Visual Function in Swine Preclinical Animal Model.

Purpose: The aim of this study was to develop and validate a test to assess visual function in pigs using the visual psychophysics contrast sensitivity function. Methods: We utilized a touchscreen along with a pellet reward dispenser to train three Göttingen pigs on a visual psychophysics test and determined their contrast sensitivity function. Images with different contrast resolutions were used as visual stimuli and presented against a control image in a two-choice test. Following animals' acclimatization and the first phase of training, the system was arranged such that animals could self-run multiple consecutive trials without human intervention. Results: All animals were trained within a week and remembered the task with 1 day of reinforcement when tested 1 month after the last visual assessment. All trained animals performed well during the trial with minimal screen side bias, especially at contrast threshold above 40%. Conclusions: Göttingen pigs are trainable for a visual psychophysics test and able to self-run the trial without human intervention. Translational Relevance: Contrast sensitivity is one of the key parameters to assess visual function in humans. The possibility of measuring the same parameters in a large animal model allows for a better translation and understanding of drug safety and efficacy in preclinical ophthalmology.

Polarized Desmosome and Hemidesmosome Shedding via Small Extracellular Vesicles is an Early Indicator of Outer Blood-Retina Barrier Dysfunction.

The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles (EVs) from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of small EVs (sEVs) including exosomes, that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral sEVs from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal-side sub-RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of sEV release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in sEV content, including basal-side specific desmosome and hemidesmosome shedding via sEVs. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases (e.g., AMD).

A biologically validated mathematical model for decoding epithelial apical, basolateral, and paracellular electrical properties.

Epithelial tissues form selective barriers to ions, nutrients, waste products, and infectious agents throughout the body. Damage to these barriers is associated with conditions such as celiac disease, cystic fibrosis, diabetes, and age-related macular degeneration. Conventional electrophysiology measurements like transepithelial resistance can quantify epithelial tissue maturity and barrier integrity but are limited in differentiating between apical, basolateral, and paracellular transport pathways. To overcome this limitation, a combination of mathematical modeling, stem cell biology, and cell physiology led to the development of 3 P-EIS, a novel mathematical model and measurement technique. 3 P-EIS employs an intracellular pipette and extracellular electrochemical impedance spectroscopy to accurately measure membrane-specific properties of epithelia, without the constraints of prior models. 3 P-EIS was validated using electronic circuit models of epithelia with known resistances and capacitances, confirming a median error of 19% (interquartile range: 14%-26%) for paracellular and transcellular resistances and capacitances (n = 5). Patient stem cell-derived retinal pigment epithelium tissues were measured using 3 P-EIS, successfully isolating the cellular responses to adenosine triphosphate. 3 P-EIS enhances quality control in epithelial cell therapies and has extensive applicability in drug testing and disease modeling, marking a significant advance in epithelial physiology.NEW & NOTEWORTHY This interdisciplinary paper integrates mathematics, biology, and physiology to measure epithelial tissue's apical, basolateral, and paracellular transport pathways. A key advancement is the inclusion of intracellular voltage recordings using a sharp pipette, enabling precise quantification of relative impedance changes between apical and basolateral membranes. This enhanced electrochemical impedance spectroscopy technique offers insights into epithelial transport dynamics, advancing disease understanding, drug interactions, and cell therapies. Its broad applicability contributes significantly to epithelial physiology research.

Polarized Desmosome and Hemidesmosome Shedding via Exosomes is an Early Indicator of Outer Blood-Retina Barrier Dysfunction.

The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of exosomes that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral exosomes from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal-side sub-RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of exosome release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in exosome content, including basal-side specific desmosome and hemidesmosome shedding via exosomes. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases, (e.g., AMD) and broadly from blood-CNS barriers in other neurodegenerative diseases.

A versatile laser-induced porcine model of outer retinal and choroidal degeneration for preclinical testing.

Over 30 million people worldwide suffer from untreatable vision loss and blindness associated with childhood-onset and age-related eye diseases caused by photoreceptor (PR), retinal pigment epithelium (RPE), and choriocapillaris (CC) degeneration. Recent work suggests that RPE-based cell therapy may slow down vision loss in late stages of age-related macular degeneration (AMD), a polygenic disease induced by RPE atrophy. However, accelerated development of effective cell therapies is hampered by the lack of large-animal models that allow testing safety and efficacy of clinical doses covering the human macula (20 mm2). We developed a versatile pig model to mimic different types and stages of retinal degeneration. Using an adjustable power micropulse laser, we generated varying degrees of RPE, PR, and CC damage and confirmed the damage by longitudinal analysis of clinically relevant outcomes, including analyses by adaptive optics and optical coherence tomography/angiography, along with automated image analysis. By imparting a tunable yet targeted damage to the porcine CC and visual streak - with a structure similar to the human macula - this model is optimal for testing cell and gene therapies for outer retinal diseases including AMD, retinitis pigmentosa, Stargardt, and choroideremia. The amenability of this model to clinically relevant imaging outcomes will facilitate faster translation to patients.

