This research was supported in part by the Intramural Research Program of the National Cancer Institute at the National Institutes of Health, and by NCI grant UA5CA152907.

1. Collection of Conditioned Media to Test for Angiogenic Potential
<ol>
	<li>Grow primary or immortalized cells to be tested for angiogenic or anti-angiogenic potential in native or low serum media and collect the conditioned media.
	<ol>
		<li>Alternatively, use conditioned media immediately, or aliquoted and stored at -80 &#176;C for several months. Use nonconditioned native or low serum media as a negative control, and use non-conditioned complete growth media (10% FBS, or appropriate concentration) as a positive control. Alternatively, to test potential stimulators of angiogenesis, supplement charcoal stripped media lacking growth factors with known concentrations of specific proteins of interest and compare to growth factor free media alone.</li>
	</ol>
	</li>
</ol>
2. Preparation of Endothelial Cells Prior to Assay
<ol>
	<li>Two days prior to assay, passage 3B-11 cells into a new T-75 flask. Passage number of cells is important. This assay works best when cells are between the second and sixth passages. The assay will not work well if endothelial cells are used before the second or after the tenth passage.</li>
	<li>Grow cells for 24 hr in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 &#956;g/ml streptomycin.</li>
	<li>One day prior to assay, serum starve 3B-11 cells as follows:
	<ol>
		<li>Aspirate media from the 3B-11 cells.</li>
		<li>Add reduced serum media of DMEM supplemented with 0.2% FBS, 2 mM L-glutamine, 1mM sodium pyruvate, 100 U/ml penicillin, and 100 &#956;g/ml streptomycin.</li>
		<li>Grow cells for an additional 24 hr.</li>
	</ol>
	</li>
</ol>
3. Preparation of Reagents Prior to Assay
NOTE: BME concentrations are highly variable dependent on lot. BME does not work well if the concentration is less than 10 mg/ml, therefore the BME manufacturer should be contacted prior to purchase to ensure acquisition of an adequately concentrated lot.
<ol>
	<li>Defrost reduced growth factor BME in the refrigerator. BME solidifies when warm, so it must be kept cold prior to use. BME can be stored under refrigeration for a few weeks, or aliquoted and frozen at -80 &#176;C.</li>
	<li>A reduced growth factor BME is preferred over traditional BME. The lack of angiogenic factors in the BME will make it easier to observe the effect that factors from the conditioned media have on tube formation.</li>
	<li>Thaw media to be tested.</li>
</ol>
4. Preparation of Endothelial Cells Immediately Prior to Assay
<ol>
	<li>Wash 3B-11 cells in DPBS.
	<ol>
		<li>If visualization with fluorescence or confocal microscopy is desired, before washing 3B-11 cells, add calcein AM to the endothelial cells in media at a final concentration of 2 &#181;g/ml. Place calcein labeled cells in the dark at 37 &#176;C and 5% CO2 prior to the start of the assay. Dissolve stock calcein AM in DMSO and once calcein is diluted in media final concentration of DMSO should not exceed 0.1%. After 30-45 min wash calcein AM from the cells (step 4.1 above) and continue with the experiment follows.</li>
	</ol>
	</li>
	<li>Trypsinize cells by adding 1 ml trypsin-EDTA to T-75 flask.</li>
	<li>After trypsinization is complete, neutralize trypsin by resuspending cells to a final volume of 10ml in DMEM, 10% FBS, 2 mM L-glutamine, 1mM sodium pyruvate, 100 U/ml penicillin, and 100 &#956;g/ml streptomycin.</li>
	<li>Filter cells through 100 &#956;m cell strainer to remove clumps.</li>
	<li>Count cells and resuspend cells to a final concentration of 7.5 x 106 cells/ml in DMEM, 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 &#956;g/ml streptomycin.</li>
</ol>
5. Tube Formation Assay
<ol>
	<li>Place 24-well plate and 1 ml tips in refrigerator or on ice for 20-30 min prior to loading the plate so that the BME does not prematurely solidify.</li>
	<li>Pipet 250&#160;&#181;l of reduced growth factor BME into each well of a 24-well plate. Avoid creating bubbles by not depressing the pipette to its final stop while dispensing. Take care that sufficient BME remains at the center of the well when the meniscus forms; if the matrix is too thin, the cells will only form a monolayer.</li>
	<li>Incubate 24-well plate at 37 &#176;C and 5% CO2 for 30 min to solidify BME.</li>
	<li>Once BME has set, mix approximately 300&#160;&#181;l of conditioned media with 10&#160;&#181;l of resuspended 3B-11 cells (approximately 75,000 cells).
	<ol>
		<li>Adjust the volume of conditioned media and number of cells plated depending on endothelial cell type used, and test the serial dilutions of the conditioned media to demonstrate activity. If comparing cells grown under different conditions, normalize conditioned media volume to the number of cells which were used to condition the media.</li>
	</ol>
	</li>
	<li>Tubes will begin to form within 2-4 hr. Depending on angiogenic factor concentration in the conditioned media, peak tube formation may occur between 3 and 12 hr and the timing of the assay will need to be optimized. If working with primary endothelial cells instead of immortalized cells, initial tube formation may be delayed by several hours.
	<ol>
		<li>Tubes will often begin to deteriorate within 18 hr and endothelial cells will undergo apoptosis.</li>
	</ol>
	</li>
	<li>At the time of peak tube formation, carefully aspirate media from the well to avoid disrupting the tube network on the BME.
	<ol>
		<li>Wash each well with 1 ml DPBS, then aspirate.
		<ol>
			<li>For immediate visualization, add 1 ml fresh DPBS to each well and image the tube network using an inverted microscope attached to a digital camera, or fluorescence/confocal microscope if the cells were stained with calcein AM as in 4.1.1.</li>
			<li>To fix the tube network for later visualization, and add 4% paraformaldehyde to each aspirated well and incubate for 15 min at room temperature.
			<ol>
				<li>Remove paraformaldehyde, wash twice with DPBS, then add 1mL fresh DPBS to each well.</li>
				<li>Microscopically image prior to fixation as few tubes may break during this procedure.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
6. Quantification of Tube Network
<ol>
	<li>Quantify the tube network in several different ways depending on the investigator&#8217;s preference, and several computer programs have capability to measure the following parameters: number of tubes; number of loops/meshes; number of branch sites/nodes; length of tubes.</li>
	<li>Use representative computer program such as Image J with the Angiogenesis Analyzer plugin16 for quantification of tube networks.</li>
</ol>

