Retinal pigment epithelial cell transplantation after subfoveal membranectomy in age-related macular degeneration: clinicopathologic correlation.

PURPOSE: To report the histopathology after retinal pigment epithelial cell transplantation and subfoveal membranectomy in age-related macular degeneration. METHODS: An 85-year-old white woman with bilateral choroidal neovascularization underwent subfoveal membranectomy combined with transplantation of a sheet of human adult retinal pigment epithelium (retinal pigment epithelium) under the foveal center in the right eye. The patient was immunosuppressed postoperatively with prednisone, cyclosporine, and azathioprine. The patient died from congestive heart failure 114 days after surgery. RESULTS: A patch of hyperpigmentation was visible at the transplant site under the foveola after surgery. Mound-like clusters of individual round, large densely pigmented cells were present in the subretinal space and outer retina in this area. There was loss of the photoreceptor outer segments and native retinal pigment epithelium in the center of the transplant bed, with disruption of the outer nuclear layer predominantly over regions of multilayered pigmented cells. Cystic spaces were present in the inner and outer retina. A residual intra-Bruchs membrane component of the original choroidal neovascular complex was present under the transplant site. CONCLUSIONS: The transplant site contained clusters of round, pigmented cells that did not form a uniform monolayer in most areas. The morphology at the transplant site is consistent with the lack of visual improvement seen after surgery in this patient.

TGF beta secretion modulates the density-dependent growth of pig retinal pigment epithelium in vitro.

BACKGROUND: We aimed to identify the cytokine(s) responsible for the density-dependent growth regulation of pig retinal pigment epithelium (RPE) in vitro. METHODS: Confluent monolayers of primary pig RPE were established on bovine corneal endothelial extracellular matrix-coated tissue culture well inserts wrapped with dialysis membranes with different molecular weight cutoffs (0.5-50 kDa). These confluent RPE monolayers were then cocultured with first passage porcine RPE plated at a density of 1 cell/mm2, so that the newly plated RPE was bathed with different molecular weight fractions of the confluent cell media. Growth rates of the newly plated RPE were determined 72 h after plating and the molecular weight fraction of the confluent cell medium that inhibits the RPE proliferation was determined. First passage pig RPE (1 cell/mm2) were cocultured with confluent monolayers of primary pig RPE on inserts in the presence of different amounts of TGF-beta neutralizing antibody (0.1-100 microg/ml). Growth rates of the newly plated RPE were calculated 72 h after plating to determine the antibody concentration that would maximize the growth rate of the newly plated RPE in the presence of an adjacent confluent RPE monolayer. RESULTS: The growth rate of the newly plated RPE decreased when RPE were bathed with the 10- to 25-kDa fractions of medium from an adjacent confluent RPE monolayer. This growth inhibition reached statistical significance with the 25- to 50-kDa fractions (p < 0.05), and was abolished by adding pan-specific neutralizing antibody against TGF-beta (0.1-5 microg/ml). Blocking greater amounts of TGF-beta in the medium with higher doses of antibody (>10 microg/ml) also inhibited the growth of the newly plated RPE, in the presence or absence of a neighboring confluent cell layer. CONCLUSION: The TGF-beta family of cytokines mediates the density-dependent growth suppression of RPE in vitro. Neutralizing the effect of these cytokines by adding anti-TGF-beta antibodies can result in more rapid growth of the RPE in vivo.

Repopulation of different layers of host human Bruch's membrane by retinal pigment epithelial cell grafts.

