The authors thank the following staff members and research personnel: Andr&#233;e Gauthier-Fisher, Matthew Librach, Tanya Barretto, Tharsan Velauthapillai, and Sarah Laronde.

All studies involving animals were conducted and reported according to ARRIVE guidelines29. All studies were performed with institutional research ethics board approval (REB number 4276). All animal procedures were approved by the Animal Care Committee of the University Health Network (Toronto, Canada), and all animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, 8th edition (National institutes of Health 2011).
1. Aortic Ring Assay Setup
<ol>
	<li>Isolate the rat aorta.
	<ol>
		<li>Euthanize 8 - 10-week-old Sprague-Dawley rats using CO2 asphyxiation: Set CO2 chambers to 20% gas replacement (flow rate = 0.2 x chamber volume/min). Confirm by the absence of pinch reflex and heart beats by palpating chest.</li>
		<li>Wet the fur on chest area using sterilized gauze with 70% ethanol. Use sterilized forceps and scissors to make an incision in the chest midline and cut through skin and muscle layers.</li>
		<li>Once the rib cage is exposed, cut the rib cage on each side and fold upwards about 5 mm from the sternum on each side. Some bleeding is expected from intercostal arteries in fresh cadavers.</li>
		<li>Push the lung tissue and heart to the anatomical right side to expose the aorta. The aorta is the white colored vessel located adjacent to the spinal column.</li>
		<li>Use forceps to pinch the aorta (after the aortic arch) and use surgical scissors to make the first incision while holding the aorta with forceps. Upon cutting the aorta, the chest cavity will quickly fill with blood, therefore it is critical to work quickly to obtain the aorta prior to blood coagulation.</li>
		<li>Use forceps to hold the aorta and gently peel the aorta downwards, away from the spinal column. The pull will sever the intercostal arteries without further incisions. Obtain approximately 10 cm of tissue and/or before the aorta divides into two branches in the abdominal cavity and cut the aorta out of the carcass. Push the diaphragm if needed to the caudal direction to gain access to lower sections of the aorta. 
		NOTE: Peel the aorta gently because intense force can cause it to tear. If the aorta tears, allow blood coagulation for a few seconds and use a paper towel to remove the coagulated blood, allowing visibility of the remaining aorta.</li>
		<li>Place the aorta in a 15 mL tube with 10 mL of ice-cold Hank&#39;s Balanced Salt Solution (HBSS) supplemented with 1% penicillin/streptomycin (P/S). Keep the tubes on ice. 
		NOTE: The aortic tissue can last for 1-2 h on ice but it is best to process it as quickly as possible.</li>
	</ol>
	</li>
	<li>Prepare the aortic ring assay culture media in a sterile biosafety cabinet (BSC).
	<ol>
		<li>Prepare the Endothelial Growth Medium (EGM) by using a filtration unit to filter 500 mL of Endothelial Basal Medium (EBM) supplemented with 2% Fetal Bovine Serum (FBS), 1% gentamicin and growth factors (see Table of Materials). Place 50 mL of the filtered media into a bead or water bath at 37 &#176;C.</li>
		<li>Use another filtration unit to filter 100 mL of EBM supplemented with 2% FBS and 1% P/S to prepared the EBM 2% FBS (EBM-FBS) and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Coat 12-well tissue culture plates with Basal Membrane Extract (BME) while working on ice.
	<ol>
		<li>Place a 12-well plate on a cold surface (large ice pack or tray). Add 200 &#181;L/well of freshly thawed BME using refrigerated pipette tips and swirl the BME to ensure even coating. Work quickly to avoid uneven polymerization of the BME. 
		NOTE: A typical aorta excision yields 20 aortic rings. To account for variability between the aortic ring assays, setup at least three aortic ring assays per treatment group and include a control. If the source permits, 6 rings per treatment group is recommended.</li>
		<li>Place the coated plates into humidified incubators (95% relative humidity, 37 &#176;C, 5% CO2; these conditions apply to all humidified incubator steps hereafter) for 30 min. Place 1,000 &#181;L pipette tips back into -20 &#176;C for step 1.5.3. 
