Introduction

R&D Systems uses HUVECs (Human Umbilical Vein Endothelial Cells) in proliferation bioassays of the following cytokines and neutralizing antibodies:

Human Cytokines

• PD-ECGF
• VEGF R1 (Flt-1)/Fc chimera
• VEGF₁₂₁
• VEGF R2 (KDR)/Fc chimera
• VEGF₁₆₅
• VEGF/PlGF

Mouse Cytokines

• VEGF₁₂₀
• VEGF R1 (Flt-1)/Fc chimera
• VEGF₁₆₄
• VEGF R2 (Flk-1)/Fc chimera

Rat Cytokines

• VEGF₁₆₄

HUVEC Bioassay Materials Cell Growth and Preparation HUVEC primary culture (Clonetics Catalog # CC-2519) Growth Medium: Endothelial Cell Growth Medium (Clonetics Catalog # CC-3024) 2% Fetal Bovine Serum (JRH Catalog # 12103-78P) Plate Preparation 96-well flat bottom microtiter plate Dulbecco's PBS (Irvine Catalog # 9240) Collagen Solution: 40 µg/mL Type I Collagen (Sigma Catalog # C7661) 0.15 M Acetic Acid Leave overnight at room temperature to dissolve Add 1/25 volume of 3.76% NaCl Store solution at 4 °C HUVEC Bioassay HUVECs 3H-thymidine (NEN Catalog # NET027E) Dulbecco's PBS (Irvine Catalog # 9240) Bovine Serum Albumin (Sigma Catalog # A-7888) 1X Trypsin Solution: Dilute 10X Trypsin EDTA (Irvine Catalog # 9342) 1:10 in PBS Assay Medium: Medium-199 1X Earle's Salts (Invitrogen # 11150-059) 10% Fetal Bovine Serum (JRH Catalog # 12103-78P), heat inactivated 10 mM HEPES 100 units/mL Penicillin 100 µg/mL Streptomycin

Procedure

Cell Growth and Preparation
As with all materials of human source, gloves and lab coats should be worn. All materials contaminated by these cells should be either decontaminated or disposed of in biohazard containers to be autoclaved. All procedures are carried out under sterile conditions.

Doubling Time: Approximately 30 hours
Appearance: Attached

1. Seed cells at 3 x 10⁵ cells/75 cm² flask.
2. Feed cells every 3-4 days with fresh Growth Medium.
3. Split cells every week and reseed in fresh Growth Medium.
   Cultures are quality assured for up to seven passages.

Plate Preparation

1. Add 50 µL/well of Collagen Solution.
2. Incubate for 2 hours at room temperature. Alternatively, incubate at 4 °C overnight.
3. Wash plate twice with PBS. Coated plates may be decanted, air dried and stored at -20 °C for up to 30 days.

HUVEC Bioassay

1. Reconstitute standards and samples in PBS + 1.0 mg/mL BSA.
2. Dilute standards and samples to working concentration with Assay Medium.
3. Add 50 µL of Assay Medium to each well of a 96-well plate.
4. Add standards and samples to the plate. Add 25 µL to first well and serial dilute. Leave last well with dilution media only, as a blank. Run samples in duplicate. Refer to figure 2 legend for neutralizing antibody assay conditions.
5. Add cells to all wells. Harvest cells by trypsinization with 1X Trypsin Solution. Wash cells twice with Medium-199. Resuspend cells in Assay Medium at 1 x 10⁵ cells/mL. Add 50 µL of cells to each well.
6. Incubate for 72 hours at 37 °C with 5% CO₂ in a humidified chamber.
7. Pulse last 18-24 hours with 0.5 µCi ³H-thymidine. Prepare 50.0 µCi/mL ³H-thymidine working stock in Assay Medium. Add 10 µL/well of working stock.
8. Harvest cells and count. Wash plate with 100 µL/well PBS. Add 100 µL/well of 1X Trypsin Solution to detach cells. Harvest on glass fiber filters using cell harvester. Count ³H-thymidine incorporated into DNA. Plot the dose response using a 4-parameter fit equation.

Troubleshooting

1. Use lower concentration of trypsin and treat for approximately 2 minutes. Do not over trypsinize.
2. Coat plate with collagen prior to assay.
3. Only use cells up to passage seven.
4. Keep timing of assay consistent.
5. Always include the correct controls.

References

1. Conn, G. et al. (1990) Proc. Natl. Acad. Sci. USA 87:1323.
2. Usuki, K. et al. (1990) Cell Regul. 1:577.

Figure 1. Human VEGF stimulates the 3H-thymidine incorporation by human umbilical vein endothelial cells in a dose-dependent manner. The ED50 for this effect is typically 2-6 ng/mL.

Figure 2. To measure the ability of the antibody to neutralize the bioactivity of rhVEGF on human umbilical vein endothelial cells, rhVEGF was incubated with various concentrations of the antibody for 1 hour at 37 °C in a 96-well microplate. Following this preincubation period, HUVECs were added. The assay mixture in a total volume of 100 µL, containing antibody at the concentrations indicated, rhVEGF at 10 ng/mL and cells at 0.5 x 104 cells/well, was incubated at 37 °C for 72 hours in a humidified CO2 incubator. 3H-thymidine was added during the final 24 hours of incubation. The cells were subsequently harvested onto glass fiber filters and the 3H-thymidine incorporated into DNA was determined. The ND50 of the antibody is approximately 3-6 µg/mL.