PROTOCOL

A protocol for isolation and culture of human

umbilical vein endothelial cells
Bruno Baudin1,2, Arnaud Bruneel2, Nelly Bosselut2 & Michel Vaubourdolle2
1Laboratoire de Biochimie et Biologie Cellulaire—UPRES JE 2493, UFR de Pharmacie, Université Paris-Sud 11, 3 rue Jean-Baptiste Clément, 92290 Châtenay-Malabry,
France. 2Biochimie A, Hôpital Saint-Antoine, AP-HP, 184 rue du Faubourg Saint-Antoine, 75571, Paris Cedex 12, France. Correspondence should be addressed to
B.B. (bruno.baudin@sat.aphp.fr).

Published online 15 March 2007; doi:10.1038/nprot.2007.54

We describe a protocol for easy isolation and culture of human umbilical vein endothelial cells (HUVECs) to supply every researcher
© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

with a method that can be applied in cell biology laboratories with minimum equipment. Endothelial cells (ECs) are isolated from
umbilical vein vascular wall by a collagenase treatment, then seeded on fibronectin-coated plates and cultured in a medium with
Earles’ salts and fetal calf serum (FCS), but without growth factor supplementation, for 7 days in a 37 1C–5% CO2 incubator. Cell
confluency can be monitored by phase-contrast microscopy; ECs can be characterized using cell surface or intracellular markers and
checked for contamination. Various protocols can be applied to HUVECs, from simple harvesting to a particular solubilization of
proteins for proteomic analysis.

INTRODUCTION
Vascular ECs organize themselves in a single cellular layer, that is,
the endothelium, at the luminal face of all blood vessels. Besides a
major function in controlling gas exchanges at the pulmonary level,
the endothelium regulates the flow of circulating blood cells and of
various bioactive molecules, for example, growth factors, coagula-
tion proteins, lipoproteins and hormones, in particular through the
presence of membrane-bound receptors. The endothelium also
plays pivotal roles in the regulation of a broad range of physiolo-
gical processes: it regulates hemostasis through the expression of
both anti-thrombotic and prothrombotic factors; in tight coopera-
tion with the underlying extracellular matrix and smooth muscle
cells, it controls vascular tone with angiotensin I conversion and
bradykinin degradation, and contributes to the metabolism of
vasoactive amines; it also participates in inflammatory and
immune responses through surface antigens and adhesive mole-
cules and through the synthesis of cytokines1–3. But ECs, which are
in direct contact with plasma and cellular components of blood, are
targets of many endogenous molecules and xenobiotics4,5.
For many years our work focused on the physiology and
pathology of ECs, in particular using (from cord of newborns),
HUVECs (for human umbilical vein endothelial cells)6–9. HUVECs
represent a model for a large community of researchers all round
the world, although properties of HUVECs certainly cannot
represent all the metabolic capacities and the responses in physio-
pathology and toxicity related to the different types of ECs found
in an organism. As an example of the interest for the scientific
community, more than 100,000 publications have cited ECs
in general, and at least 10,000 HUVECs in particular. HUVECs
model is useful for any research on general properties of human
ECs, but other sources of ECs could be better models for studies on
specific pathological areas, for example, atherosclerosis develop-
ment or metastasis dissemination in particular microvascular
areas. Nevertheless, HUVECs are the most simple and available
human EC type, accurate for the preparation of large quantities of
cells. To increase our knowledge about the protein content and the
main biological pathways of this vascular EC model, we undertook
the proteomic analysis of HUVECs in primary cultures. Our results
are available on the web at http://www.huvec.com and will be
soon completed with our recent study employing Fourier trans-
form ion cyclotron resonance mass spectrometry analysis of
HUVEC proteins fractionated in two-dimensional (2D) electro-
phoresis gels10–12.
We describe in the present paper a protocol for easy isolation and
culture of HUVECs to supply every researcher with a method that
can be applied in cell biology laboratories with minimum equip-
ment and accessories. This protocol is derived from the original
description by Jaffe et al13. The confluent primary cultures of
HUVECs can be obtained in 8 days, although you have to wait
for an additional 48 h before starting a special treatment, such as
the addition of a drug to study a particular effect. As for many other
cell types, particularly for adherent cell lines, HUVECs can be used
in subcultures, obtained with or without trypsin treatment, for
amplification of the number of cells, but most often the primary
culture is preferred because of the decrease in expression of many
proteins at each passage due to a mechanism of accelerated
senescence, with spontaneous apoptosis; for example, both angio-
tensin I-converting enzyme (ACE) and prostacyclin synthesis
decrease as a function of the number of passages14–17. Moreover,
no additional growth factor is required for this protocol, as many
growth factors and related products can modify protein synthesis
and intracellular trafficking18–20.
You can characterize HUVECs by using EC markers, such as ACE
(peptidyl-dipeptidase EC.3.4.15.1), both in activity and immuno-
fluorescence (CD-143) and Von Willebrand Factor/Factor VIII RA
(vWF, immunofluorescence), even if the latter is weakly expressed
in these particular ECs. You can also use other EC markers, such as
CD-31, CD-34, CD-54 or ICAM-1, CD-62E or E-selectin, CD-106
or VCAM-1, more or less specific for ECs and expressed often in
HUVECs after a particular stimulation. The lectin from Ulex
europaeus (anti-H blood group specificity) binds to most ECs,
normal as well as from neoplastic vessels21–24. Uptake of labeled
acetylated low-density lipoproteins is also a characteristic of EC;
but as for CD markers, this uptake is rather applied to flow-
cytometry analysis25.

