Core Facilities
Cell Biology > Protocols
Subculturing
HUVEC
PROTOCOL #1:
Subculturing Human Umbilical
Vein Endothelial Cells (HUVEC)
REAGENTS:
1. Hank's Balanced Salt Solution
without Ca++ and Mg++ (HBSS), from Biowhittaker, catalogue
#10-543.
2. Trypsin-Versene Mixture (0.5%
trypsin - 0.02% EDTA), from Gibco, Cat. #610-5300 or
Biowhittaker, Cat. #17-161. Dilute 1:1 with HBSS before
use.
3. Nutrient Medium, consisting of
20% Fetal Calf Serum from Gibco, Cat. #200-6140, 80%
Medium 199 (M199) buffered with 25mM HEPES from Biowhittaker,
Cat. #12-118, and supplemented with fresh 2mM L-Glutamine
(final concentration) from Biowhittaker, Cat. #17-605E,
and with 100 U/ml K-Penicillin G with 100 mcg/ml Streptomycin
Sulfate from Biowhittaker, Cat. #17-719R or Gibco, Cat.
#600-5140. On the day of use, add 50 mcg/ml Endothelial
Cell Growth Supplement (ECGS) from Biomedical Technologies,
Inc., Cat. #BT-203, (See Protocol #2), and 100 mcg/ml
Heparin from Sigma Chem. Co., Cat. #H-3933 (See Protocol
#3).
4. 0.1% Gelatin, from Difco,
Cat. #0143-02, dissolved in pyrogen-free, tissue culture
grade water and autoclaved.
METHOD:
1. Coat empty tissue culture flasks
(Corning or equivalent) with gelatin, draining and removing
the excess. Allow the flasks to dry (at room temperature
in a laminar flow hood).
2. Aspirate the medium from a flask
of primary HUVEC (isolated from one or more umbilical
cords) at or near confluence (see refs. 1 & 2).
3. Wash the monolayer with 5 ml of
HBSS (volumes are given for a 75 cm2 flask; use 1/2
as much for 25 cm2 flasks).
4. Aspirate.
5. Very quickly, rinse the monolayer
with 2 ml of the diluted trypsin-versene, allowing the
surface to remain covered for 10-30 seconds.
6. Aspirate.
7. Add another 2 ml of trypsin-versene
to the flask.
8. Cap the flask and agitate it briefly.
9. Examine the flask under a microscope
to determine that the cells have detached; this usually
occurs within 1-3 minutes (N.B.: monitor carefully at
this stage to avoid excessive exposure to trypsin).
10. Add nutrient medium directly
to the flask. The serum component will quench the activity
of the trypsin. The volume added is determined according
to the following scale.
Split 1 flask into 3 flasks (to be confluent in ~ 2
days). 1:3 is standard.
1:4 (~ 2-3 days)
1:5 (~ 4-5 days)
1:6 (~ 5-6 days)
(N.B. : The 1:6 ratio is the most efficient in terms
of the quantity of cells produced per number of feedings
required.)
11. Distribute the diluted cell suspension
into the gelatin pre-coated flasks.
12. Bring the final volume in each
flask up to 10-12 ml with nutrient medium.
13. Incubate the flasks at
37°C in 5% CO2 + 95% air and re-feed every 2-3 days.
- This procedure allows efficient
amplification of endothelial cell number from primary
cultures of human umbilical vein and other human vascular
sources at relatively low passage number. Continued
passage will eventually result in senescent changes
and loss of useful replicative potential.
- This procedure is currently the standard method utilized
in the Cell Culture Core Laboratory of the Vascular
Research Division, Department of Pathology, Brigham
and Women's Hospital, Boston, MA.
REFERENCES:
1. Gimbrone MA Jr., Shefton EJ and
Cruise SA: Isolation and primary culture of endothelial
cells from human umbilical vessels. Tissue Culture Association
Manual 1978; 4(2):813-818.
2. Gimbrone MA Jr: Culture of Vascular
Endothelium. Chapter 1 in: Spaet T. (ed) Progress in
Hemostasis and Thrombosis, Vol. III, Grune & Stratton,
Inc., 1976; pp. 1-28.
3. Maciag T, Hover GA, Stemerman
MB and Weinstein: Serial propagation of human endothelial
cells in vitro. J Cell Biol 1981; 91:420-428.
4. Thornton SC, Mueller SW and Levine
EM: Human endothelial cells: use of heparin in cloning
and long-term serial cultivation. Science 1983; 222:623-625.
