Human Cell Culture Protocols pp 265-274 | Cite as
The present protocol offers an economical option for the isolation and culture of human endothelial cells for vascular cell biology research due to the non-invasive collection procedure being devoid of ethical concerns and ease of the isolation technique, expansion and maintenance under standard cell culture conditions. The human umbilical vein endothelial cell (HUVEC) model is useful for any research on general properties of human endothelium, but as these cells are of foetal and venous origin, other sources could be more appropriate models for studies on specific pathological areas, for example, atherosclerosis or cancer angiogenesis. Nevertheless, HUVEC still represent the most simple and available human vascular cell type widely used in biomedical research.
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Advances in vascular endothelial cell biology over the past three decades have made a major impact on our understanding of the mechanisms underlying many pathophysiological conditions, from cardiovascular disease to cancer and inflammation. In addition, the endothelium has become a major target of human clinical therapeutics as endothelial dysfunction contributes significantly to hypertension, coronary artery disease, atherosclerosis, and tumour progression (1). Endothelial cells can alter their phenotype and contribute to atherosclerotic lesion formation and progression, not only in response to inflammatory mediators but also to mechano-transduction elicited by mechanical and fluid shear forces (2, 3). Recent studies employing gene expression profiling have shown significant endothelial cell diversity at different sites in the vascular tree with differences in the genotype of large vessel and microvascular endothelium with specific arterial and venous gene expression profiles being identified (4, 5). It has become evident that the tissue microenvironment surrounding blood vessels can control endothelial cell phenotype and recent proteomic studies have identified endothelial cell proteins restricted to specialised membrane microdomains which are expressed in specific tissues (6). Much of the growth in the field of endothelial cell biology has arisen from studies employing in vitro culture of human umbilical vein endothelial cells (HUVECs) as they can be derived from a readily available source of human tissue (umbilical cords), from which a relatively pure population of cells can be easily isolated and grown (7). The therapeutic impact of advances in understanding the molecular biology of the endothelium over the past three decades has mainly focused on agents that regulate angiogenesis to target tumour growth and metastasis (8). In addition, regulation of the synthesis and bioactivity of endothelium-derived nitric oxide has also received much interest for the development of cardioprotective strategies against atherosclerosis (9, 10). In this chapter, a protocol is provided for the isolation, culture, and characterisation of HUVEC. The technique has been adapted from the original description by Jaffe et al. (11), and has now been widely adopted in vascular cell biology laboratories. This protocol will yield confluent primary HUVEC cultures that can be obtained within a week and reliably subcultured for up to 4–5 passages for expanding cell number, cryopreservation, characterisation with endothelial specific makers such as von Willebrand factor (vWF), lectin, or uptake of acetylated low-density lipoprotein (12), and for experimental procedures in culture media containing low serum and without additional growth factors.
The most commonly used culture medium in HUVEC culture is Medium 199 (M199) but other commercially available growth media are also suitable (see Note 1). The following additions to basal M199 (without glutamine or bicarbonate) are necessary prior to use: (final concentration) 5 mM l-glutamine, 40 mM bicarbonate, 100 U/ml penicillin, 100 μg/ml streptomycin, and 20% (v/v) foetal calf serum (see Note 2). Sterile stocks of these additional components are prepared and stored as frozen aliquots as described below. The complete medium can be stored at 4°C for up to 1 month and is pre-warmed to 37°C prior to use in routine cell culture. Medium without the FCS component is used during the initial isolation procedures and the FCS content can be reduced as necessary following seeding of cells for experimental procedures.
For maintenance of proliferating HUVEC cultures, further addition of endothelial cell growth factor (ECGF, Sigma), diluted in serum-free M199 containing heparin, is necessary. Stocks solutions (concentration: 1 mg/ml ECGF, 4.5 mg/ml heparin) are filtered through a 0.5-μm prefilter and a 0.22-μm sterile filter connected in series and can be stored as 5 ml aliquots for up to 3 months at −20°C. Stocks are diluted in serum containing M199 to provide final concentrations of ECGF (20 μg/ml) and heparin (90 μg/ml). This component can be omitted from the medium following seeding of cells for experimental procedures.
Hank’s balanced salt solution (HBSS) is used for umbilical cord specimen collection. The following additions, from sterile stock solutions, are necessary (final concentration in HBSS): 100 μg/ml gentamycin, 0.025 M HEPES, 20 mM bicarbonate, and 0.001% phenol red. The final HBSS cord collection medium can be stored as aliquots at 4°C for up to 3 weeks.
l-Glutamine (200 mM) stock solution: dissolve 5.84 g l-glutamine in 200 ml tissue culture-grade deionized water and sterilise by passing through a 0.22-μm filter. Aliquots (5 ml) can be stored at −20°C for 3 months.
