Fetal Human Retinal Pigment Epithelium Research Articles

Modulation of Hypoxia-Inducible Factors and Vascular Endothelial Growth Factor Expressions by Superfood Camu-Camu (Myrciaria dubia) Treatment in ARPE-19 and Fetal Human RPE Cells.

Anti-vascular endothelial growth factor (anti-VEGF) therapy via intravitreal injection is an effective treatment for patients with abnormal ocular neovascularization, such as age-related macular degeneration (AMD) and diabetic macular edema (DME). However, prolonged and frequent anti-VEGF treatment is associated with a risk of local and systemic adverse events, including geographic atrophy, cerebrovascular disease, and death. Furthermore, some patients do not adequately respond to anti-VEGF therapy. Hypoxia-inducible factor (HIF) is a transcription factor that controls the expression of hypoxia-responsive genes involved in angiogenesis, inflammation, and metabolism. The HIF/VEGF pathway plays an important role in neovascularization, and the inhibition of HIF activation could be an effective biomolecular target for neovascular diseases. The demand for disease prevention or treatment using functional foods such as superfoods has increased in recent years. Few reports to date have focused on the antineovascular effects of superfoods in the retinal pigment epithelium (RPE). In light of the growing demand for functional foods, we aimed to find novel HIF inhibitors from superfoods worked in RPE cells, which could be an adjuvant for anti-VEGF therapy. Seven superfoods were examined to identify novel HIF inhibitor candidates using luciferase assay screening. We used the human RPE cell line ARPE-19 and fetal human RPE (fhRPE) to investigate the biomolecular actions of novel HIF inhibitors using quantitative PCR and western blotting. Under CoCl2-induced pseudohypoxic condition and 1% oxygen hypoxic incubation, camu-camu (Myrciaria dubia) showed HIF inhibitory effects determined by luciferase assays. Camu-camu downregulated HIF-1α and VEGFA mRNA expressions in a concentration-dependent manner. Camu-camu also inhibited HIF-1α protein expressions, and its inhibitory effect was greater than that of vitamin C, which is present at high levels in camu-camu. The camu-camu extract suppressed the activation of HIF and VEGF in RPE cells. This could assist anti-VEGF therapy in patients with abnormal ocular neovascularization.

Receptor mediated disruption of retinal pigment epithelium function in acute glycated-albumin exposure

Diabetic macular edema (DME) is a major cause of visual impairment. Although DME is generally believed to be a microvascular disease, dysfunction of the retinal pigment epithelium (RPE) can also contribute to its development. Advanced glycation end-products (AGE) are thought to be one of the key factors involved in the pathogenesis of diabetes in the eye, and we have previously demonstrated a rapid breakdown of RPE function following glycated-albumin (Glyc-alb, a common AGE mimetic) administration in monolayer cultures of fetal human RPE cells. Here we present new evidence that this response is attributed to apically oriented AGE receptors (RAGE). Moreover, time-lapse optical coherence tomography in Dutch-belted rabbits 48 h post intravitreal Glyc-alb injections demonstrated a significant decrease in RPE-mediated fluid resorption in vivo. In both the animal and tissue culture models, the response to Glyc-alb was blocked by the relatively selective RAGE antagonist, FPS-ZM1 and was also inhibited by ZM323881, a relatively selective vascular endothelial growth factor receptor 2 (VEGF-R2) antagonist. Our data establish that the Glyc-alb-induced breakdown of RPE function is mediated via specific RAGE and VEGF-R2 signaling both in vitro and in vivo. These results are consistent with the notion that the RPE is a key player in the pathogenesis of DME.