PURPOSE: To determine the morphology of human retinal pigment epithelium (RPE) after reattachment to different ultrastructural layers of human Bruch's membrane (BM). METHODS: Bruch's membrane explants were prepared from eyes of 23 human donors (age range, 11-89 years). The basal lamina of the RPE, inner collagenous layer, and elastin layer were removed sequentially by mechanical and enzymatic techniques. First-passage cells of human RPE (15,000 cells/6 mm explant) from three donors (ages, 52, 64, and 80 years) were plated onto different layers of human BM, and the explants were examined by scanning and transmission electron microscopy up to 21 days later. RESULTS: RPE flattened and extended footplates 6 hours after plating onto basal lamina. Cells remained round 6 and 24 hours after plating onto the inner collagenous, elastin, or outer collagenous layer. The RPE cells became confluent 14 days after plating onto basal lamina but did not become confluent up to 21 days after plating onto the inner collagenous or elastin layer. Sparse round cells were observed 21 days after plating onto deeper layers, suggesting extensive loss of RPE. CONCLUSIONS: The morphology and subsequent behavior of the RPE reattached to BM depends on the anatomic layer of BM available for cell reattachment. The results suggest that the ability of transplanted RPE to repopulate BM in age-related macular degeneration and other disorders may depend on the layer of BM available to serve as a substrate for cell reattachment.

Fate of human retinal pigment epithelial cells seeded onto layers of human Bruch's membrane.

PURPOSE: To determine the fate of human retinal pigment epithelial (RPE) cells seeded onto different layers of human Bruch's membrane (BM). METHODS: Bruch's membrane explants were prepared from 16 human cadaver eyes (7 eyes age <50 years; 9 eyes >50 years) by removing native RPE cells with ammonium hydroxide to expose the RPE cell basal lamina (BL). The inner collagenous layer (ICL) and elastin layer (EL) were exposed by removing apical layers sequentially by mechanical and enzymatic means. Synchronized first passage human RPE cells (15,000 cells/(6-mm-diameter explant) were plated onto each layer of human BM. The RPE cell reattachment and apoptosis rates at 24 hours, proliferation rates and mitotic index 24 hours after growth stimulation, and the ability of RPE cells to repopulate the explant surface were determined on each layer. RESULTS: RPE cell reattachment was highest on BL but decreased on deeper layers of BM. The apoptosis rate of attached cells increased as deeper layers of BM were exposed. The proliferation rate and mitotic index of the grafted cells were higher on BL than on deeper layers. RPE cells plated onto BL repopulated the explant surface within 14 +/- 3 days, whereas cells plated onto the ICL and EL eventually died and never reached confluence. CONCLUSIONS: The fate of RPE cells seeded onto BM depends on the ultrastructural layer of BM available for reattachment. These findings suggest that the ability of transplanted RPE cells to repopulate bare BM will depend on the layer of BM available for RPE cell reattachment.

Minimum number of adult human retinal pigment epithelial cells required to establish a confluent monolayer in vitro.

PURPOSE: To determine the minimum number of cells required to establish a confluent monolayer of retinal pigment epithelium (RPE) with an epitheloid morphology in vitro. METHODS: Primary or passaged human RPE were harvested by trypsinization from 6 donors and plated onto bovine corneal endothelium extracellular matrix-coated tissue culture plastic in 96-well plates. Plating densities ranged from 1 to 66,000 viable cells/well (0.03-2062 viable cells/mm2) for primary cells or 1 to 100,000 viable cells/well (0.03-3112 viable cells/mm2) for passaged cells. The time required to reach confluence was determined by monitoring the cultures daily until they reached confluence. Mean cell area and circularity index at confluence was calculated to determine the effect of different plating densities on final RPE morphology. RESULTS: Primary RPE plated at densities above 10 viable cells/mm2 (320 cells/well) and passaged RPE plated above 2 viable cells/mm2 (64 cells/well) reached confluence on every occasion. There was a negative correlation between the plating density and time required to reach confluence. Plating densities above 3 viable cells/mm2 (96 cells/well) and 50 viable cells/mm2 (1600 cells/well) yielded smaller, rounder cells at confluence for primary and passaged RPE, respectively. CONCLUSIONS: As few as 96 primary RPE cells and 1600 passaged RPE are required to obtain a confluent, 6mm (4-disc diameter) patch of RPE in vitro. This suggests that autologous RPE grafts can be prepared with high efficiency for subsequent transplantation into the subretinal space in vivo.

Serum-free media for culturing and serial-passaging of adult human retinal pigment epithelium.

