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S. G. Remington, J. M. Crow, J. D. Nelson; Lens Fiber Cell Proteins Expressed in Chicken Ciliary Body Cultures. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2781.
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To assess the capability of cells from the ciliary body to express characteristics of lens fiber cells.
Explant culture: Ciliary body or presumptive ciliary body was dissected from E6-E10 chickens. Diced tissue explants were plated on 35mm dishes (plastic, collagen IV or matrigel) and incubated in M199/10% chicken serum/1% penicillin/streptomycin/amphotericin B (PSA) at 37 °C in 5% CO2 for 7-8 weeks. Media was changed 2-3 times per week.Dissociated cell culture: Ciliary body or presumptive ciliary body was dissected from E6-E8 chickens. Cells were dissociated in 0.06% trypsin at 37 °C for 30 minutes, and plated at 1x106 cells per 35mm dish on plastic, collagen IV or matrigel. Cultures were maintained in M199/7.5% chicken serum/7.5% fetal calf serum/1% PSA at 37 °C in 5% CO2 for 7-8 weeks. During weeks 2 and 3, 0.1µg/ml human bFGF2 was added to the media. Media was changed 2-3 times per week.Immunofluorescence: Cultures were fixed in 4% paraformaldehyde and processed with rabbit polyclonal primary antibodies (made to AQP0 or filensin), biotinylated goat anti-rabbit secondary antibody, and AlexaFluor 488-conjugated streptavidin.
Explant culture: Lentoids developed from ciliary body explants after several weeks in culture. Many lentoids were located near the leading edges of epithelial cell sheet outgrowth from explants.Dissociated cell culture: Small groups of transparent, balloon-like cells developed above variably pigmented epithelial monolayers in ciliary body cultures.Immunofluorescence: Antibodies to lens fiber cell proteins AQP0 and filensin labeled lentoids and balloon-like cells form ciliary body cultures. These antibodies also labeled individual transparent cells within the epithelial monolayers of both explant and dissociated cell cultures.
Lens fiber cell proteins were expressed in unpigmented cells in embryonic chicken ciliary body cultures. Ciliary body cells exhibited lens fiber cell characteristics in vitro. Stem cells within the ciliary body may have the potential to differentiate into lens cells.
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