Hello all
I have cultured Rat Cardiomyocytes just regularly in a dish and on chips. I need to see their contractability and I was wondering if anyone is familiar with their post-culture process?
I am on day 4 of culture and changed the media twice so far. They have not yet started to beat. I was wondering if this is normal or should I wait for them to make connections and be mature more to beat?
Thank you.
Maedeh
Hi Maedeh.
Have you prepared neonatal or adult cardiomyocytes? They are quite different. We, and others, have found that neonatal cardiac myocytes need to be cultured at a reasonable density so that they can form networks and spontaneously beat. Adult cardiomyocytes, on the other hand tend to start beating after preparation as soon as calcium is added to their extracellular medium (usually the preparation is done with a low calcium solution). Adult cells don't make connections in culture, in our experience. Both neonatal and adult preps requires care! Adult cells are especially fragile, and you need to increase the level of extracellular calcium in steps otherwise they die and resemble popcorn rather then the lovely striated, beating cells you want.
Best,
Martin
Thank you Martin
my cells are adult myocytes.
what concentration of calcium would you recommend?
Thank you.
For adult rat ventricular myocytes, I cultured them till 48 hours for transfection purpose. Please read my paper: Article STIM1 enhances SR Ca2+ content through binding phospholamban...
I used M-199 medium without serum to prevent the cell dedifferentiation. The best way to measure contractability is to field stimulate the cells. They will not form network.
Hi Maedeh,
Have you managed to get any contractions going? Both Dr Pesl and Dr Zhao had some good suggestions. Getting the extracellular calcium concentration right is important. Like Dr Pesl, we tend to work at 1.5 - 1.8 mM extracellular calcium.
If your adult cells are alive and well, they should quite happily beat spontaneously. If they have been damaged during preparation they won't beat. You could try Dr Zhao's suggestion of using field stimulation to trigger contraction. You should see the cells twitch during each impulse. We also use field simulation to coordinate the contraction of many cells at once.
De-differentiation of adult cells can be an issue. It has been demonstrated that the expression of some proteins in adult myocytes can change in a few hours post-isolation. Over a longer time, they can also change shape. Dr Zhao's recommendation is very helpful in that regard.
Best wishes,
Martin
Thank you very much Dr. Bootman,
would you mind familiarizing me more with the field stimulation for the coordination of cells beating all at once and as a whole?
I've used the neonatal rat cardiomyoctes and they beat very nice and smoothly but I'd like to intensify or amplify the beating characteristic. Would you mind introducing any of publication within your group or anyone you might know that used the field stimulation?
Thank you very much for your great suggestion.
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