H9C2 cells - trouble when culturing H9C2 cells (Jan/17/2010 )
Hello, Im trying to culture the rat heart cell line (H9C2 rat heart cells). I'm using DMEM high glucose medium (4nm L-lactate) and I'm having the same problem: cells are too few, small with a round shape. A guy from our lab told me that I have to change my culture flasks ( i'm using T30 flasks) because , as he said, they are too large for my cells that are few, But I dont think this is the reason.
I'll really appreciate any answers from you that can help me with my work, thank you a lot
Don't discount the advice of your colleague. If the cells are too widely spaced in a flask, then they won't receive enough of the factors that cells excrete for them to survive.
If you only have a small number of cells, put them into a smaller flask, plate of dish. Go down to 96 well plates if you have to.
Thank you a lot for your answer. I really appreciate it, I have another thing to ask about, when I threw the cells, I noticed that many of them were floating, and til now, there are many cells dead somehow and they are not attached, should I use PDL or PLL to enhance their attachement? is the attachment the real reason for the cell death? thank you again for the answer !!
Have you checked that the floating cells were dead through use of a vital stain such as trypan blue?
It could be that the cells need an attachment factor, though the ATCC page doesn't mention anything about it. The ATCC says that the cells will rapidly lose the myoblasts if subcultured too densely - perhaps this happened to the tube(s) of cells that you are using, before they were frozen down.
Thank you a lot for answering my questions again ! Well I did not try to check the viability of the floating cells, you are right, maybe they are not dead, I will check this !
I don't know if the cells I thawed were sub-cultured densely or not, I got them from our lab library, others have frozen them a while ago.
My first cells died, now I'm culturing others ( the same line H9C2) and I'm having the same problem, cells are small and their growth is so slow, I reduced the surface area of culture, last time I used T30 flasks, now I'm culturing them in one well of 6-wells plate.
I hope this will work, I will let you know the result later
Thank you again for your help, I really appreciate it