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.     

Role of aquaporins -1 in corneal endothelial fluid transport

AIM: To investigate the expression of aquaporins-1 (AQP-1) in cultured bovine corneal endothelial cells and to explore the role of AQP-1 in corneal endothelial fluid transport. METHODS: The bovine corneal cells were cultured in DMEM containing 200mL/L neonate bovine serum. AQP-1 expression in the bovine corneal endothelial cells was detected with immunohistochemistry method before and after treatment of the cells with aquaporin inhibitor,p -chloromercuribenzene sulfonate. The osmotic water permeability was determined by monitoring volume changes of cultured bovine corneal endothelial cells. RESULTS: Positive staining was used to reveal the AQP-1 expression in the membrane of cultured bovine (in brown color). The reading of osmotic water permeability of the cultured bovine corneal endothelial cells before treatment with p -chloromercuribenzene sulfonate was 0.044±0.005cm/s, which significantly decreased to 0.017±003cm/s after treatment (n =15). CONCLUSION: AQP-1 expressed in the membrane of cultured bovine corneal endothelial cells may play an important role in fluid transport of corneal endothelial cells. Alteration of the AQP-1 expression may cause abnormal corneal function and corneal edema.     

The coating of bovine and rabbit corneas denuded of their endothelium with bovine corneal endothelial cells.

A method for coating corneas denuded of their endothelium has been developed. The attachment and spreading of cultured bovine corneal endothelial cells seeded upon the Descemet's membrane of corneal buttons previously denuded of their endothelium by delicately sweeping the endothelial side with a cotton swab have been analyzed. Confluent cultures of bovine corneal endothelial cells were exposed to trypsin to disrupt the cell monolayer into single cells. Increasing concentrations of endothelial cells ranging from 2·5 × 10⁴ to 3 × 10⁵ cells were then seeded on the denuded Descemet's membrane of 11 mm bovine corneal buttons. When the corneal buttons were stained with alizarin red after an incubation period of 8 hr at 37°C, the best coating was observed with 10⁵ seeded cells. In this case, no areas of denudation could be seen and the cells were clearly apposed to one another, thereby reconstituting an endothelial cell monolayer. The cultured bovine corneal endothelial cells seeded on denuded Descemet's membranes plated extremely rapidly. By 15 min, 80% of Descemet's membrane was covered with a monolayer of endothelial cells and by 30 min all of Descemet's membrane was covered.The plating of bovine corneal endothelial cells on denuded Descemet's membrane was a direct function of the trypsin concentration to which they were first exposed. Cells first treated with 0·05% trypsin plated poorly 1 hr after being seeded on a denuded Descemet's membrane. Better plating efficiency was achieved with cells first exposed respectively to 0·025% and 0·01% trypsin. The best results were consistently obtained with cells first dissociated with 0·005% trypsin.Although serum is required in vitro for the attachment of normal cells to tissue culture dishes, it was not required for the attachment of corneal endothelial cells to the denuded Descemet's membrane. Cultured corneal endothelial cells plated equally well in the presence or absence of serum. Similar results were obtained when the cells were suspended in aqueous humor instead of in tissue culture medium.When denuded rabbit corneas were used as a substrate instead of bovine corneas, all the parameters studied for the attachment and spreading of bovine corneal endothelial cells seeded on bovine corneas (cell density, time, and medium) lead to similar conclusions with respect to the interactions between corneal endothelial cells and rabbit Descemet's membrane.     

Preservation and expansion of the primate keratocyte phenotype by downregulating TGF-beta signaling in a low-calcium, serum-free medium

