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K.M. Engelsberg, B. Ehinger, F. Ghosh; Human Fetal Retinas in Culture . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2781.
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To study the development and survival of human fetal retinas kept in culture.
The work was approved by the Ethical Research Committee of the University of Lund. Human embryos were obtained from elective abortions with informed consent from the women seeking abortion. A total of 14 eyes were enucleated. The ages of the fetal retinas were 6 1/2 weeks (n=1) and 7 1/2 – 8 weeks (n=13).
Eyecups, from the 6 1/2 (n=1) and 7 1/2 (n=2) weeks retinas were fixed immediately, as references, without dissecting the neuroretina.
From the remaining 11 eyes, the neuroretina was carefully dissected while the eyes were kept in CO2–independent medium. The neuroretinal pieces, measuring approximately 2 mm in diameter were explanted on Millicell®–PCF 0.4 µm culture plate inserts (Millipore, Bedford, MA) with the future photoreceptor layer facing the membrane. The explants were cultured in Dulbecco’s Modified Eagle’s Medium F–12 (Gibco) supplemented with 10% fetal calf serum and maintained at 37°C with 95% humidity and 5%CO2. A cocktail containing 2mM L–glutamine, 100U/ml penicillin and 100ng/ml streptomycin (Sigma–Aldrich) was added. The culture medium was changed every second day.
The explants were divided into 3 groups kept for 7 (n=4), 14 (n=3), and 28 (n=4) days in vitro. After fixation and sectioning the specimens were processed for haematoxylin and eosin staining and immunohistochemistry. Antibodies against transducin (cones), recoverin (cones and rods), PKC (rod bipolar cells) and vimentin (Müller cells) were used.
The control retinas displayed no differentiation. Retinas kept 7 days in vitro displayed rosettes in two of the explants. The other two explants consisted of a single neuroblast layer. Vimentin staining displayed vertically arranged Müller cells. No other immunolabeling was found. The 3 explants kept 14 days in vitro displayed rosette formations. Dispersed cells were labeled for recoverin in 2 of the explants and some of these labeled cells had a short axon. Vimentin labeling showed cells arranged in a vertically arranged pattern. In all explants kept 7–14 days in vitro dispersed single pyknotic cells were observed. All retinas kept 28 days in vitro had several rosettes. Some parts of the explants were partially double folded. Labeling with recoverin displayed photoreceptors in both the rosettes and at the outer border of the explants. Vimentin labeling displayed vertically arranged cells throughout the explants.
We here show that human fetal human retinas can be kept in tissue culture for up to at least 4 weeks and develop at approximately their intrinsic time schedule.
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