## 3 Umbilical Cord and HUVEC
### Isolation and Culture of HUVEC
#### 3.3.3 Isolation and Culture of HUVEC Conditions  
Umbilical Cord Collection and Conservation  
According to different authors, umbilical cords should be collected from full-term infants, ideally delivered by natural birth, from healthy mothers, after an uncomplicated pregnancy, and should be free of hepatitis or human immunodeficiency virus (HIV) infection [42]. In some studies, the authors specify whether they used cords collected from natural births [51; 52; 53] or cesarean sections [29; 54; 55; 56; 57]. Occasionally, cords from pregnancies of mothers with certain diseases or traits may be required, for example, HUVEC from infants with mothers who developed gestational diabetes have been used for genetic, epigenetic, and proteomic studies of the effects of T2D on EC [58; 59]. Although the HUC is formed during the fifth week of gestation, the bioenergetic capacity and immune response of HUVEC, obtained from preterm (\(<\)37 weeks of gestation) and full-term infants (37-40 weeks of gestation), are different [60; 61; 62; 63; 64]. HUVEC, obtained from preterm infants, show an impaired mitochondrial bioenergy capacity, and increased production of reactive oxygen species (ROS) [64]. [PERSON] et al. demonstrated that HUVEC mitochondrial enzyme activity changes lead to decreased viability of cells obtained from preterm cords [62]. In addition, the expression of selectins is lower in preterm HUVEC as compared to full-term HUVEC [60; 61; 63]. All these alterations affect endothelial function [60; 61; 62; 63; 64]. Therefore, it is not recommended to use HUVEC obtained from preterm infants for the study of CVD. The cord must be collected and transported under appropriate conditions. Many authors agree that optimal storage and transportation temperature is 4\({}^{\circ}\)C. Moreover, best results are obtained when the umbilical cords are processed between 10 and 24 hours after collection [32; 42], although comparable success rates have been obtained when umbilical cords are processed up to 48 hours after birth [65]. One of the first tissue conservation studies was carried out in 1949 by [PERSON] et al., who reported that refrigerated tissues retain their viability for a certain time, depending on the type of tissue, size, oxygen availability, storage temperature and other factors [66]. Lower temperatures decreased the demand for oxygen; thus, tissues preserved at 0\({}^{\circ}\)C were able to survive for a certain period of time in oxygen-deprivation conditions. Other studies have reported that different tissues can be conserved in Ringer's solution or saline solution at temperatures between 3\({}^{\circ}\)C to 6\({}^{\circ}\)C. The preservation of blood vessels in serum or blood is also satisfactory for short periods of time; the ideal preservation medium must contain physiological concentrations of salts, buffer, glucose and small accessory molecules present in serum [50]. Finally, umbilical cord length is a parameter to consider, as some authors suggest the cord should be discarded if it is shorter than 12 cm in length or has damaged areas, as this could result in a low number of isolated cells. Similarly, if the cord has clots, it is recommended not to force them out and to cut the affected section [66]. HUVEC Culture Considerations  
HUVEC culture success depends on many factors. According to [PERSON] et al., one-third of 200 processed cords failed to reach confluent densities, and growth initiation seems to be related to the presence of multicellular aggregates in the venous effluent [29]. Various factors may decrease HUVEC viability and lead to culture failure. In this sense, [PERSON] et al. proposed some recommendations before HUVEC isolation [42]:  
1. Heat the sterile transport buffer in a water bath at 37 \({}^{\circ}\)C. This will prevent thermal shock disabling cell growth. 2. Prepare a sterile area in a laminar flow hood. This will prevent bacterial and fungal contamination of both reagents and cultures. 3. Before starting the isolation procedure, it is desirable to identify the human umbilical vein.