Porous Poly(ε-Caprolactone) Scaffolds for Retinal Pigment Epithelium Transplantation

Retinal pigment epithelium (RPE) transplantation is a promising strategy for the treatment of dry age-related macular degeneration (AMD). However, previous attempts at subretinal RPE cell transplantation have experienced limited success due to poor adhesion, organization, and function on aged or diseased Bruch's membrane. Instead, cell-based strategies may benefit from a synthetic scaffold that mimics the functions of healthy Bruch's membrane to promote the formation of a functional RPE monolayer while maintaining metabolite exchange between the vasculature and outer retina. This study evaluated the behavior of human RPE on nanopatterned porous poly(ε-caprolactone) (PCL) film as a potential scaffold for therapeutic transplantation. Fetal human RPE (fhRPE) was cultured on porous PCL, nonporous PCL, or Costar porous polyester transwells for up to 8 weeks and assessed using light microscopy, fluorescent microscopy, transepithelial resistance, quantitative PCR, ELISAs, and phagocytosis assays. fhRPE on porous PCL displayed improved markers of maturity and function compared with both porous polyester transwells and nonporous PCL, including pigmentation, increased cell density, superior barrier function, up-regulation of RPE-specific genes, and polarized growth factor secretion. This study indicates that porous PCL is an attractive scaffold for RPE transplantation. In addition to being biocompatible with the subretinal space, porous PCL also allows for trans-scaffold metabolite transport and significantly improves RPE cell behavior compared to nonporous PCL or porous polyester transwells.

Differential effects of Best disease causing missense mutations on bestrophin-1 trafficking

Mutations in bestrophin-1 (Best1) cause Best vitelliform macular dystrophy (BVMD), a dominantly inherited retinal degenerative disease. Best1 is a homo-oligomeric anion channel localized to the basolateral surface of retinal pigment epithelial (RPE) cells. A number of Best1 mutants mislocalize in Madin-Darby canine kidney (MDCK) cells. However, many proteins traffic differently in MDCK and RPE cells, and MDCK cells do not express endogenous Best1. Thus, effects of Best1 mutations on localization in MDCK cells may not translate to RPE cells. To determine whether BVMD causing mutations affect Best1 localization, we compared localization and oligomerization of Best1 with Best1 mutants V9M, W93C, and R218C. In MDCK cells, Best1 and Best1(R218C) were basolaterally localized. Best1(W93C) and Best1(V9M) accumulated in cells. In cultured fetal human retinal pigment epithelium cells (fhRPE) expressing endogenous Best1, Best1(R218C) and Best1(W93C) were basolateral. Best1(V9M) was intracellular. All three mutants exhibited similar fluorescence resonance energy transfer (FRET) efficiencies to, and co-immunoprecipitated with Best1, indicating unimpaired oligomerization. When human Best1 was expressed in RPE in mouse eyes it was basolaterally localized. However, Best1(V9M) accumulated in intracellular compartments in mouse RPE. Co-expression of Best1 and Best1(W93C) in MDCK cells resulted in basolateral localization of both Best1 and Best1(W93C), but co-expression of Best1 with Best1(V9M) resulted in mislocalization of both proteins. We conclude that different mutations in Best1 cause differential effects on its localization and that this effect varies with the presence or absence of wild-type (WT) Best1. Furthermore, MDCK cells can substitute for RPE when examining the effects of BVMD causing mutations on Best1 localization if co-expressed with WT Best1.

Subretinal Delivery of Ultrathin Rigid-Elastic Cell Carriers Using a Metallic Shooter Instrument and Biodegradable Hydrogel Encapsulation

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. 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. 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. 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.