		NOTE: While the basal membrane extract is used in stock concentration, its consistency and stiffness is strictly controlled by the polymerization time. Make sure to keep the exact same polymerization times for all parallels and independent experiments.</li>
	</ol>
	</li>
	<li>Section the aorta into uniform rings.
	<ol>
		<li>Take the aorta from the 15-mL tube and place it into a 10 cm dish with 10 mL of fresh HBSS supplemented with 1% P/S.</li>
		<li>Use two sets of forceps to carefully separate the excess connective tissue, adipose tissue, and use a scalpel for the remaining branches of the intercostal arteries connected to the aorta. This reduces variability between ring units based on differing levels of the residual tissue. Remove any residual coagulated blood from inside of the aorta.</li>
		<li>Place a ruler or grid under the 10 cm dish to precisely cut 1 - 2 mm wide sections of the aorta using sterile scalpel and forceps. Cut the sections as exact as possible to reduce variability between units.</li>
	</ol>
	</li>
	<li>Embed the aortic rings into BME. 
	NOTE: If the sectioning of aortic rings is not completed before BME polymerization incubation, remove the coated plates from humidified incubators and add 100 &#181;L of EBM to each well to terminate the polymerization. Exact timing is critical as over-polymerization of BME may interfere with the endothelial network development and cell migration. Once the aortic rings are prepared, remove the EBM from the wells and continue from step 1.5.2.
	<ol>
		<li>Remove the BME coated wells from the humidified incubators and return to the BSC.</li>
		<li>Carefully pick up individual aortic rings with forceps and place one in the middle of each BME-coated well. Repeat for the remaining aortic rings. 
		NOTE: Media transferred along with the aortic ring should be kept minimal in order to avoid polymerization irregularities.</li>
		<li>Use cooled (-20 &#176;C) 1,000 &#181;L pipette tips to add 300 &#181;L of BME on top of each aortic ring and evenly distribute the BME around the well.</li>
		<li>Return the embedded aortic rings to the humidified incubators for 30 min.</li>
		<li>Remove the plates with the embedded aortic ring from the humidified incubators and slowly add 1,000 &#181;L of EGM (prepared and warmed at step 1.2.1) by placing the pipette tip against the wall of the culture well. 
		NOTE: Directly pipetting the media onto the BME can disrupt the polymerized BME and hinder continuous endothelial network development. Use an appropriate multichannel pipette if available.</li>
		<li>Following 24 h, remove 1,000 &#181;L of EGM and replace with 1,000 &#181;L of EBM-FBS prepared in step 1.2.2, again by slowly pipetting against the side of the culture well.</li>
	</ol>
	</li>
	<li>Maintain the aortic ring assay by replacing 500 &#181;L of EBM-FBS with 500 &#181;L of fresh EBM-FBS (37 &#176;C) every 48 h. 
	NOTE: See section 2 to start the MSC culture expansion on the same day as aortic ring assay establishment. This will ensure that MSCs are ready for co-culture when the endothelial networks are ready.</li>
	<li>Use the bright field microscopy to follow aortic ring assay endothelial network development (see Figure 1 as reference for optimal network development). Once the endothelial networks aspect is as shown in Figure 2, proceed with setting up the aortic ring assay-MSC co-cultures (section 3). Refer to step 4.1 for further details of imaging the endothelial networks.</li>
</ol>
2. Tissue Culture
<ol>
	<li>Prepare stock solutions of tissue culture media.
	<ol>
		<li>Culture FTM HUCPVCs (previously established, n &#8805; 3 independent lines for each)30 and commercially available BMSCs in alpha-MEM supplemented with 10% FBS and 1% P/S.</li>
		<li>Sterilize the media using 0.2 &#181;m pore size filter bottles.</li>
		<li>Store the prepared media solutions at 4 &#176;C for up to 3 weeks.</li>
	</ol>
	</li>
	<li>Maintain FTM HUCPVC and BMSC cultures in humidified incubators and passage at 70 - 80% confluency determined by phase contrast microscopy. Use appropriate volumes of media for the size of tissue culture dish used (i.e. 10 mL in a 10 cm dish). Use these culture conditions for maintaining and passaging the MSCs.</li>