The ability of a chemically-defined serum-free culture medium to support the attachment, growth and serial passaging of primary adult human retinal pigment epithelial (RPE) cells was studied. Primary cultures of adult human RPE were established in a chemically-defined serum-free culture medium on both bare or bovine corneal endothelial extracellular matrix-coated tissue-culture plastic. Confluent cells were serially passaged in chemically-defined serum-free culture medium three times by trypsinization, and trypsin activity was quenched with aprotinin. First passage RPE cells were plated onto tissue-culture plastic precoated with bovine corneal endothelial extracellular matrix or uncoated tissue-culture plastic in 24 well plates at a density of 50 viable cells mm-2. Cells were maintained either in chemically-defined serum-free culture medium, DMEM without serum, or DMEM with 15% fetal bovine serum. For each medium plating, efficiencies were determined 24 hours after plating, and growth rates were determined on the first, third and seventh days after plating. Morphometric image analysis was performed on cells cultured for up to 6 weeks and three serial passages. Seeding efficiency on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and treated tissue-culture plastic were higher for chemically-defined serum-free culture medium (88.9+/-2.7% and 47.1+/-4.1%, respectively) and DMEM with serum (87.2+/-5.6% and 52.9+/-10.5%, respectively) than DMEM without serum (59.2+/-5.6% and 33.1+/-6.9%, respectively; P<0.01). The RPE proliferation rate in chemically-defined serum-free culture medium was comparable to DMEM with serum on both substrates within the first 3 days, although cells in DMEM with serum had a higher proliferation rate on day 7. Cells cultured in DMEM without serum, eventually decreased in number. RPE maintained in chemically-defined serum-free culture medium maintained a consistent proliferation rate, reached confluence, and retained an epitheloid morphology on either extracellular matrix or tissue-culture plastic for up to 6 weeks and three serial passages. Primary RPE reached confluence at 12+/-3 days on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and 21+/-5 days on treated tissue-culture plastic. Confluent cultures were composed of small hexagonal cells with epitheloid morphology on both substrates. We concluded that primary adult human RPE can be cultured in this chemically-defined serum-free culture medium. RPE will proliferate, reach confluence, retain their epitheloid morphology and can be serially passaged in the absence of serum.

Harvest and storage of adult human photoreceptor cells: the vibratome compared to the excimer laser.

PURPOSE: To develop a method using the vibratome and the excimer laser to harvest a sheet of human photoreceptor cells from the retinas of cadaveric donors. METHODS: Adult human photoreceptor cells were harvested as intact sheets from the retinas of cadaver eyes using a vibratome or excimer laser. The sheets were embedded in 50% gelatin (in minimum essential medium and 300 mM sucrose) and stored at 4 degrees C. The morphology, integrity, viability and sterility of the harvested photoreceptor cells was studied. RESULTS: Light and scanning electron microscopy demonstrated sheets of adult human photoreceptor cells with an outer nuclear layer and inner and outer segments with either method of harvest. The initial viability of the outer nuclear layer, harvested an average of 28.2 h after death, was > or =94.7%. Sheets stored up to 72 h after harvest maintained a viability of > or =86.5%. The sheet of cells harvested with the vibratome frequently fragmented (n = 25, 35%) during passage through the delivery cannula in contrast to the excimer laser. Harvested sheets were sterile when the gelatin powder was irradiated prior to reconstitution. CONCLUSIONS: Intact, viable adult human photoreceptor cell sheets can be isolated from the retina of a cadaver using either the vibratome or the excimer laser and stored up to 72 h at 4 degrees C. With the vibratome, there is damage to the outer segments of the photoreceptors, the sheets are fragile, and the harvest of specimens is time-consuming as only one or two specimens can be harvested from a single donor retina. These technical limitations are avoided with the excimer laser.

Posterior vitreous detachment with dispase.

