Abstract
A particularly powerful culture method for the retina is the explant assay, which consists in culturing a small piece of retina on an organotypic filter. Retinal explants can be prepared any time between embryonic day 13 (E13) and postnatal day 4 (P4). Although retinal ganglion cells tend to degenerate shortly after they are generated in explants, and photoreceptor cells do not grow extended outer segments, the explants will develop very similarly to a retina in vivo and generate all the different retinal cell types that will migrate to the appropriate layer. The retinal explant culture assay is particularly useful in cases where a mouse mutant is embryonic lethal and its retinal development cannot be studied in vivo. Because retinal explants can be prepared from embryonic animals and electroporated or infected with viral vectors, it is also a useful approach for the study of gene function at embryonic stages. Here, we present a retinal explant culture method that we have used extensively in various publications (Kechad et al., 2012; Cayouette et al., 2003; Cayouette and Raff, 2003; Elliott et al., 2008).
Keywords: Cell lineage, Neural progenitor, Asymmetric division, Organotypic culture, Differentiation
Materials and Reagents
Equipment
Procedure
Day of Experiment
Recipes
Acknowledgments
Funding for this work was provided by the Canadian Institutes of Health Research and the Foundation Fighting Blindness Canada. This protocol was adapted from procedures published in Kechad et al. (2012) and Elliott et al. (2008). The authors wish to thank members of the Cayouette lab, past and present, for continuous support and improvements on the protocol over the years.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
Hi Maryam,This can happen. Make sure you are using room temperature PFA (very important). You should first remove the medium from under the filter and replace it with PFA. Fix for 5 minutes and than add PFA on top of the explants. If your explants still detach easily it is often a sign that they are not very healthy.
Thanks Christine, I will make sure to follow these steps next time. What are the reasons that my culture might be unhealthy? Do I have to switch the media to different one?Thanks
Hi Maryam, sometimes there is some contamination that can be visible around the explants. It looks like a white ring. The explants still grow and this contamination doesn't take over but it is not optimal to grow explants in these conditions. For the medium, no need to change the composition but make sure you change it twice a week. The evaporation of the medium is quit substential and it depend on the kind of incubator you have. We have an incubator the monitor the humidity to 90% and we calculated that 30 microliters of medium evaporate every day. The protocol says that 80% of the medium should be change but you can change the totality and it won't affect your culture. Hope it helps! Good luck, Christine
Thank you, I am starting new explant tomorrow, hopefully work this time:)
Thanks for your interest in our protocol. You should be able to see the retinal tissue in the OCT block when you start sectioning (it has a slightly different colour than the white OCT). I suspect you may not be sectioning the tissue in the proper orientation if you cannot see it. Make sure to rotate the OCT block by 90 degrees when you section so that you will cut across the explant (i.e. perpendicular to it). Each explant is at least 2-3 mm wide, so there should be enough tissue for you to generate many cryosections (2000 microns explant = 100 sections at 20 microns!). Good luck.