We thank Drs. Victoria Bautch and Joshua Boucher for helpful discussions and advice on optimizing standard bead sprouting assay conditions and a sprouting assay staining protocol.&#160;S.H.A. was supported in part by a grant from the National Institute of General Medical Sciences under award 5T32 GM007092.

Day 1:
1. Transient Transfection of Endothelial Cells
<ol>
	<li>If desired, perform a transient transfection of Human Umbilical Vein Endothelial Cells (HUVEC) using gene-regulatory oligonucleotides (e.g. micro-RNAs or small interfering RNA (siRNA)) and the appropriate lipofectamine reagent according to manufacturer&#39;s instructions. 
	NOTE: This protocol has had great success performing reverse transfections with custom siRNA sequences and a commercial transfection reagent (See Materials Table). The authors have not tested other transfection protocols and reagents with this assay. The steps for the reverse transfection using the reagents listed are as follows:</li>
	<li>Reconstitute lyophilized siRNAs or microRNAs at a concentration of 20 &#181;M in nuclease free water.</li>
	<li>Make transfection solutions at the following ratio: 1 mL of any serum free media with 9 &#181;L of siRNA or microRNA.</li>
	<li>Agitate the solution and allow it to incubate for 5 min at room temperature.</li>
	<li>Add 18 &#181;L of transfection reagent to the solution and agitate.</li>
	<li>Allow the solution to incubate at room temperature for 20-40 min, agitating the solution every 10 to 15 min for the duration of the incubation.</li>
	<li>While the transfection solution is incubating, detach HUVEC using a cell detachment solution (e.g., Accutase). For a T175 flask, wash the flask with 10 mL of Phosphate Buffered Saline (PBS), and then add 5 mL of cell detachment solution and incubate the flask at 37 &#176;C for 10 min.</li>
	<li>Add 5 mL of complete media to the T175 flask, remove the cell suspension from the flask and centrifuge at 1200 rpm for 3 min.</li>
	<li>Remove the supernatant using a vacuum aspirator and resuspend the cell pellet at a density of 1 X 106 cells per mL in EGM2. 
	NOTE: This protocol has had great success using Endothelial Cell Growth Medium (EGM2) containing the standard growth factor bullet kit as well as an additional 10% FBS.</li>
	<li>Once the transfection solution has finished incubating, plate 1 mL of solution in a 10-cm plate, and add 1 mL of HUVEC suspension on top.</li>
	<li>Add an additional 7 mL of complete media to bring the final volume in the 10-cm plate to 9 mL.</li>
	<li>Make up at least two 10 cm plates worth of cells for each transfection condition to ensure that there are enough cells available for later steps in the assay. 
	NOTE: These conditions result in a final working concentration of gene regulatory oligonucleotide of 20 nM. The authors have found these conditions to result in a 60-80% knockdown of gene expression for their targets of interest. Other researchers may need to optimize transfection conditions according to their experimental needs.</li>
	<li>Exchange the media 4-6 h post addition of siRNA complexes with fresh complete media. 
	NOTE: This protocol was developed using passage 1 - 2 HUVEC.</li>
</ol>
Day 2
2. Coating of Microcarrier Beads with Endothelial Cells
<ol>
	<li>Reconstitute microcarrier beads (e.g., Cytodex 3) at a concentration of 60,000 beads/mL in PBS and autoclave. 
	NOTE: microcarrier beads come at a concentration of approximately 3 x 106 beads per gram. Reconstitute the beads at a ratio of 20 mg of beads per mL of PBS to get a bead density of approximately 60,000 beads/mL.</li>
	<li>Aliquot 1 mL of complete media into the desired number of round-bottomed Fluorescence Activated Cell Sorting (FACS) tubes.</li>
	<li>Pipette 20 &#181;L of microcarrier bead suspension into each tube (take care to ensure that the bead solution is well resuspended prior to pipetting). 
	NOTE: There will likely be dramatic variability in the final working concentration of beads present in a given microcarrier bead suspension. Beads can easily adhere to the sides of plastic and glass containers which can greatly alter bead concentration in the solution. The most appropriate volume of bead solution added to the FACS tube for each batch of microcarrier bead suspension will need to be determined empirically by the investigator. The numbers presented here are meant to serve more as a starting point. Ideally, conditions should be optimized so there are 10-20 beads embedded within the fibrin gel per well in the 24 well plate at the end of the assay. See further discussion in the discussion section below regarding optimization of beads numbers for the assay.</li>
	<li>Detach transfected HUVEC 24 h post transfection using the cell detachment solution as follows:
	<ol>
		<li>Wash each 10-cm plate of cells with 5 mL of PBS, and then add 2 mL of the cell detachment solution and incubate the cells at 37 &#176;C for 10 min.</li>
		<li>Resuspend the cells at a concentration of 1 X 106 cells/mL in complete media.</li>
	</ol>
	</li>
	<li>Add 1 mL of cell suspension (i.e. 1 X 106 HUVEC) to each FACS tube containing beads.</li>
	<li>Gently agitate the FACS tubes every 20 min for the duration of 4 h at 37 &#176;C (if proceeding to step 3.4) or 2.5 to 3 h (if proceeding to step 3.2). 
