Isolation and culture of Sprague-Dawley rat aortic smooth muscle cells

The intact mature arterial media is composed of at least four phenotypically unique cell subpopulations that reside in distinct medial layers.  The sub-endothelial media is predominately populated by non muscle-like cells; the middle media, by smooth muscle cells; and the outer media, by two phenotypically distinct cell subpopulations, one smooth muscle and the other non muscle-like. 

In order to isolate the four cell subtypes identified in vivo, the arterial media are separated into the three layers:
(1) A very thin sub-endothelial layer (termed here L1);
(2) An intermediate-sized middle layer (termed L2);
(3) A thick outer layer (termed L3);
After separation of the media into three layers, cells were grown from each layer by explant techniques.

Primary cultures of rat aortic smooth muscle cells were obtained by enzymatic dissociation (Primary Cell Isolation Kit) of thoracic aortae from 250-300 g male Sprague-Dawley rats.

1. Sprague-Dawley rat aortic smooth muscle cells were isolated from thoracic aortas.
2. The adventitia and connective tissue were removed; the remaining arterial intima and media were cut into 1-cm2 segments and placed in culture dishes with Primary Cell Isolation Solution.
3. Cells were grown in Primary Cell Culture Medium including with Primary Cell Culture Supplement, 10% heat-inactivated calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin…
4. These cultures were harvested twice a week with primary cell culture solution and passaged at a 1:4 ratio in T-flasks.
5. The cells were then seeded at a density of 5x10⁵/cm² and were cultured at 37°C in 95% air/5% CO_².
6. Cells from passages 2 through 4 were used in all experiments.
7. The phenotype of the cultured cells was determined by staining the cells for -actin and desmin.
8. Antibodies for muscle-specific -actin and desmin were purchased.

References
Haller H, Lindschau C, Quass P, Distler A, Luft FC. Differentiation of Vascular Smooth Muscle Cells and the Regulation of Protein Kinase C-. Circ. Res. 1995; 76: 21–29.