Bioprinted 3D outer retina barrier uncovers RPE-dependent choroidal phenotype in advanced macular degeneration.

Age-related macular degeneration (AMD), a leading cause of blindness, initiates in the outer-blood-retina-barrier (oBRB) formed by the retinal pigment epithelium (RPE), Bruch's membrane, and choriocapillaris. The mechanisms of AMD initiation and progression remain poorly understood owing to the lack of physiologically relevant human oBRB models. To this end, we engineered a native-like three-dimensional (3D) oBRB tissue (3D-oBRB) by bioprinting endothelial cells, pericytes, and fibroblasts on the basal side of a biodegradable scaffold and establishing an RPE monolayer on top. In this 3D-oBRB model, a fully-polarized RPE monolayer provides barrier resistance, induces choriocapillaris fenestration, and supports the formation of Bruch's-membrane-like structure by inducing changes in gene expression in cells of the choroid. Complement activation in the 3D-oBRB triggers dry AMD phenotypes (including subRPE lipid-rich deposits called drusen and choriocapillaris degeneration), and HIF-α stabilization or STAT3 overactivation induce choriocapillaris neovascularization and type-I wet AMD phenotype. The 3D-oBRB provides a physiologically relevant model to studying RPE-choriocapillaris interactions under healthy and diseased conditions.

Transcriptional mapping of the macaque retina and RPE-choroid reveals conserved inter-tissue transcription drivers and signaling pathways.

The macula and fovea comprise a highly sensitive visual detection tissue that is susceptible to common disease processes like age-related macular degeneration (AMD). Our understanding of the molecular determinants of high acuity vision remains unclear, as few model organisms possess a human-like fovea. We explore transcription factor networks and receptor-ligand interactions to elucidate tissue interactions in the macula and peripheral retina and concomitant changes in the underlying retinal pigment epithelium (RPE)/choroid. Poly-A selected, 100 bp paired-end RNA-sequencing (RNA-seq) was performed across the macular/foveal, perimacular, and temporal peripheral regions of the neural retina and RPE/choroid tissues of four adult Rhesus macaque eyes to characterize region- and tissue-specific gene expression. RNA-seq reads were mapped to both the macaque and human genomes for maximum alignment and analyzed for differential expression and Gene Ontology (GO) enrichment. Comparison of the neural retina and RPE/choroid tissues indicated distinct, contiguously changing gene expression profiles from fovea through perimacula to periphery. Top GO enrichment of differentially expressed genes in the RPE/choroid included cell junction organization and epithelial cell development. Expression of transcriptional regulators and various disease-associated genes show distinct location-specific preference and retina-RPE/choroid tissue-tissue interactions. Regional gene expression changes in the macaque retina and RPE/choroid is greater than that found in previously published transcriptome analysis of the human retina and RPE/choroid. Further, conservation of human macula-specific transcription factor profiles and gene expression in macaque tissues suggest a conservation of programs required for retina and RPE/choroid function and disease susceptibility.

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.

Cell-autonomous lipid-handling defects in Stargardt iPSC-derived retinal pigment epithelium cells.

Stargardt retinopathy is an inherited form of macular degeneration caused by mutations in gene ABCA4 and characterized by the accumulation of lipid-rich deposits in the retinal pigment epithelium (RPE), RPE atrophy, and photoreceptor cell death. Inadequate mechanistic insights into pathophysiological changes occurring in Stargardt RPE have hindered disease treatments. Here, we show that ABCA4 knockout and induced pluripotent stem cell-derived RPE (STGD1-iRPE) from patients with Stargardt differentiate normally but display intracellular lipid and ceramide deposits reminiscent of the disease phenotype. STGD1-iRPE also shows defective photoreceptor outer segment (POS) processing and reduced cathepsin B activity-indicating higher lysosomal pH. Lipid deposits in STGD1-iRPE are lowered by increasing the activity of ABCA1, a lipid transporter, and ABCA4 ortholog. Our work suggests that ABCA4 is involved in POS and lipid handling in RPE cells and provides guidance for ongoing gene therapy approaches to target both RPE and photoreceptor cells for an effective treatment.

Oxidation of DJ-1 Cysteines in Retinal Pigment Epithelium Function.