PURPOSE: To demonstrate whether the original keratocyte phenotype is maintained with proliferative activity by suppressing TGF-beta signaling in rhesus monkey keratocytes expanded in a serum-free and low-[Ca2+] medium. METHODS: Rhesus monkey keratocytes were isolated from central corneal buttons by collagenase digestion for 16 hours, seeded on plastic in Dulbecco's modified Eagle's medium (DMEM) containing insulin-transferrin-sodium selenite (ITS) supplement (DMEM/ITS) or 10% fetal bovine serum (DMEM/10% FBS), or in a defined keratinocyte serum-free medium (KSFM). After confluence, cells in KSFM were continuously subcultured at a 1-to-3 split. Cellular proliferation was analyzed by immunostaining for Ki67 and the MTT assay. The cellular phenotype was determined by immunostaining for aldehyde dehydrogenase (ALDH), keratocan, and CD34 and by the expression of keratocan promoter-driven enhanced cyan fluorescent protein (ECFP). The stability of the keratocyte phenotype was examined by switching KSFM to DMEM/ITS and DMEM/10% FBS. TGF-beta signaling was monitored by measuring the promoter activity of TGF-beta1, -beta2, and -beta RII after transient adenoviral transfection, and cytolocalization of Smad2 and Smad4. RESULTS: In KSFM, monkey keratocytes proliferated while maintaining the expression of keratocan, CD34, and ALDH proteins and keratocan promoter-driven ECFP for at least 15 passages. The nuclear accumulation of Smad2 and Smad4 and the promoter activities of TGF-beta1 and -beta RII were significantly downregulated in KSFM compared with DMEM/10% FBS. In KSFM, an increase of [Ca2+] to 1.8 mM and addition of 10% FBS synergistically downregulated the keratocan promoter activity, facilitated Smad2 and Smad4 nuclear translocation, and upregulated TGF-beta1 and -beta RII promoter activities. CONCLUSIONS: The normal monkey keratocyte phenotype can be maintained in a low-calcium, serum-free medium by downregulating Smad-mediated TGF-beta signaling.     

Eurosol versus fetal bovine serum-containing corneal storage medium

PURPOSE: To evaluate the usability of Eurosol, a new medium-term corneal storage medium without components of bovine origin. METHODS: Ten pairs of human donor corneas were placed in tissue culture at 31 degrees C for 7, 14, 21, 28, or 35 days. One cornea of each pair was cultivated in conventional storage medium on Earls' minimum essential medium base containing 2% fetal bovine serum; the other one was stored in Eurosol. Corneas were examined with inverse light microscopy; corneal thickness was measured; and scanning electron microscopy was performed. RESULTS: No significant difference in corneal thickness and endothelial cell count was found at any time. Scanning electron microscopy showed a complete endothelial cell layer on all corneas. CONCLUSION. The findings indicate a potential clinical applicability of the tested serum-free medium-term storage medium, offering a safer alternative to conventional media containing fetal bovine serum.     

Partial restoration of the keratocyte phenotype to bovine keratocytes made fibroblastic by serum

PURPOSE: To determine whether keratocytes made fibroblastic in vitro by addition of fetal bovine serum to the medium regain the keratocyte phenotype after culture in serum-free medium. METHODS: Collagenase-isolated keratocytes from bovine corneas were plated in DMEM/F-12 containing 1% horse plasma, to allow cell attachment, and then cultured until day 4 in either DMEM/F-12 alone, to retain the keratocyte phenotype, or in DMEM containing 10% fetal bovine serum, to cause the keratocytes to become fibroblastic. Medium for the fibroblastic cells was replaced on day 4 with serum-free medium, and cells were cultured until day 12. Cell phenotypes were determined on days 4 to 5 and 11 to 12 of culture as follows: (1) by the morphologic appearance on phase-contrast microscopy; (2) by the levels of aldehyde dehydrogenase in the cells, determined by SDS-PAGE and Coomassie blue staining; (3) by the relative synthesis of collagen types I and V, determined by (14)C-proline radiolabeling; (4) by pepsin digestion and analysis of collagen types by SDS-PAGE autoradiography; (5) by relative synthesis of cornea-specific proteoglycan core proteins determined by analysis of chondroitinase- or endo-beta-galactosidase-generated radiolabeled core proteins by SDS-PAGE autoradiography; and (6) by the relative synthesis of keratan sulfate and chondroitin sulfate determined by (35)SO(4) radiolabeling and measuring the sensitivity to endo-beta-galactosidase and chondroitinase ABC. RESULTS: Keratocytes cultured in serum-free medium appeared dendritic and became fibroblastic in appearance when exposed to medium containing serum. Keratocytes and fibroblasts synthesized a similar proportion of collagen types I and V. However, compared with the keratocytes, the fibroblasts possessed no aldehyde dehydrogenase and synthesized significantly higher levels of decorin and significantly lower levels of prostaglandin D synthase (PGDS) and keratan sulfate. Subsequent culture of the fibroblasts in serum-free medium did not restore aldehyde dehydrogenase to keratocyte levels but did restore the cell morphology to a more dendritic appearance and returned the synthesis of decorin, PGDS, and keratan sulfate to keratocyte levels. CONCLUSIONS: The results of these studies indicate that primary cultures of keratocytes made fibroblastic by exposure to serum can return to their keratocyte phenotype in synthesizing extracellular matrix. These results also indicate that the differences in the organization of the collagenous matrix produced by keratocytes and fibroblasts may be related more to the different proteoglycan types than to the collagen types produced.     