NATURE PROTOCOLS | VOL.2 NO.3 | 2007 | 481

 PROTOCOL
MATERIALS
REAGENT
Umbilical cord ! CAUTION Appropriate consent must be obtained before
processing human tissue.
EQUIPMENT
. Hood for cell culture with vertical laminar flow and equipped with UV light
for decontamination
. Water-bath with temperature control
. Centrifuge (no temperature control is needed)
. Incubator with both temperature and gas composition controls
. Optical microscope with phase-contrast equipment
. A procedure for material sterilization (Poupinel oven for example)
. Cannulae (i.d. 1 mm
60 mm) m CRITICAL Cannulae that are not
fitted can lead to various problems, see TROUBLESHOOTING;
manufacturers specialized in surgical materials can provide appropriate
© 2007 Nature Publishing Group http://www.nature.com/natureprotocols
gently stir for 10 min at laboratory temperature (20 1C) and in the dark (cover
with an aluminum foil); then adjust the pH at 7.4 with 1 N NaOH; filter through
0.22 mm as before; fractionate in 10 ml aliquots and freeze at 20 1C until used.
m CRITICAL The use of another collagenase or another proteolytic enzyme
may modify the conditions for ECs isolation, such as concentration of the
enzyme and time for incubation (Step 10).
FCS Thaw the FCS (Biosepra, a reserved lot checked for the absence of toxicity
toward HUVECs); heat for 30 min at 56 1C (for decomplementation; keep the
bottle closed); fractionate in 25 ml aliquots and freeze at 20 1C until used.
m CRITICAL The lot of the FCS can be critical for ECs growth; thus, keep
the same lot for one set of experiments and check each new lot with a
viability test.
Culture medium M199 ‘‘stock’’: dilute 100 ml of 10
M199 with Earles’ salts26
(Gibco, cat. no. 21180-021) in 900 ml of distilled water.

