Primary cardiac fibroblast and cardiomyocyte isolation
Primary cardiac fibroblast and cardiomyocyte isolation
1. Ventricles were removed under sterile conditions.
2. Ventricles were placed in cold sterile primary cell culture medium, minced into approximately 2 mm cubes and treated with solution of primary cell isolation.
3. Tissue fragments retained on 105 μm nylon mesh were placed in primary cell culture medium containing 1% bovine serum albumin (BSA) and 100 μM CaCl2, teased apart with sterile forceps and dispersed by trituration using a wide-bore pipette.
4. Tissue fragments were removed and the cell suspension was adjusted to 500 μM CaCl2 and centrifuged over a 5 ml cushion of primary cell culture medium containing 6% BSA.
5. The resulting cardiomyocyte-enriched pellet was resuspended in primary cell culture medium containing primary cell culture supplement and 10% fetal bovine serum and seeded on 100 mm collagen-coated tissue culture plates for 3 h at 37 °C in a CO2 incubator to remove non-myocyte cells.
6. Non-adhered cardiomyocytes were collected and stored at −80 °C.
7. For cardiac fibroblast isolations, ventricles were removed as noted above, minced and incubated in Hank's buffer containing trypsin (0.1 mg/ml) and collagenase (50 units/ml) for 10 consecutive 10 min treatment periods at 37 °C.
8. Cells from each digestion period were pooled, resuspended in primary cell culture medium containing primary cell culture supplement and 10% FBS and seeded in standard culture dishes for 8 h.
9. Non-adherent debris was discarded and the attached fibroblasts were maintained in primary cell culture medium incuding 10% FBS, scraped into ice-cold PBS, pelleted and stored at −80 °C for EMSA and Western blot analysis.
Reference
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2. Bashey RI, Donnelly M, Insinga F, Jimenez SA. Growth properties and biochemical characterization of collagens synthesized by adult rat heart fibroblasts in culture. J. Mol. Cell. Cardiol. 1992; 24: 691-700.
3. Subramanian SV, Kelm RJ Jr, Polikandriotis JA, Orosz CG, Strauch AR. Reprogramming of vascular smooth muscle actin gene expression as an early indicator of dysfunctional remodelling following heart transplant. Cardiovasc. Res. 2002; 54: 539–548.