Culture of retinal capillary endothelial cells

The culture of retinal capillary endothelial cells involves certain problems concerning contamination by pericytes, the maintenance of differentiation and the duration of culture viability. A procedure for the isolation and culture of capillary endothelial cells from bovine retina which overcomes these difficulties, is described. Microvessel fragments isolated by mechanical dispersion and filtration techniques adhere strongly to dishes coated with extracellular matrix produced by bovine corneal endothelial cells. The first migrating cells emerge from the original microvessel fragments two days after plating. This technique and subsequent cloning provides migrating and proliferating cells derived only from the retinal capillaries and uncontaminated by other cell types such as pericytes. Endothelial cells were grown on gelatin coated dishes in a serum supplemented medium (10% calf serum). Cell proliferation was significantly enhanced by the addition of basic fibroblast growth factor (1 ng/ml) to the culture medium. In these culture conditions, retinal capillary endothelial cells can be repeatedly passing without the loss of their principal morphological characteristics and some of the differentiated properties of endothelial cells. Primary cultures and subcultures, at least up to the 8th passage, formed a monolayer of small, elongated, tightly-packed, contact inhibited cells which expressed Factor VIII-related antigen. Ultrastructural examination by transmission electron microscopy of confluent bovine retinal capillary endothelial cells showed many tight junctions and Webel Palade granules. These studies provide new means for the isolation and culture of retinal capillary endothelial cells and presents evidence for growth factor requirements for the ability of cells to be repeatedly passing.     

Increased endothelial survival of organ-cultured corneas stored in FGF-2-supplemented serum-free medium

PURPOSE: To investigate the effect of FGF-2 on corneal endothelial cell survival in porcine and human corneas during corneal storage in a serum-free medium. METHODS: Porcine and paired human corneas were stored at 32 degrees C for 9 and 22 days, respectively. One cornea of each pair was stored in a serum-free culture medium, and the mate was preserved in the same medium supplemented with 10 ng/mL FGF-2. Quantitative analysis of corneal damage after storage was determined by the Janus green photometry technique. 5-Bromo-2-deoxyuridine (BrdU) labeling of the endothelium determined the effect of FGF-2 on endothelial proliferation during storage. Additional cell culture studies were performed to elucidate the role of FGF-2 on the incidence of endothelial apoptosis after serum deprivation. RESULTS: When FGF-2 was added to the serum-free medium, the damage rates of porcine endothelia were reduced from 15.1% +/- 8.7% (control) to 6.4% +/- 2.0% after 9 days and from 25.3% +/- 10.2% to 13.6% +/- 4.2% after 22 days of storage. In the human corneas stored during 22 days in FGF-2-supplemented medium, the amount of endothelial damage was 11.8% +/- 3.2%, which was significantly less damage than in the control fellow corneas stored in unsupplemented serum-free medium (19.3% +/- 6.3%; P < 0.01). DNA synthesis was not enhanced in corneas stored in serum-free medium, serum-free medium+FGF-2, or medium containing 10% FCS. Only a few (3.8%) TUNEL-positive endothelial cells were detected in cultures maintained in FGF-2-supplemented serum-free medium compared with a high number (48%) of apoptotic cells in control cultures. CONCLUSIONS: FGF-2 efficiently reduces human corneal endothelial damage that occurs during organ culture storage in a serum-free medium. This effect is truly protective, because no proliferative activity and a decreased rate of apoptosis were determined. FGF-2 emerges as an important component of a future serum-free corneal organ-culture medium established to replace fetal calf serum (FCS) as a potential source of recipient infection.     

