Engineering high-density endothelial cell monolayers on soft substrates

This study demonstrates that a confluent monolayer of endothelial cells (ECs) can be tissue engineered on a soft substrate with a cell density and morphology that approximates in vivo conditions. We achieved formation of a confluent EC monolayer on polydimethylsiloxane (PDMS) elastomer by microcontact printing of fibronectin (FN) in a square lattice array of 3 μm diameter circular islands at a 6 μm pitch. Uniform coatings of FN or serum proteins on PDMS or on tissue-culture-treated polystyrene failed to support the equivalent EC density and/or confluence. The ECs on the FN micropatterned PDMS achieved a density of 1,536 ± 247 cells mm−2, close to the 3,215 ± 336 cells mm−2 observed in vivo from porcine pulmonary artery and significantly higher (2- to 5-fold) than EC density on other materials. The probable mechanism for enhanced EC adhesion, growth and density is increased focal adhesion (FA) formation between the ECs and the substrate. After 14 days culture, the micropatterned FN surface increased the average number of FAs per cell to 35 ± 10, compared to 7 ± 6 for ECs on PDMS uniformly coated with FN. Thus, microscale patterning of FN into FA-sized, circular islands on PDMS elastomer promotes the formation of EC monolayers with in vivo-like cell density and morphology.