I've been trying to expand the CMV strain AD169 in fibroblasts without success. I'm following the protocol provided by the NIH AIDS reagent program: I'm infecting the fibroblasts monolayer using a MOI of 0.01 and 0.1 for one hour and after 10-14 days I don't really see any CPE, the cells usually begin to detach from the plate, but I don't see any clear CPE.
I have tried HFF and an in house fibroblast cell line available in my lab, but none of those have work.
Thanks for any tips you can provide me with!
Check reference below for your answer:
Methods Mol Biol. 2014;1119:47-57.
'Use of diploid human fibroblasts as a model system to culture, grow, and study human cytomegalovirus infection.'
Fortunato EA.
Thank you very much Amit, I'll try that protocol. It is not that different from the one I'm doing, but we'll see.
Hi Rodrigo,
If both your HFF (not too old) and AD169 are okay, this shouldn't be a problem. I haven't checked the protocols that are being suggested, but we use 2hr incubations on a rocker in a minimal volume (eg. 1 ml for a T25 flask) followed by taking off excess virus and replacement with standard media. This works with all the CMVs we use including low passage strains such as Merlin. I would be tempted to check your AD169 stock using a much higher MOI, just to see what level of infection you can achieve. An MOI of 10 should give close to 100% infection. If it doesn't, then your titres are likely to be down. Are you storing your AD169 at -70˚C? HCMVs are very temperature sensitive - they fall apart at -50˚C or less....
Kind regards
Eddie
Wasn't particularly accurate with my last comment. I meant -50˚C or higher (ie. -70˚C is fine).
When I was a graduate student, if we wanted to store HCMV long term (either Towne or AD169) we would always freeze down infected cells (harvest the cells when they are about 50-75% CPE). The cell freezing protocol is the same you would use for any other cell line (20%FBS, 10% DMSO). When you thaw the cells, add them to a culture of HFF at around 50% confluent. Of course you will still need to amplify the virus in HFF once you have it back up. We felt that supernatants with HCMV lost titer slowly at -80C so over long periods of time, your virus might become inactivated. Freezing the virus in cells eliminates the risk of totaling losing the virus. Other than that, I agree with all of the suggestions above. Though checking out different isolates of HFF might be worth doing since some isolates replicate virus to a higher titer. And if your HFFs are beyond passage 20, you might want to get fresh anyway.
Good Luck!
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