The retina and RPE cells are regularly exposed to chronic oxidative stress as a tissue with high metabolic demand and ROS generation. DJ-1 is a multifunctional protein in the retina and RPE that has been shown to protect cells from oxidative stress in several cell types robustly. Oxidation of DJ-1 cysteine (C) residues is important for its function under oxidative conditions. The present study was conducted to analyze the impact of DJ-1 expression changes and oxidation of its C residues on RPE function. Monolayers of the ARPE-19 cell line and primary human fetal RPE (hfRPE) cultures were infected with replication-deficient adenoviruses to investigate the effects of increased levels of DJ-1 in these monolayers. Adenoviruses carried the full-length human DJ-1 cDNA (hDJ) and mutant constructs of DJ-1, which had all or each of its three C residues individually mutated to serine (S). Alternatively, endogenous DJ-1 levels were decreased by transfection and transduction with shPARK7 lentivirus. These monolayers were then assayed under baseline and low oxidative stress conditions. The results were analyzed by immunofluorescence, Western blot, RT-PCR, mitochondrial membrane potential, and viability assays. We determined that decreased levels of endogenous DJ-1 levels resulted in increased levels of ROS. Furthermore, we observed morphological changes in the mitochondria structure of all the RPE monolayers transduced with all the DJ-1 constructs. The mitochondrial membrane potential of ARPE-19 monolayers overexpressing all DJ-1 constructs displayed a significant decrease, while hfRPE monolayers only displayed a significant decrease in their ΔΨm when overexpressing the C2S mutation. Viability significantly decreased in ARPE-19 cells transduced with the C53S construct. Our data suggest that the oxidation of C53 is crucial for regulating endogenous levels of ROS and viability in RPE cells.

Systemic immunosuppression promotes survival and integration of subretinally implanted human ESC-derived photoreceptor precursors in dogs.

Regenerative therapies aimed at replacing photoreceptors are a promising approach for the treatment of otherwise incurable causes of blindness. However, such therapies still face significant hurdles, including the need to improve subretinal delivery and long-term survival rate of transplanted cells, and promote sufficient integration into the host retina. Here, we successfully delivered in vitro-derived human photoreceptor precursor cells (PRPCs; also known as immature photoreceptors) to the subretinal space of seven normal and three rcd1/PDE6B mutant dogs with advanced inherited retinal degeneration. Notably, while these xenografts were rejected in dogs that were not immunosuppressed, transplants in most dogs receiving systemic immunosuppression survived up to 3-5 months postinjection. Moreover, differentiation of donor PRPCs into photoreceptors with synaptic pedicle-like structures that established contact with second-order neurons was enhanced in rcd1/PDE6B mutant dogs. Together, our findings set the stage for evaluating functional vision restoration following photoreceptor replacement in canine models of inherited retinal degeneration.

Single-cell-resolution map of human retinal pigment epithelium helps discover subpopulations with differential disease sensitivity.

Regional phenotypic and functional differences in the retinal pigment epithelium (RPE) monolayer have been suggested to account for regional susceptibility in ocular diseases such as age-related macular degeneration (AMD), late-onset retinal degeneration (L-ORD), and choroideremia (CHM). However, a comprehensive description of human topographical RPE diversity is not yet available, thus limiting the understanding of regional RPE diversity and degenerative disease sensitivity in the eye. To develop a complete morphometric RPE map of the human eye, artificial intelligence­based software was trained to recognize, segment, and analyze RPE borders. Five statistically different, concentric RPE subpopulations (P1 to P5) were identified using cell area as a parameter, including a subpopulation (P4) with cell area comparable to that of macular cells in the far periphery of the eye. This work provides a complete reference map of human RPE subpopulations and their location in the eye. In addition, the analysis of cadaver non-AMD and AMD eyes and ultra-widefield fundus images of patients revealed differential vulnerability of the five RPE subpopulations to different retinal diseases.

Leukocyte chemotactic receptor Fpr1 protects against aging-related posterior subcapsular cataract formation.

Cataracts are a common consequence of aging; however, pathogenesis remains poorly understood. Here, we observed that after 3 months of age mice lacking the G protein-coupled leukocyte chemotactic receptor Fpr1 (N-formyl peptide receptor 1) began to develop bilateral posterior subcapsular cataracts that progressed to lens rupture and severe degeneration, without evidence of either systemic or local ocular infection or inflammation. Consistent with this, Fpr1 was detected in both mouse and human lens in primary lens epithelial cells (LECs), the only cell type present in the lens; however, expression was confined to subcapsular LECs located along the anterior hemispheric surface. To maximize translucency, LECs at the equator proliferate and migrate posteriorly, then differentiate into lens fiber cells by nonclassical apoptotic signaling, which results in loss of nuclei and other organelles, including mitochondria which are a rich source of endogenous N-formyl peptides. In this regard, denucleation and posterior migration of LECs were abnormal in lenses from Fpr1-/- mice, and direct stimulation of LECs with the prototypic N-formyl peptide agonist fMLF promoted apoptosis. Thus, Fpr1 is repurposed beyond its immunoregulatory role in leukocytes to protect against cataract formation and lens degeneration during aging.