The anterior lens capsule used as support material in RPE cell-transplantation

PURPOSE: To investigate the use of an ocular basement membrane as support material for transplanted porcine RPE cells. METHODS: Porcine RPE cells were grown on bovine corneal extracellular matrix (ECM), isolated bovine- and porcine lens capsules, and tissue culture plastic. Cell density, and cell morphology were studied by phase contrast microscopy and transmission electron microscopy. RESULTS: RPE cells grown on porcine anterior lens capsule and on ECM obtained better morphology and higher final cell density than cells grown on plastic and on bovine anterior lens capsule. It was possible to transplant the porcine anterior lens capsule to the subretinal space in pigs. Within two weeks of observation, the lens capsule was well tolerated in the subretinal space. CONCLUSION: The anterior lens capsule seems to be promising as support material for use in RPE cell-transplantation.     

Apical and basal regulation of the permeability of the retinal pigment epithelium

PURPOSE: The functional characteristics of tight junctions in the outer blood-retinal barrier change during embryonic development and in the presence of disease. A culture model of developing retinal pigment epithelium (RPE) was used to examine the regulation of the tight junctions. METHODS: RPE from chick embryos was cultured on filters that separated the apical and basal medium compartments. Cultures were maintained in various combinations of serum-free medium, serum-free medium that was conditioned by neural retinas, or serum-free medium that was supplemented with bovine pituitary extract, serum, or various hormones. Function was monitored by the transepithelial electrical resistance (TER) or the permeation of small organic tracers. Structure was monitored by immunofluorescence and freeze-fracture electron microscopy. RESULTS: Functional analysis indicated differences in permeability among RPE of different embryonic age and culture conditions. In serum-free medium, the tight junctions were leaky or failed to form. Barrier properties increased if pituitary extract was added to the basal medium chamber or retina-conditioned medium was added to the apical chamber. Retina-conditioned medium was more effective at organizing tight junctional strands into a continuous network, but bovine pituitary extract appeared to modulate the permeability of that network. In combination, they synergistically elevated the TER to physiological levels. Although the thyroid hormone T3 had no effect, serum in the apical medium chamber inhibited the ability of RPE cells to respond to retina-conditioned medium. CONCLUSIONS: Diffusible factors secreted by the neural retina acted synergistically with basolateral stimulation to regulate the structure and function of RPE tight junctions. Serum on the apical side of the RPE monolayer inhibited the ability of retinal factors to upregulate the tight junction barrier.     

Human corneal endothelial cells: isolation, characterization and long-term cultivation

Long-term cultures of human corneal endothelial cells have been established. In culture, these cells form a dense monolayer (about 500,000 cells cm-2), similar to that found in vivo, and synthesize an extracellular matrix containing laminin, entactin, and fibronectin. Factor VIII and angiotensin-converting enzyme were not found in either the cultured or native corneal endothelium. Cells were obtained by scraping corneal buttons that had been preincubated in the culture medium supplemented with endothelial cell mitogen. The human corneal endothelium was grown under conditions virtually the same as those used for cultivation of human vascular endothelial cells, namely, on fibronectin- or gelatin-coated tissue culture plastic in Medium 199 supplemented with 20% human serum and 400 micrograms ml-1 endothelial cell growth supplement. Human corneal endothelial cells from the culture obtained can be used for transplantation onto human corneas, for studying repair of damaged corneal endothelium in situ, as well as for in vitro studies of cell growth regulation.     

Lens epithelium-derived growth factor: increased survival and decreased DNA breakage of human RPE cells induced by oxidative stress.

