Retinal pigment epithelium cell culture on thin biodegradable poly(DL-lactic-co-glycolic acid) films

Thin films of 50 : 50 and 75 : 25 poly(DL-lactic-co-glycolic acid) (PLGA) were manufactured with a controlled thickness of less than 10 μm. The effect of PLGA copolymer ratio on in vitro cell attachment, proliferation, morphology, and tight junction formation was evaluated using a human D407 retinal pigment epithelium (RPE) cell line. Almost complete cell attachment was achieved on both PLGA films after 8 h of cell seeding, which was comparable to that on tissue culture polystyrene (TCPS) controls. The initial cell seeding density affected attachment, and the optimal value for 50: 50 PLGA was 25 000 cells cm^-2. After 7 days of in vitro culture, cell density on 50 : 50 and 75 : 25 PLGA films increased 45 and 40 folds, respectively, and a 34-fold increase was observed on TCPS. The RPE cells cultured on PLGA films at confluence had a characteristic cobblestone morphology. Confluent RPE cells also developed normal tight junctions in vitro which were concentrated mainly at the apical surfaces of cell-cell junctions. These results demonstrated that thin biodegradable PLGA films can provide suitable substrates for human RPE cell culture, and may serve as temporary carriers for subretinal implantation of organized sheets of RPE.     

Retinal pigment epithelium cell culture on surface modified poly(hydroxybutyrate-co-hydroxyvalerate) thin films

There is currently no effective treatment for the retinal disorders caused by retinal pigment epithelium (RPE) degeneration. Transplantation of allografts is the main strategy towards correction of this malady. Tissue engineering could offer hope and involve the use of biodegradable polymeric templates to replace diseased or lost RPE. In this study PHBV8 film was chosen as a temporary substrate for growing retinal pigment epithelium cells as an organized monolayer before their subretinal transplantation. The surface of the PHBV8 film was rendered hydrophilic by oxygen plasma treatment to increase the reattachment of D407 cells on the film surface. Power and duration was changed, from 50 W, 10 min to 100 W, 20 min during plasma treatment. The effect of these two parameters on surface hydrophilicity, morphology, topography, surface composition of PHBV8 thin films was studied using AFM, SEM, and phase contrast microscopy. The effect of changes in surface characteristics on cell reattachment, spreading and cell growth rate was investigated. It was found that as the treatment level was increased the surface hydrophilicity increased and roughness was decreased probably due to ablation. The PHBV8 film treated with 100 W 10 min was found to be the most suitable for 24 h reattachment of D407 cells. The cells were also grown to confluency as an organized monolayer suggesting PHBV8 film as a potential temporary substrate for subretinal transplantation to replace diseased or damaged retinal pigment epithelium.     

Retinal pigment epithelium adenoma in vitreous fluid cytology

Ocular cytology specimens are relatively uncommon, adding to the difficulty of their evaluation by cytopathologists. While melanomas account for a majority of primary intraocular pigmented lesions, other diagnostic considerations must be included in the differential. This brief report highlights a case of a pigmented ocular lesion in a 24‐year‐old man and key morphologic, immunohistochemical, and clinical differences between melanoma, melanocytoma, choroidal nevus, and retinal pigment epithelium (RPE) adenoma.     

Retinal pigment epithelium engineering using synthetic biodegradable polymers

Retinal pigment epithelium (RPE) plays a key role in the maintenance of the normal functions of the retina, especially photoreceptors. Alteration in RPE structure and function is implicated in a variety of ocular disorders. Tissue engineering strategies using synthetic biodegradable polymers as temporary substrates for RPE cell culture and subsequent transplantation may provide a promising new therapy. In this review article, the manufacture of thin biodegradable poly(DL-lactic-co-glycolic acid) (PLGA) films and their degradation behavior in vitro are discussed. RPE cell proliferation and differentiation on these PLGA films are reviewed. The fabrication of model substrates with desired chemical micropatterns in the micrometer scale is discussed and the effects of surface patterning on RPE morphology and function are assessed. Finally. the preparation of biodegradable micropatterns with adhesive PLGA and non-adhesive poly(ethylene glycol)/PLA domains to modulate RPE cell adhesion is presented.     

