<p class="jove_step">PREPARING CELLS</p>
<ol>
<li>Bring up HUVEC and fibroblasts in M199/10% FBS/Pen-Strep (1:100) 1-2 days before beading.</li>
<li>Switch medium to EGM-2 (Clonetics) the day before beading for HUVEC and the day before embedding for fibroblasts.</li>
<li>A&nbsp;concentration of ~ 400 HUVEC per bead is needed.</li>
<li>20,000 fibroblasts per well is needed.</li>
</ol>
<p class="jove_step">COATING THE BEADS WITH HUVEC - DAY -1</p>
<ol>
<li>Trypsinize HUVEC.</li>
<li>Allow beads to settle (DO NOT CENTRIFUGE!). Aspirate the superna...

<ol>
	<li>Nehls, V., Drenckhahn, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microcarrier-based+cocultivation+system+for+the+investigation+of+factors+and+cells+involved+in+angiogenesis+in+three-dimensional+fibrin+matrices+in+vitro.">A microcarrier-based cocultivation system for the investigation of factors and cells involved in angiogenesis in three-dimensional fibrin matrices in vitro.</a> <em style='font-style: italic'>Histochem Cell Biol</em>. <b>104</b>, 459-466 (1995).</li><li>Nehls, V., Drenckhahn, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel,+microcarrier-based+in+vitro+assay+for+rapid+and+reliable+quantification+of+three-dimensional+cell+migration+and+angiogenesis.">A novel, microcarrier-based in vitro assay for rapid and reliable quantification of three-dimensional cell migration and angiogenesis.</a> <em style='font-style: italic'>Microvasc Res</em>. <b>50</b>, 311-322 (1995).</li></ol>

<p class="jove_content">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 op...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cytodex-3 Beads</td>
<td>Reagent</td>
<td>Amersham Pharmacia</td>
<td>17-0485-01</td>
<td>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 115&#xF0B0;C.
6.	Store it at 4&#xF0B0;C.
</td>
</tr>
<tr>
<td>Aprotinin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-1153</td>
<td>10mg/bottle. Reconstitute lyophilized aprotinin at 4 U/mL in DI water. Sterile filter. Make aliquots of 1 mL each. Store at -20C.
</td>
</tr>
<tr>
<td>Fibrinogen Type I</td>
<td>Reagent</td>
<td>Sigma</td>
<td>F-8630</td>
<td>1g. Dissolve 2 mg/mL fibrinogen in DPBS
Note clottable protein % and adjust accordingly 
Heat in a 37&#xF0B0;C-water bath to dissolve the fibrinogen. Mix by inverting the tube. Do not vortex.
Sterile filter through 0.22 um
</td>
</tr>
<tr>
<td>Thrombin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T-3399</td>
<td>22 mg=1000 units. Reconstitute in sterile water at 50 U/mL. Make aliquots of 0.5 mL each. Store at -20C.
</td>
</tr>		</tbody>
		</table>
		</div>
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optimized fibrin gel bead assay for the study of angiogenesis

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.

Watch this Scientific Journal Video about Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis at JoVE.com

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

<p class="jove_content">This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis.  Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.</p>

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

optimized-fibrin-gel-bead-assay-for-the-study-of-angiogenesis

Research

JoVE Journal

Biology

31.3K Views.  University of California, Irvine (UCI). This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.