Human Retinal Pigment Epithelium Cells as Functional Models for the RPE In Vivo

The two most commonly used in vitro models of the retinal pigment epithelium (RPE) are fetal human RPE (fhRPE) and ARPE-19 cells; however, studies of their barrier properties have produced contradictory results. To compare their utility as RPE models, their morphologic and functional characteristics were analyzed. Monolayers of both cell types were grown on permeable membrane filters. Barrier function and cellular morphology were assessed by transepithelial resistance (TER) measurements and immunohistochemistry. Protein expression was evaluated by immunoblotting and ELISA assays, and retinoid metabolism characterized by HPLC. Both cultures developed tight junctions. However, only the fhRPE cells were pigmented, uniform in size and shape, expressed high levels of RPE markers, metabolized all-trans retinal, and developed high TER (>400 Ωcm(2)). The net secretion of pigment-epithelium-derived factor (PEDF) was directed apically in both cultures, but fhRPE cells exhibited secretion rates a thousand-fold greater than in ARPE-19 cells. The net secretion of vascular endothelial growth factor (VEGF) was significantly higher in fhRPE cultures and the direction of this secretion was basolateral; while net secretion was apical in ARPE-19 cells. In fresh media, VEGF-E reduced TER in both cultures; however, in conditioned media fhRPE cells did not respond to VEGF-E administration, but retreatment of the conditioned media with anti-PEDF antibodies allowed fhRPE cells to fully respond to VEGF-E. Properties of fhRPE cells align with a functionally normal RPE in vivo, while ARPE-19 cells resemble a pathologic or aged RPE. These results suggest a utility for both cell types in understanding distinct, particular aspects of RPE function.

MicroRNA‐204/211 alters epithelial physiology

MicroRNA (miRNA) expression in fetal human retinal pigment epithelium (hfRPE), retina, and choroid were pairwise compared to determine those miRNAs that are enriched by 10-fold or more in each tissue compared with both of its neighbors. miRs-184, 187, 200a/200b, 204/211, and 221/222 are enriched in hfRPE by 10- to 754-fold compared with neuroretina or choroid (P<0.05). Five of these miRNAs are enriched in RPE compared with 20 tissues throughout the body and are 10- to 20,000-fold more highly expressed (P<0.005). miR-204 and 211 are the most highly expressed in the RPE. In addition, expression of miR-204/211 is significantly lower in the NCI60 tumor cell line panel compared with that in 13 normal tissues, suggesting the progressive disruption of epithelial barriers and increased proliferation. We demonstrated that TGF-beta receptor 2 (TGF-betaR2) and SNAIL2 are direct targets of miR-204 and that a reduction in miR-204 expression leads to reduced expression of claudins 10, 16, and 19 (message/protein) consistent with our observation that anti-miR-204/211 decreased transepithelial resistance by 80% and reduced cell membrane voltage and conductance. The anti-miR-204-induced decrease in Kir7.1 protein levels suggests a signaling pathway that connects TGF-betaR2 and maintenance of potassium homeostasis. Overall, these data indicate a critical role for miR-204/211 in maintaining epithelial barrier function and cell physiology.

Integrin α5β1 Mediates Attachment, Migration, and Proliferation in Human Retinal Pigment Epithelium: Relevance for Proliferative Retinal Disease

The aim of this study was to determine the expression and localization of integrin alpha5beta1 in human retinal pigment epithelium (RPE) and its ability to modulate RPE cell attachment, proliferation, migration, and F-actin cytoskeleton distribution. Expression and localization of alpha5beta1 were analyzed on human RPE by immunoblot/immunofluorescence. Polarized secretion of fibronectin was measured. RPE attachments to different substrates were determined using cell attachment screening kits. BrdU incorporation and wound-healing assays were used to test hfRPE proliferation and migration. F-actin cytoskeleton was visualized with phalloidin. Integrin alpha5beta1 was detected in native adult and fetal human RPE. The alpha5-subunit is predominantly localized at the apical membrane of hfRPE, whereas the beta1-subunit is uniformly detected at the apical/basolateral membranes. The authors also found that hfRPE cultures secrete significant amounts of fibronectin to the apical bath. JSM6427, a specific integrin alpha5beta1 antagonist, significantly inhibited hfRPE cell attachment to fibronectin, but not laminin, or collagen I or IV. JSM6427 also showed a strong inhibitory effect on bFGF, PDGF-BB, and serum-induced cell migration and proliferation. Furthermore, JSM6427 induced significant disruption of the F-actin cytoskeleton of dividing RPE cells but had no effect on quiescent cells. The apical localization of alpha5beta1 and the secretion of fibronectin to the apical bath suggest the presence of an autocrine loop that can guide the migration of RPE. The strong inhibitory effects of JSM6427 on human RPE cell attachment, proliferation, and migration is probably mediated by F-actin cytoskeletal disruption in proliferating cells and suggests a potential clinical use of this compound in proliferative retinopathies.