Retinal degeneration triggered by inactivation of PTEN in the retinal pigment epithelium

Adhesion between epithelial cells mediates apical–basal polarization, cell proliferation, and survival, and defects in adhesion junctions are associated with abnormalities from degeneration to cancer. We found that the maintenance of specialized adhesions between cells of the retinal pigment epithelium (RPE) requires the phosphatase PTEN. RPE-specific deletion of the mouse pten gene results in RPE cells that fail to maintain basolateral adhesions, undergo an epithelial-to-mesenchymal transition (EMT), and subsequently migrate out of the retina entirely. These events in turn lead to the progressive death of photoreceptors. The C-terminal PSD-95/Dlg/ZO-1 (PDZ)-binding domain of PTEN is essential for the maintenance of RPE cell junctional integrity. Inactivation of PTEN, and loss of its interaction with junctional proteins, are also evident in RPE cells isolated from ccr2^−/− mice and from mice subjected to oxidative damage, both of which display age-related macular degeneration (AMD). Together, these results highlight an essential role for PTEN in normal RPE cell function and in the response of these cells to oxidative stress.     

Retinal pigment epithelial cell function on substrates with chemically micropatterned surfaces

Model substrates with desired chemical micropatterns were fabricated using a microcontact printing technique. The substrate surfaces contained organized arrays of circular glass domains with a diameter of either 10 or 50 microm surrounded and separated by regions modified with octadecyltrichlorosilane (OTS) self-assembled monolayers (SAMs). The effects of surface patterning on in vitro cell attachment, proliferation, morphology, and cytoskeletal organization were evaluated using a human retinal pigment epithelium (RPE) cell line. Both micropatterns affected initial RPE cell attachment, limited cell spreading, and promoted the characteristic cuboidal cell morphology throughout the culture period. In contrast, RPE cells on plain glass control were elongated and appeared fibroblast-like prior to confluence. In addition, cells seeded at 30,000 cell/cm2 on the patterned surfaces maintained a normal pattern of actin and cytokeratin expression, and formed confluent monolayers within 4 days of culture. The cell density increased about 30-fold on both micropatterns by day 7. These results show that it is feasible to control RPE cell shape and expression of differentiated phenotype using micropatterned surfaces.     

Characterization of collagen gel solutions and collagen matrices for cell culture

The influence of glutaraldehyde as a crosslinking agent to increase the strength of collagen matrices for cell culture was examined in this study. Collagen solutions of 1% were treated with different concentrations (0-0.2%) of glutaraldehyde for 24 h. The viscoelasticity of the resulting collagen gel solution was measured using dynamic mechanical analysis (DMA), which demonstrated that all collagen gel solutions examined followed the same model pattern. The creep compliance model of Voigt-Kelvin satisfactorily described the change of viscoelasticity expressed by these collagen gel solutions. These crosslinked collagen gel solutions were freeze-dried to form a matrix with a thickness of about 0.2-0.3 mm. The break modulus of these collagen matrices measured by DMA revealed that the higher the degree of crosslinking. the higher the break modulus. The compatibility of fibroblasts isolated from nude mouse skin with these collagen matrices was found to be acceptable at a cell density of 3 x 10(5) cells/cm2 with no contraction, even when using a concentration of glutaraldehyde of up to 0.2%.     

Retinal dystrophy resulting from ablation of RXR alpha in the mouse retinal pigment epithelium

Vitamin A (retinol) actions in eye development are mediated by retinoic acid receptors (RARs and RXRs). Using the Cre/loxP system, we have selectively ablated RXR alpha in the retinal pigment epithelium (RPE), a cell monolayer critically involved in visual retinoid renewal and phagocytosis of photoreceptor outer segments. In the mutant (RXR alpha (rpe-/-)) mice, RPE cells are morphologically and functionally abnormal and display decreased expression of proteins involved in the visual retinoid cycle, namely RPE65, CRALBP, and RGR. RXR alpha (rpe-/-) mice also show alterations of photoreceptor cells including: 1) decrease in their number; 2) outer segment shortening and disorganization, and 3) reduced light responses in electroretinograms. These results indicate that RXR alpha is required for normal maturation of the RPE, which is known to play essential roles in photoreceptor cell function and survival, and point to a possible involvement of RXR alpha signaling pathways in the RPE in human retinal diseases.     