PURPOSE: To induce a posterior vitreous detachment (PVD) in porcine and human cadaver eyes in vitro with Dispase (Gibco, Grand Island, NY). METHODS: Dispase (0.5 mL) was injected into the vitreous cavity of enucleated porcine (0.05-25 U/mL) and human (5 U/mL) eyes. After incubation at 37 degrees C for 15-120 minutes, the globes were hemisected and the extent of PVD was graded as complete, partial, or absent. The structural integrity of the retina was estimated by measuring the elastic constant and maximal stretching before fracture. Retinal cell viability was determined by an intracellular esterase assay. Light, transmission, and scanning electron microscopy were performed to examine the ultrastructure of the vitreoretinal interface. RESULTS: After 15 minutes, a partial or total PVD was present in 7/10, 8/10, or 9/10 enucleated porcine eyes incubated with 1, 5, or 10 U/mL Dispase, respectively, versus 3/10 control eyes (P < 0.05). A partial or complete PVD was present in 3/5, 4/5, 5/5, or 14/15 porcine eyes after 15, 30, 60, or 120 minutes of treatment with 0.1 U/mL Dispase, respectively. Similarly, 19/20 human cadaver eyes developed a complete and 1/20 an incomplete PVD after incubation with 5 U/mL Dispase for 15 minutes. Microscopy demonstrated that Dispase cleaved the attachment of the posterior hyaloid to the internal limiting membrane with minimal damage to the inner retina. Retinal cell viability and the mechanical properties of the retina were similar for Dispase-treated and control eyes. CONCLUSION: Dispase disrupts the attachment of the posterior hyaloid to the inner limiting membrane with minor morphologic changes in the inner retina. The enzyme may be useful in removing cortical vitreous during vitreous surgery.

Reattachment rate of human retinal pigment epithelium to layers of human Bruch's membrane.

OBJECTIVES: To determine the reattachment rate of human retinal pigment epithelium (RPE) to different layers of human Bruch's membrane (BM). METHODS: Explants of BM were prepared from 10 human cadaver eyes by removing native RPE. The RPE basal lamina, inner collagenous layer, elastin layer, and outer collagenous layer were exposed by sequentially removing each apical layer by mechanical or enzymatic means. First-passage human RPE was plated onto the surface and the RPE reattachment rate to each layer of BM was determined. RESULTS: Retinal pigment epithelial cell reattachment was highest to the inner aspects of BM and decreased as deeper layers of BM were exposed (ie, reattachment rate to basal lamina was higher than to the inner collagenous layer, which was higher than to the elastin layer, which was higher than to the outer collagenous layer). The reattachment rate to the inner collagenous layer, elastin layer, and outer collagenous layer harvested from elderly donors (age >60 years) was less than the reattachment rate to corresponding layers harvested from younger (age <50 years) donors. CONCLUSIONS: Retinal pigment epithelial cell reattachment depends on the anatomical layer of BM present in the host tissue. Age-related changes in BM may interfere with RPE reattachment. Our observations may have implications for understanding the pathogenesis of age-related macular degeneration and its potential treatment with RPE transplantation techniques.

Human photoreceptor transplantation in retinitis pigmentosa. A safety study.

OBJECTIVE: To establish the technical feasibility and safety of photoreceptor transplantation in retinitis pigmentosa. METHODS: A sheet of human photoreceptor cells was harvested from 2 human cadaveric eyes with a vibratome and transplanted into the subretinal spaces of 2 patients with advanced retinitis pigmentosa and visual acuity of no light perception by means of submacular surgery techniques. Preoperative and postoperative electrophysiologic testing, fundus photography, fluorescein angiography, and scanning laser ophthalmoscopy were performed. RESULTS: Twelve months after photoreceptor transplantation, the visual acuity of each patient remained no light perception. The temporal edge of the retinotomy in 1 patient was folded but was not associated with a retinal detachment. The patients were not immunosuppressed, and there was no evidence of rejection of the allogeneic transplant. Cystoid macular edema, uveitis, and macular pucker were not observed. CONCLUSION: A sheet of adult human photoreceptor cells can be harvested from human cadaveric eyes and safely transplanted to the subretinal spaces of patients with retinitis pigmentosa without systemic immunosuppression.

Harvest and storage of adult human retinal pigment epithelial sheets.