	<li>Dissociate the MSC monolayers for passaging or aortic ring assay-MSC co-cultures using a dissociation enzyme solution (4 mL/10 cm dish) and incubate in the humidified incubators for 3 min. Ensure detachment of the cells using bright field microscopy; incubate for an additional 1 - 2 min if required.</li>
	<li>Transfer the dissociated cells into a 15 mL tube and centrifuge at 400 x g for 5 min.</li>
	<li>Aspirate the supernatant without disturbing the cell pellet and resuspend the cells in 1 mL of an appropriate culture media (alpha-MEM complete media for passaging cells or EBM-FBS for aortic ring assay/MSC co-cultures) for counting using a cell counter.</li>
</ol>
3. Preparation of Aortic Ring Assay/MSC Co-cultures
<ol>
	<li>Seed 104 MSCs on each embedded aortic ring (9,000 cells/cm2/12-well plate). Based on the number of aortic ring assays, pre-stain the MSCs with viable, nontransferable fluorescent dye. Maintain at least three wells of aortic rings without MSCs for a control group.
	<ol>
		<li>To stain 105 MSCs/mL EBM-FBS media, add 2.5 &#181;L of viable, non-transferable fluorescent dye/mL media (5 &#181;M) into a 15 mL tube and place in the humidified incubator for 30 min. Gently mix the tube halfway through the incubation.</li>
		<li>Following the 30 min incubation, add 1 volume of fresh EBM-FBS to the stained cells and spin at 400 x g for 5 min.</li>
		<li>Remove the supernatant and re-suspend the cell pellet in 1 mL of EBM-FBS media. Confirm successful staining of the MSCs using fluorescence microscopy.</li>
	</ol>
	</li>
	<li>Remove the aortic ring-containing plates from the humidified incubator and remove 500 &#181;L of EBM-FBS from each well.</li>
	<li>Carefully seed 104 MSCs in 500 &#181;L of EBM-FBS on each embedded aortic ring by evenly pipetting around the endothelial networks. Ensure even distribution by gently shaking the culture plate.
	<ol>
		<li>Add additional EBM-FBS to ensure that the total volume of media on the aortic ring assay/MSC co-cultures is 1,000 &#181;L.</li>
	</ol>
	</li>
	<li>Use fluorescence microscopy to visualize the fluorescently labelled MSCs in the aortic ring assay.</li>
</ol>
4. Microscopy
<ol>
	<li>Use bright-field microscopy to image the rat endothelial networks prior to the aortic ring assay/MSC co-cultures for baseline (day 0) measurements of endothelial network properties (e.g., network length, network loops). Image 4 quadrants per well from the aortic ring to the furthest section of the endothelial network within that quadrant.
	<ol>
		<li>Image the aortic ring networks following the aortic ring assay/MSC co-cultures at days 3, 5 and 7. Use these images to quantify the MSC effect on the endothelial network development.</li>
	</ol>
	</li>
	<li>Use fluorescence microscopy to image pre-labelled MSCs in the aortic ring assay.
	<ol>
		<li>Following 24 h of aortic ring assay/MSC co-cultures, use fluorescence microscopy to visualize MSC homing, elongation and integration with the endothelial networks. Overlap the fluorescent images of MSCs with the bright field images of endothelial cells to observe the colocalization of both cell types.</li>
		<li>Image the MSCs localized within the developed endothelial networks proximal to the aortic ring tissue, and the MSCs localized within newly developing networks distal to the aortic ring tissue (Figure 2).</li>
	</ol>
	</li>
</ol>
5. Flow Cytometry and qPCR
<ol>
	<li>One day prior to dissociating the aortic ring endothelial networks, thaw frozen dispase at 4 &#176;C O/N. Section 5.3 is identical for both flow cytometry and qPCR.</li>
	<li>Warm 50 mL of dispase and 50 mL of 0.5% trypsin in 37 &#176;C bead or water bath.</li>
	<li>Retrieve the cells for analysis from the aortic ring assay/MSC co-cultures.
	<ol>
		<li>After 1 week of aortic ring assay/MSC co-culture, remove the culture media and add 1 mL of Phosphate-Buffered Saline (PBS) slowly to each well for 3 min and remove. Repeat this wash twice more.</li>
		<li>Add 800 &#181;L of pre-warmed dispase to each well and incubate in the humidified incubators for 15 min.</li>
		<li>Remove the plates from the humidified incubators and suspend dispase by pipetting (5 - 10x) to break up the BME, and transfer the contents of each well into separate 15 mL tubes.</li>