基因转染的虹膜色素上皮细胞移植后RCS鼠视网膜BDNF表达观察

目的探讨脑源性神经营养因子（brain-derived neurotrophic factor,BDNF）基因转染的虹膜色素上皮细胞（AAV-BDNF-IPE）移植入皇家外科学院（royal college of surgeons,RCS）大鼠视网膜下腔后,不同时期视网膜组织BDNF表达变化。方法通过外路途径将BDNF基因转染的虹膜色素上皮细胞移植到RCS大鼠视网膜下腔,术后3、5、7、9、11周分别取RCS大鼠手术眼及对照组动物眼视网膜组织,用酶联免疫吸附法（Elisa）检测视网膜组织中BDNF的表达水平,比较分析这些数据。结果对照组RCS大鼠出生后3周龄时视网膜组织中BDNF仍保持较高水平,其后迅速降低,其中3周龄组与其它周龄组比较,P〈0.01;手术组RCS大鼠术时、术后3、5、7、9、11周各组间两两比较,BDNF表达无显著差异（P〉0.05）;出生后6周龄直到14周龄的不同时期,AAV-BDNF-IPE移植手术组RCS大鼠视网膜BDNF表达水平均明显高于对照组（其中6周龄组P〈0.05,其它各周龄组P〈0.01）。结论 BDNF基因转染的虹膜色素上皮细胞在RCS大鼠视网膜下腔移植后,视网膜组织中BDNF可以持续稳定高水平表达,这为临床开发新的神经营养因子给药方式提供了实验依据。     

Retinal pigment epithelial cell plasma membrane: a monoclonal antibody study

The plasma membranes of retinal pigment epithelial cells are highly specialized organelles with multiple functions including nutritional and metabolic support of the photoreceptor cells. Using purified bovine retinal epithelial cell plasma membranes as antigen, we produced two monoclonal antibodies, MAbD1-C6 and MAbD1-C8, that cross react with the plasma membranes from bovine, rat and human retinal pigment epithelial cells. In radioimmunoassay both MAbD1-C6 and MAbD1-C8 had similar affinities for bovine plasma membranes. Both monoclonal antibodies identified a protein of 72 Kd with an apparent subunit of 32-35 Kd. The protein was localized to the cell surface of human and bovine retinal pigment epithelium by immunocytohistochemistry. In the normal eye the antigen identified by the monoclonal antibodies was strongly associated with the retinal pigment epithelium and weakly associated with lens tissue. Using either monoclonal antibody, components of purified bovine or rat retinal pigment epithelial plasma membranes were precipitated from solution. Based on these results, we conclude that both monoclonal antibodies are closely related and that they may be useful for the isolation and study of retinal pigment epithelial cell structure.     

Microfluidic alignment of collagen fibers for in vitro cell culture

Three dimensional gels of aligned collagen fibers were patterned in vitro using microfluidic channels. Collagen fiber orientation plays an important role in cell signaling for many tissues in vivo, but alignment has been difficult to realize in vitro. For microfluidic collagen fiber alignment, collagen solution was allowed to polymerize inside polydimethyl siloxane (PDMS) channels ranging from 10–400 μm in width. Collagen fiber orientation increased with smaller channel width, averaging 12 ± 6 degrees from parallel for channels between 10 and 100 μm in width. In these channels 20–40% of the fibers were within 5 degrees of the channel axis. Bovine aortic endothelial cells expressing GFP-tubulin were cultured on aligned collagen substrate and found to stretch in the direction of the fibers. The use of artificially aligned collagen gels could be applied to the study of cell movement, signaling, growth, and differentiation.     

