Maybe you can find some answers here Viero, C. et al.  (2008). A primary culture system for sustained expression of a calcium sensor in preserved adult rat ventricular myocytes. Cell Calcium, 43(1), 59–71. doi:10.1016/j.ceca.2007.04.001

or here

Tian, Q. et al. (2012). Functional and morphological preservation of adult ventricular myocytes in culture by sub-micromolar cytochalasin D supplement. Journal of Molecular and Cellular Cardiology, 52(1), 113–124. doi:10.1016/j.yjmcc.2011.09.001

Good luck and best regards

Benjamin

Hello Benjamin,

Thanks for the reply and references.

I am already using ITS in my culture medium and I have done few experiments with electric stimulation , doing analysis of those.

Cytochalasin D have shown to have some effect on calcium transience. But I can try for experiments and see if I can get improved cell culture and also check for calcium transience.

Thanks for the suggestions.

Best Regards

Ketaki

Cytochalasin D or another cross-bridge cycling inhibitor (such as BDM) do indeed increase the viability of the cells. Also make sure that the [Ca] of your culturing medium is not too high (and not too different from the buffer in which you bring them up to [Ca} after isolation).

Another important determinant is the quality of the isolation. If you start out with only 30-40% rod-shaped cells, these cells also tend to survive less well.

Good luck,

Diederik

 Generally I start experiments around 70-80% cell viability, but the viability drops to around 40% in 24 hrs and approx. 20% after 48 hrs, which I guess overshadow the results.

Did you try to culture at a physiological oxygen tension (2-3%O2)?

Best regards, Willem van der Laarse

I culture cardiomyocytes in M199 medium with ITS and Hepes and in normal cell culture conditions . But during Isolation by langendorf I do Oxygenate all the perfusion and enzyme solutions with oxygen.

These conditions (including normal culture conditions) are hyperoxic, so your cells may die of oxidative stress. Perhaps addition of 10 mM N-acetyl cysteine during isolation helps.