	NOTE: This duration and frequency of agitation has been empirically determined by previous researchers who have optimized the basic bead sprouting assay.4&#160;&#160;Other frequencies of agitation and durations of the bead coating step have not been tested by the authors.</li>
</ol>
3. Sequential Coating of Pericytes on HUVEC-Coated Microcarrier Beads
<ol>
	<li>Begin detaching 10T1/2 cells (pericytes) using the cell detachment solution (trypsin) as follows:
	<ol>
		<li>Wash a T175 flask with 10 mL of PBS, then add 5 mL of trypsin and incubate at 37 &#176;C for 10 min.</li>
		<li>Resuspend 10T1/2 at a concentration of 2 X 105 cells/0.5 mL in complete media. 
		NOTE: Cells were purchased from the American Type Culture Collection (ATCC). 
		NOTE: Culture 10T1/2 cells in Minimum Essential Medium (MEM) supplemented with 10% FBS and 1% Penicillin Streptomycin.</li>
	</ol>
	</li>
	<li>After 2.5 to 3 h of agitating the bead + HUVEC solution, wait 5 min to allow the beads and free-floating HUVEC to settle to the bottom of the tube.</li>
	<li>Remove 0.5 mL of complete media from each tube using a P1000 pipette and add 2 x 105 10T1/2 in complete media in a volume of 0.5 mL to each FACs tube. Agitate the cell and bead solution and then continue to incubate at 37 &#176;C. Continue to agitate every 20 min until the beads have been agitated for a total of 4 h. 
	NOTE: The ratio of 5 HUVEC: 1 pericyte on the beads is essential to maintain appropriate pericyte coverage of the vessels. 
	NOTE: The following alternative steps can be followed instead at this point in the protocol:</li>
	<li>Agitate beads and HUVEC only for 4 h.</li>
	<li>Once the 4 h of HUVEC coating of the microcarrier beads is complete, agitate the tubes one last time, wait 30-60 s, then immediately remove 1.5-1.75 mL of solution from the FACS tube. 
	NOTE: This should allow enough time for most of the beads to settle while many free-floating HUVEC in the media will be removed.
	<ol>
		<li>Add 2 X 105 10T1/2 in complete media and bring the volume of the FACS tube up to 2 mL.</li>
		<li>Gently agitate the FACS tubes every 20 min for an additional hour.</li>
	</ol>
	</li>
	<li>Once the agitating is complete, gently agitate the tubes one last time and immediately remove the entire solution (2 mL), and add it to a 6-cm plate. Add an additional 3 mL of complete media to each plate and place the plates in the 37 &#176;C incubator overnight.</li>
</ol>
Day 3
4. Preparation of Fibrin Gel Solutions
<ol>
	<li>Prepare working solutions of aprotinin at a concentration of 1 mg/mL in PBS and thrombin at 50 units/mL. 
	NOTE: A large stock of each of these solutions can be made and stored at 4 &#176;C for at least a year.</li>
	<li>Make a fresh solution of fibrinogen at a concentration of 2 mg/mL. Make enough solution to have 2.5 mL of solution per agitated FACS tube.
	<ol>
		<li>Warm the solution in a 37 &#176;C water bath for 10 min to allow all of the fibrinogen to go into solution.</li>
		<li>Add 37.5 &#181;L of aprotinin solution per 1 mL of fibrinogen solution.</li>
		<li>Sterile-filter the solution and set aside at room temperature.</li>
	</ol>
	</li>
</ol>
5. Preparing Beads for Gel Implantation
<ol>
	<li>Examine the dishes containing the microcarrier-coated beads under the microscope using the 20X objective to ensure that all beads have been sufficiently coated by endothelial cells.</li>
	<li>Vigorously pipette the plated solution of cells to detach them from the 6-cm plate and place the solution into a&#160;tube. Wash the plate 2X additional times with complete media and transfer to the tube to collect all residual beads that may be adhered to the plate.