Cellular proliferation on desamidated collagen matrices

The attachment and growth of human skin keratinocytes (KR) and fibroblasts (FB) on collagen modified by desamidation were investigated. Unlike type I native collagen, desamidated collagen has a sharp iso-ionic pH (5.1) thereby making available either negative or positively charged biomolecules as and when required for a specific bio-application. The cytocompatibility of desamidated collagen prepared from bovine achilles tendons (DCA) and hide trimmings (DCH) was compared with the bovine type I collagen (NC). KR or FB were seeded onto the wells of tissue culture plates coated with the collagen preparations (DCA, DCH and NC). Cellular proliferation on each of these substrates was monitored by reduction of MITT ([3-(4,5-dimethyl-2-thiazolyl)-2,5-dimethyltetrazolium bromide] and by determining the DNA synthesis with [3H]thymidine. In both cases cells proliferated well indicating that DCA and DCH serve as good substrata and are biocompatible. Cell growth occurred without any morphological changes in cell appearance as observed from the phase-contrast microscopy of the cultures, indicating no toxicity of the material during the period studied.     

Retinal pigment epithelial cell adhesion on novel micropatterned surfaces fabricated from synthetic biodegradable polymers

Novel synthetic biodegradable polymer substrates with specific chemical micropatterns were fabricated from poly(DL-lactic-coglycolic acid) (PLGA) and diblock copolymers of poly(ethylene glycol) and poly(DL-lactic acid) (PEG/PLA). Thin films of PLGA and PEG/PLA supported and inhibited, respectively, retinal pigment epithelial (RPE) cell proliferation, with a corresponding cell density of 352,900 and 850 cells/cm2 after 7 days (from an initial seeding density of 15,000 cells/cm2). A microcontact printing technique was used to define arrays of circular (diameter of 50 microm) PLGA domains surrounded and separated by regions (width of 50 microm) of PEG/PLA. Reversed patterns composed of PEG/PLA circular domains surrounded by PLGA regions were also fabricated. Both micropatterned surfaces were shown to affect initial RPE cell attachment, limit cell spreading, and promote the characteristic cuboidal cell morphology during the 8-h period of the experiments. In contrast, RPE cells on plain PLGA (control films) were elongated and appeared fibroblast-like. The reversed patterns had continuous PLGA regions that allowed cell-cell interactions and thus higher cell adhesion. These results demonstrate the feasibility of fabricating micropatterned synthetic biodegradable polymer surfaces to control RPE cell morphology.     

Production of ordered collagen matrices for three-dimensional cell culture.

The aim of this study was to produce collagen gels with controlled fibrillar order as matrices for cell culture. Their structural characterization and colonization by human dermal fibroblasts arc presently reported. Ordered matrices are obtained by using the property of type I collagen monomers to self-assemble in liquid crystalline arrays by slow evaporation of acidic solutions at high concentrations. Induction of fibrillogenesis concomittent with the stabilization of the supramolecular order is then obtained, within petri dishes, by gelation of the viscous preparations under ammoniac vapours. For comparison, dermal equivalents, in which collagen compaction depends on fibroblasts contraction, are made according to the method of Bell et al. (Proc. Natl. Acad. Sci. 76(3) (1979) 1274). The fibrillar arrangement of the collagen network in the samples is determined by polarizing optical microscopy and by transmission electron microscopy. Whereas dermal equivalents exhibit heterogeneous distributions of fibrils, two differents types of order are obtained in the stabilized liquid crystalline collagen samples, namely aligned, i.e. nematic, at 20 mg/ml, or crimped, i.e. precholesteric, at 40 mg/ml. The morphology and behaviour of fibroblasts seeded on the surface of the matrices are analysed from day 1 to day 21. The cells are viable, proliferate at the surface of ordered matrices and migrate up to 400 microm in depth. Production of concentrated and ordered collagen matrices provides new perspectives to study the behaviour of cells in a valorized three-dimensional context where the fibrillar organization becomes close to in vivo situations.     

