3. ISOLATION OF ENDOTHELIAL PROGENITOR CELLS

Mononuclear cells from the blood are centrifuged on Ficoll™ and seeded for 24 hrs in wells coated with fibronectin and containing culture medium EndoCult® Liquid Medium; population of adhering cell is removed. Non-adhering cells are yielded after 2 days and seeded in culture dishes coated with fibronectin. After 3 following days, the first colonies appear (see Fig. 4). Founder cells of the colonies, endothelial progenitor cells, are described as CFU-EC (colony-forming unit-endothelial cells). From 1 ml of blood up to 35,000 endothelial progenitor cells can be isolated.
Fig. 4. Isolation of endothelial progenitor cells. Shortly after cell seeding (Fig. In left – 2 days) cell culture contains single cells only. Four days after seeding (middle image) CFU-EC proliferate and give rise to the first colonies that increase their size in the following dates (Fig. In right – 5 days). Photographs were taken from the brochure on Endocult Liquid Medium.
Endothelial progenitors express specific markers and they also are able to form tubular structures (similar to blood capillaries) in 3D gels (formed e.g. by collagen type I) (Fig. 5).
Fig. 5. In vitro differentiation of endothelial cells. Phase contrast of tubular structures formed by endothelial cells grown in 3D gels (Fig. on left). A central part has a clear lumen whereas edge parts (arrows – middle image) have not been luminized so far. In a semithin section, the endothelium looks like a simple squamous epithelium lining an empty lumen (Fig. on right).