TISSUE ENGINEERING OF HEART VALVES – IN VITRO EXPANSION OF ENDOTHELIAL CELL BY DIFFERENT VESSELS REVEALS THE BETTER AUTOLOGOUS CELLULAR SOURCE FOR TISSUE REPLACEMENT

Background. Consistent expansion of primary human endothelial cells in vitro is critical in the development of engineered tissue. A variety of complex culture media and techniques developed from different basal media have been reported with alternate success. Incongruous results are further confounded by donor-to-donor variability and cellular source of derivation. Our results demonstrate the overcome of these limitations using soluble CD54 (sCD54) as additive to conventional culture medium. Methods. Isolated primary fragment of different vessel types were expanded in Ham’s F12 DMEM, enriched with growth factors, foetal calf serum and conditioned medium of human umbilical vein endothelial cells (HUVEC) collected at different passages. Cytokines content of culture media were analysed in order to identify the soluble factors correlating with better proliferation profile. Results. sCD54 was found to induce the in vitro expansion of human endothelial cells (HEC) independently from the vessels source and even in the absence of HUVEC-conditioned medium. The HECs cultivated in presence of sCD54 (50ng/ml), resulted positive for the expression of CD146 and negative for CD45, and lower fibroblast contamination. Cells were capable to proliferate with an S phase of 25%, to produce VFGF (10 ng/ml) and to give origin to vessel-like tubule in vitro. Conclusions. Our results demonstrate that sCD54 is essential factor for the in-vitro expansion of HEC without donor and vessels-source variability. Resulting primary cultures can be useful, for tissue engineering in regenerative medicine (e.g. artificial micro tissue generation, coating artificial heart valve etc.) and bio-nanotechnology applications.