cannulae.
REAGENT SETUP
13 PBS Dilute 100 ml of 10
PBS, without calcium nor magnesium (Eurobio,
cat. no. CS3PB01-04), in 900 ml of distilled water plus 2 g of glucose (Sigma, cat.
no. G7520); pass through a syringe equipped with sterile Millex GV 0.22 mm
filter (Millipore, cat. no. SLGV025BS) fractionate in 50 ml aliquots and freeze
at 20 1C until used.
Buffer for conservation and transport of umbilical cords Add 1 ml of
1 million unit penicillin G (Peni G, Panpharma) and 1 ml of 1 million unit
colistine (Colimycine, Aventis) to 50 ml of 1
PBS; prepare fresh buffer for each
cord collection.
Collagenase solution (0.2% wt/vol) Dissolve 0.2 g of collagenase from
Clostridium histolyticum (Roche, cat. no. 103586) in 100 ml of 1
PBS and
‘‘Complete M199 medium’’ (with 20% FCS) Make up to 100 ml with
M199 ‘‘stock’’ a solution containing 1 ml of 0.2 M glutamine (Sigma, cat. no.
P0781), 1.5 ml of 1 M HEPES (Eurobio, cat. no. CSTHEPOO-OP), 1.8 ml of 7%
(w/v) NaHCO3 (Eurobio, cat. no. CSTBICOO-OU), 1 ml of penicillin/
streptomycin (10,000 U per 10,000 U; Sigma, cat. no. P0781) and 20 ml of
FCS (the defrost aliquot is centrifuged for 10 min at 1,500g at 15 1C before
using 20 ml of the supernatant). ‘‘Complete M199 medium’’ can be conserved
for 1 week at 4 1C.
Fibronectin solution Dissolve in 32 ml of distilled water 1 mg of fibronectin
from human plasma (Sigma, cat. no. F2006). Keep the solution at 4 1C for
6 months; do not freeze. In place of fibronectin, collagen can also be used
(for example from rat tail, type I) or, for certain studies, no coating is
preferred; nevertheless, fibronectin obviously gives the best results27.

PROCEDURE
The day before isolation of HUVECs
1| Coat 35-mm-diameter Petri dishes with one drop of fibronectin solution per plate. Allow seven dishes per umbilical cord.
Dry the dishes under the hood, with lid open.
2| Take a sterile container, with 50 ml of buffer for conservation and transport, to the maternity hospital. One container is
needed for each umbilical cord of 10–30 cm; the container must be kept at 4 1C until cord collection and again immediately
afterwards. Provide enough containers, if necessary, for preparing several umbilical cords, and processing for culture within 6 h.

The day of HUVECs isolation and culture

3| Fill up another container with 500 ml of sterile 0.15 M NaCl (‘‘physiologic serum’’ or 0.09% saline) and maintain at 37 1C in
a water-bath.

4| Under the hood, prepare (for one cord) two syringes of 50 ml, one syringe of 30 ml and one syringe of 10 ml, one surgical
clamping clip, two cannulae, string, sterile compresses, sterile scalpels and sterile gloves.

5| Spread onto the working area, under the hood, for the subsequent cord manipulation one aluminum foil, one paper sheet
and the sterile paper envelope of gloves (strictly in this order).

6| Check whether cords are suitable for use and then treat each cord one after another as follows.
m CRITICAL STEP Hematic or damaged cords should be discarded and also those from infected parturient women (such as HBV+,
HIV+, and so on)28.

7| Tidily cut both ends of the cord with a scalpel.

8| Introduce a cannula at each extremity of the vein and tightly maintain them with string.
m CRITICAL STEP Umbilical vein (the widest vessel) must not be confounded with the two arteries29 and both cannulae must be
carefully fitted.
? TROUBLESHOOTING

9| Wash the umbilical vein with 1
PBS using the 50 ml syringe until the effluent buffer is transparent or slightly pink.
m CRITICAL STEP All red blood cells must be removed.

10| Inject the 0.2% collagenase solution at one extremity of the vein using the 30 ml syringe; when it leaks out of the other
extremity, tightly clamp it with the surgical clamp; maintain the syringe and protect both cord extremities with a clean
aluminum foil; incubate the cord in the water-bath for 10 min.

482 | VOL.2 NO.3 | 2007 | NATURE PROTOCOLS

 PROTOCOL

11| Afterwards, under the hood, onto the sterile envelope of gloves, gently squeeze the cord to facilitate cell detachment.

12| Fill up a sterile 50 ml tube with 10 ml of ‘‘complete M199 medium’’; collect the cells in this tube by washing the vein with
40 ml of 1
PBS.
m CRITICAL STEP Do not lose cell suspension during this manipulation.

13| Centrifuge the closed tube at 750g for 10 min without temperature control.

14| Carefully discard the supernatant and suspend the pellet of cells in 14 ml of ‘‘complete M199 medium’’; dissociate the cells
by gentle aspiration and repulsing (three times) with the 30 ml syringe equipped with a needle (i.d. 0.7 mm
30 mm).