		<li>Repeat step 5.3.3 again with fresh pre-warmed dispase until no residual BME is observed in the culture well. Add 1 volume of PBS supplemented with 3% FBS to the dispase-cell suspension to inactivate the dispase. Remove the aortic rings floating in the cell suspension using pipette tips.</li>
		<li>Spin the cell suspensions at 400 x g for 5 min. Remove the supernatant carefully, leaving 3 mL of cell suspension in the 15 mL tube. 
		NOTE: An obvious, solid pellet may not be visible but cells and BME are present.</li>
		<li>Add 3 mL of prewarmed 0.5% trypsin to the cell suspension and vigorously resuspend (5 - 10x by pipetting) cells. Incubate the trypsinized cell solutions in the humidified incubators for 10 min.</li>
		<li>Remove the trypsinized cell suspensions from humidified incubators and add 6 mL of PBS supplemented with 3% FBS and resuspend a few times. Spin the cell suspensions at 400 x g for 5 min.</li>
		<li>Carefully remove all the supernatant, leaving the cell pellet in the tube and resuspend cells in 1 mL PBS supplemented with 3% FBS in each tube.</li>
		<li>Filter the 1 mL cell suspension using a 70 &#181;m cell strainer to remove aggregated cells and residual BME from the cell suspension. Count the cells in 1 mL of cell suspension using a cell counter.</li>
	</ol>
	</li>
	<li>Prepare the cells for flow cytometry.
	<ol>
		<li>Incubate the cell suspensions (105 cells in 200 &#181;L PBS supplemented with 3% FBS) with fluorophore-conjugated (FITC or APC) primary antibodies (TRA-1-85-, CD146, CD31) concentration = 1:40) at 4 &#176;C for 30 min, protected from light. 
		NOTE: Flow cytometry for MSCs was optimized by Hong et al., 201328. A minimum of 10,000 cells is required for accurate readings.</li>
		<li>Following the 30 min incubation, resuspend the cells in 2 mL of PBS supplemented with 3% FBS and centrifuge (400 x g, 5 min).</li>
		<li>Keep cells at 4 &#176;C protected from light until analyzing by flow cytometry (at least 1 x 104 events) within 1 h. For the gating strategy, see Supplementary Figure 1. Sort human MSCs using anti-TRA-1-85 (APC) (concentration: 1:40 and in 0.5% FBS/PBS) and sort cells into 350 &#181;L of cell lysis buffer for qPCR analysis. 
		NOTE: Lysed cells can be stored at -80 &#176;C for up to 3 months for future qPCR analysis.</li>
	</ol>
	</li>
	<li>Prepare the cells for qPCR analysis.
	<ol>
		<li>Isolate the RNA from lysed cells using column-based RNA isolation kits and determine the RNA concentration and quality.</li>
		<li>Prepare cDNA from up to 5 &#181;g of RNA per 100 &#181;L reverse transcriptase reaction. Perform a pre-amplification step if the RNA yield is low (&#60; 10 ng/&#181;L). Use of less than 100 ng of RNA will result in a high rate of false negatives. 
		NOTE: The cDNA templates can be stored at -20 &#176;C for further analysis for up to 4 months.</li>
		<li>Perform the qPCR using angiogenesis expression profiler arrays to detect changes in gene expression. Use 5 ng of cDNA per reaction (40 cycles, 60 &#176;C annealing/extending temperature). Express fold change of expression compared to undifferentiated MSC derived cDNA samples. Run the appropriate negative controls (aortic ring without human MSCs).</li>
	</ol>
	</li>
</ol>
6. Network Quantification Following Aortic Ring Assay/MSC Co-cultures
<ol>
	<li>Download imaging software such as ImageJ along with Angiogenesis Analyzer plugin31.</li>
	<li>Import the endothelial network images taken and open using the software.</li>
	<li>Convert the image scales based on the specifications of the microscope used.</li>
	<li>Use a straight-line tool to measure the radial network growth.</li>
	<li>Count the endothelial network loops using the cell counter tool (loops with at least 4 sides should be quantified).</li>
	<li>For additional quantification of network properties, use the Angiogenesis Analyzer plugin or other similar plugins to analyze master segments, total segment length, total network areas, and number of junctions. Use the blurred mask tool to blur aortic ring tissue and empty spaces to avoid false positive calculations.</li>
	<li>Transfer the values to a statistics program and graph endothelial network properties following the aortic ring assay/MSC co-cultures.</li>
</ol>

