I want to culture Adult Rat Ventricular Cardiomyocytes upto 48 hrs. The problem is cell viability which falls rapidly after first 6 hrs of Isolation. My aim is to induce hypertrophy in this culture so cells are Serum starved during culturing. I wanted to ask whether preconditioning the cells in Serum will help in better cell viability?
Dear Ketaki
We have had the same problems with adult cardiomyocytes. In first place, it is necessary to ensure that the heart is perfused properly and with solutions with decreasing calcium concentrations. Assumed this, consider the serum batch that you are using, because the yield varies a lot among different batches. Finally, I strongly recommend culturing neonatal cardiomyocytes. It is easier and accurate for your work
Hector
I've only isolated adult mouse cardiomyocytes, but to prevent early apoptosis I use blebistatin 25uM in the plating and culture medium. First I use a plating medium with 10% FBS to allow attaching to the plate, and after 3 hours I change it to culture medium with 10% BSA (if you want to check hypertrophy or any stimulation this is important as FBS is a potent growth factor). With this blebbistatin you can keep cardiomyocytes alive for up to 4 days.
Thanks for the input!
We use BCS during heart perfusion-digestion and then remove dead cells by increasing Calcium concentration to 1.8 M . But once cells are isolated I dont use serum at all. So was thinking about preconditioning cells with serum, then serum starve them and do the treatement.
I am a bit skeptical about use of Blebbistatin as its inhibitor of Myosin II, will it not affect downstream hypertrophy signaling?
Depends on what your goal is - can also use BDM for longer healthier looking cells, albeit uncoupled. As said above your cells should be lasting longer better, may need to alter your collagenase protocol. Many culture in PC1, nonsera containing pricey media, helps with longevity/morphology. Is the goal to induce hypertorphy or make better cells, and what for, contraction studies, viability morphology etc.
I'm not sure about the effect of Blebbistatin on hypertrophy. Also, as Jody says, using BDM is another alternative.
I want to induce hypertrophy in these cells and for readout I am using cell size and hypertrophy marker gene expressions. So for both the readouts I require good viability cells at 24 and 48 hrs of culture. And I am already using BDM for perfusion of heart during cell isoaltion.
You can try to culture the cardiomyocytes with a serum free rich medium. To the basic serum free medium you add 2 mg/mL albumin, 5 mM creatine, 2 mM L-carnitine, 5 mM taurine. This is the original formulation from J.C. Perriard and H.M. Eppenberger.
This medium allows cardiomyocytes to better survive in culture up to 72 hours without inducing all the changes typical of the Serum containing medium.
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