Human Umbilical Vein Endothelial Cells (HUVECs) Tube Formation Assay

Angiogenesis is essential for tumorigenesis and metastasis. Angiogenesis inhibition is one of the main therapeutic targets in cancer drug discovery. During angiogenesis, endothelial cells (ECs) are activated by binding of angiogenic factors to their receptors, releasing proteases to dissolve the basement membrane, migrating towards angiogenic signals, proliferating, and increasing in cell numbers for new blood vessel formation.

HUVECs tube formation assay is one of the simple, but well-established in vitro angiogenesis assays based on the ability of ECs to form three-dimensional capillary-like tubular structure, which represents the later stage of the angiogenic process. During the assay, HUVEC cells differentiate, directionally migrate to align, branch, and form the polygonal networks of blood vessels.

Effect of cell number on HUVECs tube formation Figure 1. Effect of cell number on HUVECs tube formation.

Materials

Primary human umbilical vein endothelial cells (HUVECs)
Growth factor-reduced matrigel
Medium 200PRF
Low serum growth supplement (LSGS)
Cell-permeable dye (e.g., Calcein AM)
Appropriate treatment (angiogenesis factors/regulators and/or inhibitors)

Equipment

T25 or T75 tissue culture flask
96-well plate
Cell counter
Centrifuge
Light or fluorescence microscope

Preparation of Human Umbilical Vein Endothelial Cells (HUVECs)

  • Prepare the HUVECs medium 200PRF supplement with LSGS according to the product insert.
  • Seed HUVEC cells in T25 or T75 flask using LSGS-supplemented medium 200PRF.
  • Change the medium every other day thereafter, until they are 80-90% confluent on the day of the experiment.

Coating of 96-Well Plate with Growth Factor-Reduced Matrigel

  • Thaw an appropriate volume of growth factor-reduced matrigel one day before use at 4°C.
  • Pre-chill the 96-well plate and the pipet tips at -20°C for 2-3 h.
  • Before counting HUVEC cells, add 50 μL of growth factor-reduced matrigel per well of the pre-chilled 96-well plate on ice. Swirl the plate until the gel is evenly distributed over the whole well. It is very important to avoid bubble formation. Allow it to polymerize at room temperature for 1 h on a level surface.
    Note: Background control well = no growth factor-reduced matrigel.

Tube Formation

  • Trypsinize to dislodge the cells from the surface of the flask, pellet down the cells at room temperature by centrifugation at 1,200 rpm for 3 min, resuspend in 2-3 mL of non-supplemented medium 200PRF, determine the concentration of HUVECs and pipet up and down several times to ensure a homogeneous single-cell suspension.
  • Plate 1-2 x 104 cells/well onto the solidified extracellular matrix gel and the background control well.
  • Add appropriate treatment (angiogenesis factors/regulators and/or inhibitors) to the desired wells.
    Note: For a positive inducer control, we suggest diluting the cells in LSGS-supplemented media 200PRF containing 2% (v/v) FBS and bFGF (3 ng/mL). For a positive inhibitor control, we suggest diluting cells in LSGS-supplemented medium and 30 µM vinblastine.
  • Incubate the plate for 4-18 h in a 37°C incubator containing 5% CO2.

Tube Staining (Optional)

  • If cells were not pretreated with a dye before harvesting, they can be stained at the end of the incubation period after the tube network has formed using a cell-permeable dye (e.g., Calcein AM).
  • Carefully remove incubation medium using a pipette without disturbing the cells or the extracellular matrix gel.
  • Add the dye to the cells and incubate for 30 min at 37°C (protect from light).
  • Gently remove the dye-containing media with a pipette, and replace with an equivalent volume of warm medium 200PRF. The replacement media should be identical to the media the cells were incubated in during the tube formation.
  • Visualize the cells using light or fluorescence microscopes. Take pictures of the capillary network and count the tube lengths.

Troubleshooting

ProblemCauseSolution
Low or no tube network formation in positive controlDead or stressed (overcrowded) cellsUse only healthy cells or cells from an earlier passage.
Make sure that cells are in the log growth phase.
Insufficient inducer concentrationDetermine the appropriate concentration of inducer for the cell lines used in the study.
Low or no fluorescent signal in samplesInappropriate time point of detectionEnsure time of detection is optimized and samples are prepared immediately.
Cell density is too lowCheck cell count to confirm proper cell density.
High background fluorescenceInappropriate cell conditionsEnsure the inducer treatment does not kill the cells during the time frame of the experiment.
Omitting wash stepMake optional wash steps mandatory.

References

  1. Tahergorabi Z, et al.; A review on angiogenesis and its assays. Iranian Journal of Basic Medical Sciences, 2012, 15(6): 1110-1126.
  2. Donovan D, et al.; Comparison of three in vitro human ‘angiogenesis’ assays with capillaries formed in vivo. Angiogenesis, 2001, 4(2): 113-121.

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