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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+mesenchymal+stem+cells+for+tissue+engineering+versus+regenerative+medicine.">Adult mesenchymal stem cells for tissue engineering versus regenerative medicine.</a> J Cell Physiol. 213 (2), 341-347 (2007).</li><li>Caplan, A. I., Dennis, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+as+trophic+mediators.">Mesenchymal stem cells as trophic mediators.</a> J Cell Biochem. 98 (5), 1076-1084 (2006).</li><li>Keating, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cells:+new+directions.">Mesenchymal stromal cells: new directions.</a> Cell Stem Cell. 10 (6), 709-716 (2012).</li><li>Kilroy, G. E., 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=Cytokine+profile+of+human+adipose-derived+stem+cells:+expression+of+angiogenic,+hematopoietic,+and+pro-inflammatory+factors.">Cytokine profile of human adipose-derived stem cells: expression of angiogenic, hematopoietic, and pro-inflammatory factors.</a> J Cell Physiol. 212 (3), 702-709 (2007).</li><li>Tang, Y. L., 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=Paracrine+action+enhances+the+effects+of+autologous+mesenchymal+stem+cell+transplantation+on+vascular+regeneration+in+rat+model+of+myocardial+infarction.">Paracrine action enhances the effects of autologous mesenchymal stem cell transplantation on vascular regeneration in rat model of myocardial infarction.</a> Ann Thorac Surg. 80 (1), 229-236 (2005).</li><li>Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All+MSCs+are+pericytes?.">All MSCs are pericytes?.</a> Cell Stem Cell. 3 (3), 229-230 (2008).</li><li>Hong, S. H., 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=Ontogeny+of+human+umbilical+cord+perivascular+cells:+molecular+and+fate+potential+changes+during+gestation.">Ontogeny of human umbilical cord perivascular cells: molecular and fate potential changes during gestation.</a> Stem Cells Dev. 22 (17), 2425-2439 (2013).</li><li>Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M., Altman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+bioscience+research+reporting:+The+ARRIVE+guidelines+for+reporting+animal+research.">Improving bioscience research reporting: The ARRIVE guidelines for reporting animal research.</a> J Pharmacol Pharmacother. 1 (2), 94-99 (2010).</li><li>Sarugaser, R., Ennis, J., Stanford, W. L., Davies, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+propagation,+and+characterization+of+human+umbilical+cord+perivascular+cells+(HUCPVCs).">Isolation, propagation, and characterization of human umbilical cord perivascular cells (HUCPVCs).</a> Methods Mol Biol. 482, 269-279 (2009).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9 (7), 671-675 (2012).</li><li>Gelati, M., Aplin, A. C., Fogel, E., Smith, K. D., Nicosia, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+angiogenic+response+of+the+aorta+to+injury+and+inflammatory+cytokines+requires+macrophages.">The angiogenic response of the aorta to injury and inflammatory cytokines requires macrophages.</a> J Immunol. 181 (8), 5711-5719 (2008).</li></ol>

