Endothelial cell seeding with rotation of a ventricular blood sac

Successful establishment of a durable endothelial cell (EC) monolayer inside a ventricular blood sac requires homogeneous coverage of the entire luminal surface with attached cells. For this purpose, a new device was developed that slowly rotates a fully assembled cardiac prosthesis with three degrees of freedom. We seeded ECs derived from human adipose tissue at a density of approximately 3.5 x 10(4) cells/cm2 onto the surfaces of polyurethane-made blood sacs and "ersatz" bladders (consisting of T-25 tissue culture flasks). The kinetics of cell attachment, spreading, and proliferation were determined using video microscopy combined with image analysis and cell viability assays. After 60 min of seeding at 5-10 rotations/hr, the plating efficiency inside the blood sacs was 35.7 +/- 11%, with cell viability remaining approximately 90 +/- 5%. After 3 hr, when the plating efficiency reached a plateau (approximately 70%), the rotation was stopped and the ECs were allowed to spread and proliferate under static conditions. Within 48 hr, the entire luminal surface was evenly covered by a confluent EC monolayer. Our long-term studies show that with a proper feeding schedule, such an EC monolayer can be maintained intact in vitro for more than 2 weeks.