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Abstract Background Light exposure and more specifically the spectrum of blue light contribute to the oxidative stress in Age-related macular degeneration (AMD). The purpose of the study was to establish whether blue light filtering could modify proangiogenic signaling produced by retinal pigmented epithelial (RPE) cells under different conditions simulating risk factors for AMD. Methods Three experiments were carried out in order to expose ARPE-19 cells to white light for 48 h with and without blue light-blocking filters (BLF) in different conditions. In each experiment one group was exposed to light with no BLF protection, a second group was exposed to light with BLF protection, and a control group was not exposed to light. The ARPE-19 cells used in each experiment prior to light exposure were cultured for 24 h as follows: Experiment 1) Normoxia, Experiment 2) Hypoxia, and Experiment 3) Lutein supplemented media in normoxia. The media of all groups was harvested after light exposure for sandwich ELISA-based assays to quantify 10 pro-angiogenic cytokines. Results A significant decrease in angiogenin secretion levels and a significant increase in bFGF were observed following light exposure, compared to dark conditions, in both normoxia and hypoxia conditions. With the addition of a blue light-blocking filter in normoxia, a significant increase in angiogenin levels was observed. Although statistical significance was not achieved, blue light filters reduce light-induced secretion of bFGF and VEGF to near normal levels. This trend is also observed when ARPE-19 cells are grown under hypoxic conditions and when pre-treated with lutein prior to exposure to experimental conditions. Conclusions Following light exposure, there is a decrease in angiogenin secretion by ARPE-19 cells, which was abrogated with a blue light - blocking filter. Our findings support the position that blue light filtering affects the secretion of angiogenic factors by retinal pigmented epithelial cells under normoxic, hypoxic, and lutein-pretreated conditions in a similar manner.

Purpose: A cell line spontaneously derived from human retinal pigment epithelium (hRPE) was cultured on alginate film gelatinized with different concentrations of neurobasal cell culture medium (NCCM) to assess its growth and morphological behavior on this naturally occurring polysaccharide. Methods: Neonatal human globes were used to isolate hRPE cells. They were cultured in Dulbecco's modified Eagle's-medium-and-Ham's-F12-medium-(DMEM/F12) supplemented with 10% fetal bovine serum (FBS). Cultures were continuously studied using phase contrast microscopy. After the nineth passage, cells were characterized through immunocytochemical analysis for Oct4, Chx10, and Pax6 and Ki67 markers. In each well of a 6-well microplate, 1 and 2% weight/volume (w/v) alginate in deionized water was added and gelatinized using 1× and 10× NCCM. hRPE cells were cultured at a density of 2 × 105 cells/well in alginate-coated microplates. After 5 days, hRPE colonies were harvested and re-plated on polystyrene substrates. Morphology and growth of hRPE cultures were determined during the next 2 weeks. Results: The first few passages of the cultures were purely hRPE cells that revealed typical morphological features of the pigmented epithelium. They made spaces, devoid of cells, between hRPE cell monolayer and fill in the unoccupied spaces. They grew faster than native RPE cells and rapidly overgrew. Immunocytochemical test revealed that the founded cells expressed Chx10, Pax6, Ki67 and Oct4. The hRPE cells survived unlimitedly on alginate film and formed giant adjoining colonies. After re-plating, hRPE colonies adhered quickly on polystyrene and displayed native hRPE morphological features. Conclusion: Alginate film can support the survival and growth of hRPE cells and induce the cells to re-organize in tissue-like structures.

Background: Light exposure and more specifically the spectrum of blue light contribute to the oxidative stress in Age-related macular degeneration (AMD). The purpose of the study was to establish whether blue light filtering could modify proangiogenic signaling produced by retinal pigmented epithelial (RPE) cells under different conditions simulating risk factors for AMD.Methods: Three experiments were carried out in order to expose ARPE-19 cells to white light for 48 h with and without blue light-blocking filters (BLF) in different conditions. In each experiment one group was exposed to light with no BLF protection, a second group was exposed to light with BLF protection, and a control group was not exposed to light. The ARPE-19 cells used in each experiment prior to light exposure were cultured for 24 h as follows: Experiment 1) Normoxia, Experiment 2) Hypoxia, and Experiment 3) Lutein supplemented media in normoxia. The media of all groups was harvested after light exposure for sandwich ELISA-based assays to quantify 10 pro-angiogenic cytokines.Results: A significant decrease in angiogenin secretion levels and a significant increase in bFGF were observed following light exposure, compared to dark conditions, in both normoxia and hypoxia conditions. With the addition of a blue light-blocking filter in normoxia, a significant increase in angiogenin levels was observed. Although statistical significance was not achieved, blue light filters reduce light-induced secretion of bFGF and VEGF to near normal levels. This trend is also observed when ARPE-19 cells are grown under hypoxic conditions and when pre-treated with lutein prior to exposure to experimental conditions.Conclusions: Following light exposure, there is a decrease in angiogenin secretion by ARPE-19 cells, which was abrogated with a blue light - blocking filter. Our findings support the position that blue light filtering affects the secretion of angiogenic factors by retinal pigmented epithelial cells under normoxic, hypoxic, and lutein-pretreated conditions in a similar manner. [ABSTRACT FROM AUTHOR]