CO2-induced ion and fluid transport in human retinal pigment epithelium

In the intact eye, the transition from light to dark alters pH, [Ca2+], and [K] in the subretinal space (SRS) separating the photoreceptor outer segments and the apical membrane of the retinal pigment epithelium (RPE). In addition to these changes, oxygen consumption in the retina increases with a concomitant release of CO2 and H2O into the SRS. The RPE maintains SRS pH and volume homeostasis by transporting these metabolic byproducts to the choroidal blood supply. In vitro, we mimicked the transition from light to dark by increasing apical bath CO2 from 5 to 13%; this maneuver decreased cell pH from 7.37 ± 0.05 to 7.14 ± 0.06 (n = 13). Our analysis of native and cultured fetal human RPE shows that the apical membrane is significantly more permeable (≈10-fold; n = 7) to CO2 than the basolateral membrane, perhaps due to its larger exposed surface area. The limited CO2 diffusion at the basolateral membrane promotes carbonic anhydrase–mediated HCO3 transport by a basolateral membrane Na/nHCO3 cotransporter. The activity of this transporter was increased by elevating apical bath CO2 and was reduced by dorzolamide. Increasing apical bath CO2 also increased intracellular Na from 15.7 ± 3.3 to 24.0 ± 5.3 mM (n = 6; P < 0.05) by increasing apical membrane Na uptake. The CO2-induced acidification also inhibited the basolateral membrane Cl/HCO3 exchanger and increased net steady-state fluid absorption from 2.8 ± 1.6 to 6.7 ± 2.3 µl × cm−2 × hr−1 (n = 5; P < 0.05). The present experiments show how the RPE can accommodate the increased retinal production of CO2 and H2O in the dark, thus preventing acidosis in the SRS. This homeostatic process would preserve the close anatomical relationship between photoreceptor outer segments and RPE in the dark and light, thus protecting the health of the photoreceptors.

Impaired RPE survival on aged submacular human Bruch's membrane

Resurfacing of diseased or iatrogenically damaged Bruch's membrane with healthy retinal pigment epithelium (RPE) has been proposed as adjunctive treatment for age-related macular degeneration (AMD). The purpose of this study was to determine whether cultured fetal human RPE cells can attach and differentiate on aged submacular human Bruch's membrane. Bruch's membrane was debrided to expose native RPE basement membrane, the superficial inner collagenous layer directly below the RPE basement membrane, or the deep inner collagenous layer. These are three surfaces that transplanted RPE cells will encounter in situ. Approximately 3146 cultured fetal RPE cells mm −2 were seeded onto these three surfaces and grown in organ culture for 1, 7, or 14 days. Explants were bisected and examined histologically or analyzed with a scanning electron microscope. RPE nuclear density was measured on stained sections. Morphology and cell density were compared to cells seeded onto bovine corneal endothelial cell-extracellular matrix (BCE-ECM). In situ submacular RPE nuclear density was also measured in tissue sections of donor eyes ranging from 18 weeks gestation to 88 years of age to determine the effect of age on RPE density. Compared to cells seeded onto BCE-ECM at similar density, RPE cell coverage and cellular morphology on aged submacular human Bruch's membrane was poor at all time points. In contrast to cells on BCE-ECM, RPE cell density on Bruch's membrane decreased with time. In general, cell morphology on all three Bruch's membrane surfaces worsened by day-7 compared to day-1. Although some cells were more pigmented on RPE basement membrane and the deep inner collagenous layer at day-7, poor cellular morphology indicated the remaining cells were not well differentiated. At day-14, the cells were uniform and cuboidal on BCE-ECM, with cell density similar to that at day-7 and similar to in situ density of young donors (<age 30 years). The morphology of cells on Bruch's membrane was variable, and the nuclear density declined over time. A Bruch's membrane explant from a donor with large soft drusen showed the poorest resurfacing at day-7 in organ culture. These data indicate that aged submacular human Bruch's membrane does not support transplanted RPE survival and differentiation readily. The formation of localized RPE defects, cell death, and worsening cellular morphology on aged Bruch's membrane indicates that modification of Bruch's membrane may be necessary to prevent graft failure in AMD patients receiving RPE transplants.