K. C./M. Gimbrone
7/1985 (5/19/00)
Vascular Research Division
Department of Pathology
Brigham & Women's Hospital
Boston, MA
PROTOCOL #2:
Preparing Endothelial Cell Growth
Supplement Stock Solution (5 mg/ml)
MATERIALS:
1. Endothelial cell mitogen (aka
ECGS), obtained from Biomedical Technologies, Inc.,
Stoughton, MA, Cat. #BT-203
2. Hank's Balanced Salt Solution
(HBSS), Cat. #10-543 from Biowhittaker
3. 1 sterile disposable plastic syringe
(e.g., 20 ml) with or without a large-bore needle (without
is preferred for safety reasons)
4. Sterile Millex 0.22 micron syringe
filter units (Millipore, Cat. #SLGS02505 or #SLGP033RB)
5. 2 sterile culture tubes
(plastic, disposable)
METHOD:
1. Use 50.0 mg of ECGS per liter
of medium you plan to utilize within one week (see steps
9 and 10). Continue the procedure in a sterile hood.
2. For each liter of medium to be
made, place 5 ml of HBSS at 37°C into the sterile
bottle of ECGS.
3. Cap and swirl the bottle gently,
avoiding bubbling.
4. Transfer the liquid to one of
the sterile tubes.
5. Rinse the bottle with an additional
5 ml volume of HBSS and add it to the liquid in the
tube.
6. Draw up the solution into a syringe.
7. If used, replace the needle with
a sterile Millex 0.22 micron filter, and filter the
solution into the second sterile tube.
8. Change the filter after every
20 ml to prevent filter overload.
9. Store this stock solution at 4°C.
(DO NOT FREEZE). Also note that the activity of ECGS
drops after it has been in solution for 7 - 10 days.
10. To feed subcultured human umbilical
vein endothelial cells, dilute this 5 mg/ml ECGS stock
solution 1:100 in 20% Fetal Calf Serum in M199 with
100 micrograms/ml Heparin (see Protocol #3) in HBSS
on the day of feeding. (The activity of ECGS in nutrient
medium deteriorates after approximately 48 hours.) Do
not store medium supplemented with ECGS and heparin
for more than two days.
K. C./M. Gimbrone
7/1985 (5/19/00)
PROTOCOL #3:
Preparing a Stock Solution
of Heparin (10 mg/ml).
MATERIALS:
1. Powdered heparin sodium salt (Grade
1, from porcine intestinal mucosa), Sigma, Cat. #H-3393.
2. Hank's Balanced Salt Solution
(HBSS) from Biowhittaker, Cat. #10-543.
3. 1 sterile plastic disposable syringe
(e.g. 20cc) with or without a large-bore needle (without
is preferred for safety reasons).
4. Sterile Millex 0.22 micron syringe
filter units (Millipore, Cat. #SLGS02505 or #SLGP033RB).
5. 2 sterile plastic culture
tubes with caps (e.g. 50 ml).
METHOD:
1. Weigh out 100 mg of heparin per
liter of nutrient medium to be made for use within the
next 7 days.
2. In a sterile hood, add the heparin
to HBSS at 37°C (10 ml HBSS per liter of medium
to be made) in sterile tube; cap and swirl.
3. If the heparin is slow to go into
solution, vortex it briefly, then leave it in a 37°C
water bath for 10 minutes.
4. Draw up the heparin sterilely
with a syringe and needle.
5. Replace the needle, if used, with
a 0.22 micron filter and collect the filtrate in another
sterile tube.
6. See Methods: Steps 9 and 10 in
Protocol #2. Heparin may not be subject to the same
decrease of activity over time in solution, but is routinely
treated the same way as the ECGS.
7. Dilute this stock solution of
heparin 1:100 in 20% Fetal Calf Serum in M199 with 50
micrograms per ml of ECGS in HBSS for use in feeding
subcultured HUVEC.
In general, it is easiest to make
up larger volumes of 20% FCS in M199 with 2mM L-Glutamine,
plus 100 U/ml of K-Penicillin G and 100 mg/ml of Streptomycin
sulfate in advance, as this can be stored for 10 days
or more at 4°C, then, simply dilute the ECGS and
Heparin stock solutions 1:100 into the volume of medium
to be used on a given day.
K. C./M. Gimbrone
7/1985 (5/19/00) |
