Tissue culture of cat retinal pigment epithelium (original) (raw)

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

Journal of Visualized Experiments, 2010

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

Protein synthesis and secretion by cultured retinal pigment epithelia

Biochimica et Biophysica Acta (BBA) - General Subjects, 1995

Protein synthesis and secretion by post-natal sheep and calf retinal pigment epithelial (RPE) cells was investigated following labelling of choroidal pieces, isolated RPE cells and RPE cells in tissue culture with L-[U-14C] leucine. We show that RPE cells secrete a specific set of proteins that includes retinol binding protein (RBP) and transthyretin (TTR), which are both involved in retinol transport in blood. Using a two-chambered culture system we show that protein secretion by the post-natal RPE cells occurs predominantly across the apical pole of the cells, i.e., across the surface of the cells which, in vivo, faces the retina. In agreement with results of others using foetal RPE cells (Ong, D.E., Davis, J.T., O'Day, W.T. and Bok, D. (1994) Biochemistry 33, 1835-1842) we show that RBP and, to a lesser extent, TTR are also secreted predominantly across the apical pole of the cell. We have developed a cell culture model for the RPE that may be used as an in vitro model for studying transport across the blood-retinal barrier.

Human retinal pigment epithelium in long term explant culture

Acta Ophthalmologica, 2009

Explants of human retinal pigment epithelium were maintained in culture in various types of media, and examined by light and transmission electron microscopy. After one month in vitro, the central areas showed a monolayered configuration with distinct polarity and presence of ruthenium red stainable material on the apical surface. On the peripheral areas of Bruch's membrane, multilayered lesions were observed to develop and to extend from the monolayered epithelium 2nd past the cut edge in Bruch's membrane. Cells in these lesions contained little melanin and generally lacked an apico-basal polarity. Ruthenium red staining revealed the presence of electron dense material on the apical surface of the lesions as well as in the extracellular space between cells in the various layers. Development of multilayered lesions with deposition of extracellular material are seen in various chorio-retinal disorders, including senile macular degenerations and also subsequent to laser and cryo-therapy. The findings in the present study point to the explant culture system as a valuable tqol in the study of important aspects of chorio-retinal pathology.

In vitro culture of human retinal pigment epithelium for biochemical and metabolic study

Vision Research, 1981

proper functioning of the many types of ocular epithelium, most notably the retinal pigment epithelium (RPE), is critical to the sensory functions of the human eye. We have developed new techniques for the in vitro culture of human RPE using collagen coated tissue culture dishes and Epithelial Growth Factor. These techniques increased culture success using pairs of donor eyes from 25':{, to 871';. Human RPE has been cultured for up to 4 months while retaining marked pigmentation and epithelioid characteristics, Use of pure cultures of human RPE will allow biochemical study of this important tissue

The cell biology of the retinal pigment epithelium

Progress in Retinal and Eye Research, 2020

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A proper protocol for isolation of retinal pigment epithelium from rabbit eyes

Advanced Biomedical Research, 2014

Background: Retinal pigment epithelium (RPE) is a hexagonal monolayer of pigmented cells located between the neural retina and the choroid with an essential role for visual function. So, isolation, propagation and maintenance of their functional integrity of RPE are crucial for research in vitro which next used for cell transplantation. The evaluation of features of RPE cells as a sheet after 14 days has not been reported yet. This study aimed to examine and compare three protocols for RPE isolation from rabbit eyes and obtain a proper protocol, which illustrated isolated RPE cells as a sheet cause to preserve their characterize even after 2 weeks. Materials and Methods: RPE cells were prepared from eyes of 24 rabbit eyes. After enucleating of eyes, anterior segment discarded and posterior segment cut to small pieces. Two of these procedures are based on the enzymatic digestion, but third protocol based on mechanical dissection. The culture cells harvested and morphological feature of cells assessed by phase-contrast microscope and then analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry. Results: Evaluation of morphological feature showed that isolation of RPE cells as a sheet lead to preserve their hexagonal morphology. Immunocytochemistry and RT-PCR assessment demonstrated RPE cell cultured in sheet maintained their phenotypic feature, tight junction and the distribution of actin and cytokeratin filament. Comparison of three protocols showed that dissociation of RPE cells as a sheet was superior in the preserve of RPE characteristic. Conclusions: Isolation of RPE cells as a sheet maintains the integrity of these cells, this procedure promising a therapeutic approach, which is important for some retinal diseases.

Reactive Changes in the Human Retinal Pigment Epithelium in Vitro

Acta Ophthalmologica, 2009

Explants from the retinal pigment epithelium and the underlying choroid and sclera were dissected from human eyes and transfered to culture wells. The mechanical trauma caused by the dissection and removal of the explants, and the changes in biological milieu caused by transfer of the tissue to an in vitro system causes injury, necrosis and detachment of cells from Bruchs membrane. In the retinal pigment epithelium, cells adjacent to damaged, spherical and detaching cells and smaller cell free zones form rosettes. At the periphery of big defects, the cells spread out to cover the denuded areas of Bruch's membrane. The present work has shown that cell injury in the human retinal pigment epithelium is followed by reactive cellular changes in vitro. The result of these reactive changes are increased variation in cellular form and magnitude and in pigment concentration per unit area.

Absence of phosphoglucose isomerase-1 in retinal photoreceptor, pigment epithelium and Muller cells

European Journal of Neuroscience, 2004

Macroarray analysis was used to compare equal amounts of cDNA from wild-type and rd/rd (retinal degeneration) mice, collected at P90 when photoreceptor degeneration is virtually complete. A stronger signal for the glycolytic enzyme phosphoglucose isomerase (Gpi1) was observed in the rd/rd sample. Extracellularly, Gpi1 may act as a cytokine, independently described as neuroleukin and autocrine motility factor. Retinal Gpi1 expression was investigated by Northern and Western blot analysis and immunohistochemistry. Double-labelling was performed with antibodies against Gpi1 and calbindin-D, glutamine synthetase, RPE65, calretinin and ultraviolet opsin in order to provide positive cell type identi®cation. Northern and Western blots showed double expression levels per microgram of RNA and protein, respectively, in the rd/rd retina compared with wild-type. However, the total amount of Gpi1 protein per retina was indistinguishable. Gpi1 immunoreactivity was found in ganglion, amacrine, horizontal and bipolar cells, but not in rods, cones, pigment epithelium and Muller cells. This distribution explains why the absolute amounts of Gpi1 protein were not appreciably different between wild-type and the rd/rd phenotype, where rods and cones are absent, whilst the relative contribution of Gpi1 to the total protein and RNA pools differed. Some extracellular immunoreactivity was observed in the photoreceptor matrix around cones in freshly ®xed tissue only, which could possibly re¯ect a role as a cytokine. We propose that glycolysis in Gpi1-negative cells proceeds entirely through the pentose phosphate pathway, creating NADPH at the cost of organic carbon. We hypothesize that the unique metabolic needs of photoreceptors justify this trade-off.