Retinal Pigment Epithelial Cells Secrete and Respond to Hepatocyte Growth Factor

Hepatocyte growth factor (HGF) is normally expressed by mesenchymal cells while its receptor, c-Met, is expressed in epithelial cells. Since HGF is critically involved in epithelial-mesenchyme interactions and the retinal pigment epithelium (RPE) is present at the interface between the retina and choroid, this study was initiated to determine whether the RPE expresses or responds to HGFin vitro.Cultured adult and fetal human RPE expressed mRNA for HGF and c-Met by RT-PCR. ELISA assay demonstrated the secretion of HGF into RPE culture supernatants. Tyrosine phosphorylation of c-Met was constitutively found in 72 hour RPE cultures and could be rapidly induced in serum-starved cells by concentrated RPE supernatants. HGF was mitogenic for cultured RPE (100 ng/ml.) and stimulated their chemotaxis (maximal response at 50 ng/ml). RPE are one of only a very limited number of epithelia that express both HGF and its receptor, suggesting the possibility of an autocrine action for this growth factor.

In vitro Transplantation of Fetal Human Retinal Pigment Epithelial Cells onto Human Cadaver Bruch's Membrane

Retinal pigment epithelium transplantation has been proposed as adjunctive treatment for age-related macular degeneration following surgical excision of choroidal neovascular membranes. The goal of this study was to develop a model to evaluate retinal pigment epithelium transplantation onto human Bruch's membrane in vitro. We investigated the ability of cultured fetal human retinal pigment epithelium to colonize human cadaver Bruch's membrane, determined the incubation time needed to form a monolayer and to exhibit apical microvilli and tight junctions, and assessed the production of basement membrane. Freshly enucleated (less than 48 hours old) human eyes were cut through the pars plana, and the anterior segment, vitreous, and retina were removed. The native retinal pigment epithelium was debrided with a surgical sponge. Bruch's membrane and choroid at the macula were trephined with a 7.0 mm diameter trephine and then incubated with 1/2 ml of Dulbecco's modified Eagle's medium +15% fetal calf serum+basic fibroblast growth factor (1 ng ml−1), and fetal human retinal pigment epithelium at a concentration of 242,000 cells ml−1. Specimens were incubated for 1, 4, 6, 8, 12, or 24 hours. The specimens were fixed in half strength Karnovsky's fixative, processed, and analysed with scanning and transmission electron microscopy. The retinal pigment epithelium covered the debrided macular specimens to different degrees at different incubation times. After 1 hour, the cells started to attach and flatten (median percent coverage: 78%). The extent of Bruch's membrane coverage by fetal retinal pigment epithelium varied greatly between specimens. After 4–6 hours, the cells covered the entire debrided surface in a monolayer (median percent coverage: 97.2% at 4 hours, 99.8% at 6 hours). Tight junctions were observed, and the cells had few apical microvilli. The lateral cell borders were obliquely oriented with respect to Bruch's membrane, and the nuclei were elongated, exhibited prominent nucleoli, and were oriented parallel to Bruch's membrane. After 6–8 hours, cells started to become hexagonal (median percent coverage at 8 hours: 99.97%). Cells attached to the inner collagenous layer tended to be flatter than cells attached to residual native basement membrane. At 12 and 24 hours, expression of hexagonal shape, tight junctions, and apical microvilli were observed more frequently (median percent coverage: 99.87% at 12 and 100% at 24 hours). No newly formed basement membrane was observed at these time points. In separate experiments comparing attachment in the presence and absence of native RPE basement membrane, the presence of native retinal pigment epithelial basement membrane promoted the early attachment of the cells and more rapid expression of normal morphology. This in vitro system provides a reproducible way to study the adherence of retinal pigment epithelium to normal and diseased human Bruch's membrane.

