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 or inhibit angiogenesis. Although we used 3B-11 mouse endothelial cells for our experiments, the assay can be performed with a variety of human or murine endothelial lines, including HUVEC and SVEC.
To perform an endothelial cell tube formation assay, endothelial cells are mixed with conditioned media which contains pro or antiangiogenic factors and plated over reduced growth factor basement membrane matrix. Endothelial cells begin to align themselves within 1&#160;hr and lumen-containing tubules start to appear within 2&#160;hr. Tube formation can be visualized using a phase contrast inverted microscope, or calcein AM can be added at the conclusion of the assay and tubes visualized using fluorescence or confocal microscopy.
Results may be optimized in several ways. First, primary cells should be ideally between the second and sixth passages. Using cells prior to the second or after the tenth passage is not be conducive for maximum tube formation. Second, the cell number needs to be optimized based on cell type and line used. For mouse 3B-11, we found maximal response using 75,000-100,000 cells/well in a 24-well plate grown with mouse keratinocyte and fibroblast conditioned media. Using too few cells results in minimal or no tube formation, and too many cells results in formation of a monolayer or clumping. Finally, the quantity of conditioned media used should be normalized to cell number instead of protein concentration. If using normal media with 10% FBS, the FBS will account for the majority of the protein concentration measured through a standard protein assay. The concentration will look nearly identical even though number of cells, and therefore the concentrations of the proteins secreted during conditioning, between treatments may be highly variable.
In summary, the tube formation assay is a fast, reproducible and sensitive method for in vitro measurement of angiogenesis. It has advantages over other assays and is a useful method to determine genes and pathways that potentially play an important role in the activation or inhibition of angiogenesis.

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><tbody><tr><td>Costar 24-well tissue culture-treated plate</td><td>Corning</td><td>3524</td><td></td></tr><tr><td>BD Matrigel basement membrane matrix, growth factor reduced</td><td>BD Biosciences</td><td>354230</td><td></td></tr><tr><td>Gibco 0.05% trypsin-EDTA</td><td>Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>Gibco L-glutamine 200 mM (100x)</td><td>Life Technologies</td><td>25030-081</td><td></td></tr><tr><td>Gibco pen strep</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Gibco DMEM (1x)</td><td>Life Technologies</td><td>11965-092</td><td></td></tr><tr><td>Gibco DPBS (1x)</td><td>Life Technologies</td><td>14190-144</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Atlas Biologicals</td><td>F-0500-A</td><td></td></tr><tr><td>Calcein AM</td><td>Life Technologies</td><td>C3100MP</td><td></td></tr><tr><td>DMSO</td><td>ATCC</td><td>4-X</td><td></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, some branches remained, but many of the cells became apoptotic and tubes began to disconnect.
Cell number has a profound impact on tube formation (Figure 2). When an insufficient number of cells are seeded on the matrix, few tubes form and the network is minimal. As more cells are seeded, the tube network becomes more extensive. However, at too high of a concentration, the cells begin to clump or form monolayers, and differences between treatment groups become masked.
Tube formation with 3B-11 cells works the best with cells between the second and sixth passages. After the sixth passage, cells do not form as extensive a network; cells will not form any major network after the tenth passage. Cells should have a minimum of two passages after reviving from liquid nitrogen storage to ensure adequate tube formation (Figure 3). The growth rate of 3B-11 is so rapid that 25% of cells passaged one day will yield a confluent flask the following day.
Images can be easily documented through phase contrast microscopy using objectives between 4X and 20X. However, if fluorescent images are desired, calcein AM may be added and the tube network visualized through fluorescence or confocal microscopy (Figure 4). There are several different ways to detect and quantify tube network formation. The most common methods of analysis involve quantification of number of tubes, nodes, loops/meshes, or length of tubes. These parameters can be counted by hand or can be done through various imaging programs, including NIH ImageJ with the Angiogenesis Analyzer plugin. Examples of each of these types of analysis are shown in Figure 4.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51312/51312fig1highres.jpg" width="500" /> 
Figure 1.&#160;Mouse 3B-11 endothelial cell tube formation over time. A total of 1 x 105 cells were seeded per well on reduced growth factor BME with 350&#160;&#181;l of conditioned media from either fibroblasts or keratinocytes. Tube formation was recorded over the course of 24 hr.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51312/51312fig2highres.jpg" width="500" /> 
Figure 2.&#160;The effect of cell number on endothelial cell tube formation. 3B-11 cells were seeded in wells at concentrations ranging from 5 x 104 to 2 x 105 with 350&#160;&#181;l of fibroblast or keratinocyte conditioned media, and then followed over time. Note the lack of tube formation in the wells plated with 5 x 104 cells and the crowding of cells in the wells plated with 2 x 105 cells.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51312/51312fig3highres.jpg" width="500" /> 
Figure 3.&#160;The effect of passage number on endothelial cell tube formation. A total of 1 x 105 cells were seeded per well on reduced growth factor BME at passage 0, 1, or 2. Little tube formation was seen before passage 2.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51312/51312fig4highres.jpg" width="500" /> 
Figure 4.&#160;Fluorescence microscopy imaging and quantification of tube formation. Prior to the start of the assay, the endothelial cells can be treated with calcein AM in order to visualize the cells using fluorescence or confocal microscopy. There have been several reported methods for quantifying the formed tube network, including counting the number of tubes, loops/meshes, branch sites/nodes, or the length of tubes. Here, we show how NIH Image J with the Angiogenesis plugin software can be used on a calcein AM stained tube network (A) to detect total nodes (red)/tubes (pink)/mesh (blue) in combination (B) and merged on network (C). Additionally, this program can be used to individually detect nodes (D), tubes (E), or mesh (F) which can then be quantified (G).