PURPOSE: Lens epithelium-derived growth factor (LEDGF) has been shown to be a growth and survival factor and to be present in a wide variety of cell types. The purpose of this study was to determine whether LEDGF enhances survival of human retinal pigment epithelial (RPE) cells when challenged by oxidative stress or by ultraviolet (UVB) irradiation in a culture system. METHODS: Primary RPE cells were cultured in standard Dulbecco's modified Eagle's medium (DMEM) containing 15% fetal bovine serum. Protein blot analysis with antibodies to LEDGF was used to detect LEDGF in RPE cells. Initially, RPE cells were cultured in the standard medium for 1 day to allow attachment to the culture plates and then cultured in serum-free DMEM, with and without LEDGF. The trypan blue exclusion method was used to test RPE cell viability. Single-cell electrophoresis was used to evaluate single strand breaks of genomic DNA after exposure to H(2)O(2) or irradiation by UVB. RESULTS: LEDGF was present in RPE cells, predominantly in the nucleus. RPE cells grew for 1 week and survived for 3 weeks in the presence of LEDGF. In the absence of LEDGF, they increased in number for the first week and gradually died in the following 2 weeks. LEDGF protected RPE cells against H(2)O(2) exposure and UVB irradiation. DNA damage induced by H(2)O(2) exposure or UVB irradiation was lower in the presence than in the absence of LEDGF. The expression of heat shock protein (Hsp)27 was elevated by LEDGF. CONCLUSIONS: LEDGF enhanced survival of RPE cells in culture when challenged by oxidative stress and UVB irradiation. LEDGF protected DNA from single-strand breakage and upregulated the expression of Hsp27. These results suggest that LEDGF may be a potential agent for protecting RPE cells under various stress conditions.     

Cell culture conditions affect RPE phagocytic function.

BACKGROUND: Changes in the phenotype of retinal pigment epithelium (RPE) cells in vitro are associated with medium conditions and changes in function. Main goals in RPE tissue engineering are cell propagation in serum-free defined culture conditions, resulting in cells exhibiting differentiated morphology and functioning in vitro. METHODS: To compare the effects of various media and supplements on cell function, an optimized high-throughput phagocytosis assay was developed. Adult human SV40-RPE cells were cultured. Test media included: MEM(E), DMEM, F99, SFM and hSFM, with or without supplements. SNAFL-2 labelled OS were added to RPE in vitro for 4 h and phagocytic binding and uptake were measured. RESULTS: RPE phagocytosis was of different magnitude depending on the serum-free basic cell culture media in the following order: hSFM, SFM > DMEM, MEM > F99. Choroid-conditioned medium (ChCM) decreased phagocytosis dose dependently. Whereas 1% retinal extract (RE) supplementation increased, higher concentrations decreased phagocytosis. Addition of 10% FCS increased phagocytosis. 15% ChCM quenched the stimulation induced by 10% FCS, an effect which could be reversed by the addition of 1% RE. CONCLUSIONS: Cell culture media and RPE environmental factors exert substantial and differential alteration of RPE phagocytic ability. Phagocytosis in a serum-free defined medium is superior to unsupplemented basic media, but still differs from serum-supplemented media (F99RPE) designed for cell propagation. We conclude that media SFM or hSFM promoted phagocytosis most, and application of FCS or 1% RE supports phagocytosis. Unknown factors from neighbouring tissues (retina and choroid) affect phagocytosis differently, suggesting a role in retinal pathogenesis. The results will support identification of specific environmental factors and facilitate design of cell culture media.     