Transplantation of retinal pigment epithelium using viable cryopreserved cells

Transplantation of retinal pigment epithelium (RPE) may have potential clinical application for the surgical treatment of RPE-specific retinal degeneration, including age-related macular degeneration. The feasibility of an RPE storage bank has been investigated by experimenting with transplantation using viable, cryopreserved RPE cells. Fresh and cultured fetal human and bovine RPE cells were cryopreserved in 90% fetal bovine serum containing 10% dimethyl sulfoxide. The viability of the cells before and after cryopreservation was evaluated by trypan blue dye exclusion test, microculture tetrazolium assay (MTA), tissue culture, and transplantation after cryopreservation. The origin of RPE cells before and after cryopreservation was assessed by immunocytochemistry, immunoblotting, and indirect ELISA of RPE-marker protein using cytokeratin for cultured fetal human RPE cells and by immunocytochemistry of cellular retinaldehyde-binding protein (CRALBP) for cultured bovine RPE cells. Freshly isolated and cryopreserved uncultured bovine RPE cells were transplanted by posterior transscleral approach into the subretinal spaces of adult albino rabbits and 23-day-old Royal College of Surgeons (RCS) rats with a 33 gauge Hamilton syringe. Following surgery, artificial retinal blebs were confirmed by fundus examination. Morphologic examination was performed postoperatively by light and electron microscopy in albino rabbits and by light microscopy in RCS rats up to 3 mo. Control subretinal injections using vehicle solution also were performed in RCS rats. Cultured fetal human and bovine RPE cells after cryopreservation were found to be viable, based on the results of trypan blue dye exclusion test, MTA, tissue culture, and transplantation. Expression and reexpression of cytokeratin intermediate filaments in cultured fetal human RPE were demonstrated by immunocytochemistry, immunoblotting, and indirect ELISA before and after cryopreservation. Immunocytochemistry of CRALBP before and after cryopreservation in uncultured bovine RPE cells disclosed expression and reexpression of RPE cell marker protein. No uncultured fetal human RPE cells showed proliferation in tissue culture after cryopreservation. In rabbits, light and electron microscopy disclosed xenografted RPE cells residing on Bruch's membrane of the host retina. No sign of graft vs. host reaction was observed. No morphologic difference was noted between the fresh and 10-day-old cryopreserved RPE cells in situ following transplantation at day 25. In RCS rats, subretinal injection of 3-wk-old cryopreserved bovine RPE cells partially rescued photoreceptor cells locally at the transplanted area observed at 3 mo postoperatively. The retinal photoreceptors at the inferior hemisphere of the transplanted eye and the eye injected with vehicle solution showed no rescue effect. We found that cryopreserved cultured fetal human RPE cells and uncultured and cultured bovine RPE cells can be used for RPE transplantation studies. The ability to create an RPE storage bank as a source of donor cells may result in several clinical advantages.

Transplantation of retinal pigment epithelium using viable cryopreserved cells.

