To support my in vitro experimental data, I start to try retina explant culture to check the axonal outgrowth of retina ganglia cell. I have tried many times with WT E18 mouse. The isolation of retina is good, and were cut into pieces round 200~500um. retina was put on the glass surface coated with laminin- (40 µg/ml) and poly-D-lysine- (100 µg/ml). The maintenance medium is containing Neural Basal medium and 1% N2 neuronal supplement (Gibco BRL), 1% penicillin-streptomycin (Gibco BRL), and 5 µg/ml insulin, 100 µg/ml transferrin, 20 nM progesterone, 0.1 mM putrescine, 30 nM selenium, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.4 mM glutamine. The problem is that the outgrowth rate of axon is very very low ( round 200~300 um round DIV10) compared to the previous report. some report said after 24h there is quite obvious axon out. some literatures used the collagen as matrix. some literatures just used glass coverslip as growth surface. I tried DRG explant culture with the same coating condition and medium condition, and the result was pretty good.
Our key has always been to incubate the ex vivo retina at the air-water interface in the tissue culture dish. So we have never put the retina directly on glass but rather on a transwell membrane. The retina is grown photoreceptor side down. Attached is a recent publication which gives most of the details of our methods.
We have not assayed RGC axon growth - the Bonhoeffer stripe assay was developed specifically for this purpose - there is a recent review of it - Knöll et al Nature Protocols 2, - 1216 - 1224 (2007) Published online: 10 May 2007 | doi:10.1038/nprot.2007.157
Hope some of this helps -
Our key has always been to incubate the ex vivo retina at the air-water interface in the tissue culture dish. So we have never put the retina directly on glass but rather on a transwell membrane. The retina is grown photoreceptor side down. Attached is a recent publication which gives most of the details of our methods.
We have not assayed RGC axon growth - the Bonhoeffer stripe assay was developed specifically for this purpose - there is a recent review of it - Knöll et al Nature Protocols 2, - 1216 - 1224 (2007) Published online: 10 May 2007 | doi:10.1038/nprot.2007.157
Hope some of this helps -
Though being no expert on retina epithelium I have a suggestion. For coating do you really need poly-D-lysine? It may be too sticky for your cells and severly impede cell migration and thus also proliferation - I'm just guessing.Furthermore, as William J Brunken describes, it should be crucial to maintain cell/tissue polarity (by growing the epithelium on a filter support) and to allow access to the growth medium at the proper site.
My expertise is in other water-air culture tissues, though not retine itself, and I agree with William J.B. that that air-water interface is a key factor, in the case of embryonic frog skin (mucosal epithelia) explants, it was important to have very low level of media for slightly covering the explants, so high air exchange but not exposed directly to air which is dreadful for that tissue. Then, for marine fish testis explants it was useful to have also a 1-2 mm thick layer of 0.8% agarose and also a very low level media, since that tissue did not like the glass nor plastics that we tried (bacteriological, primary and cell line culture materials).
Thanks all you guys! The reason I like to culture on glass coverslip is easy to perform living neuron imaging. It seems like air supply is pretty important for this tissue culture. I will try to put the retina explant on the tissue culture membrane. BTW, 5% CO2 is sufficient? and do need extra O2 supply?
We use a conventional tissue culture incubator - with CO2 controlled and no added O2. For retina and skin, the air-water interface is critical. This is very different than the conditions for the DRG cultures you have been using as controls. You should be able to image in the transwell dish as well.
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