Bicarbonate (4.4%, 0.52 M)–Phenol red (0.03%) solution: dissolve 44 g NaHCO3 and 30 mg phenol red in 1,000 ml tissue culture-grade deionized water, and sterilise by autoclaving for 10 min at 115°C. Aliquots of 15 ml are stored at 4°C for up to 3 months.
Penicillin and streptomycin stock solution (80× concentrate): dissolve 480 mg penicillin (G sodium salt) and 1.5 g streptomycin sulphate in 200 ml tissue culture-grade deionized water and sterilise by passing through a 0.5-μm pre-filter and a 0.22-μm filter. Aliquots of 5 ml are stored at −20°C and one aliquot used in 400 ml of medium.
Gentamycin solution (80× concentrate): dissolve 750 mg gentamycin sulphate in 100 ml tissue culture-grade deionized water and sterilise by passing through a 0.22-μm filter. Aliquots of 5 ml are stored at −20°C and one aliquot used in 400 ml of HBSS.
HEPES solution (1 M): dissolve 47.6 g of HEPES in 200 ml tissue culture-grade deionized water and sterilise by passing through a 0.22-μm filter. Aliquots of 5 ml can be stored at −20°C and two aliquots used in 400 ml of HBSS.
Sterile Dulbecco’s phosphate-buffered saline (PBS).
Trypsin solution (2.5%): trypsin from porcine pancreas (Sigma) is dissolved (2.5 g/100 ml) in PBS and sterilised by passing through a 0.22-μm filter. Aliquots of 10 ml are stored at −20°C.
EDTA solution (1%): EDTA disodium salt is dissolved (500 mg/50 ml) in tissue culture-grade deionized water and sterilised through a 0.22-μm filter. Aliquots of 5 ml are stored at 4°C.
Trypsin (0.1%)–EDTA (0.02%, 0.5 mM) solution is prepared by adding 10 ml Trypsin (2.5%) and 5 ml EDTA (1%) to 250 ml sterile PBS. This solution is pre-warmed to 37°C before use to detach cells from culture flasks and stored at 4°C for up to 2 months.
Gelatin solution (1%): dissolve gelatin (Sigma G9382) in tissue culture-grade deionised water and sterilise by autoclaving for 20 min. Aliquots can be stored at 4°C for up to 3 months.
Collagenase, Type II (Sigma): dissolve collagenase in serum-free medium (0.5 mg/ml) on ice. Particulate material is removed by filtering the solution through a 0.5-μm pre-filter and then sterilise by passing through a 0.22-μm filter. This enzyme solution can be stored for long term as 5–10 ml aliquots at −20°C until use in the proportion of 10 ml per 20 cm of umbilical cord.
Monoclonal antibody against human von Willebrand factor (Sigma).
Normal rabbit serum (Sigma).
Fluorescein isothiocyanate or Alexa Fluor-488 conjugated rabbit anti-mouse antibody (Dako).
Methanol (100%).
25 and 75 cm2 tissue culture flasks. Culture vessels should be coated with warmed gelatin solution (∼6 ml for a 75-cm2 flask) for ∼10 min prior to use. The gelatin is removed by aspirating using a sterile glass Pasteur pipette before the addition of cell suspensions. Flasks and plates can be stored for a week at 4°C after coating.
Sterile glass Pasteur, 5 and 10 ml pipettes.
Sterile 50 and 15 ml centrifuge tubes.
Sterile scalpel blades, scissors, watchmakers forceps, strong thread, and cannulae.
Lab-Tek chamber slides (Nunc).
In this laboratory, human endothelial cells are routinely isolated from umbilical veins. Umbilical cords should be obtained with informed consent and local ethics approval. As human tissues should be treated as potentially hazardous, it is recommended that those engaged in isolating HUVEC should be immunised against hepatitis B as a precaution. To ensure sterility and operator safety, all work should be performed in a Class II biological safety cabinet using sterile techniques. As soon as possible after delivery, the whole umbilical cord is placed in the HBSS collection medium and stored at 4°C. Cords that are collected in this way can be used for HUVEC isolation up to 48 h after delivery (see Note 3).
All cell culture media and solutions are prepared and warmed to 37°C in a water bath.
Sufficient collagenase solution is made up (or defrosted) for the lengths of cords to be processed.