Amniotic membrane maintains the phenotype of rabbit retinal pigment epithelial cells in culture

The success of surgical removal of choroidal neovascularisation followed by transplantation of autologous retinal pigment epithelial cells (RPE) for age-related macular degeneration (ARMD) may be limited by damage in Bruch's membrane. We investigated whether amniotic membrane (AM) might be used as an alternative basement membrane-containing matrix to support RPE growth and differentiation. Primary RPE plastic cultures were established from freshly enucleated Dutch belted rabbit eyes in DMEM/F12 containing 0.1 mM Ca(++) and 10% dialysed FBS. Upon subconfluence, cells were subcultured at 5000-9000 cells cm(-2) in the above-mentioned culture medium on intact AM (iAM), epithelially denuded AM (dAM) or plastic. After confluence, the Ca(++) concentration in the medium was increased to 1.8 mm for 4 weeks. Growth and morphology were monitored by phase contrast microscopy, and the phenotype by immunostaining with antibodies against cytokeratin 18, tight junction protein ZO-1, and RPE65 protein, and by transepithelial resistance (TER) measurement. Immunostaining to cytokeratin 18 confirmed the epithelial origin of isolated cells in both primary culture and subcultures. Compared to plastic cultures, RPE increased pigmentation within 24 hr after seeding on AM, with iAM being more pronounced than dAM. RPE adopted a hexagonal epithelial phenotype with more organised pigmentation, strong expression of ZO-1 and RPE65, and a significantly higher TER 4 weeks after Ca(++) switch on dAM. Our results indicate that AM may be used as a basement membrane-containing matrix to maintain RPE phenotype in vitro, and may facilitate subsequent transplantation to treat ARMD.     

人胚胎视网膜色素上皮细胞在微孔聚酰胺纳米纤维膜上的培养

目的研究人胚胎视网膜色素上皮（RPE）细胞在微孔聚酰胺纳米纤维膜（EPN）上的生长和分化情况,观察EPN能否作为一种理想的膜性支架而支持RPE细胞的生长和分化,为后续Bruch膜替代物的研究奠定基础。方法将培养的人胚胎RPE细胞分别以5×105/cm2的密度种植在EPN和常规塑料（对照组）的Transwell植入物的24孔培养板中,低钙培养基（Ca2＋浓度0.1mmol/L）中培养,待细胞贴壁生长后,改用正常钙培养基（Ca2＋浓度1.8mmol/L）培养4周。用相差显微镜每天观察2组细胞的形态及生长情况;分别以免疫荧光染色及Westernblot法鉴定RPE细胞中CK-18、细胞连接蛋白ZO-1及RPE65的表达,观察培养的RPE细胞遗传表型的维持情况。结果培养在EPN上的RPE细胞与对照组一样,能够较好地贴壁生长,形成单细胞层。EPN较常规塑料更适于RPE细胞贴附生长。EPN的纤维排列类似Bruch膜内胶原层纤维排列。EPN上培养的人胚胎RPE细胞CK-18表达阳性,与对照组相比,ZO-1和RPE65均呈较强表达,较好地保持了原代RPE细胞的表型。结论生长在EPN上的人胚胎RPE细胞能够较好地生长、分化并形成单细胞层,有效地维持了原代RPE细胞的表型。为后续Bruch膜替代物组织工程学研究提供了新的思路。     

Serum-free cultivation of human Tenon's capsule fibroblasts

PURPOSE: In the present study, the effect of three different serum-free and one serum-containing control medium on adhesion, proliferation, cryopreservation and PDGF-induced effects on cell proliferation of human Tenon's capsule fibroblasts (HTF) was compared. MATERIALS AND METHODS: Third passage HTF were suspended in four different culture media (WM/F12, WM/F12/FCS 1%, LR-1, DMEM) and plating efficiency was determined after 24h using a cell counter system. Subsequently, cells were seeded at a density of 50/mm2 and cultured for ten days using the different culture media. Cell number was determined at day 2, 4, 7 and 10 after seeding. Furthermore, HTF cultured under the different conditions were stimulated by PDGF-BB [50 ng/ml]. Additionally, cell vitality after two weeks cryopreservation in five different culture media (WM/F12, WM/F12/FCS 1%, WM/F12/FCS 20%, LR-1, DMEM) was determined. RESULTS: The plating efficiency of HTF when seeded in serum-free medium ranged from 55.3% to 59.6%. Using serum containing WM/F12/FCS 1% a slightly higher plating efficiency of 74.8% was obtained. Proliferation assays revealed population doublings of 0.77 with WM/F12/FCS 1% after an incubation period of 10 days. Cultivation of HTF using serum-free conditions did not cause significant cell proliferation but a slight cell loss which ranged from 23.1% to 34%. Addition of PDGF-BB resulted in a significant increase in cell proliferation with WM/F12/FCS 1%, WM/F12 and DMEM. After two weeks of cryopreservation in WM/F12, LR-1, DMEM, WM/F12/FCS 1% and WM/F12/FCS 20%, only the application of high serum concentrations led to sufficient preservation of cell vitality with a plating efficiency of 82.9%. DISCUSSION: The results of the present study demonstrate that the use of serum-containing media is mandatory for cryopreservation of HTF. Seeding of cells can be performed either with serum or without serum. HTF cultured under serum-free conditions can be maintained quiescent with a sufficient number of cells remaining vital. The serum-free media used in this study can be applied for the investigation of cytokine effects on HTF.     