Dr. Clifford L. Librach is joint holder of the patent: Methods of isolation and use of cells derived from first trimester umbilical cord tissue. Granted in Canada and Australia.

There are several critical stages in setting up a successful aortic ring assay MSC co-culture experiment. First, the most important steps when isolating and sectioning aorta are: 1) obtaining exclusively the thoracic segment of aorta; 2) carefully removing branching blood vessels, connective and adipose tissue and; 3) cutting even sections of the aorta (~1 mm) to limit variability between each assay. Second, the successful embedding of aortic rings into BME is critical for this assay. If the BME is not completely polymer...

The complex process of angiogenesis improves and maintains tissue perfusion by promoting new blood vessel development from pre-existing vasculature1. It is a tightly regulated, balanced process by pro-angiogenic and anti-angiogenic factors. Any deficiency in this system may lead to insufficient vessel maintenance or growth, causing severe ischemic diseases including myocardial disease, stroke, and neurodegenerative disorders. However, exaggerated vascular development is characteristic for conditions including cancer and inflammatory disorders2.
Developing therapies that aim to control angiogenesis to achieve favorable tissue regeneration is of key importance. Despite extensive preclinical and clinical investigations, attempts to stimulate angiogenesis using pro-angiogenic factors and microRNAs have failed to achieve desired outcomes3,4,5. Possible reasons for the transient effects include: limited longevity of angiogenic proteins and nucleic acids, and the finite number of targeted growth factors6,7. Although soluble angiogenic factors are essential for initiating angiogenesis, the maintenance and functionality of vasculature depend on supporting cell types including pericytes and smooth muscle cells8. The field of pro-angiogenic therapies is now exploring potential stem cell and progenitor cell sources that may provide angiogenic factors locally, while physically supporting newly developed vasculature, self-renewing or even differentiating into endothelial-like cells9,10. Finding the optimal angiogenic cell types with the capability to fulfill these functional requirements holds a great promise for ischemic tissue regeneration.
In order to successfully translate potential cell-based therapies into clinical trials, pre-clinical studies need to demonstrate their efficacy and highlight the underlying angiogenic mechanisms. Despite the high number of established angiogenesis assays, the field lacks a &#34;gold-standard&#34; in vitro assay that could reliably evaluate the efficacy of potential candidate cell types11,12,13. Most in vitro angiogenesis assays (including the endothelial proliferation, migration and tube formation assays) typically assess the effects of cells or compounds on endothelial cells&#39; phenotypical changes or differentiation into tubular and network structures14,15. While these features are critical for angiogenesis, a &#34;translatable&#34; assay should also evaluate: 1) the augmentation or replacement of the supporting cell types including pericytes or smooth muscle cells, 2) the processing of ECM and/or basement membrane, and 3) the efficiency to promote the formation of functional microvasculature. In vivo angiogenesis models, including the corneal assay and Matrigel plug assay, recapitulate the unique in vivo microenvironment but are challenged by the difficulty of tracking administered cells to observe physical interactions. Furthermore, in in vivo models, xeno-immune rejection can occur while testing potential allogeneic cell therapy candidates16. Ex vivo angiogenesis models, particularly the aortic ring assay can provide: 1) easy observation and quantification of tubular structures, 2) accessory supportive cells, 3) ECM from host and artificial supplies, 4) exclusion of inflammatory components, and 5) quick and inexpensive setup17,18. Typically, the aortic ring assay can test the angiogenic potential of small secretory proteins, pharmacological agents, and transgenic rodent models19,20,21.
MSCs are promising candidates for vascular regeneration primarily through their paracrine-mediated effects22,23,24. MSCs have been shown to secrete key angiogenic factors including Vascular Endothelial Growth Factor (VEGF), Hepatocyte Growth Factor (HGF), Insulin-like Growth Factor-1 (IGF-1), basic Fibroblast Growth Factor (bFGF), and angiopoeitin-1 (Ang-1)25,26. MSCs can also detect and home to ischemic or inflamed tissues, however, the exact mechanisms are still under investigation. Increasingly, the literature supports the hypothesis that most MSCs arise from perivascular cells, co-express pericyte markers, and can behave like pericytes27. HUCPVCs are a young source of MSCs derived from the perivascular region of the human umbilical cord. They represent a population of MSCs with pericyte-like properties and have been characterized from both FTM and term umbilical cords. FTM HUCPVCs demonstrate a high expression of pericyte markers including CD146 and NG2, high proliferative and multilineage potential, immune-privileged properties, and display a robust paracrine profile28. FTM HUCPVCs are an ideal candidate cell type to promote regeneration of injured tissue through the promotion of new vasculature via their pericyte-like properties.
To test the angiogenic potential and pericyte-like properties of human MSCs, a very limited number of angiogenesis assays are available where positive angiotropic migration (hereafter referred to as &#34;homing&#34;), ECM processing, and development of physical interactions between cell types can be investigated, while obtaining quantitative data on microvasculature development.
Hereby we present a protocol that describes a novel application of the aortic ring assay. Human MSCs were co-cultured with developing rat-derived aortic endothelial networks to assess their contribution to tube formation, maturation, and homeostasis. This version of the aortic ring assay assesses the ability and potency of cell therapy candidates to home to sites of angiogenesis, perform and mediate ECM processing, and contribute to endothelial tubular development through establishing pericyte-like physical interactions. In addition to quantifying the net effect of MSCs on in vitro endothelial network formation and observing intercellular interactions, we also optimized a protocol to isolate MSCs from co-cultures. By performing flow cytometry and qPCR, it is possible to characterize changes in MSC phenotype and gene expression following co-culture. As model cell types, we compared ontogenetically early (prenatal) and late (adult) sources of human MSCs: FTM HUCPVCs and human bone marrow-derived MSCs (BMSC), respectively, in the aortic ring assay. We propose that the aortic ring assay can be used to study the angiogenic potential of any physically supporting cell type when under investigation for angiogenic regenerative applications.