The cord is removed from the collection solution, and paper towel (see Note 4) is used to blot the cord for removal of blood and excess medium.
Make a clean transverse cut across one end of the cord to expose the two umbilical arteries and the umbilical vein. The latter can be identified by its thinner wall and larger, stretchable lumen.
Insert a cannula into the vein, and remove tissue and arteries to expose about 1 cm of vein. It is important to remove a short section of the adjacent arteries; otherwise, it may be difficult to tie the ligature tightly enough, and the cannula may slip from the vein during cell isolation. The cannula is secured by tightly tying it to the vein, above the barb, with strong thread.
The process is repeated at the other end of the cord. Very long cords can be divided to give two shorter lengths of about 20 cm.
Fill a 20-ml syringe with warmed PBS and attach it to the cannula at one end, and then use this solution to flush the vein free from blood, clots, and so forth, collecting the eluate in a beaker.
Repeat the procedure from the other end (gentle massage may facilitate the removal of clots). Before expelling all the wash solution, occlude the cord at one end, and check for leaks from perforations made when blood samples were taken. Any holes found can be closed using suitable “crocodile” clips.
Attach an empty syringe to one cannula, and then use another syringe to fill the vein with sufficient pre-warmed collagenase solution to ensure that the lumen is distended.
Loosely cover the cord in cling-film or aluminium foil (see Note 4) and, with the syringes still firmly attached, place the cord in an incubator at 37°C for 10 min.
Remove the cord from the incubator, and massage it gently to assist the detachment of cells from the vessel wall.
Apply suction with one syringe while exerting gentle pressure along the cord in the direction of flow to draw out as much of the cell suspension eluate as possible in to the syringe, which is then transferred in to a sterile 50 ml centrifuge tube.
Leaving one syringe still attached to the cord, take up 20 ml of pre-warmed sterile PBS into a syringe, and use this to flush free any further loosened cells, working the solution backward and forward between the two syringes a few times. Draw off as much of this second cell suspension as possible, and add to the same 50 ml tube containing the first eluate.
Pellet the cells by centrifugation at 1,000 rpm (∼l50 × g) in a bench top centrifuge for 5 min at room temperature.
Carefully aspirate or decant the supernatant and resuspend the cells in 5 ml pre-warmed serum containing culture medium.
Transfer the cell suspension to a gelatin-coated 25 cm2 tissue culture flask, and place in an incubator at 37°C in a humidified atmosphere of 5% CO2/95% air. At this point, the flask will contain large numbers of erythrocytes in addition to endothelial cells (see Note 5).
The next day all the medium is aspirated from the culture flask using a sterile glass Pasteur pipette, the cells washed with pre-warmed serum-free medium to remove any residual erythrocytes and non-adherent cells, and 5 ml of fresh serum containing culture medium is added.
Every second day, cells should be fed by removing and discarding the medium in the flask and replacing it with fresh warmed serum containing culture medium. The cells should reach confluence in 5–7 days and are then ready for use or subculture to obtain larger numbers of cells.
Once a confluent monolayer has been attained in a 25-cm2 flask, the HUVEC can be subcultured (passaged) into further 25 or 75 cm2 flasks, plates, or dishes (see Note 6). The culture medium is removed and cells are washed twice with pre-warmed sterile PBS to remove traces of serum.
Pre-warmed trypsin/EDTA solution (0.5 ml for 25 cm2 flask or 1 ml for a 75-cm2 flask) is added to cover the cells and the flask incubated at 37°C for up to 4 min. The cell layer is periodically examined under the microscope to ensure cells have fully detached. This can also be facilitated by vigorous tapping of the side of the flask to dislodge cells and disassociate aggregates (see Note 7).
Serum containing culture medium (∼5–7 ml) is added to stop the action of the trypsin which can reduce cell viability through prolonged exposure. The cell suspension is then drawn up and down a sterile Pasteur or 5 ml pipette 4–6 times to further break up any cell aggregates.
The cell suspension is then transferred into new culture flasks at a dilution ratio of ∼1:3 and sufficient serum containing medium added to the new flasks (5 ml in a 25-cm2 and 12 ml in a 75-cm2 flask). The flasks are returned to the 37°C, 5% CO2 humidified incubator and the serum containing culture medium changed every 2 days.
The subcultured cells, which are now at passage 1, should again become confluent within 4–7 days. If required, cells can be further subcultured by dividing the contents of a 75-cm2 flask between three new flasks or in other culture plates/dishes. Further subculture beyond passages 4–5 is not advisable, since the cell growth rate may decline and phenotypic changes become evident.