<ol>
	<li>Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+life,+disease+and+medicine.">Angiogenesis in life, disease and medicine.</a> Nature. 438, 932-936 (1038).</li><li>Carmeliet, P., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+and+clinical+applications+of+angiogenesis.">Molecular mechanisms and clinical applications of angiogenesis.</a> Nature. 473, 298-307 (2011).</li><li>Potente, M., Gerhardt, H., Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+and+therapeutic+aspects+of+angiogenesis.">Basic and therapeutic aspects of angiogenesis.</a> Cell. 146, 873-887 (2011).</li><li>Coultas, L., Chawengsaksophak, K., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+and+VEGF+in+vascular+development.">Endothelial cells and VEGF in vascular development.</a> Nature. 438, 937-945 (2005).</li><li>Bouis, D., Kusumanto, Y., Meijer, C., Mulder, N. H., Hospers, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+pro-+and+anti-angiogenic+factors+as+targets+of+clinical+intervention.">A review on pro- and anti-angiogenic factors as targets of clinical intervention.</a> Pharmacological research : the official journal of the Italian Pharmacological Society. 53, 89-103 (2006).</li><li>Ausprunk, D. H., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+and+proliferation+of+endothelial+cells+in+preformed+and+newly+formed+blood+vessels+during+tumor+angiogenesis.">Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis.</a> Microvascular research. 14, 53-65 (1977).</li><li>Chung, A. S., Lee, J., Ferrara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+tumour+vasculature:+insights+from+physiological+angiogenesis.">Targeting the tumour vasculature: insights from physiological angiogenesis.</a> Nature reviews. Cancer. 10, 505-514 (2010).</li><li>Chung, A. S., Ferrara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+and+pathological+angiogenesis.">Developmental and pathological angiogenesis.</a> Annual review of cell and developmental biology. 27, 563-584 (2011).</li><li>Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+angiogenesis.">Tumor angiogenesis.</a> The New England journal of medicine. 358, 2039-2049 (2008).</li><li>Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+angiogenesis:+therapeutic+implications.">Tumor angiogenesis: therapeutic implications.</a> The New England journal of medicine. 285, 1182-1186 (1056).</li><li>Kubota, Y., Kleinman, H. K., Martin, G. R., Lawley, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+laminin+and+basement+membrane+in+the+morphological+differentiation+of+human+endothelial+cells+into+capillary-like+structures.">Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures.</a> The Journal of cell biology. 107, 1589-1598 (1988).</li><li>Arnaoutova, I., George, J., Kleinman, H. K., Benton, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+cell+tube+formation+assay+on+basement+membrane+turns+20:+state+of+the+science+and+the+art.">The endothelial cell tube formation assay on basement membrane turns 20: state of the science and the art.</a> Angiogenesis. 12, 267-274 (2009).</li><li>Arnaoutova, I., Kleinman, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+angiogenesis:+endothelial+cell+tube+formation+on+gelled+basement+membrane+extract.">In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract.</a> Nature protocols. 5, 628-635 (2010).</li><li>Walter-Yohrling, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+endothelial+cell+lines+as+models+of+tumor+endothelial+cells.">Murine endothelial cell lines as models of tumor endothelial cells.</a> Clinical cancer research : an official journal of the American Association for Cancer Research. 10, 2179-2189 (2004).</li><li>Connell, K. A., Edidin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+lymphoid+endothelial+cell+line+immortalized+by+simian+virus+40+binds+lymphocytes+and+retains+functional+characteristics+of+normal+endothelial+cells.">A mouse lymphoid endothelial cell line immortalized by simian virus 40 binds lymphocytes and retains functional characteristics of normal endothelial cells.</a> Journal of immunology. , 144-521 (1950).</li><li>Carpentier, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ+contribution:+Angiogenesis+Analyzer.">ImageJ contribution: Angiogenesis Analyzer.</a> ImageJ News. , (2012).</li></ol>