	<ol>
		<li>Avoid introducing air bubbles and use a p1000 pipette to minimize shear stress on the endothelial cell-coated beads.</li>
		<li>If assessing siRNA knockdown efficiency in the endothelial cells is needed, in step 2, be sure to have a plate of HUVEC-only coated beads. Then during this step (5.2), detach the beads and collect the residual HUVEC attached to the plate for RNA isolation and qPCR analysis.</li>
	</ol>
	</li>
	<li>Wait 3 min to allow the beads in the solution to settle to the bottom of the tube.</li>
	<li>Remove as much of the supernatant as possible without disturbing the bead pellet using a p1000 pipette tip (do not use a vacuum aspirator).</li>
	<li>Add 1 mL of complete media to each tube and resuspend the beads.</li>
	<li>Wait 30-60 s to allow the beads to settle to the bottom. Then remove as much of the supernatant as possible using a P1000 pipette without disturbing the bead pellet. Do not use a vacuum aspirator as the likelihood of accidentally removing the bead pellet is greatly increased.</li>
	<li>Repeat Steps 5.5 and 5.6 two additional times. The goal is to remove as much of the free-floating HUVEC that may have been detached from the 6-cm plate as possible, while not removing too many HUVEC-coated beads, which should settle in the FACS tube more quickly than free-floating cells.</li>
	<li>Add 1 mL of complete media to the beads.</li>
</ol>
6. Embedding Coated Beads in a Fibrin Gel
<ol>
	<li>Remove as much media as possible without disturbing the bead pellet in each FACS tube using a P1000 pipette.</li>
	<li>Resuspend the beads in 2.5 mL of fibrinogen solution.</li>
	<li>Pipette 13 &#181;L of thrombin solution into each desired well of a 24-well glass-bottomed plate. Plating in a glass-bottomed plate is essential for enabling optimal imaging of the sprouts. 
	NOTE: Do not leave thrombin sitting in a well for more than 5-10 min before the next step.</li>
	<li>Add 0.5 mL of bead/fibrinogen solution to each well.
	<ol>
		<li>Be sure to resuspend the bead solution well prior to pipetting the solution each time.</li>
		<li>Take caution to carefully pipette directly into the thrombin already present in the well. Slowly pipette up and down 2-3 times to thoroughly mix the solution, taking caution to not introduce any air bubbles.</li>
		<li>Be sure to change the pipette tip between wells.</li>
		<li>Take caution to not move or disturb the plate at any point during initial fibrin clotting, as any movement may disrupt the gel formation.</li>
	</ol>
	</li>
	<li>Leave the plate sitting in the hood for 30 min at room temperature.</li>
	<li>Carefully transfer the plate to a 37 &#176;C incubator for an additional 1.5-2 h to allow the gel to fully solidify</li>
</ol>
7. Plating Fibroblasts on Top of the Fibrin Gel
<ol>
	<li>During the last 30 min of the gel solidifying, detach Normal Human Lung Fibroblasts (NHLF) as follows:
	<ol>
		<li>Wash a T175 flask of cells with 10 mL of PBS, then add 5 mL of cell detachment solution and incubate at 37 &#176;C for 10 min.</li>
		<li>Resuspend NHLF at a concentration of 20,000 cells/mL in complete media. 
		NOTE: NHLF cells can be cultured with Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% Penicillin Streptomycin.</li>
	</ol>
	</li>
	<li>Plate 1 mL of the NHLF cell suspension onto the gel of each well and place back in the incubator.</li>
	<li>Change complete media every other day for the duration of the assay.</li>
</ol>
8. Observe sprouting over the course of 2-7 days following bead implantation in the fibrin gel. The length of time required for sprouting will depend on the passage and lot number of HUVEC.
9. Fixation of Sprouting Assay
<ol>
	<li>Once sufficient sprouting has occurred to be able to determine a phenotypic difference between treatment groups, wash all wells one time with 2 mL of PBS.</li>
	<li>Add 0.5 mL of cell detachment solution to each well and place the plate in a 37 &#176;C incubator for 5 min.</li>
	<li>Remove the plate from the incubator and tap the sides of the plate. 
	NOTE: The purpose of this step is to remove as many of the NHLF cells growing on top of the fibrin gel as possible to facilitate antibody penetration into the gel as well as imaging of the gel. The total incubation time with cell detachment solution may need to be adjusted to optimize NHLF removal. Researchers are cautioned to not incubate the gel for excessive periods of time as the cell detachment solution may penetrate into the gel and begin disrupting the endothelial structures within.</li>
	<li>Once a sufficient amount of NHLF cells have been removed, quench the cell detachment solution with 1 mL of complete media.</li>
	<li>Aspirate the detached NHLF and media using a vacuum aspirator, and wash the wells once with 1 mL of PBS.</li>
	<li>Add 300 &#181;L of 2% paraformaldehyde solution in PBS to each well and incubate at room temperature for 10 min.</li>
	<li>Remove the 2% paraformaldehyde solution and wash each well 3 times with 1 mL of PBS for 3 min at room temperature. 
