## 3 Umbilical Cord and HUVEC
### Isolation and Culture of HUVEC
#### 3.3.3 Isolation and Culture of HUVEC Conditions
Umbilical arteries are more rigid and difficult to dilate (Figure 1A,B). 4. It is recommended to store collagenase in 10-12 mL aliquots at -20 \({}^{\circ}\)C to avoid denaturation. The culture flasks must be previously covered with gelatin or fibronectin to ensure cell adhesion. The culture medium supplies amino acids used for protein synthesis in cell culture. The most used medium is M199 (Table 2). However, MCDB 131 medium with high levels of magnesium (10 mM) increases cell proliferation two to three-fold, as compared to M199 medium (1.6 mM). Artificial media containing growth factors are also efficient [42]. The use of enriched media, such as M199 or Dulbecco-Vogt medium is ideal for promoting cell growth, but glutamine deficient media or minimum essential medium of Eagle are not suitable for the cultivation of HUVEC [42]. HUVEC can survive in an FBS-free medium for up to 12 h without losing their phenotypic characteristics. However, in experiments taking longer than 24 hours, the absence of growth factors promotes apoptosis. This can be avoided by using an enriched medium, but with a low concentration of proteins, such as the commercial medium Ultrose G at 1% in M199 without FBS where HUVEC can survive for 48 h [29]. For optimal growth, freshly isolated cells require medium supplemented with 20-30% of FBS, although 10% FBS is sufficient to maintain the growth density of primary cultures and subcultures [11]. Vascular endothelial growth factor (VEGF) or vasculotropin supplementation stimulates the selective growth of EC in serum-free medium. To date, it is the only EC-specific growth factor. VEGF is an angiogenic growth factor, stable to heat and acidic environment, belonging to a small family of chemically similar dimeric heparin-binding glycoproteins with an approximate molecular weight of 46 kD [67]. This factor is essential in the development and formation of blood vessels in mammals, both in normal and pathological conditions. It is a highly selective and potent mitogen for EC [49]. The concentration of FBS and growth factors can modify the synthesis of some EC molecules; particularly, progressive deprivation of FBS decreases the secretion of IL-8 into the medium [42]. Heparin, a sulfated mucopolysaccharide, increases EC proliferation in the presence of heparin-binding growth factors, such as endothelial cell growth factor (ECGF) [42]. In 1983 heparin was used for the first time in culture to enhance the stimulatory effect of ECGF for HUVEC proliferation. It is likely that heparin binds to the cell surface, where it facilitates intracellular communication and accessibility to membrane receptors [68]. Heparin concentrations used in HUVEC cultures vary from 10 ug/mL to 90 ug/mL [35; 43; 50; 59; 68; 69; 70; 71]. Finally, the use of fibronectin or gelatin in the culture is very important because it enhances cell adhesion, while ECGF supplementation and serum-enriched medium allow long-term growth. Under these conditions, it was possible to perform 15 to 21 passages before HUVEC became quiescent, while cultures without these elements ceased their growth after the second passage [72].