[Experimental study on culture method of human umbilical vein endothelial cells]

To establish an efficient and stable culture method of human umbilical vein endothelial cells (HUVECs) in vitro so as to provide good source of seed cells for tissue engineered vascular grafts and for preclinical research.

The umbilical cords were harvested from full-term normal delivered neonates, which were perfused with 0.1% collagenase II by self-made needle and were digested at 37 degrees C and 5% CO2 humidified incubator. The HUVECs were cultured in endothelial culture medium (ECM) containing 5% fetal bovine serum (FBS) and 1% endothelial cell growth factor (ECGS). HE staining of the umbilical cords before and after digestion was used to observe the detachment of HUVECs, flow cytometry to detect the purity of primary HUVECs, and inverted phase contrast microscope to observe the morphology of the cultured HUVECs. The growth of the 3rd passage cells was measured by MTT assay; immunocytochemical technique and matrigel-based capillary-like tube formation assay were carried out to identify the function of HUVECs.

After digestion of 0.1% collagenase II, marked HUVECs detachment was observed with complete digestion. The purity of the HUVECs was 99.56% by digestion of 0.1% collagenase II at 37 degrees C and 5% CO2 humidified incubator for 15 minutes. Primary HUVECs showed a cobblestone or pitching stone-like appearance in vitro, forming a confluent monolayer cells after 2-3 days of culture. MTT assay demonstrated that HUVECs showed the fastest growth speed at 3 to 4 days, and showed growth of cell fusion at about 5 days. Immunocytochemistry showed that HUVECs highly expressed endothelial marker factor VIII. Matrigel based capillary-like tube formation assay showed that it could form endothelial-like tube structures after 24 hours of culture.

Using improved method and ECM could obtain high quantity and high quality primary HUVECs, which might be a kind of promising seed cells for tissue engineering and preclinical research.