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JoVE: Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis (Video Protocol)

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Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Martin N. Nakatsu, Jaeger Davis, Christopher C.W. Hughes
Department of Molecular Biology and Biochemistry, University of California, Irvine
0:00 Estimation of bead concentrations
0:10 Introduction
0:46 Reagents
1:27 Coating beads with HUVEC cells
10:22 Layering lung fibroblasts atop the fibrin gel
3:15 Washing beads
5:34 Estimation of bead concentrations
8:02 Forming the fibrin matrix
Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo. Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel. Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads. After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.


  1. Bring up HUVEC and fibroblasts in M199/10% FBS/Pen-Strep (1:100) 1-2 days before beading.
  2. Switch medium to EGM-2 (Clonetics) the day before beading for HUVEC and the day before embedding for fibroblasts.
  3. A concentration of ~ 400 HUVEC per bead is needed.
  4. 20,000 fibroblasts per well is needed.


  1. Trypsinize HUVEC.
  2. Allow beads to settle (DO NOT CENTRIFUGE!). Aspirate the supernatant and wash the beads briefly in 1 mL of warm EGM-2 medium.
  3. Mix 2500 beads w/ 1X106 HUVEC in 1.5 mL of warm EGM-2 medium in a FACS tube. Place it vertically in the incubator. (This will be enough for ~10 wells. Scale up if needed)
  4. Incubate for 4 hours at 37°C, shaking the tube every 20 min. (Good coating is crucial for sprouting.)
  5. After 4 hours, transfer the coated beads to a T25 flask in 5mL of EGM-2 and leave O/N.


  1. Prepare the 2.0 mg/mL fibrinogen solution (See recipe section).
  2. Add 0.15 Un