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

Nanyang Technological University

Research

JoVE Journal

Biology

59.5K 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

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 ...

Fibroblast-Derived 3D Matrix System Applicable to Endothelial Tube Formation Assay

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ECM also participates in cell migration, proliferation, and differentiation. Fibroblasts are the main type of cells modifying ECM fiber arrangement and production. In cancer, CAFs (cancer associated fibroblasts) are in permanent activation status, participating in ECM remodeling, facilitating tumor cell migration, and stimulating tumor-associated angiogenesis, among other pro-tumorigenic roles. The objective of this method is to create a three-dimensional matrix with a fiber composition that is similar to in vivo matrices, using immortalized fibroblasts or human primary CAFs. Fibroblasts are cultured in pre-treated cell culture plates and grown under ascorbic acid stimulation. Then, fibroblasts are removed and matrices are blocked for further cell seeding. In this ECM model, fibroblasts can be activated or modified to generate different kinds of matrix, whose effects can be studied in cell culture. 3D matrices are also shaped by cell signals, like degradation or cross-linking enzymes that might modify fiber distribution. In this context, angiogenesis can be studied, along with other cell types such as epithelial tumor cells.

The aim of this method is to obtain fibroblast-derived 3D matrices as a natural scaffold for subsequent cellular assays. Fibroblasts are seeded in a pre-treated culture plate and stimulated with ascorbic acid for matrix generation. Matrices are decellularized and blocked to culture relevant cells (e.g., endothelial cells).

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ...

Cancer Research

HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the formation of new blood vessels and involve several signal transduction pathways. Here we show that the tube formation assay is a simple in vitro method to evaluate the impact of natural products on angiogenesis and to investigate the molecular mechanisms involved. In particular, in the presence of the water extract of Ruta graveolens (RGWE), endothelial cells are no longer able to form a cell-cell network and that the RGWE effects on human umbilical vein endothelial cell (HUVEC) tube formation is abolished by the constitutive activation of MEK.

Here, we evaluate the effects of the water extract of Ruta graveolens on vessel network formation by using a tube formation assay on a gelled basement matrix.

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the ...

Developmental Biology

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

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 ...

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

Medicine

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current in vitro screening models that have sufficient throughput only have limited physiological relevance, which hinders the translation of findings from in vitro to in vivo. On the other hand, microfluidic cell culture platforms have shown unparalleled physiological relevancy in vitro, but often lack the required throughput, scalability and standardization. We demonstrate a robust platform to study angiogenesis of endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) in a physiological relevant cellular microenvironment, including perfusion and gradients. The iPSC-ECs are cultured as 40 perfused 3D microvessels against a patterned collagen-1 scaffold. Upon the application of a gradient of angiogenic factors, important hallmarks of angiogenesis can be studied, including the differentiation into tip- and stalk cell and the formation of perfusable lumen. Perfusion with fluorescent tracer dyes enables the study of permeability during and after anastomosis of the angiogenic sprouts. In conclusion, this method shows the feasibility of iPSC-derived ECs in a standardized and scalable 3D angiogenic assay that combines physiological relevant culture conditions in a platform that has the required robustness and scalability to be integrated within the drug screening infrastructure.

This method describes the culture of iPSC-derived endothelial cells as 40 perfused 3D microvessels in a standardized microfluidic platform. This platform enables the study of gradient-driven angiogenic sprouting in 3D, including anastomosis and stabilization of the angiogenic sprouts in a scalable and high-throughput manner.

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current ...

Bioengineering

Analyzing Angiogenesis: In Vitro Assay for Tube Formation on BM Matrix

Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of mesenchymal to endothelial-like transition. Traditionally, this process is evaluated by an in vitro tube-formation assay, where the cells organize themselves into tube-like structures when seeded over a polymerized basement membrane preparation. Although several structural features can be measured in photomicrographs of the structures, to assess the real commitment of EVT to the endothelial-type phenotype, biochemical analysis of cell extracts is required. Scraping the cells from the culture dish to obtain RNA and/or protein extracts is not an alternative since the tube-like structures are severely contaminated by the bulk of proteins from the polymerized basement membrane. Thus, a strategy to separate the cells from the basement membrane proteins prior to the preparation of cell extracts is needed. Here, a simple, fast, and cost-effective method to recover the tube-like structures from the in vitro tube-formation assay and the subsequent analysis by biochemical techniques is presented. Tube-like structures formed by HTR8/SVneo cells, an EVT cell line, were liberated from the polymerized basement membrane by a short incubation with PBS supplemented with ethylenediaminetetraacetic acid (EDTA). After serial washes, a ready-to-use pellet of purified tube-like structures can be obtained. This pellet can be subsequently processed to obtain RNA and protein extracts. qPCR analysis evidenced the induced expression of VE-cadherin and alphav-integrin, two endothelial cell markers, in EVT-derived tube-like structures compared to control cells, which was consistent with the induction of the endothelial cell marker, CD31, evaluated by immunofluorescence. Western blot analysis of the tube-like structures' protein extracts revealed the overexpression of RECK in transfected HTR8/SVneo cells. Thus, this simple method allows to obtain cell extracts from the in vitro tube-formation assay for the subsequent analysis of RNAs and protein expression.

The present protocol allows the recovery of tube-like structures formed in the classical in vitro angiogenesis-tube formation assay where cells, such as trophoblast cells, organize in capillary-like structures over a layer of extracellular basement membrane matrix. The recovered structures are used for RNA and protein isolation for various biochemical analyses.

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of ...

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for ...

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

Summary

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Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Fibroblast-Derived 3D Matrix System Applicable to Endothelial Tube Formation Assay

HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

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

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing