Particle-Mediated Gene Transfection and Organotypic Culture of Postmortem Human Retina

Published on Mar 1, 2019in Translational Vision Science & Technology2.112
· DOI :10.1167/TVST.8.2.7
Rania A. Masri5
Estimated H-index: 5
(USYD: University of Sydney),
Sammy C.S. Lee14
Estimated H-index: 14
(USYD: University of Sydney)
+ 1 AuthorsUlrike Grünert49
Estimated H-index: 49
(USYD: University of Sydney)
Sources
Abstract
Purpose: Particle-mediated gene transfer has been used in animal models to study the morphology and connectivity of retinal ganglion cells. The aim of the present study was to apply this method to transfect ganglion cells in postmortem human retina. Methods: Postmortem human eyes from male and female donors aged 40 to 76 years old were obtained within 15 hours after death. In addition, two marmoset retinas were obtained immediately after death. Ganglion cells were transfected with an expression plasmid for the postsynaptic density 95 protein conjugated to green or yellow fluorescent protein. Retinas were cultured for 3 days, fixed and then processed with immunohistochemical markers to reveal their stratification in the inner plexiform layer. Results: The retinas maintained their morphology and immunohistochemical properties for at least 3 days in culture. Bipolar and ganglion cell morphology was comparable to that observed in noncultured tissue. The quality of transfected cells in human retina was similar to that in freshly enucleated marmoset eyes. Based on dendritic field size and stratification, at least 11 morphological types of retinal ganglion cell were distinguished. Conclusions: Particle-mediated gene transfer allows efficient targeting of retinal ganglion cells in cultured postmortem human retina. Translational Relevance: The translational value of this methodology lies in the provision of an in vitro platform to study structural and connectivity changes in human eye diseases that affect the integrity and organization of cells in the retina.
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