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Jose Hurst, Jan Halbmann, Johanna Hofmann, Martin Stephan Spitzer, Sebastian Thaler, Sven Schnichels; New Method for Isolation of Retinal Ganglion Cells- via Retrograde Transferred Magnetic Beads. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3235. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Due to the limited access of retinal ganglion cells (RGCs) it is our goal to establish a new method for RGC isolation. The concept includes the transport of magnetic beads via retrograde transfer to the RGCs and the isolation of the cells from the retina via magnetic columns. Through the successful establishment of our introduced method, molecular biological standard methods could be used more efficient in developing therapies, which would result in a huge progress in RGC research.
Based on the publication of Thaler et al. 2006 demonstrating the retrograde transport of labeled DNA from the brain to the RGCs, originated the idea to transport magnetic beads. Beads with 100 nm of diameter were selected, which carry glucuronic acid, lipid sheath, casein cover, poly aspartate acid or PEI cover. Additional to their coating the beads are fluorescently labeled. For all beads toxicity and the optimum isolation method was determined in 3 different cell lines. Also the uptake of the beads was evaluated by electron microscopy. The most promising beads were selected and injected into the brain of adult rats. After the designated time-points (7, 10, 14 days) the rats were euthanized and the brain as well as the eyes were removed. Fluorescence microscopy of retina and brain sections was used to compare the location and degree of transport. For the isolation retinae were dissociated. The cell suspension was applied on the super magnetic columns and the cell eluate was recultured.
24 hours after application 5 out of 6 magnetic beads were well tolerated in all tested concentrations. From three isolation methods a magnetic plate, a magnetic fixture and mini columns with a so-called “super magnetic”, the isolation with the super magnetic columns turned out to be the most effectual technique. The uptake was proven with electron microscopy. With this optimized protocol an isolation of 80-95% of the originally cell numbers was received. Injections into the S. colliculus resulted in transfection of the tractus opticus and the papilla 7 days after treatment the beads were detected.
Suitable beads were selected successfully and the optimum isolation method was determined. First in vivo experiments demonstrated that retrograde transfer of magnetic beads is feasible and well tolerated. In the next experiments, the time span and the concentration will be optimized to improve the isolation method.
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