The extracellular matrix and the control of proliferation of corneal endothelial and lens epithelial cells

When rabbit or bovine corneal endothelial cells were plated at low cell density in the presence of high (10%) concentrations of serum, cells maintained on plastic proliferated slowly and after a few days enlarged considerably. If the cultures were exposed to FGF, the cells proliferated actively and, after a week, a confluent monolayer of closely apposed mononucleated cells was formed. In contrast to cells maintained on plastic, cells maintained on an extracellular matrix (ECM) produced by corneal endothelial cells proliferated even faster than cells maintained on plastic and exposed to FGF and no longer required the presence of FGF to reach confluence. Addition of FGF to such cultures did not decrease the mean doubling time, which was already at a minimum (16 hr), nor did it result in a higher final cell density, which was already at a maximum (700–1000 cells/mm²). It can therefore be concluded that, when the proliferation of corneal endothelial cells from two different species is compared, cells maintained on plastic proliferate poorly and FGF is needed in order for the cultures to become confluent. In contrast, when similar cultures are maintained on ECM, they proliferate actively and no longer require FGF in order to become confluent. Similar results were observed when lens epithelial cells were maintained on plastic versus an ECM. Although cells maintained on plastic hardly proliferated, yielding within a few days a population composed of large and binucleated cells, cells plated on an ECM proliferated actively, with an average doubling time for lens epithelial cells of 15 hr during their logarithmic growth phase.The ability of plasma vs. serum to sustain cell proliferation was analyzed using corneal endothelial cells maintained on plastic versus an ECM. It was observed that cultures maintained on plastic proliferate poorly when exposed to plasma. When exposed to serum they proliferate more actively. Nevertheless, in both cases cultures required the presence of FGF in order to become confluent. When similar cultures were maintained on an ECM, they proliferated equally well regardless of whether they were exposed to plasma or serum and no longer required FGF in order to become confluent. One can therefore conclude that the simple change of substrate from plastic to ECM will restore the sensitivity of these cells to agents present in plasma.     

Cell culture conditions affect RPE phagocytic function

Background Changes in the phenotype of retinal pigment epithelium (RPE) cells in vitro are associated with medium conditions and changes in function. Main goals in RPE tissue engineering are cell propagation in serum-free defined culture conditions, resulting in cells exhibiting differentiated morphology and functioning in vitro. Methods To compare the effects of various media and supplements on cell function, an optimized high-throughput phagocytosis assay was developed. Adult human SV40-RPE cells were cultured. Test media included: MEM(E), DMEM, F99, SFM and hSFM, with or without supplements. SNAFL-2 labelled OS were added to RPE in vitro for 4 h and phagocytic binding and uptake were measured. Results RPE phagocytosis was of different magnitude depending on the serum-free basic cell culture media in the following order: hSFM, SFM > DMEM, MEM > F99. Choroid-conditioned medium (ChCM) decreased phagocytosis dose dependently. Whereas 1% retinal extract (RE) supplementation increased, higher concentrations decreased phagocytosis. Addition of 10% FCS increased phagocytosis. 15% ChCM quenched the stimulation induced by 10% FCS, an effect which could be reversed by the addition of 1% RE. Conclusions Cell culture media and RPE environmental factors exert substantial and differential alteration of RPE phagocytic ability. Phagocytosis in a serum-free defined medium is superior to unsupplemented basic media, but still differs from serum-supplemented media (F99_RPE) designed for cell propagation. We conclude that media SFM or hSFM promoted phagocytosis most, and application of FCS or 1% RE supports phagocytosis. Unknown factors from neighbouring tissues (retina and choroid) affect phagocytosis differently, suggesting a role in retinal pathogenesis. The results will support identification of specific environmental factors and facilitate design of cell culture media. Support: Werner-Otto Foundation, Pro Retina Germany e.V. (M.O.K.).     