Microgrooved fibrillar collagen membranes as scaffolds for cell support and alignment

For several years, microgrooved substrates have been evaluated as a means to orient cells in engineered tissues. Recently, we fabricated thin (0.1-5.3 microm) planar and tubular collagen membranes (CMs) from air-dried hydrogels of native, fibrillar type I collagen (Vernon et al., Biomaterials 2004;26:1109-17). The CMs were strong, stable, and permeable and, hence, of potential use as scaffolds for tissue engineering. In the present study, planar CMs supported a robust attachment, spreading, and proliferation of human dermal fibroblasts (HDFs) and human umbilical artery smooth muscle cells (HUASMCs). Collagen hydrogels were air-dried onto microgrooved templates and subsequently removed in the form of grooved CMs with the potential to align cells. The grooved CMs were highly effective at inducing HDFs and HUASMCs to elongate and align, as revealed by scanning electron microscopy and by assays of f-actin and nuclear orientation. Alignment of cells was maintained at high cell densities. CMs with grooves of substantially different widths and depths were similarly effective in causing cell alignment; however, cells aligned poorly on CMs that had grooves less than 1 microm in depth. Grooved CMs with the capability to align cells might be of considerable use in the fabrication of tissue substitutes.     

Fibroblast populated dense collagen matrices: cell migration, cell density and metalloproteinases expression

Dense collagen matrices obtained by using the property of type I collagen to form liquid crystals at high concentrations, were shown to be colonized by human dermal fibroblasts (Biomaterials 23 (2002) 27). In order to evaluate them as possible tissue substitutes, we investigated in this study the mechanism of cell colonization. Fibroblasts were seeded at the surface of collagen matrices at concentrations of 5 and 40 b mg/ml. Cell density and migration were estimated from histological sections over 28 days within 500 microm thick matrices. At day 14, migration started in the 40 mg/ml matrices, attaining 320 microm in distance and 5500 cell/mm(3) in density at day 28. As zymography and western blot techniques demonstrated production of collagenase 1 (MMP1) and gelatinase A (MMP2) in culture medium, collagen hydrolysis was required for cells to penetrate the collagen network. Furthermore, the presence of MMP1 and MMP2 and their tissue inhibitors TIMP1 and TIMP2 was revealed by immunohistochemistry. We presently show that 40 mg/ml collagen matrices are colonized by human dermal fibroblasts and reach, at day 28, a density close to that measured in human dermis.     

Measurement of cell proliferation and cell mediated contraction in 3-dimensional hydrated collagen matrices

Summary This report describes simplified methods for the preparation of reconstituted collagen gels containing a uniform distribution of anchorage dependent cells in 96-well microtitration plates, their dissolution with collagenase allow harvesting of either cells or medium, and objective analytical methods for measuring cell mediated contraction of collagen gels and [³ H]thymidine incorporation rates.To whom requests for reprints should be addressed at Opthalmic Research Unit, University of Western Australia, Royal Perth Hospital, GPO Box X2213, PERTh 6001, Western Australia.     

Retinal pigment epithelium. Interrelations of endoplasmic reticulum and melanolysosomes in the black mouse and its beige mutant.

By cytochemistry (acid phosphatase and tyrosinase activities) GERL, a specialized hydrolase-rich region of endoplasmic reticulum (ER), can be visualized in the cells of the mouse retinal pigment epithelium. Previously catalase cytochemistry permitted us to identify microperoxisomes, with numerous continuities to the ER. The present report reveals the extensive continuities of the ER to pigment granules in various stages of maturation. When the pigment granules, which we consider to be "melanolysosomes," first appear they consist of electron-opaque grains within dilated areas of the ER. As the dilations enlarge, fine fibrils appear in the ER cisternae. Thicker fibers develop from the fibrils; these fibers are generally obscured when melanin deposition occurs. At all stages, the melanolysosomes appear to be connected to the ER.