Transplantation of retinal pigment epithelium (RPE) may have potential clinical application for the surgical treatment of RPE-specific retinal degeneration, including age-related macular degeneration. The feasibility of an RPE storage bank has been investigated by experimenting with transplantation using viable, cryopreserved RPE cells. Fresh and cultured fetal human and bovine RPE cells were cryopreserved in 90% fetal bovine serum containing 10% dimethyl sulfoxide. The viability of the cells before and after cryopreservation was evaluated by trypan blue dye exclusion test, microculture tetrazolium assay (MTA), tissue culture, and transplantation after cryopreservation. The origin of RPE cells before and after cryopreservation was assessed by immunocytochemistry, immunoblotting, and indirect ELISA of RPE-marker protein using cytokeratin for cultured fetal human RPE cells and by immunocytochemistry of cellular retinaldehyde-binding protein (CR-ALBP) for cultured bovine RPE cells. Freshly isolated and cryopreserved uncultured bovine RPE cells were transplanted by posterior transscleral approach into the subretinal spaces of adult albino rabbits and 23-day-old Royal College of Surgeons (RCS) rats with a 33 gauge Hamilton syringe. Following surgery, artificial retinal blebs were confirmed by fundus examination. Morphologic examination was performed postoperatively by light and electron microscopy in albino rabbits and by light microscopy in RCS rats up to 3 mo. Control subretinal injections using vehicle solution also were performed in RCS rats. Cultured fetal human and bovine RPE cells after cryopreservation were found to be viable, based on the results of trypan blue dye exclusion test, MTA, tissue culture, and transplantation. Expression and reexpression of cytokeratin intermediate filaments in cultured fetal human RPE were demonstrated by immunocytochemistry, immunoblotting, and indirect ELISA before and after cryopreservation. Immunocytochemistry of CRALBP before and after cryopreservation in uncultured bovine RPE cells disclosed expression and reexpression of RPE cell marker protein. No uncultured fetal human RPE cells showed proliferation in tissue culture after cryopreservation. In rabbits, light and electron microscopy disclosed xenografted RPE cells residing on Bruch's membrane of the host retina. No sign of graft vs. host reaction was observed. No morphologic difference was noted between the fresh and 10-day-old cryopreserved RPE cells in situ following transplantation at day 25. In RCS rats, subretinal injection of 3-wk-old cryopreserved bovine RPE cells partially rescued photoreceptor cells locally at the transplanted area observed at 3 mo postoperatively. The retinal photoreceptors at the inferior hemisphere of the transplanted eye and the eye injected with vehicle solution showed no rescue effect. We found that cryopreserved cultured fetal human RPE cells and uncultured and cultured bovine RPE cells can be used for RPE transplantation studies. The ability to create an RPE storage bank as a source of donor cells may result in several clinical advantages.

Chloride Transport in Cultured Fetal Human Retinal Pigment Epithelium

This study identifies bumetanide-sensitive chloride transport in cultured fetal human retinal pigment epithelium (RPE). Ion flux and electrophysiologic studies were performed on fetal human RPE grown to confluence on microporous culture wells mounted in modified Ussing chambers. Unidirectional transepithelial Cl -fluxes were measured along with the transepithelial potential (TEP), resistance ( R t), and short circuit current ( I sc). In separate experiments the apical and basal membrane voltages ( V apand V ba) and membrane resistance ratio ( R ap/ R ba) were measured using standard electrophysiological techniques. The average electrical parameters under control conditions were TEP=3.6±2.1mV, R t=385±98ohmcm 2, and I sc=8.8±3.6μAcm -2. Apical bumetanide (10 -5 m) reduced the TEP and I scan average of 0.6±0.4mV and 1.6±0.8μ Acm -2, respectively. Under open-circuit conditions, we measured a net apical to basal 36Cl flux of 0.30μEqcm -2hr -1. Bumetanide reduced this net Cl -flux by 83% by reducing the unidirectional apical-to-basal Cl -flux. Apical bumetanide hyperpolarized V bafrom -58±14 to -66±17mV and reduced R ap/ R bafrom 1.17±0.50 to 0.85±0.38, consistent with blockade of Cl -uptake and reduction of the Cl -equilibrium potential at a basal membrane Cl -channel. Basal bumetanide had no effect on electrical parameters. We identified a bumetanide sensitive Cl -uptake mechanism at the apical membrane which may represent the Na–K–2Cl cotransporter reported in explant and other cultured preparations of mammalian RPE.