<table><tbody><tr><td>Alpha-MEM</td><td>Gibco</td><td>12571071</td><td>For FTM HUCPVC and BMSC culture media.</td></tr><tr><td>APC-conjugated anti human TRA-1-85</td><td>R&amp;amp;D Systems</td><td>FAB3195A</td><td>Human-specific cell marker for flow cytometry and cell sorting</td></tr><tr><td>Basal membrane extract (BME) (Matrigel)</td><td>Corning</td><td>354234</td><td>For aorta embedding</td></tr><tr><td>Bullet-kit</td><td>Lonza</td><td>CC-3162</td><td>Includes: Gentamicin/Amphotericin-B&lt;br/&gt; (GA)human Epidermal&lt;br/&gt; Growth Factor (hEGF); Vascular&lt;br/&gt; Endothelial Growth Factor (VEGF); R3-&lt;br/&gt; Insulin-like Growth Factor-1 (R3-IGF-1);&lt;br/&gt; Ascorbic Acid; Hydrocortisone;&lt;br/&gt; human Fibroblast Growth Factor-Beta (hFGF-&amp;beta;); Heparin; Fetal Bovine Serum&lt;br/&gt; (FBS). Required to prepare EGM</td></tr><tr><td>CellTracker Green CMFDA Dye</td><td>Thermo-fisher</td><td>C2925</td><td>For staining MSCs, green is picked up optimally by MSCs</td></tr><tr><td>CKX53 Culture Microscope</td><td>Olympus</td><td></td><td>For bright-field imaging of endothelial network development</td></tr><tr><td>Countess automated cell counter</td><td>Invitrogen</td><td>C10227</td><td>Cell counting for MSC culture, flow cytometry and qPCR</td></tr><tr><td>Dispase</td><td>StemCell technologies</td><td>7923</td><td>For dissociating aortic ring-MSC co-cultures (pre-warm at 37 &amp;deg;C)</td></tr><tr><td>Disposable sterile scalpels</td><td>VWR</td><td>21909-654</td><td>For sectioning aorta</td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline</td><td>Sigma-Aldrich</td><td>D8537</td><td>PBS. 1X, Without calcium chloride and magnesium chloride</td></tr><tr><td>Endothelial basal media (EBM)</td><td>Lonza</td><td>CC-3156</td><td>Basal media required for culturing aortic ring assay-MSC co-cultures (warm at 37 &amp;deg;C before use). Required for EGM and EBM-FBS</td></tr><tr><td>Ethanol, 70%, Biotechnology Grade</td><td>VWR</td><td>97064-768</td><td>To sterilize surfaces</td></tr><tr><td>EVOS</td><td>Life Technologies</td><td></td><td>In-house fluorescent microscope to track MSC migration and integration</td></tr><tr><td>Fetal bovine serum (FBS) (Hyclone)</td><td>GE Healthcare</td><td>SH3039603</td><td>Serum component of cell culture medium</td></tr><tr><td>FITC-conjugated anti-CD31 antibody</td><td>BD</td><td>558068</td><td>Human endothelial marker for flow cytometry</td></tr><tr><td>FITC-conjugated anti-CD146antibody</td><td>BD</td><td>560846</td><td>Human pericyte marker for flow cytometry</td></tr><tr><td>Forceps</td><td>Almedic</td><td>7727-A10-704</td><td>For handing rat tissue. Can use any similar forceps</td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Life Technologies</td><td>14175-094</td><td>1X Without calcium chloride and magnesium chloride</td></tr><tr><td>HERAcell 150i CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Thermo Fisher Scientific</td><td>51026410</td><td>For incubating cells</td></tr><tr><td>Human Angiogenesis RT2 profiler PCR array</td><td>Qiagen</td><td>PAHS-024Z</td><td>Human specific and includes primers for 84 genes involved in angiogenesis. Each well is 1 primer reaction</td></tr><tr><td>ImageJ</td><td></td><td></td><td>Open source image processing software. Require Angiogenesis analyzer plugin</td></tr><tr><td>LSR II</td><td>BD</td><td></td><td>UHN SickKids FC Facility. For flow cytometry.</td></tr><tr><td>MoFlo Astrios</td><td>Beckman Coulter</td><td></td><td>UHN SickKids FC Facility. For cell sorting.</td></tr><tr><td>Penicillin/streptomycin</td><td>Gibco</td><td>15140122</td><td>Antibiotic component to buffers and cell culture medium</td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>74104</td><td>For RNA purification. Includes cell lysis buffer</td></tr><tr><td>RT&lt;sup&gt;2&lt;/sup&gt;Easy First Strand Kit</td><td>Qiagen</td><td>330421</td><td>For preparation of cDNA for qPCR</td></tr><tr><td>RT&lt;sup&gt;2&lt;/sup&gt;PreAMP cDNA Synthesis Kit</td><td>Qiagen</td><td>330451</td><td>Pre-amplification of cDNA if low-yield RNA</td></tr><tr><td>Surgical scissors</td><td>Fine Science Tools</td><td>14059-11</td><td>For cutting skin, muscle and aorta</td></tr><tr><td>Sterile gauze</td><td>VWR</td><td>3084</td><td>To dampen and sterilize chest fur</td></tr><tr><td>TrypleE</td><td>Thermo Fisher Scientific</td><td>12605036</td><td>MSC dissociation enzyme pre-warm at 37 &amp;deg;C</td></tr><tr><td>0.2 &amp;mu;m pore filtration unit</td><td>Thermo Fisher Scientific</td><td>566-0020</td><td>To sterilize tissue culture media</td></tr><tr><td>0.25% Trypsin/EDTA</td><td>Gibco</td><td>25200056</td><td>For cell dissociation, pre-warm at 37 &amp;deg;C</td></tr><tr><td>10 cm tissue culture dishes</td><td>Corning</td><td>25382-428</td><td>For cleaning and sectioning aorta and MSC cell culture</td></tr><tr><td>12 well-cell culture plates</td><td>Corning-Sigma Aldrich</td><td>CLS3513</td><td>For setting up aortic ring assay-MSC co-cultures</td></tr><tr><td>15 mL tube</td><td>BD Falcon</td><td>352096</td><td>For general tissue culture procedures</td></tr><tr><td>70 &amp;mu;m cell strainer</td><td>Fisherbrand</td><td>22363548</td><td>To ensure a single cell suspension before flow cytometry or sorting</td></tr></tbody></table>

