Identification of key factors in deep O2 cell perfusion for vascular tissue engineering

Blood vessel engineering requires an understanding of the parameters governing the survival of resident vascular smooth muscle cells. We have developed an in vitro, collagen-based 3D model of vascular media to examine the correlation of cell density, O2 requirements, and viability. Dense collagen sheets (100 micron) seeded with porcine pulmonary artery smooth muscle cells (PASMCs) at low or high (11.6 or 23.2x10(6) cells/mL) densities were spiraled around a mandrel to create tubular constructs and cultured for up to 6 days in vitro, under both static and dynamic perfusion conditions. Real-time in situ monitoring showed that within 24 hours core O2 tension dropped from 140 mmHg to 20 mmHg and 80 mmHg for high and low cell density static cultures, respectively, with no significant cell death associated with the lowest O2 tension. A significant reduction in core O2 tension to 60 mmHg was achieved by increasing the O2 diffusion distance of low cell density constructs by 33% (p<0.05). After 6 days of static, high cell density culture, viability significantly decreased in the core (55%), with little effect at the surface (75%), whereas dynamic perfusion in a re-circulating bioreactor (1 ml/min) significantly improved core viability (70%, p<0.05), largely eliminating the problem. This study has identified key parameters dictating vascular smooth muscle cell behavior in 3D engineered tissue culture.