## 4 HUVEC Identification
### Morphological Description  
The morphology of HUVEC was first characterized by [PERSON] in 1973. After five days of culture, EC grew, forming a monolayer with a poorly defined spiral pattern. The cells were homogeneous, closely opposite, long and polygonal with an oval nucleus in the center. Contrast microscopy revealed that the cell borders were different, and in some areas, interdigitations connecting separated cells appeared. TEM observations showed homogeneous flat cells, 30x50 um in size, that grew in a monolayer with varied edges and prominent nuclei. EC observed by TEM in situ in the umbilical cord vein, were thin and flat with pinocytic vesicles and several cytoplasmic vesicles. The cytoplasm also contained clusters of free ribosomes, smooth and rough endoplasmic reticulum, agglomerations of fine filaments and prominent microtubules [28]. Additionally, the cytoplasm of these cells contained numerous oval rod-shaped bodies, described previously by [PERSON] (WPB) [45]. HUVEC form a uniform monolayer of polygonal cells in approximately one week. They are characterized as EC by the presence of WPB, pinocytic vesicles, small amounts of 100 A fibrils located near the nucleus, mitochondria with tubular shapes which rarely showed ramifications, an ellipsoid nucleus with a fine granular pattern of condensed chromatin and one to three nucleoli present in each nucleus [81]. As primary EC cultures grow and expand, cell-cell contacts are created and always appear circumscribed by a basement membrane layer. After three to four weeks, the cells acquire their typical endothelium form in a confluent monolayer, that is, in a hexagonal or cuboidal form with long, round and slightly overlapping nuclei (Figure 3). If the cells remain in confluence, the monolayer decreases and elongated rather than cuboidal cells are observed [82]. Other exclusive ultrastructures found in EC are prominent nucleoli, perinuclear regions rich in mitochondria, endoplasmic reticulum and Golgi complexes, micropinocytic vesicles and the presence of abundant microfilaments in the cytoplasm [29].  
The peripheral cytoplasm shows complex interdigitations with adjacent cells, often forming small spaces in cross-sections [82].  
The WPB are ovoid rod-shaped granules, with a membrane containing parallel arrangements of 6-26 tubular structures, of approximately 150 A width. These are deposit organelles for factor VIII, rarely found in microcapillaries. The density of WPB in EC decreases depending on the distance from the heart [49]. These structures are used to identify cultured cells as endothelium, since these organelles have not been found in fibroblasts or smooth muscle cells [29].  
EC have also been described as confluent cobblestone-shaped layers [11; 49], simulating the morphology of the endothelium in vivo. When the culture has been maintained for long periods of time, the monolayer begins to disorganize. Likewise, cobblestone morphology can also be affected by exposure to a dynamic flow [11]. Thus, shear stress can induce elongation of the cells in the flow direction. Another important feature of EC is their ability to form capillary-like structures when placed in a Matrigel matrix, fibrin or collagen, and stimulated with serum or growth factors [49; 83].  
Even though EC show numerous morphological characteristics, EC identification cannot be achieved by optic microscopy examination alone. WPB can be identified only by electron microscopy, and are often difficult to locate, so the identification of other factors mentioned below is suggested.