For experimental protocols, once HUVEC monolayers are confluent, the growth medium can be replaced with either low (1% v/v FCS) serum or serum-free medium for up to 24 h before cells exhibit an altered apoptotic phenotype due to the absence of growth factors.
Endothelial cells are subcultured into Lab-Tek chamber slide wells and characterised after 48 h.
The culture medium is removed from the wells and cells gently washed three times with serum-free culture medium before being fixed with ice-cold methanol (100%) for 45 s, and then further washed three times with ice-cold PBS.
Cells are then incubated with a mouse monoclonal anti-vWF antibody at 1:100 dilution in PBS for ∼60 min at room temperature. As a negative control, some cells are incubated with PBS only at this stage.
The primary antibody or PBS is then removed and cells washed three times with PBS and incubated for 20 min with normal rabbit serum at 1:50 dilution.
After a single wash with PBS, cells are then further incubated for 60 min at room temperature with FITC or Alexa Fluor-488 conjugated rabbit anti-mouse IgG diluted 1:100 in PBS.
Finally, cells are washed three times with PBS and viewed under a microscope equipped for epifluorescence with appropriate fluorescence filters. Images are captured using a CCD camera.
Cells at passage 2 should be detached from a 75-cm2 culture flask as described in Subheading 3.3.
Following centrifugation of the cell suspension for 5 min at 1,000 rpm (∼l50 × g), the supernatant is aspirated and the cell pellet resuspended in serum containing culture medium (∼1.5 ml) with an additional 10% (v/v) dimethyl sulfoxide (DMSO) and transferred to a suitable cryovial (Nunc).
To facilitate a slow freezing rate (∼1°C/min), the cryovial is then placed at 4°C for 30 min, transferred to −20°C for 30 min and at −80°C for 2 h before being immersed into liquid nitrogen for long-term (∼6 months to 1 year) storage. Alternatively a “Mr. Frosty” cryovial freezing chamber (Nalgene) containing isopropanol is placed at −80°C to facilitate the slow cooling procedure.
To thaw cells, the cryovial should be rapidly warmed to room temperature in a water bath and the cell suspension (∼1.5 ml) transferred to a new gelatincoated 25 cm2 culture flask. Pre-warmed serum containing culture medium (∼10 ml) is added and cells incubated overnight at 37°C in a 5% CO2 humidified incubator to facilitate cell attachment, with a change in medium to fresh serum containing medium the next day.
Other “endothelial cell optimised” culture media are commercially available, such as Endothelial Cell Growth Media (Promocell or Lonza) which are based on MCDB131 medium. These media may help to promote more rapid growth of cells, but it should be noted that they may not contain antibiotics, have lower concentrations of serum and include components which could alter HUVEC gene expression, such as insulin and vascular endothelial growth factor.
Alternatively, M199 containing 10% (v/v) foetal calf serum (Sigma) and 10% (v/v) new born calf serum (Sigma) can be used to reduce long-term costs. Serum batches should be tested prior to bulk purchase to ensure they support consistent cell proliferation.
For maximal HUVEC yield and viability, cells should be isolated from the umbilical cord as soon as possible after child birth. This also reduces the risk of infections arising on initial isolation of cells since the tissues are often handled and excised under non-sterile conditions.
Paper towels, cling film, and aluminium foil should be sterilised using 70% ethanol spray to reduce the risk of bacterial and fungal infections.
Should infections frequently occur following isolation, additional antibiotics, and fungicides can be supplemented to the culture medium for the initial 24 h following isolation and then the medium replaced. Gentamycin (20 μg/ml) and amphotericin B (2 μg/ml) are commonly used and aliquots can be stored at −20°C as 2 mg/ml and 200 μg/ml stocks respectively.
HUVEC cultures left confluent more than 2–3 days will start to detach from the flask and cease to be viable. Primary cultures that start to proliferate but fail to become confluent in 7 days can be sometimes stimulated to grow if detached and seeded in a new culture flask.
The trypsin/EDTA solution should be pre-warmed to 37°C only immediately prior to use and not left in a heated water bath for extended periods to prevent loss of activity. Cells should not need incubation with trypsin/EDTA at 37°C for longer than 5 min to detach from the flask and this may indicate that a fresh solution should be prepared.
The endothelial cell and vascular biology research in this laboratory is supported by the British Heart Foundation and Heart Research UK. We are grateful to the midwives at the Maternity Unit, St. Thomas’ Hospital, Guy’s, and St. Thomas’ NHS Foundation Trust, for their routine collection of human umbilical cords.
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