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
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0:00 Estimation of bead concentrations0 0:10 Introduction10 0:21 Subscription Lock21 0:46 Reagents46 1:27 Coating beads with HUVEC cells87 3:15 Washing beads195 5:34 Estimation of bead concentrations334 8:02 Forming the fibrin matrix482 10:22 Layering lung fibroblasts atop the fibrin gel622
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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.
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PREPARING CELLS
- Bring up HUVEC and fibroblasts in M199/10% FBS/Pen-Strep (1:100) 1-2 days before beading.
- Switch medium to EGM-2 (Clonetics) the day before beading for HUVEC and the day before embedding for fibroblasts.
- A concentration of ~ 400 HUVEC per bead is needed.
- 20,000 fibroblasts per well is needed.
COATING THE BEADS WITH HUVEC - DAY -1
- Trypsinize HUVEC.
- Allow beads to settle (DO NOT CENTRIFUGE!). Aspirate the supernatant and wash the beads briefly in 1 mL of warm EGM-2 medium.
- 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)
- Incubate for 4 hours at 37°C, shaking the tube every 20 min. (Good coating is crucial for sprouting.)
- After 4 hours, transfer the coated beads to a T25 flask in 5mL of EGM-2 and leave O/N.
EMBEDDING COATED BEADS IN FIBRIN GEL - DAY 0
- Prepare the 2.0 mg/mL fibrinogen solution (See recipe section).
- Add 0.15 Units/mL of aprotinin to the fibrinogen solution.
- Transfer coated beads to a 15mL conical tube and let beads settle. Resuspend beads in 1mL of EGM-2 and transfer to a 1.5mL centrifuge tube.
- Wash the beads 3X with 1mL of EGM-2 by pipeting up and down SLOWLY.
- Count beads on a coverslip and resuspend in fibrinogen solution at a concentration of ~500 beads/mL.
- Add 0.625 Units/mL of thrombin to each well.
- Add 0.5 mL of the fibrinogen/bead suspension to each well of a 24-well plate.
Change the pipette tip for each well !!!
- Mix the thrombin and the fibrinogen by going up and down gently with the pipette tip ~ 4 to 5 times. Be careful not to make large bubbles.
- Leave the plate for 5 min in the hood, then place it in the 37°C-incubator for 10-15 min to generate a clot.
- While waiting for the clot, trypsinize fibroblasts.
- Add 1 mL of EGM-2 per well drop wise.
- Seed fibroblasts on top of fibrin gel at a concentration of 20,000 cells per well.
NOTES:
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.
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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.
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| Cytodex-3 Beads |
Reagent |
Amersham Pharmacia |
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 or
50 mL -> 10 mg/mL => 30000 beads/ mL
4. Place the bead suspension in a siliconized glass bottle (Windshield Wiper or Sigmacote).
5. Sterilize the beads by autoclaving for 15 min at 115C.
6. Store it at 4C.
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| Aprotinin |
Reagent |
Sigma |
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.
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| Fibrinogen Type I |
Reagent |
Sigma |
F-8630 |
1g. Dissolve 2 mg/mL fibrinogen in DPBS
Note clottable protein % and adjust accordingly
Heat in a 37C-water bath to dissolve the fibrinogen. Mix by inverting the tube. Do not vortex.
Sterile filter through 0.22 um
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| Thrombin |
Reagent |
Sigma |
T-3399 |
22 mg=1000 units. Reconstitute in sterile water at 50 U/mL. Make aliquots of 0.5 mL each. Store at -20C.
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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)
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Nakatsu MN.., Davis J, Hughes CC.W.. (2007). Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis. JoVE. 3. http://www.jove.com/index/details.stp?id=186, doi: 10.3791/186
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Allowed tags: i, b, u, sup, sub 
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| | 05/03/2007 1:00:38 PM
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emesri responded with a statement of type: Neutral
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Excellent video. Very useful and informative. Excellent video script, recording and editing. Very good job.
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| | 01/06/2008 11:20:13 AM
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Anonymous responded with a statement of type: Neutral
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Thank you for the amazing job you guys at the Hughes lab have been doing. I have recently started working in angiogenesis and your protocols have helped immensely in getting me to be autonomous. Again, thank you.
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| | 06/26/2008 11:35:27 AM
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Deb responded with a statement of type: Neutral
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In forming the fibrin matrix, a concentration of .625 units/mL of thrombin is mentioned. Is this concentration for the final gel, or for some unknown, unmentioned volume of thrombin? |
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| | 06/11/2009 4:34:06 PM
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Chris Hughes responded with a statement of type: Neutral
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This is final conc
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| | 07/16/2008 8:02:50 AM
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karen responded with a statement of type: Question
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Thank you for a very informative video and protocol. How do you visualise your experiments at the end? Have you tried any staining methods and if so which ? I have found the clot to be very fragile during media changes - do you use collagen plates?
Many thanks
Karen |
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| | 01/19/2009 6:23:18 PM
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Wenda Ramma responded with a statement of type: Neutral
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Hi. Really good video. I would like to try this experiment. Can someone please tell me if the fibroblasts are primary cells or are they cell lines? Where can I get them ? Thank you. |
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| | 01/20/2009 1:53:15 PM
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chris hughes responded with a statement of type: Neutral
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Glad you like it. The cells are primary cultures and we get them from ATCC. I'm afraid not all lines work so every so often we have to order in a couple to try...
Good luck,
Chris Hughes
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| | 01/20/2009 1:58:26 PM
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chris hughes responded with a statement of type: Neutral
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We recently published some additional protocols that might be useful, including how to stain the cultures for immunofluorescence:
Nakatsu MN and Hughes CCW 2008. An optimized three-dimensional in vitro model for the analysis of angiogenesis. Methods in Enzymology 443: 65-82
Chris Hughes
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| | 06/11/2009 6:19:14 AM
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Anonymous responded with a statement of type: Neutral
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What is the evidence that the microvessel have patent lumens?
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