15| Count the cells from an EC aliquot under an optical microscope with a Malassez’s cell; adjust the volume for seeding each
Petri dish with 40,000 cells. Typically, when one 20–30 cm cord can be used entirely, you can isolate enough ECs for seeding
© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

seven dishes with 14 ml, thus allowing 2 ml per dish. For some studies needing several cords, pool the cells and mix before
seeding; but, in some cases, pooling is rejected because HUVECs express HLA-DR antigens that may cause problems of
immunocompatibility30. You can use smaller dishes (24- or 96-well plates), or larger dishes or Falcon flasks (Becton-Dickinson);
however, the 35 mm Petri dishes are particularly adapted to fibronectin coating with a cell scraper, examination by both
optical and fluorescent microscopy and lysis of the cells with a cell scraper or a lysis solution.

16| Incubate ECs cultures at 37 1C in a 95% air/5% CO2 atmosphere saturated with H2O.

The days after isolation of HUVECs

17| The following day (after less than 24 h), remove non-adherent cells by changing the culture medium. Replace with 1.5 ml
culture medium instead of 2 ml. The result must be examined under phase-contrast microscopy; if some remaining blood clots
contaminate the ECs, change the culture medium again. Check for contamination. At this stage, you can also count the
adherent EC islets or determine a yield of adhesion.
? TROUBLESHOOTING

18| Change culture medium every 2 days (replacing with 1.5 ml medium each time). Typically, cell confluency is achieved in
6–8 days, although it is dependent on the number of cells seeded, the yield of adhesion and the growth capabilities of these
peculiar cells, in particular when mixtures of several umbilical cords are used. When confluent ECs show contact inhibition,
the EC cultures will present a ‘‘cobblestone appearance’’ (tightly packed polygonal ECs) in phase-contrast microscopy (Fig. 1).
Often you have to wait 2 days more before treating the cells, because real confluency (with no mitosis) is achieved later than
can be seen via microscope. Typically, in a 35 mm Petri dish, the constituted endothelium (organotypical culture) contains
about 1 million cells.

HUVECs treatment
19| If you wish to harvest the cells, wash three times with
PBS. Then scrape the cells into PBS, water or lysis buffer. ECs
adhere onto fibronectin and secrete at their basal membrane
molecules of the extracellular matrix, that is, collagens and
glycosaminoglycans; thus, this is also harvested with the
cells31. Use of a lysis solution is recommended, in particular
for the solubilization of membranes. An example of a suitable
lysis solution for the recovers of proteins and enzymatic
activities (cell proteins and enzymes) is 1% Triton X-100
solution in PBS containing protease inhibitors, 2 mM
phenymethyl-sulfonylfluoride to inhibit serine-proteases
and 1 mM N-ethylmaleinimide to inhibit cysteine-
proteases6,8,23. For our proteomic analysis of HUVECs10,11
we washed three times with PBS and then harvested directly
in 2D electrophoresis buffer (6 M urea, 2 M thiourea,
2% w/v CHAPS, 0.3% w/v dithiothreitol, 0.5% immobilized
pH gradient buffer, 2 mM phenymethyl-sulfonylfluoride
and 5 mM N-ethylmaleinimide).
20| If you wish to subculture the cells, use a solution with
0.25% trypsin and 0.02% EDTA. At the end of the treatment Figure 1 | HUVECs at confluency observed under phase-contrast microscopy.

NATURE PROTOCOLS | VOL.2 NO.3 | 2007 | 483

 PROTOCOL

inhibit trypsin activity by washing the harvested cells in complete medium (calcium and proteins in FCS inhibit trypsin and
trypsin-like activities).

 TIMING
Day 1, Steps 1 and 2, preparation of plates: 15 min for seven dishes
Day 2, Steps 3–15, initial processing of the cord: 2 h for one umbilical cord, add 30 min for each additional cord
Days 3–8, Steps 17 and 18,replacing medium and monitoring cell growth: 10 min for seven dishes without counting or particular
washing