Objective: Retinal pigment epithelium (RPE) is a highly polarized and specialized monolayer epithelium that placed at the interface between the photoreceptors of the neural retina and the vascular layer of the choroid. Almost all cells in the human body grow within organized 3D matrices. Alginate is naturally derived polysaccharide that, is an ideal bioMaterials for tissue engineering. culturing cells on alginate as a substrate provides a more in vivo-like environment. A spontaneously derived line of human RPE (hRPE) was found in our routine hRPE cultures. It revealed reasonable expression of stem/progenitor cell markers. We cultured the aforesaid hRPE line on alginate films and its apparent characteristics were investigated. Materials and Methods: 940 μl of 1% and 2% (v/w) alginate in deionized water were added in every well of 6 well micro plates for preparing a 1mm alginate film. Alginate substrate was gelatinized using 1X and 10X neurobasal cell culture medium (NCCM). hRPE cell line was isolated from neonatal human globes and cultured in DMEM/F12 supplement with 10% FBS. We characterized these cultures trough immunocytochemistry analysis for Oct4, Chx10, Pax6 and Ki67 markers. Cell were cultivated at a density of 200000 on each well of alginate coated 6 well micro plates. After 5 days RPE colonies were harvested and re-cultured on polystyrene substrates. Results: HRPE cell line survived for more than ten days on alginate film. Viability of the cultures was 99%. They grew very well on alginate films; total number of cells increased eleven times respect to controls. The cells formed big sticky colonies on alginate and exhibited bigger colonies on 2 %( v/w) alginate gelatinized with 10X (NCCM). When we harvested hRPE colonies and re-cultured them on polystyrene, cells did not switch their morphologic features and maintained their spheroidal structure. Conclusion: We found that alginate can support survival and growth of hRPE cell line and force them to organize in giant colony structures like spheroids. We are going to characterize hRPE cultures more precisely and investigate their differentiating potential into other retinal cells. [ABSTRACT FROM AUTHOR]

Objective: RPE is a monolayer of cells located between vessels of the choriocapillaris and light-sensitive outer segments of the photoreceptors. During the embryonic development of the eye, retinal stem cells in the optic vesicle give rise to multipotent progenitor cells that generate all the neurons and the Muller glia of the mature retina.This study describes morphology, viability, growth and characterization of a spontaneously arising human RPE (hRPE) cell line that have been found in our routine hRPE cell culture. Materials and Methods: RPE was isolated from neonatal human globes and cultured in DMEM/F12 supplement with 10% FBS. Cultures were continuously studied using phase contrast microscopy. After the 9th passage cultured cells were characterized trough immunocytochemistry analysis for Oct4, Chx10, and Pax6 and Ki67 markers. Results: The first few passages of the cultures were purely RPE cells, those revealed usual morphological features of the pigmented epithelium. Then suddenly alternative variant of cells appeared They made sizeable holes between RPE cells' monolayer and push surround of this holes. They finally push all of the culture vessels and RPE cells' features totally disappeared. The new founded cells were smaller than RPE cells and showed an appreciable tendency to form giant macroscopic colonies. After sequential passages they got smaller than before with a high nucleocytoplasmic ratio, representing thin layer of cytoplasm around the nucleus. The aforementioned cultures were passaged for 13 times until now. They grew faster than native RPE cells and rapidly covered culture surface .When we assessed for viability they showed more than 99% viable. Immunocytochemistry test revealed that 100% of the obtained cells expressed Chx10 whereas just 4% were pax6+ .60% of the studied nuclei were Ki67+ and 50% expressed Oct4. Conclusion: Our human RPE cultures gave rise to a cell line of hRPE that exhibited retinal cell stemness markers. [ABSTRACT FROM AUTHOR]