We have a transgenic mouse line in the laboratory from which we are looking to culture retinal cells from. Our experiments indicate we might be able to produce stable cultures from these cells that could be used for many passages.
However, our current limiting factor is cell survival after extraction of the retina. Currently, I am planning on following a protocol I designed from several sources:
1. Extract retina from eye tissue and incubate in HBSS/EBSS containing DNase and papain for 60 minutes at 37 C whilst shaking at 700 rpm.
2. Pipette remaining tissue gently to dissociate further and place in DMEM with 10% FBS to inhibit papain action.
3. Culture in 24-well plates in retinal maturation media.
Any recommendations, improvements or notes would be highly appreciated.
Thank you for your question
But this is not the scope of my work
Greetings to you
The retina contains many cell types creating a primary culture for each of the types requires a different approach and different cultures. Your papain dissociation approach is correct although I don't recommend shaking as it reduces the viability of the cells. I can tell you more if you let me know what cells you are tying to culture.
Anton Lennikov I am actually attempting to culture all cells types together in an attempt to replicate the retinal signalling environment in vitro.
I could possibly extract different cell types seperately and then combine later on, rather than culturing all cells together?
Ideally I am looking at culturing retinal ganglion cells, biopolar cells and Muller glials although the other cell types would be very valuable too!
Thank you
Thank you for your question
But this is not the scope of my work
Greetings to you
Jack Hopkins Unfortunately it doesn't work like this. You can't just seed the retinal dissociate on DMEM and expect that dissociated cells to organize themselves into something it takes the whole embryonic development process to develop into a retina. From the crude dissociative you may get some microglia and pericyte growing but most of the cell types will simply die as each requires its own growth factor that is usually provided by complex cell interaction within the tissue. Isolating just one type of the retinal cell type is a complex task requiring its own protocol. You may have better luck using retinal explants just pieces of retina small enough that they can get the nutrition and oxygenation from the media. While explants will eventually deteriorate and die (lack of RPE cell support, damage to ganglion cells through severed optic nerve connections but still you can have several weeks of semi-functional retina tissue in your culture for the study of your interest.
Anton Lennikov thank you for your reply. To confirm, I am not expecting the cells to spontaneously organise themselves as in the retina - only to increase their survival. Your suggestion of using explants is very interesting and I will look into this further.
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