the aortic ring co-culture assay: a convenient tool to assess the angiogenic potential of mesenchymal stromal cells in vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The schematic work-flow for establishing the aortic ring/MSC co-culture assay is demonstrated in Figure 1. The main steps include: rat aorta isolation, sectioning and embedding of the aortic rings, monitoring the endothelial sprouting and network development, and finally labelling and administering the MSCs. The timeline of the endothelial network analysis outlines the window for the analyses feasible for each period of co-culturing: day 1, 5 &#38;&#160;7. Ad...

Watch this Scientific Journal Video about The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro at JoVE.com

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

the-aortic-ring-co-culture-assay-convenient-tool-to-assess-angiogenic

Research

JoVE Journal

Developmental Biology

11.0K Views.  Create Fertility Centre. Hier presenteren we een nieuwe toepassing van de bepaling van de aorta ring waar prelabelled mesenchymale cellen zijn mede gekweekte met rat aorta afkomstige endothelial netwerken. Deze nieuwe methode kunt visualisatie van mesenchymale stromale cellen (MSCs) homing en integratie met endothelial netwerken, kwantificering van netwerkeigenschappen en evaluatie van MSC immunophenotypes en gen-expressie.

Video: The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

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 ...

Biology

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

Bioengineering

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including embryogenesis and development, wound healing, tumor growth and metastasis, and ocular and cardiovascular diseases. Effective in vitro models that recapitulate the biology of angiogenesis are needed to appropriately study this process and identify mechanisms of regulation that can be ultimately targeted for novel therapeutic strategies. The bead angiogenesis assay has been previously demonstrated to recapitulate the multiple stages of endothelial sprouting in vitro. However, a limitation of this assay is a lack of endothelial &#8211; mural cell interactions, which are key to the molecular and phenotypic regulation of endothelial cell function in vivo. The protocol given here presents a methodology for the incorporation of mural cells into the bead angiogenesis assay and demonstrates a tight association of endothelial cells and pericytes during sprouting in vitro. The protocol also details a methodology for effective silencing of target genes using siRNA in endothelial cells for mechanistic studies. Altogether, this protocol provides an in vitro assay that more appropriately models the diverse cell types involved in sprouting angiogenesis, and provides a more physiologically-relevant platform for therapeutic assessment and novel discovery of mechanisms of angiogenesis regulation.

This protocol presents a novel in vitro bead assay that more appropriately models the process of in vivo sprouting angiogenesis by incorporating pericytes. This modification enables the bead assay to more faithfully recapitulate the heterotypic cellular interactions between endothelial cells and mural cells that are critical for angiogenesis.

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including ...

Aortic Ring Assay

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis assays involve the study of individual endothelial cells in culture conditions (1). The aortic ring assay is an angiogenesis model that is based on organ culture. In this assay, angiogenic vessels grow from a segment of the aorta (modified from (2)). Briefly, mouse thoracic aorta is excised, the fat layer and adventitia are removed, and rings approximately 1 mm in length are prepared. Individual rings are then embedded in a small solid dome of basement matrix extract (BME), cast inside individual wells of a 48-well plate. Angiogenic factors and inhibitors of angiogenesis can be directly added to the rings, and a mixed co-culture of aortic rings and other cell types can be employed for the study of paracrine angiogenic effects. Sprouting is observed by inspection under a stereomicroscope over a period of 6-12 days. Due to the large variation caused by the irregularities in the aortic segments, experimentation in 6-plicates is strongly advised. Neovessel outgrowth is monitored throughout the experiment and imaged using phase microscopy, and supernatants are collected for measurement of relevant angiogenic and anti-angiogenic factors, cell death markers and nitrite.

Angiogenesis, the sprouting of blood vessels from pre-existing vasculature, is associated with both natural and pathological processes. Here we demonstrate an aortic ring assay that allows angiogenic potentiators and inhibitors to be directly added to aortic rings in culture. Sprouting and neovessel outgrowth can be determined by inspecting the aortic rings over a period of 6-12 days.

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis ...

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 ...

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

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

3D Co-Culture Assay: Vascularized Osteogenic Niches for Cancer Drug Screening

Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models recapitulating bone- and bone marrow-specific functions, including vascularization, that are compatible with drug screening are highly desirable. Such models can bridge the gap between simplistic, structurally irrelevant two-dimensional (2D) in vitro models and the more expensive, ethically challenging in vivo models. This article describes a controllable three-dimensional (3D) co-culture assay based on engineered poly(ethylene glycol) (PEG) matrices for the generation of vascularized, osteogenic bone-marrow niches. The PEG matrix design allows the development of 3D cell cultures through a simple cell seeding step requiring no encapsulation, thus enabling the development of complex co-culture systems. Furthermore, the matrices are transparent and pre-cast onto glass-bottom 96-well imaging plates, rendering the system suitable for microscopy. For the assay described here, human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are cultured first until a sufficiently developed 3D cell network is formed. Subsequently, GFP-expressing human umbilical vein endothelial cells (HUVECs) are added. The culture development is followed by bright-field and fluorescence microscopy. The presence of the hBM-MSC network supports the formation of vascular-like structures that otherwise&#160;would not form and that remain stable for at least 7 days. The extent of vascular-like network formation can easily be quantified. This model can be tuned toward an osteogenic bone-marrow niche by supplementing the culture medium with bone morphogenetic protein 2 (BMP-2), which promotes the osteogenic differentiation of the hBM-MSCs, as assessed by increased alkaline phosphatase (ALP) activity at day 4 and day 7 of co-culture. This cellular model can be used as a platform for culturing various cancer cells and studying how they interact with bone- and bone marrow-specific vascular niches. Moreover, it is suitable for automation and high-content analyses, meaning it would enable cancer drug screening under highly reproducible culture conditions.

Author Spotlight: Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(Ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate  

An in vitro model of the bone marrow vascular niche is established by seeding mesenchymal and endothelial cells onto pre-cast 3D PEG hydrogels. The endothelial networks, ECM components, and ALP activity of the niches vary depending on the growth factor used. The platform can be used for advanced cancer models.

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

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