Hi,
I am trying to culture cardiomyocytes from 6th day chick embryo. Cells are beating till 5-6 days. After that it changed to round morphology, and big vacuole kind of things are presented inside the cells.Culture medium i am using DMEM/F12, with 10% FBS +1%Pen-strep.One more thing i wanted to know that is there any specific number of cells we have to seed in T25 flask for the maintenance of cells . I am not using any growth factors or coating for cell attachment. Can any one help me to solve this issue?
I have attached the video below for better understanding...
I can't comment specifically on the chicken embryo cardiac myocytes, but mammalian cardiac myocytes are typically maintained in serum-free media (as serum contributes to their de-differentiation) and do not require maintenance cells. With that said, groups typically coat their plates with extracellular proteins such as laminin or matrigel. Less frequently used are mouse embryonic fibroblasts, which presumably deposit an ECM. Although, depending on your isolation, you likely are carrying along fibroblasts which may continue to proliferate in culture unlike myocytes.
Mammalian myocytes will typically stop beating in culture within 24-96hrs depending on the species. Neonatal myocytes will beat longer, but rapidly de-differentiate into a beating sheet and lose many mature aspects of their morphology.
Insulin-Transferrin-Sodium Selenite has been shown by some to help maintain sarcomeres in cultures, although it's not requisite. Other groups have also suggested supplementing with Taurine may have similar effects and help maintain contractility. DMEM/F12 also typically has 1mM calcium, whereas typical serum concentrations are 1.8mM. This could be contributing to changes in calcium handling resulting in decreased contractility. You could try a complex media such as M199, although supplementing DMEM/F12 will likely yield similar results.
Regarding cell plating density, myocytes seem to respond positively to relatively high confluent densities. Take care not to go so high such that plated myocytes will overlap, this will result in poor adherence to their substrate.
What are you trying to accomplish with these cardiac myocytes?
Article Isolation and Culture of Neonatal Mouse Cardiomyocytes
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