I suspect that it could be due to the loss of pacemaker cells during the isolation or is it due to the culture conditions.
Hi Krishna I did similar exp many years ago. What we did was to isolate cells from 5 day old rats using trypsinization. Cells were then centrifuged and suspended in culture medium. The cells were incubated in a culture dish to allow mesenchymal cells to bind to dish. I cannot remember the incubation time as this was about 35 years ago. After the initial incubation, the cells that were not bound in the initial incubation were counted and seeded into plates. Medium was changed every 3 days. After 5-7 days a person see synchronous beating of the cardiomyocyte clusters. The method used is described in the following paper: T. Chajek, O. Stein, Y. Stein, Lipoprotein lipase of cultured mesenchymal rat heart cells, Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, Volume 528, Issue 3, 1978, Pages 466-474
Hi Krishna, I isolated neonatal rat cardiomyocytes every weeks with a kit of extraction. After 3 days, every cells, covering the culture surface, beats synchronously.
The cells which don't beat may be fibroblasts? Do you pre-plating the cell suspension to avoid cardiac fibroblasts?
What is the size of your culture dish? We noticed that the beats are best in plastic 6-well plate. It seems that in the smaller wells (96 well-plate or 24), the pacemaker cells may not be enough numerous to allow good beats. Therefore your hypothesis is plausible.
Do you seeding the cells at a concentration sufficient? Have you tested a growing range of seeding concentration?
Good luck !
Hi Krishna, I have used collagen dispersion method to isolate neonatal rat cardiomyocytes. The cells not beating in plates are not necessarily nonmyocytes. Beating of cells usually occurs when the cells are present in clusters or clumps., forming connections with each other. Isolated single cells usually do not beat. If you have any doubt, stain your cells with alpha sarcomeric actinin and count the number of actinin positive cells. A good isolation generally shows more than 90% actinin positive cells (myocytes).
Prof. Edmund Pool, thanks for the literature suggestion, I will read them for my reference. Hélène Peyret, I culture them on dishes of size of 35 mm dia with circular opening at bottom. I belive as you mentioned, the concentration of cells could have been very low. But this week the isolation was good and i had a good concentration of cells. 80000-100,000 cells per dish looks like a good concentration.
Ritwik Datta, yes that is a fair point, however when i isolate and culture them onto plates most cells are not single cells, they form clusters as soon as they adhere to the surface. And yes i will try count of actinin positive cells soon.
Thanks to all of your for valuable and to-the-point suggestions. I wish you all well.
- Badges
- Science topic