To begin, take the pellet of the GFP-HUVECs grown to a confluence level of 80 to 100%and resuspend the cells in an appropriate volume of basal medium to achieve a concentration of one times 10 to the seventh cells per milliliter of stock solution. Prepare 50-milliliter conical centrifuge tubes containing the required volume of basal medium supplemented with the respective growth factors. Add the GFP-HUVECs from the stock solution at a dilution of one to 66.67 to achieve a concentration of 1.5 times 10 to the fifth cells per microliter.
Then aspirate the medium from the plate containing the stromal monoculture and add 200 microliters per well of the prepared GFP-HUVECs suspension. Incubate at 37 degrees Celsius in 5%carbon dioxide in a humidified atmosphere, changing the medium every three to four days. Monitor the development of the culture by bright-field and fluorescence microscopy using a five times objective.
Differences between the cultures could be observed from the bright-field and fluorescence images showing only the GFP-HUVECs. The cultures appeared less developed without any growth factor, However, faster development was observed in the presence of FGF-2. In contrast, the bright-field images showed the densest cultures in the presence of BMP-2.
However, vascular like networks formed in both growth factor-containing conditions and the most extensive and interconnected networks were formed with FGF-2.
