HUVECs can be serially propagated for 30-70 population doublings, but after about 5-7 passages the cells gradually start to increase in size, to grow more slowly and to lose specific functions. Experiments should be done with cells between 2-4 passages.
HUVEC medium: complete endothelial cell growth medium (EGM-2; Lonza).
Basal medium: Ham’s F12:DMEM 50:50 (Sigma #D8062) supplemented with 0.1% fatty acid-free BSA (Sigma #A7030).
Freezing medium: 70% EGM-2, 20% FBS, 10% DMSO.
Cells are grown on tissue culture plastic coated with fibronectin. Recombinant human fibronectin is from Sigma (#F0895). Dishes are coated by covering with a thin layer of 10 µg/ml fibronectin and incubating at room temperature for at least 30 min. The coating medium is then aspirated and the cells seeded immediately onto this dish.
- Grow cells to 80% confluence.
- Aspirate medium, wash with PBS, aspirate.
- For a T75 T-flask (15ml), add 1.5 ml of trypsin solution (0.05 % trypsin, 0.53 mM EDTA).
- Add 4 ml basal medium containing 10% FBS.
- Centrifuge cells 5 min at 200 g and resuspend in EGM-2.
- Split cells into new coated dishes at 1:4.
HUVEC can be stored for long periods in liquid nitrogen:
- Wash cells in PBS and trypsinise.
- Neutralise the trypsin with 5X volume basal medium/10% FBS.
- Centrifuge cells 5 min at 200 g.
- Resuspend cells in 0.5 ml freezing medium.
- Transfer cell suspension to cryotube and freeze slowly to -70 °C, using a specialised freezing container, or by wrapping the tubes heavily in tissue and placing overnight in a -70 °C freezer.
- Transfer to liquid nitrogen.
- Thaw cells rapidly in a 37 °C waterbath.
- Dilute with 9 ml culture medium.
- Centrifuge 5 min at 200 g.
- Resuspend cells in culture medium and seed onto appropriate flasks/plates.
Different coating affect endothelial cells in different ways. We use the triple-coat when we really need the cells to stick down and behave. It gives the best monolayers (least permeability).
Collagen I: 100 µg/ml bovine type I collagen (PureCol; Advance BioMatrix #5409) in PBS for 1 h at 37 °C.
Triple coat: 50 µg/ml fibronectin, 30 µg/ml collagen I, 0.1 % gelatin (Sigma #G1393) in PBS for 1 h at 37 °C.
When we are short of cash, we make our own endothelial growth medium. This works pretty well, but not as well as the commercial medium. Basically, the cells grow a bit slower. The most expensive component of endothelial cell medium is FGF-2 (bFGF). In some formulations, this comes from bovine pituitary extract – which is basically a crude source of FGF. You can only save significant money if you find a cheap source of FGF-2. The R&D Systems tissue culture grade FGF-2 is very good value.
|DMEM/F12 50:50||Sigma #D8062|
|2% heat-inactivated FBS|
|1 µg/ml hydrocortisone||Sigma #H0135|
|5 ng/ml EGF||R&D Systems #236-EG|
|10 ng/ml FGF-2||R&D Systems #4114-TC|
|20 µg/ml heparin sulphate||Sigma #H3393|
|250 ng/ml insulin||Sigma #I3536|
|100 U/ml penicillin|
|100 µg/ml streptomycin|
Curing HUVEC Infections
HUVEC sometimes come with infections. Delivery rooms are not the most sterile of environments. Infections are usually bacterial and often show resistance to pen/strep. The Lonza medium contains amphotericin B (an antifungal) and gentamycin (an antibiotic that also has some effectiveness against mycoplasma). Bacterial infections that are resistant to gentamycin can usually be treated by swapping the antibiotic mix to 10 µg/ml tetracycline, 5 µg/ml chloramphenicol. This combination targets both Gram-negative and Gram-positive bacteria, and tetracycline is also effective against mycoplasma. The infection should clear up within a day, but requires much longer (weeks) to remove all traces of infection. If you are not sure if you have an infection (or whether you have cured it), DAPI staining will tell you.