? TROUBLESHOOTING
Contamination
Bacteria and fungi can be identified by regular optical microscopy; however, it might be necessary to detect low-level infection
© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

by incubation of cell cultures in microbiological broth (fluid thioglycolate medium and tryptone soya broth). This test procedure
should be carried out in a microbiology laboratory away from the cell culture laboratory.
Mycoplasma, which is not usually detected by light microscopy because it is intracellular, can be detected either by culture on
specific medium (also in microbiology laboratory, giving a result in 3–4 weeks) or by indirect DNA stain (Hoechst 33258) and
fluorescence examination (in the cell culture laboratory, giving a result in 2–3 days). Using Hoechst is quicker, but less sensitive.
Fibroblasts and vascular smooth muscle cells can contaminate EC cultures. They have characteristic features that enable
them to be identified on microscopic examination. The use of a panel of markers (such as vimentin, a-smooth muscle actin,
desmin and calponin) is difficult as HUVECs also express these proteins10,11; only fibroblast specific protein-1, the matrix
metalloproteinases-2 and -3, and CD-90 could be more specific for fibroblastic cells. Moreover, HUVECs retain the potential
to differentiate into smooth muscle-like cells by deprivation of growth factors32.

Culture of HUVECs without serum

HUVECs can survive in serum-free medium (M199 without FCS) for up to 12 h without losing their phenotypic features. However,
for longer experiments, in particular those lasting 24 h, the absence of growth factors triggers apoptosis, an irreversible
mechanism. This process can be circumvented by the use of an enriched medium but with a low concentration of proteins
such as the commercial medium Ultroser G (Biosepra, cat. no. 259515) (see refs. 6,8,23) at 1% in M199 without FCS; in this
medium, HUVECs can survive for 48 h. Some other low-serum media that support HUVECs viability can also be used.
Troubleshooting advice can be found in Table 1.

TABLE 1 | Troubleshooting table.

Problem
No cells before seeding
No adherent cells 24 h after
seeding
No confluency after 8 days and/or
detached cells
Rapid senescence (many detached
and dead cells after confluency or
subconfluency)
Both macroscopic and microscopic
mycelium
Elongated cells, in particular after
HUVECs confluency
Possible cause and repair
Bad collagenase (type, concentration, and so on); prepare fresh solution
Bad fibronectin, HLA incompatibility, bad conditions in incubator (hypoxia, high CO2 level, no water);
verify
HLA incompatibility, bad conditions in incubator as above, bacterial contamination; verify incubator as
above and check for contamination
Various contaminations, multiple passages; discard the cultures, check for contamination and use primary
cultures
Fungi contamination; discard the cultures and decontaminate using antifungal drugs
Contamination by smooth muscle cells or fibroblasts; discard the cultures

ANTICIPATED RESULTS
In a series of experiments (n¼4), 2 days after confluency, HUVECs contained 1.56 ± 0.49 U mg1 ACE activity, as determined on
synthetic substrate23,33 and expressed as a function of protein content, whereas they secreted in culture medium (as determined
on supernatant) 0.643 ± 0.054 U per 24 h per 106 cells. These results correlate with immunofluorescence examination showing
fine fluorescent granulates on cell surface using a personal anti-ACE antibody and after paraformaldehyde fixation. In these
conditions but after methanol cell permeation, HUVECs were poorly stained with anti-vWF antibody. Vascular smooth muscle
cells, as cultured from porcine aortic or pulmonary artery explants, did not contain or secrete ACE activity and were not stained
by either anti-ACE or anti-vWF antibodies.

484 | VOL.2 NO.3 | 2007 | NATURE PROTOCOLS

 PROTOCOL

Typically, after isolation of HUVECs from three or four umbilical cords, we could seed twenty-four 35-mm Petri dishes allowing
experiments in quadruplates, that is, six conditions such as one control and five concentrations of a drug or a particular
molecule, or three controls versus three times of exposition with this particular molecule, that we have made for studying
the toxicity and/or apoptosis induced by anticancer drugs7–9. For our proteomic study using 2D electrophoresis and mass
spectrometry, we plated HUVECs on the same dishes but we pooled cell lysates in specific solution, as stated above, from
eight dishes (control). The other 16 dishes were used for the examination of apoptosis induced by etoposide, an anticancer
drug, at two concentrations, with eight dishes for each concentration, thereafter pooled such as for controls11.

COMPETING INTERESTS STATEMENT The authors declare that they have no
competing financial interests.
© 2007 Nature Publishing Group http://www.nature.com/natureprotocols
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Published online at http://www.natureprotocols.com

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