PURPOSE: To describe a method for the harvesting and storing of intact viable sheets of adult human retinal pigment epithelial (RPE) cells. METHODS: Adult human RPE cells were harvested as intact sheets from 21 cadaver eyes, using the enzyme Dispase. The sheets were embedded in 50% gelatin containing 300 mM sucrose and stored at 4 degrees C. The viability of the cells, as well as their ability to proliferate in vitro, was studied for 96 hours after harvesting. Light microscopy (LM), transmission (TEM) and scanning electron microscopy (SEM) were performed to determine the integrity and ultrastructural features of the cells. Microbiologic culture of the harvested sheets was performed to exclude contamination. RESULTS: LM, TEM and SEM showed intact RPE cells with well-developed microvilli, basal infoldings and intercellular connections. The initial viability of intact RPE sheets was 86%, with a progressive decline in viability with increased storage time. Cells harvested within 24 hours after death maintained greater viability than those harvested after 24 hours (p < 0.05). Harvested RPE cells were free of microbial contamination and rapidly proliferated when cultured in vitro. CONCLUSION: Intact sheets of adult human RPE can be isolated using the enzyme Dispase. The cells appeared suitable for retinal transplantation if harvested within 24 hours of death and maintained 82% viability for as long as 48 hours if stored at 4 degrees C.

Reattachment to a substrate prevents apoptosis of human retinal pigment epithelium.

BACKGROUND: Epithelial cells generally fail to survive in suspension. Harvesting human retinal pigment epithelium (RPE) for transplantation may separate the cells from their extracellular matrix and induce apoptosis. We investigated whether reattachment of RPE to a substrate will prevent apoptosis. METHODS: Second-passage human RPE cells were plated onto tissue culture plastic precoated with extracellular matrix, fibronectin or laminin, uncoated tissue culture plastic, untreated plastic and untreated plastic coated with 4% agarose. Reattachment rates were determined for each substrate 24 h after plating. The TUNEL technique was used to determine apoptosis rates in attached cells, unattached cells and the entire cell population. RESULTS: Attachment rates were as follows: ECM-coated tissue culture plastic-->fibronectin-coated tissue culture plastic-->laminin-coated tissue culture plastic-->uncoated tissue culture plastic-->untreated plastic-->agarose-coated untreated plastic. Apoptosis rates for the entire cell population increased as the RPE cell attachment rate decreased. The proportion of apoptotic cells in the entire population was inversely related to the percent attached cells (r = -0.95). CONCLUSION: Reattachment of harvested RPE to a substrate decreased the rate of RPE apoptosis in vitro. RPE cells which are removed from their substrate prior to transplantation must reattach rapidly to a substrate to prevent apoptosis.

Correlation between scanning laser ophthalmoscope microperimetry and anatomic abnormalities in patients with subfoveal neovascularization.

PURPOSE: The purpose of the study is to identify the anatomic abnormalities associated with an absolute scotoma and the location and stability of fixation in patients with subfoveal neovascularization in age-related macular degeneration, presumed ocular histoplasmosis syndrome, and other disorders. METHODS: Scanning laser ophthalmoscope microperimetry was superimposed on color fundus photographs and fluorescein angiograms of 21 eyes with subfoveal neovascular membranes secondary to age-related macular degeneration (14 eyes) and presumed ocular histoplasmosis syndrome (7 eyes). The authors determined the location and the area occupied by the absolute scotoma and each of the following subretinal lesions: subretinal hemorrhage, neurosensory retinal detachment, retinal pigment epithelial (RPE) atrophy, RPE hyperplasia, atrophy of the choriocapillaris, hard exudates, and the subfoveal neovascular membrane. The area of absolute scotoma determined by scanning laser ophthalmoscope microperimetry was superimposed on the anatomic lesions. The authors calculated the relative risk ratio (RR) of an absolute scotoma occurring in regions corresponding to each anatomic abnormality, and determined the preferred location and stability of fixation in each eye. RESULTS: An absolute scotoma was present in areas of chorioretinal scar (RR = 107.61), RPE atrophy (RR = 9.97), subretinal hemorrhage (RR = 2.88), and the neovascular membrane (RR = 1.86). Fixation was stable in all patients with presumed ocular histoplasmosis syndrome but only 29% of patients with age-related macular degeneration. Fifty-five percent of patients with stable fixation fixated over an area of RPE hyperplasia. CONCLUSION: The relative risk of an absolute scotoma is highest over areas of chorioretinal scars, RPE atrophy, subretinal hemorrhage, and the neovascular membrane. Fixation is more stable in patients with subfoveal neovascularization from presumed ocular histoplasmosis syndrome than with age-related macular degeneration and frequently is present over an area of RPE hyperplasia.

