## 3 Umbilical Cord and HUVEC
### Isolation and Culture of HUVEC
#### 3.3.2 HUVEC Cultures  
Once the EC have been isolated, they should be kept in a suitable medium that allows them to grow and reach confluence, either to study metabolic functions, or to be used as an in vitro model in different investigations. Table 2 summarizes the reagents and conditions necessary for their culture, according to the most relevant reports on the subject.  
\begin{table}
\begin{tabular}{l l l l} \hline \hline
**Solution** & **Composition** & **Conservation time** & **References** \\ \hline
**Cord Buffer, pH 7.4** & NaCl 0.14 M & \textless{3 h} & [28] \\  & KCl 0.004 M & & \\  & Phosphate buffer solution 0.001 & & \\  & M & & \\  & Glucose 0.011 M & & \\ \hline
**Phosphate buffer** & Calcium, magnesium, & 2-3 h & [41] \\
**solution (cold)** & streptomycin and penicillin. & & \\ \hline
**Transportation buffer** & NaCl 1.4M & \textless{24 h} & [42] \\  & Na2 HPO4 5.2 mM & & \\  & KCl 40 mM. & & \\ \hline
**Transportation and** & 50 mL of PBS 1X & \textless{6 h} & [11] \\
**conservation buffer** & 1 mL of penicillin & & \\  & 1 mL of colistin & & \\ \hline
**HEPES (1X) buffer** & NaCl 137 mM & \textless{3 h} & [43] \\  & KCl 4 mM & & \\  & HEPES 10 mM & & \\  & Glucose 11.1 mM. & & \\ \hline
**Supplemented PBS** & PBS supplemented with 1.5\% & \textless{3 h} & [44] \\  & antibiotic-antimycotic. & & \\ \hline \hline \end{tabular} Abbreviations: HEPES, (4-(2-hydroxyethyl))-1-piperazine ethanesulfonic acid); KCl, potassium chloride; NaCl, sodium chloride; Na2 HPO4, Sodium phosphate dibasic; PBS, phosphate buffered saline.  
\end{table}
Table 1: Solutions used for umbilical cord conservation.  
\begin{table}
\begin{tabular}{p{113.8 pt} p{113.8 pt} p{113.8 pt} p{113.8 pt} p{113.8 pt}} \hline
**Culture Medium** & **Additional reagents** & **Temperature and humidity** & **Periodicity of change of medium** & **References** \\ \hline
**TC199** & FBS, penicillin (200 U/ml), streptomycin (200 \(\mu\)g/mL), L-glutamine (2 mM). & 37\({}^{\circ}\)C, 5\% CO\({}_{2}\) & Twice a week & [28] \\ \hline
**M199** & 15 mg/L of HEPES, penicillin (60 mg/mL), streptomycin (129 mg/mL), FBS & & \\ \hline
**M199 or RPMI** **1640** & HEPES 25 mM, 20\% of FBS, L-glutamine (0.3 mg/mL), penicillin (100 U/mL), streptomycin (100 \(\mu\)g/mL) and anti-PPLO agent (0.03 mg/mL). & 37\({}^{\circ}\)C, 5\% CO\({}_{2}\) & Twice a week & [41] \\ \hline
**Minimum Essential Medium** & 10\% of FBS, L-glutamine (1\%) & 37\({}^{\circ}\)C, 5\% CO\({}_{2}\) & Every 72 hours & [46] \\ \hline
**M199** & 20\% of FBS, penicillin (100 U/ml), streptomycin (100 U/ml), L-glutamine (2 mM), ECGF. & 37\({}^{\circ}\)C, 5\% CO\({}_{2}\) & Every 48 hours & [42] \\ \hline
**MCDB 131** & 20\% of FBS, penicillin and streptomycin (50 U/mL-50 U/mL), 20 \(\mu\)g/mL of ECGF, heparin (16 U/mL). & & \\ \hline
**M199** & 20\% of FBS, 1 mL of penicillin (10,000 U by 10,000 U). & 37\({}^{\circ}\)C, 5\% CO\({}_{2}\) & Every 48 hours & [11] \\ \hline
**M199** & 20\% of FBS, 50 ug/ mL of ECGF, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 \(\mu\)g/mL). & & \\ \hline \end{tabular} Abbreviations: FBS, fetal bovine serum; CO\({}_{2}\), carbon dioxide; HEPES, (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid); ECGF, endothelial cell growth factor; PPLO, pleuropneumonia-like organism.  
\end{table}
Table 2: HUVEC culture conditions.