Hello,
I am trying to isolate cardiomyocytes. But i am facing issue while culturing the cardiomyocytes after 3-5 days other cell types are dominating over the cardiomyocytes. So, there is a gradual reduction in the beating of cardiomyocytes. I followed adhesion based method to get enriched cardiomyocytes. But that protocol also doesn't work properly. Anyone knows how to eliminate cells other than cardiomyocytes from culture? Is there any reagents which kill the smooth muscle cells and fibroblasts in culture?
Thank You..
You can initially pre-plate cardiomyocytes (CM) primary culture for two hours, during this time fibroblast will attach, while CM don't, collect the CM and culture them again in new dish.
You can add horse serum in primary culture, it will inhibit the growth of fibroblast.
You can selectively detach the CM, as fibroblast show strong adherent properties, during trypsinization the CM will detach within 1-2 minutes, while fibroblast don't, collect the CM and re-plate them.
You can try adding 100 µM cytosine β-D-arabinofuranoside (a.k.a. Ara-C) to your culture medium. Ara-C prevents DNA replication and kills dividing cells, but has no effect on post-mitotic cardiac myocytes.
Thank you very much Christophe. But neonatal rats or chick cardiomyocytes have some proliferation ability. So there is a chance for the death of those cardiomyocytes.
Irfan Khan Thank you Very much. I am facing big problem with the smooth muscles cells. These cells are more proliferating than any other cells in culture. Whether this adhesion based protocol only for fibroblasts or it works for SMC's also.
I used the same protocol for the neonatal cardiomyocytes. I always get the pure CM with this method, characterized with immuno, and flow. I grow them in DMEM-F12. If you have a sorter in the lab, you can purify the CM. Which kind of growth medium you are using for neonatal growth?
I am using DMEM-F12 only. Can you give the adhesion protocol what you used.
Thank you very much Irfan Khan. What type of makers are prefferable for sorting cardiomyocytes? We have Connexin-43 is this enough to sort the cardiomyocytes or any other suggestions.
Connexin is expressed in other cells as well, I will suggest to go for GATA-4, troponin T or I, myosin heavy chain, .....
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