Density-dependent growth regulation of pig retinal pigment epithelial cells in vitro.

BACKGROUND: Transplantation of the retinal pigment epithelium (RPE) involves delivering donor RPE onto a bare area of host Bruch's membrane that is surrounded by a confluent monolayer of RPE. We investigated the effects of different plating densities and the presence of an adjacent confluent monolayer on the growth characteristics and final morphology of pig RPE in vitro. METHODS: In single-well experiments, porcine RPE were plated in 24-well plates at densities varying from 1 to 75 cells/mm2. Triplicate plates were counted on the 3rd and 10th days after plating and at confluence. A multiwell chamber was built to allow cells plated at different densities to be bathed with conditioned media from adjoining wells. RESULTS: In single-well experiments, plating at low densities increased the time to reach confluence and resulted in fewer, larger and more fusiform RPE at confluence. In multiwell experiments, the growth rate of cells plated at low density decreased as the amount of high-density medium increased in communicating wells and led to smaller, rounder cells at confluence. The presence of low-density RPE in adjoining wells increased the growth rate of RPE plated at high density and produced fewer, larger and more fusiform cells at confluence. Newly plated RPE grew more slowly when confluent monolayers of RPE were present in adjoining wells. CONCLUSIONS: Plating density is a critical factor in determining the growth rate and the final morphology of RPE in tissue culture. The presence of a neighboring confluent monolayer of RPE inhibits the growth rate of newly plated RPE in vitro.

Morphometric analysis of exudative retinal arterial macroaneurysms: a geometrical approach to exudate curves.

We studied morphometrically the color transparencies and fluorescein angiograms of 5 patients with six exudative retinal arterial macroaneurysms. Our aim was to express the dependence of the exudate morphology upon the location of the macroaneurysm with an algebraic polynomial analysis. We derived the second-degree conical equations of the exudate curves and computed the location of their cardinal descriptive parameters. The relationship between the site of the macroaneurysms and the computed hypothetical parameters of their exudate curves revealed that the structural features of the exudates are dependent upon the distance of the macroaneurysm to the foveola, the gravitational force and the clearing capacity of the venous net. The point where the macroaneurysm develops approximately 3 mm from the center of the macula seems to be a 'crucial point'. Macroaneurysms located closer than this point may cause the exudates that occupy the fovea and create severe visual loss.

The comparative analysis of optic disc damage in exfoliative glaucoma.

To compare the initial and follow-up discernible glaucomatous neuroretinal rim (NRR) and parapapillary chorioretinal changes in patients with primary open-angle glaucoma (POAG) and exfoliative glaucoma (EG), we analyzed 40 optic discs from 40 patients with POAG, 40 optic discs from 40 patients with EG and 20 optic discs from 20 normal subjects. The mean intraocular pressure was higher in the EG group when compared with POAG (p < 0.001). Although the mean disc areas in both groups were not significantly different, the mean initial NRR area to disc area ratio was significantly smaller in patients with EG (p < 0.001). At the initial diagnosis the most prominent NRR defects of the patients with POAG were at the inferotemporal and superotemporal sectors, followed by the temporal and nasal sectors. However, the NRR in the EG group was noticed to decrease diffusely without such a sectorial preference. There was no significant difference between the mean loss of the NRR to disc area ratio in both groups at the end of the follow-up period of 1 year (p > 0.05). The values of parapapillary chorioretinal atrophy areas in POAG and EG patients were not significantly different from each other, neither at the initial examination nor during the follow-up period (p > 0.05). Those results suggest that high intraocular pressure in eyes with EG may constitute a major risk for rapid and progressive glaucomatous optic disc damage.