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K. M. Engelsberg, F. Ghosh; Culture of the Human Embryonic Retina. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5898. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To study the development and survival of human embryonic retinas kept in culture as whole eyes.
All proceedings were in accordance to Declaration of Helsinki.Human embryos were obtained from elective abortions with informed consent from the women seeking abortion. A total of 20 eyes were enucleated.The ages of the embryonic retinas were 6-7½ weeks. The age was determined by measuring the length of the embryo with ultrasound and by use of the Carnegie staging table. Eyecups from 2 eyes were fixed immediately, to be used as controls. The remaining 18 eyes were carefully placed on culture plate inserts with the posterior pole facing the membrane. The eyes were cultured in Dulbecco’s Modified Eagle’s Medium F-12 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 explants were divided into 3 groups kept for 7 (n=5), 14 (n=7), and 28 (n=6) days in vitro (DIV). After fixation and sectioning the specimens were processed forhaematoxylin and eosin staining and immunohistochemistry. Antibodies against recoverin (cones and rods), PKC (rod bipolar cells) and vimentin (Müller cells) were used.
The control retinas consisted of large neuroblastic cell layer (NBL) and a marginal zone. The retinas kept 7 DIV displayed a NBL and a marginal zone. Only a few pyknotic cells were found and these cells were localised to the inner part of the retina. The 7 eyes kept 14 days in vitro consisted of a NBL, but no marginal zone was found. Pyknotic cells were seen at the inner part of the retina and there was a gradient of pyknotic cells with highest concentration in the posterior pole and almost none at ora serrata. After 28 days in culture several pyknotic cells were observed at the inner part of the retinas, however, in the outer part no pyknotic cells were seen. Explants kept 28 DIV were partially double folded and in one of the retinas rosette formations were found. Vimentin labeling showed cells arranged in a vertically arranged pattern in all explants Labeling with recoverin displayed photoreceptors in one of the explants kept 14 DIV and in all retinas kept 28 days in culture. PKC labeling displayed cells with small axons in 3 of the retinas kept in culture for 28 days.
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