Toll-like Receptor 2 Facilitates Oxidative Damage-Induced Retinal Degeneration.

Retinal degeneration is a form of neurodegenerative disease and is the leading cause of vision loss globally. The Toll-like receptors (TLRs) are primary components of the innate immune system involved in signal transduction. Here we show that TLR2 induces complement factors C3 and CFB, the common and rate-limiting factors of the alternative pathway in both retinal pigment epithelial (RPE) cells and mononuclear phagocytes. Neutralization of TLR2 reduces opsonizing fragments of C3 in the outer retina and protects photoreceptor neurons from oxidative stress-induced degeneration. TLR2 deficiency also preserves tight junction expression and promotes RPE resistance to fragmentation. Finally, oxidative stress-induced formation of the terminal complement membrane attack complex and Iba1+ cell infiltration are strikingly inhibited in the TLR2-deficient retina. Our data directly implicate TLR2 as a mediator of retinal degeneration in response to oxidative stress and present TLR2 as a bridge between oxidative damage and complement-mediated retinal pathology.

Retinal Pigment Epithelium Replacement Therapy for Age-Related Macular Degeneration: Are We There Yet?

Pluripotent stem cells (PSCs) are a potential replacement tissue source for degenerative diseases. Age-related macular degeneration (AMD) is a blinding disease triggered by degeneration of the retinal pigment epithelium (RPE), a monolayer tissue that functionally supports retinal photoreceptors. Recently published clinical and preclinical studies have tested PSC-derived RPE as a potential treatment for AMD. Multiple approaches have been used to manufacture RPE cells, to validate them functionally, to confirm their safety profile, and to deliver them to patients either as suspension or as a monolayer patch. Since most of these studies are at an early regulatory approval stage, the primary outcome has been to determine the safety of RPE transplants in patients. However, preliminary signs of efficacy were observed in a few patients. Here, we review the current progress in the PSC-derived RPE transplantation field and provide a comparative assessment of various approaches under development as potential therapeutics for AMD.

High-yield, automated intracellular electrophysiology in retinal pigment epithelia.

BACKGROUND: Recent advancements with induced pluripotent stem cell-derived (iPSC) retinal pigment epithelium (RPE) have made disease modeling and cell therapy for macular degeneration feasible. However, current techniques for intracellular electrophysiology - used to validate epithelial function - are painstaking and require manual skill; limiting experimental throughput. NEW METHOD: A five-stage algorithm, leveraging advances in automated patch clamping, systematically derived and optimized, improves yield and reduces skill when compared to conventional, manual techniques. RESULTS: The automated algorithm improves yield per attempt from 17% (manually, n = 23) to 22% (automated, n = 120) (chi-squared, p = 0.004). Specifically for RPE, depressing the local cell membrane by 6 µm and electroporating (buzzing) just prior to this depth (5 µm) maximized yield. COMPARISON WITH EXISTING METHOD: Conventionally, intracellular epithelial electrophysiology is performed by manually lowering a pipette with a micromanipulator, blindly, towards a monolayer of cells and spontaneously stopping when the magnitude of the instantaneous measured membrane potential decreased below a predetermined threshold. The new method automatically measures the pipette tip resistance during the descent, detects the cell surface, indents the cell membrane, and briefly buzzes to electroporate the membrane while descending, overall achieving a higher yield than conventional methods. CONCLUSIONS: This paper presents an algorithm for high-yield, automated intracellular electrophysiology in epithelia; optimized for human RPE. Automation reduces required user skill and training while, simultaneously, improving yield. This algorithm could enable large-scale exploration of drug toxicity and physiological function verification for numerous kinds of epithelia.

Regulation of phagolysosomal activity by miR-204 critically influences structure and function of retinal pigment epithelium/retina.