The authors have nothing to disclose.

Angiogenesis is involved in both physiological and pathological processes. Studying mechanisms involved in angiogenesis requires the use of assays that recapitulate the major steps in angiogenesis. The endothelial cell tube formation assay offers several advantages over other assays. It is easy to set-up, is relatively inexpensive, produces tubules within hours, and is quantifiable. Further, it can be completed in 24- or 96-well plates, and thus can be used for high throughput screening to identify factors that stimulate...

Angiogenesis, the development of new blood vessels from preexisting vessels, is vital for a variety of processes including organ growth, embryonic development, and wound healing1-3. Newly developed blood vessels, lined by endothelial cells, supply oxygen and nutrients to tissues, promote immune surveillance by hematopoietic cells and remove waste products2,4. Angiogenesis is of key importance during embryonic and fetal development. However, this process remains dormant in the adult except during times of wound healing, skeletal growth, pregnancy or during the menstrual cycle1-3.
Over the last two decades, key molecular mechanisms that regulate angiogenesis have begun to emerge. Angiogenesis is a tightly regulated event, balanced by pro and antiangiogenic signals including integrins, chemokines, angiopoietins, oxygen sensing agents, junctional molecules and endogenous inhibitors5. Once proangiogenic signals such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), and epidermal growth factor (EGF) activate endothelial cell receptors the endothelial cells release proteases to degrade the basement membrane. The endothelial cells then proliferate and migrate, forming sprouts at a rate of several millimeters per day6,7.
Angiogenesis is associated with various pathological conditions including cancer, psoriasis, diabetic retinopathy, arthritis, asthma, autoimmune disorders, infectious diseases, and atherosclerosis8-10. Due to the importance of angiogenesis in various diseases, understanding the genes and pathways that regulate this process is critical to the design of better therapeutics.
The tube formation assay is a rapid and quantitative method for determining genes or pathways involved in angiogenesis. First described in 1988, the principles underlying this assay are that endothelial cells retain the ability to divide and migrate rapidly in response to angiogenic signals11-13. Further, endothelial cells are induced to differentiate and form tube-like structures when cultured on a matrix of basement membrane extract (BME). These tubes contain a lumen surrounded by endothelial cells linked together through junctional complexes. Tube formation occurs quickly with most tubes forming in this assay within 2-6 hr depending on quantity and type of angiogenic stimuli.
Several types of endothelial cells can be used for this assay including both primary cells and immortalized cell lines14,15.&#160;The cell line used for this article was mouse 3B-11, but the same methodology can be applied with other endothelial cell lines such as SVEC4-10 (mouse) or primary endothelial cells such as HUVEC (human) cells. Depending on which cell line is used and whether the endothelial cells are transformed or non-transformed, optimization will need to be conducted to identify the ideal time needed for proper tube formation.

