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
1. He Q, Cahill CJ, Spiro MJ. Suspension culture of
differentiated rat heart myocytes on non-adhesive surfaces. J. Mol.
Cell. Cardiol. 1996; 28: 1177-1186.
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.