Purchase this article with an account.
J. Sanderson, N. Niyadurupola, P. Sidaway, L. Wang, D. C. Broadway; Characterisation of Organotypic Cultures From Human Retina. Invest. Ophthalmol. Vis. Sci. 2009;50(13):79.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Retinal organotypic explant cultures are valuable experimental models, since cellular architecture and neuronal connectivity is retained. The aim of these experiments was to develop organotypic cultures using human retina.
Human retina was obtained from donor human globes within 24 hours post mortem. The retina was dissected, then 4mm circular sections were taken for analysis and/or culture. Thy-1 expression was measured using Taqman QRT-PCR. Morphology was assessed by haemotoxylin/eosin staining and immunocytochemistry.
Thy-1 mRNA was measured as an index of retinal ganglion cell (RGC) number in explants taken from different regions of the retina. In keeping with the known distribution of RGCs in the retina, Thy-1 levels were highest in the macular and lowest in peripheral regions. Levels in regions directly surrounding the macular showed comparable Thy-1 expression, allowing 5 para-macular samples per retina to be used for organotypic culture. Culture in serum-free DMEM/HamF12 medium for 96 hours showed no change in gross morphology of the explants: size, colour and integrity were maintained over this time period. Histology showed the retinal layers to be intact at the time points investigated. Immunocytochemistry using the ganglion cell marker NeuN indicated the presence of RGCs in explants cultured for up to 96 hours. Measurement of Thy-1 mRNA levels showed a progressive decrease in Thy-1 levels with time, decreasing to a baseline level at 48 hours. Similar data was obtained using Neurobasal A medium supplemented with B27/N2.
This PDF is available to Subscribers Only