<table border="0" cellpadding="0" cellspacing="0" width="651">
	<tbody>
		<tr height="21">
			<td height="42" rowspan="2" style="height:42px;width:339px;">Item</td>
			<td rowspan="2" style="width:189px;">Manufacturer</td>
			<td rowspan="2" style="width:124px;">Catalog #</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="93">
			<td height="114" rowspan="2" style="height:114px;width:339px;">Costar 24-Well Tissue Culture-Treated Plate</td>
			<td rowspan="2" style="width:189px;">Corning</td>
			<td rowspan="2" style="width:124px;">3524</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="169">
			<td height="190" rowspan="2" style="height:190px;width:339px;">BD Matrigel Basement membrane matrix, Growth factor reduced</td>
			<td rowspan="2" style="width:189px;">BD Biosciences</td>
			<td rowspan="2" style="width:124px;">354230</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="55">
			<td height="76" rowspan="2" style="height:76px;width:339px;">Gibco 0.05% Trypsin-EDTA</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">25300-054</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="74">
			<td height="95" rowspan="2" style="height:95px;width:339px;">Gibco L-Glutamine 200mM (100X)</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">25030-081</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="36">
			<td height="57" rowspan="2" style="height:57px;width:339px;">Gibco Pen Strep</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">15140-122</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="36">
			<td height="57" rowspan="2" style="height:57px;width:339px;">Gibco DMEM (1X)</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">11965-092</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="36">
			<td height="57" rowspan="2" style="height:57px;width:339px;">Gibco DPBS (1X)</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">14190-144</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="36">
			<td height="57" rowspan="2" style="height:57px;width:339px;">Fetal Bovine Serum</td>
			<td rowspan="2" style="width:189px;">Atlas Biologicals</td>
			<td rowspan="2" style="width:124px;">F-0500-A</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Calcein AM</td>
			<td style="width:189px;">Life Technologies</td>
			<td style="width:124px;">C3100MP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DMSO</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:124px;">4-X</td>
		</tr>
	</tbody>
</table>

endothelial cell tube formation assay for the in vitro study of angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Mouse 3B-11 endothelial cells were seeded on solidified reduced growth factor BME &#8211; in this assay, the product Matrigel was used - and followed over time. As shown in Figure 1, angiogenic factors secreted by either mouse keratinocytes or fibroblasts are capable of inducing tube formation over time. Endothelial cells migrate and begin to form small branches within 1-2 hr of plating. Maximum tube formation was reached by 4-6 hr using conditioned media previously obtained from keratinocytes. By 24 hr,...

Watch this Scientific Journal Video about Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis at JoVE.com

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

endothelial-cell-tube-formation-assay-for-vitro-study

Research

JoVE Journal

Biology

59.0K Views.  American University. The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Video: Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Badania wizualne Wrażliwość na prędkość i kierunek ruchu w Jaszczurki

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

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.

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.

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

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.