I'm working with H9C2 cells for the first time and I've been following protocols I could find on papers and research gate. For the sub-culture, I seed 10k cells/cm2 and split the cells every 2-3 days as needed. I've then continued with a differentiation protocol in which I added 1 uM or 10 nM RA in dark daily for 5-7 days, and measured cardiomyogenic markers such as Actc1, Myl2, and Tnnt2. However, I saw no difference in gene expression (qPCR) in RA-treated cells compared to DMSO-treated although there were morphological differences. For the differentiation experiment, I seeded 30k cells/cm2 on a 12-well plate.
Is there anything I'm doing not right? For example too high seeding density? Can anyone experienced with handling H9C2 cells please help?
Hi. This problem has been described here in greater detail: https://doi.org/10.24170/14-2-2498
Is there a specific reason that you want to use H9C2? Other cardiac cell models might be more physiological and/or reproducible. Depending on what you want to measure e.g. iPSCs or neonatal myocyte might be suitable.
Anja Riedel hi! Thank you for your answer. Yes, I've read that paper before and took that into account.
I eventually would like to use a transfectable cardiomyocyte model to do some measurements (qPCR, induce hypertrophy, etc). I've considered the primary neonatal myocytes as well but we don't have the expertise in our lab to work with such a model.
I have used rat neonatal cardiac myocytes and performed hypertrophic stimulation and RNA analysis as well. Its quite a nice model (of course I am heavily biased here). Once you have a working assay and are familiar with the isolation process it is quite robust. A good technician that has general experience with cell isolations can perform that assay very well after being supervised thrice, as far as my teaching experience goes in that matter, so that is not the tricky part if you can get somebody on site. Getting there in my own took me about two years, though, so I would defintely advise on teaming up with someone xD then again If that H9C2 would also take 2 years to get up and running, the neos might be the more accepted model anyways. Also of course primary cells mean paperwork.
One thing to keep in mind generally is that transfection works rather poorly in cardiac myocytes (single digit percentages). I do not know whether the H9C2 cells are easier in that regard, but especially if you want to do RNA analysis you might want to sort your cells beforehand and/or consider AAV oder AdV Gene transfer, that should get about 60+% of them, depending on the virus quality.
Best of Luck!!
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