Department of Molecular Biology and Biochemistry, University of California, Irvine (UCI)
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Nakatsu, M. N., Davis, J., Hughes, C. C. Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis. J. Vis. Exp. (3), e186, doi:10.3791/186 (2007).
COATING THE BEADS WITH HUVEC - DAY -1
EMBEDDING COATED BEADS IN FIBRIN GEL - DAY 0
Usually, when the fibrin gel is formed, you will see tiny bubbles in the gel. Don't worry, they will disappear in 3-4 days.
Change the media every other day, i.e., Day 2, 4, 6, etc...
By day 3 or 4 you should start to see sprouting.
There is a growing consensus that three-dimensional (3D) in vitro angiogenesis assays offer a model which is much closer to the actual environment in vivo than can be achieved using 2D cultures. It is apparent that superior 3D systems should be reproducible, and be able to mimic several of the major steps of angiogenesis. While several previous 3D assays have been developed, many of these either use hard-to-obtain microvascular cells, or only recapitulate some of the stages. In this video, we describe and perform an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC, which are easily obtainable and the most commonly used EC in vascular research. The assay, over the course of several days, consistently reproduces long vessels with clear, patent intercellular lumens surrounded by polarized EC. Later stages of EC branching and fusion of vessels (anastomosis) are also observed. Importantly, in these cultures the HUVEC undergo all of the morphological changes that are seen with microvascular EC, either in vivo or in vitro, including sprouting, migration, alignment, proliferation, tube formation, branching and anastomosis. The gene expression profile of the HUVEC changes, in parallel, to more closely match that of microvascular EC. In conclusion, we present an optimized protocol for an in vitro angiogenesis model that recapitulates several important stages of this process.
|Cytodex-3 Beads||Reagent||Amersham||17-0485-01||10 g/bottle. 1.0.5 g of dry beads are hydrated and swollen in 50 mL PBS (pH=7.4) for at least 3 hours at RT.Use a 50 mL tube and place it on the rocker.2.Let the beads settle down (~ 15 min). Discard the supernatant and wash the beads for a few minutes in fresh PBS (50 mL).3.Discard the PBS and replace with fresh PBS:25 mL -> 20 mg/mL => 60000 beads/mL or50 mL -> 10 mg/mL => 30000 beads/ mL4.Place the bead suspension in a siliconized glass bottle (Windshield Wiper or Sigmacote).5.Sterilize the beads by autoclaving for 15 min at 115ï‚°C.6.Store it at 4ï‚°C.|
|Aprotinin||Reagent||Sigma-Aldrich||A-1153||10mg/bottle. Reconstitute lyophilized aprotinin at 4 U/mL in DI water. Sterile filter. Make aliquots of 1 mL each. Store at -20C.|
|Fibrinogen Type I||Reagent||Sigma-Aldrich||F-8630||1g. Dissolve 2 mg/mL fibrinogen in DPBSNote clottable protein % and adjust accordingly Heat in a 37ï‚°C-water bath to dissolve the fibrinogen. Mix by inverting the tube. Do not vortex.Sterile filter through 0.22 um|
|Thrombin||Reagent||Sigma-Aldrich||T-3399||22 mg=1000 units. Reconstitute in sterile water at 50 U/mL. Make aliquots of 0.5 mL each. Store at -20C.|
1. Nehls, V. and Drenckhahn, D. A microcarrier-based cocultivation system for the investigation of factors and cells involved in angiogenesis in three-dimensional fibrin matrices in vitro. Histochem Cell Biol 104:459-66. (1995)
2. Nehls, V. and Drenckhahn, D. A novel, microcarrier-based in vitro assay for rapid and reliable quantification of three-dimensional cell migration and angiogenesis. Microvasc Res 50:311-22. (1995)