Culture medium effects on vascular smooth muscle cell contractile protein expression and morphology in 2D v. 3D

Vascular tissue engineering is dependent on achieving both tissue strength and contractility. Proliferative, synthetic smooth muscle cells (SMCs) are necessary for producing strong, engineered vascular tissue; however, in vivo, healthy SMCs are quiescent and contractile. In this study, we evaluated the effects of culturing cells in a high serum, growth medium (GM), compared to a low serum, quiescence medium (QM). QM increases contractile protein expression and decreases proliferation in 2D primary human SMC cultures. We observed an increase in population doubling time within 5 days (from 23±1 to 47±3 hrs; n=6) in 2D human SMC cultures, consistent with a contractile phenotype. After 3 days in QM, smooth muscle α-actin (SMaA) increased 10-fold with similar increases in calponin (compared to SMCs cultured in GM). The cellular response was different when applied to 3D cultures, which were created by human SMC self-assembly into ring-shaped tissue constructs. SMC rings were cultured for 24 hours in GM followed by 13 days in GM or QM. The rings grown in QM were significantly thinner than rings grown in GM (400±30 v 540±90μm; n=5,4) with a visible increase in collagen deposition by picrosirius red staining. There was no detectable difference in SMaA or calponin expression within SMC ring samples cultured in GM or QM. This difference in SMC response indicates the need for other approaches to SMC differentiation in 3D constructs.