1. Hearts were removed from newborn rats (day 0), put into calcium- and bicarbonate-free HEPES-buffered Hanks’ medium, cut into pieces and digested with solution of primary cell digestion under constant stirring.

2. The collected primary cells were passed through a cell strainer (40 mm) and then seeded onto uncoated plastic dishes and incubated for 60 min at 37℃.

3. The supernatant (containing the cardiomyocytes) was collected and the adherent cells were washed several times, and then cultured in primary cell culture system containing 5% fetal calf serum (FCS).

4. These cultures contained almost exclusively primary cardiac fibroblasts, as >95% of the cells were stained with antibodies directed against the fibroblast-specific antigen prolyl-4-hydroxylase, and >95% of the cells were negative for the cardiomyocyte-specific marker α2-actinin, the endothelial cell marker platelet/endothelial cell adhesion molecule (CD31 antigen), as well as smooth muscle α2-actinin.

5. The cells in the supernatant that was collected were plated in MEM containing vitamin B12, NaHCO^₃ and 5% FCS.

6. This cell population was almost exclusively cardiomyocytes, as >95% of the cells stained positive for α2-actinin.

7. Separation of cardiomyocytes was achieved by sedimentation at room temperature (22℃) for 10 min followed by a pre-plating step to further deplete fibroblasts.

8. The supernatant of the sedimentation step contained fibroblasts that were pelleted by low-speed centrifugation (250g for 10 min).

9. The resulting preparation was seeded on tissue culture dishes and vigorously washed after 2 h.

10. These cultures contained 95% fibroblasts, based on staining for cell-type-specific markers.

References
Thum T et al. MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts. Nature. Vol 456: 18/25 December 2008. doi: 10.1038/nature07511