Stimulation of DNA synthesis in human and bovine RPE by peptide growth factors: the response to TNF-alpha and EGF is dependent upon culture density

A range of concentrations of several peptide mitogens was tested for growth activity on bovine and human RPE cells under serum free conditions by analysis of 3H-thymidine incorporation within the first 24 hrs of exposure to the agents. For cultures which were subconfluent or in early confluence, TNF-alpha, a product of activated macrophages, was the most effective mitogen; little or no growth stimulation was observed for PDGF, EGF, NGF, IGF-1, IL-1B, bFGF or TGF-beta 1. For TNF-alpha and EGF the growth response was analyzed in cultures of varying density. TNF-alpha was more active in sparse RPE cultures whereas EGF stimulation was greater in dense cultures. The response to growth factors was similar in RPE cells from the two species sources, but the apparent magnitude of the response was greater for bovine cells because the growth rate in serum free medium, which was used as the basal reference, was lower for bovine RPE. It is concluded that culture conditions, especially the timing of the assay and the level of confluence of the cells, affect the detection of a growth response to peptide mitogens. Although several of the agents which were tested did not stimulate DNA synthesis in RPE in this study, they may nonetheless promote growth when assayed in combination with other agents or they may affect other biological functions of RPE cells.     

Retinal pigment epithelium rescues vascular endothelium from retinoic acid-induced apoptosis

PURPOSE: To determine whether retinoids are capable of inducing vascular endothelial cell apoptosis and whether the presence of an intact RPE monolayer can block retinoid-induced vascular endothelial cell death. METHODS: Confluent fetal bovine aortic endothelial (FBAE) cells were incubated with various concentrations of all-trans or 9-cis retinoic acid (an analogue of 11-cis retinoic acid). Apoptosis rates were determined at 24 hours, and the effect of inhibition of protein synthesis and activation of protein kinase C on apoptosis was investigated by supplying culture medium with 0.1 mg/mL cycloheximide and 10 nM phorbol myristate acetate. To investigate the impact of RPE on retinoid-induced apoptosis, confluent FBAE cells were cultured with a confluent layer of RPE in inserts where retinoids were added to the upper compartment. A confluent bovine corneal endothelium monolayer was used as the control. The permeabilities of the RPE and bovine corneal endothelium monolayers to fluorescein (20 microg/mL) and 9-cis retinoic acid (3 x 10(-4) M) were also determined. RESULTS: 9-cis Retinoic acid induced higher rates of apoptosis in FBAE cells than did all-trans retinoic acid and the control (P = 0.004). This effect was dose-dependent, with an ED(50) of 1.4 microM (r = 0.99, P = 0.004). Cycloheximide did not inhibit 9-cis retinoic acid-induced apoptosis, but phorbol myristate acetate significantly decreased the apoptosis rate (P = 0.005). The presence of a confluent RPE monolayer reduced the 9-cis retinoic acid-induced apoptosis rate (P = 0.002), but the presence of a bovine corneal endothelial monolayer did not (P > 0.05). Both cell types established a similar diffusion barrier against fluorescein and 9-cis retinoic acid. CONCLUSIONS: 9-cis Retinoic acid is an important mediator of vascular endothelial apoptosis. A confluent monolayer of RPE can prevent endothelial cell apoptosis, and this effect is not due simply to establishment of a diffusion barrier by the RPE.