## 3 Umbilical Cord and HUVEC
### Isolation and Culture of HUVEC  
#### 3.3.1 First HUVEC Isolation Techniques  
EC cultures began in the 20 th century with the work of [PERSON] [27] and [PERSON] [40]; [PERSON] successfully isolated these cells and established the means to identify endothelial cultures by morphological, immunohistochemical, and serological criteria. The isolation technique established by [PERSON] was as follows:  
One end of the umbilical cord is cannulated with a 14G needle and secured with Nylon thread. Next, the vein is perfused with 100 mL of cord buffer (Table 1) to remove traces of blood and drain the vessel. The other end of the cord is cannulated with a 12G needle. The vein is then perfused with 10 mL of 0.2% collagenase, diluted in cord buffer, the cord is placed in a water bath with the same buffer, and incubated at 37 \({}^{\circ}\)C for 15 minutes. Afterwards, the collagenase solution (containing the EC) is drained by introducing 30 mL of cord buffer into the vein. The effluent is collected in sterile 50 mL tubes containing 10 mL of Medium 199 (M-199) enriched with 20% fetal bovine serum (FBS). The cells are centrifugated at 250 g for 10 minutes and washed with 20 mL of enriched M-199. Finally, the pellet is resuspended in 5 mL of fresh culture medium. The yield of this procedure is in the range of 0.5-1.5 \(\times\) 10\({}^{6}\) cells per cord [28].  
Figure 2: Timeline of human umbilical vein endothelial cell (HUVEC) cultures in science. The most important events since the first description of embryonic tissues are described, including the endothelial cell of the umbilical vein, the first isolation of HUVEC and cultures, as well as applications of the model to study cardiovascular disease (CVD) and other pathological and physiological processes [27; 28; 29; 30; 31; 32; 33; 34; 35; 36; 37; 38; 39].  
[PERSON] et al. obtained EC that grew in a homogeneous monolayer of polygonal cells with poorly defined borders. In addition, morphological comparison with smooth muscle cells and fibroblasts, and Transmission Electron Microscopy (TEM) revealed that EC contain rod-shaped cytoplasmic organelles, such as those described by [PERSON] (Weibel-Palade bodies or WPB) in 1963 [45], absent from both fibroblasts and smooth muscle cells [28].  
In 1974, [PERSON] et al. characterized the population growth behavior of primary and subcultures of HUVEC using \({}^{3}\)[H] thymidine to analyze DNA synthesis. EC cultures were found to behave as a density-dependent population with respect to DNA synthesis, useful to study cell growth and endothelial regeneration. In addition, the effluent of the umbilical vein contained small clusters of rounded cells, which adhered to the surface, was cultured and spread in the form of small epithelioid clusters within the first 2-6 hours. These agglomerations increased in size and gradually coalesced to form incomplete monolayers in 3-5 days [29]. Since 1974, reports on EC isolation emerged, aiming to study EC physiology, most based on the descriptions made by these two authors [46; 47; 48; 32; 41; 42].  
EC tend to be quiescent in vivo. However, after injury, they can change their phenotype, migrate and proliferate to heal the lesion in a few days. Much progress has been achieved, due to the culture of these cells, even though the culture process may introduce certain artifacts, cause the loss of specific functions and introduce new metabolic characteristics, which are not present in vivo [9]. For example, in vitro growth rate far exceeds in vivo rate. However, HUVEC cultures have led to many interesting findings, such as the identification of nitric oxide (NO), the relaxation factor derived from the endothelium [49].