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Vascular Research - Cell Biology Core - Protocols
Vascular Research Division, Center for Excellence in Vascular Biology, BWH   Brigham and Women's Hospital, a teaching hospital affiliated with Harvard Medical School
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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.

  1. 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.
  2. 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)

 

 

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