MicroRNA-204 (miR-204) is expressed in pulmonary, renal, mammary and eye tissue, and its reduction can result in multiple diseases including cancer. We first generated miR-204-/- mice to study the impact of miR-204 loss on retinal and retinal pigment epithelium (RPE) structure and function. The RPE is fundamentally important for maintaining the health and integrity of the retinal photoreceptors. miR-204-/- eyes evidenced areas of hyper-autofluorescence and defective photoreceptor digestion, along with increased microglia migration to the RPE. Migratory Iba1+ microglial cells were localized to the RPE apical surface where they participated in the phagocytosis of photoreceptor outer segments (POSs) and contributed to a persistent build-up of rhodopsin. These structural, molecular and cellular outcomes were accompanied by decreased light-evoked electrical responses from the retina and RPE. In parallel experiments, we suppressed miR-204 expression in primary cultures of human RPE using anti-miR-204. In vitro suppression of miR-204 in human RPE similarly showed abnormal POS clearance and altered expression of autophagy-related proteins and Rab22a, a regulator of endosome maturation. Together, these in vitro and in vivo experiments suggest that the normally high levels of miR-204 in RPE can mitigate disease onset by preventing generation of oxidative stress and inflammation originating from intracellular accumulation of undigested photoreactive POS lipids. More generally, these results implicate RPE miR-204-mediated regulation of autophagy and endolysosomal interaction as a critical determinant of normal RPE/retina structure and function.

Longitudinal adaptive optics fluorescence microscopy reveals cellular mosaicism in patients.

The heterogeneity of individual cells in a tissue has been well characterized, largely using ex vivo approaches that do not permit longitudinal assessments of the same tissue over long periods of time. We demonstrate a potentially novel application of adaptive optics fluorescence microscopy to visualize and track the in situ mosaicism of retinal pigment epithelial (RPE) cells directly in the human eye. After a short, dynamic period during which RPE cells take up i.v.-administered indocyanine green (ICG) dye, we observed a remarkably stable heterogeneity in the fluorescent pattern that gradually disappeared over a period of days. This pattern could be robustly reproduced with a new injection and follow-up imaging in the same eye out to at least 12 months, which enabled longitudinal tracking of RPE cells. Investigation of ICG uptake in primary human RPE cells and in a mouse model of ICG uptake alongside human imaging corroborated our findings that the observed mosaicism is an intrinsic property of the RPE tissue. We demonstrate a potentially novel application of fluorescence microscopy to detect subclinical changes to the RPE, a technical advance that has direct implications for improving our understanding of diseases such as oculocutaneous albinism, late-onset retinal degeneration, and Bietti crystalline dystrophy.

Clinical-grade stem cell-derived retinal pigment epithelium patch rescues retinal degeneration in rodents and pigs.

Considerable progress has been made in testing stem cell-derived retinal pigment epithelium (RPE) as a potential therapy for age-related macular degeneration (AMD). However, the recent reports of oncogenic mutations in induced pluripotent stem cells (iPSCs) underlie the need for robust manufacturing and functional validation of clinical-grade iPSC-derived RPE before transplantation. Here, we developed oncogenic mutation-free clinical-grade iPSCs from three AMD patients and differentiated them into clinical-grade iPSC-RPE patches on biodegradable scaffolds. Functional validation of clinical-grade iPSC-RPE patches revealed specific features that distinguished transplantable from nontransplantable patches. Compared to RPE cells in suspension, our biodegradable scaffold approach improved integration and functionality of RPE patches in rats and in a porcine laser-induced RPE injury model that mimics AMD-like eye conditions. Our results suggest that the in vitro and in vivo preclinical functional validation of iPSC-RPE patches developed here might ultimately be useful for evaluation and optimization of autologous iPSC-based therapies.

Validation of iPS Cell-Derived RPE Tissue in Animal Models.

Previous work suggests that replacing diseased Retinal Pigment Epithelium (RPE) with a healthy autologous RPE sheet can provide vision rescue for AMD patients. We differentiated iPSCs into RPE using a directed differentiation protocol. RPE cells at the immature RPE stage were purified and seeded onto either electrospun poly(lactic-co-glycolic acid) (PLGA) scaffolds or non-biodegradable polyester cell culture inserts and compared the two tissues. In vitro, PLGA and polyester substrates produced functionally similar results. Following in vitro evaluation, we tested RPE tissue in animal models for safety and function. Safety studies were conducted in RNU rats using an injection composed of intact cells and homogenized scaffolds. To assess function and develop surgical procedures, the tissues were implanted into an acute RPE injury model pig eye and evaluated using optical coherence tomography (OCT), multifocal ERG (mfERG), and histology. Subretinal injection studies in rats demonstrated safety of the implant. Biodegradability and biocompatibility data from a pig model demonstrated that PLGA scaffold is safe, with the added benefit of being resorbed by the body over time, leaving no foreign material in the eye. We confirmed that biodegradable substrates provide suitable support for RPE maturation and transplantation.