Manufacture and characterization of poly(α-hydroxy ester) thin films as temporary substrates for retinal pigment epithelium cells

For many disorders of the retinal pigment epithelium (RPE) for which there are no effective treatments, transplantation of RPE cells may provide a viable means of restoring function. Using a solvent casting technique, we have manufactured thin films of poly( l-lactic acid) and poly( dl-lactic-co-glycolic acid) 75:25 and 50:50. Non-porous, flexible films with controlled thickness as thin as 12 ± 3 μm and reproducible surface morphologies and flexural properties were produced. Fetal human RPE cells were found to attach to these substrates when cultured in vitro. The films made using this technique may provide a means of transplanting allogeneic RPE cells as a therapy for a number of ocular diseases related to RPE dysfunction.

An improved method of isolating fetal human retinal pigment epithelium.

The purpose of this study was to develop an improved method of isolating fetal human retinal pigment epithelium (RPE) for tissue culture or transplantation. Fetal human eyes ranging from 8 to 20 wks of gestation were collected and stored in Optisol solution. Under a dissecting microscope, an incision was made behind the ora serrata and extended circumferentially to remove the anterior segment. The vitreous was withdrawn, and the neural retina was carefully detached from the RPE. The sclera then was teased away from the choroid-RPE. The choroid-RPE was treated with 2% dispase in DMEM + 20 mM HEPES at 37 degrees C for 25 min. While still in dispase, the RPE was separated from the choroid using a pair of fine tipped jeweler's forceps under dark-field. An intact sheet of RPE could be separated from the choroid after treatment with dispase. No choroidal contamination was present as determined by light microscopy or cell culture. In vitro, the isolated RPE cells demonstrated classic cobblestone phenotype and expressed cytokeratin. This technique provides an easy and reliable method for isolating pure sheets of fetal human RPE. It also allows utilization of the neural retina of the same eye for other purposes, as the neural retina is not exposed to the enzymatic digestion. These features make this method especially useful for RPE and retinal transplantation; such an application is already underway.

Influence of zinc on selected cellular functions of cultured human retinal pigment epithelium.

Zinc is a necessary micronutrient, usually abundant in human RPE. Our study was undertaken to determine the effects of short-term, zinc deficiency on human retinal pigment epithelium (RPE) using a culture model of fetal human RPE cells. Human fetal RPE cells were isolated and cultured in Coon's modified Ham's F-12 medium. For zinc depletion studies, cells were cultured for 1 week in Chelex-treated Dulbecco's modified Eagle's medium containing low (0.25 microM) or physiologic (11 microM) total zinc concentrations as determined by flame atomic absorption spectroscopy. Protein synthesis was determined by incorporation of 35S-cysteine/methionine and labeled proteins analysed by polyacrylamide gel electrophoresis. Several cell parameters and enzymes were significantly reduced below control when cultured in low zinc: zinc content (40%), proliferation (63%), protein/well (50%), catalase activity (68%), alkaline phosphatase activity (61%), alpha-mannosidase activity (68%), and metallothionein (82%). No statistically significant decline was seen in acid phosphatase activity, superoxide dismutase activity, glutathione peroxidase activity and dexamethasone induction of metallothionein. Zinc repletion (100 microM, 1 h) increased catalase and alpha-mannosidase activities from 32% and 33% of control to 75% and 73%, respectively. Cycloheximide did not inhibit this short-term zinc-induced repletion of catalase or alpha-mannosidase. Protein synthesis in low zinc medium was depressed, but not significantly, as shown by incorporation of radiolabeled 35S-cysteine/methionine into newly synthesized proteins. The effects of zinc deficiency in cultured human RPE are selective. Adequate intracellular zinc was required for maximal activity of some enzymes. The dependence of catalase activity on zinc was not predicted and may help explain the observed decline in catalase activity seen with age in RPE. Our model of zinc deficiency should prove useful in elucidating the complex effects of zinc deficiency and repletion in human RPE.