	NOTE: Please dispose of 2% paraformaldehyde solution appropriately in accordance with Environmental Health and Safety (EHS) guidelines.
	<ol>
		<li>Add 1 mL of PBS to each well and store at 4 &#176;C until ready to stain or image the sprouts.</li>
	</ol>
	</li>
</ol>
10. Staining of Sprouting Assay
<ol>
	<li>Add 300 &#181;L of 0.5% Triton-X 100 in PBS to each well and incubate for 30 to 45 min at room temperature.</li>
	<li>Using a vacuum aspirator, remove Triton-X100 and wash 3 times in PBS for 5 min each.</li>
	<li>Add 500 &#181;L of blocking solution to each gel and incubate overnight at 4 &#176;C. 
	NOTE: The blocking solution is made up of the following components: 1% bovine serum albumin (BSA), 5% FBS, 0.1% tween-20, and 1% antiserum (use the same species from which your secondary antibodies are derived) in PBS.</li>
	<li>Remove blocking solution from each well and add 300 &#181;L of staining solution containing primary antibody to each well. 
	NOTE: The staining solution is the same as the blocking solution, but without the antiserum present. The authors have had success incubating with primary antibodies at dilutions of 1:200 to 1:400.</li>
	<li>Incubate primary antibodies in staining solution for 24 h at 4 &#176;C.</li>
	<li>Aspirate primary antibody solution and wash 3 times in TBS with 0.5% tween for 20 min each at room temperature with gentle rocking. After the third wash, replace with fresh wash buffer again, and store at 4 &#176;C overnight.</li>
	<li>Incubate with fluorescent secondary antibody diluted 1:1000 in staining solution for 2 h at room temperature.</li>
	<li>Remove secondary antibody staining solution and wash 5 times for 20 min each with TBS containing 0.5% tween at room temperature with gentle rocking.</li>
	<li>Dilute nuclear stain in PBS and incubate overnight at 4 &#176;C. 
	NOTE: This protocol has had great success with Hoechst diluted 1:10000 in PBS. 
	NOTE: There is no need to remove nuclear staining solution prior to imaging.</li>
	<li>Image within a few days of completing the staining to capture optimal fluorescent signal.</li>
</ol>
11. Sprouting Assay Quantification
<ol>
	<li>After the sprouts have been imaged, import the image files into ImageJ and use the Count tool to count the number of sprouts, or the Line tool to measure sprout lengths. 
	NOTE: Sprouts are defined as endothelial protrusions with a length greater than or equal to the diameter of the bead from which they are sprouting.11 Sprout length can be measured as the distance from the point at which the sprout begins protruding off of the bead to the tip of the sprout. Average sprout length per bead and average number of sprouts per bead are useful metrics to assess phenotypic effects of gene silencing in sprouting angiogenesis. The average number of beads containing at least one sprout per treatment group can also be used as a metric to assess the robustness of sprouting.</li>
</ol>

<ol>
	<li>Hanahan, D., 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=Patterns+and+emerging+mechanisms+of+the+angiogenic+switch+during+tumorigenesis.">Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.</a> Cell. 86 (3), 353-364 (1996).</li><li>Semenza, G. L. <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+ischemic+and+neoplastic+disorders.">Angiogenesis in ischemic and neoplastic disorders.</a> Annu Rev Med. 54, 17-28 (2003).</li><li>Zhang, Z. G., Chopp, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurorestorative+therapies+for+stroke:+underlying+mechanisms+and+translation+to+the+clinic.">Neurorestorative therapies for stroke: underlying mechanisms and translation to the clinic.</a> Lancet Neurol. 8 (5), 491-500 (2009).</li><li>Nakatsu, M. N., Hughes, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+three-dimensional+in+vitro+model+for+the+analysis+of+angiogenesis.">An optimized three-dimensional in vitro model for the analysis of angiogenesis.</a> Methods Enzymol. 443, 65-82 (2008).</li><li>Nehls, V., Drenckhahn, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel,+microcarrier-based+in+vitro+assay+for+rapid+and+reliable+quantification+of+three-dimensional+cell+migration+and+angiogenesis.">A novel, microcarrier-based in vitro assay for rapid and reliable quantification of three-dimensional cell migration and angiogenesis.</a> Microvasc Res. 50 (3), 311-322 (1995).</li><li>Sims, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pericyte--a+review.">The pericyte--a review.</a> Tissue Cell. 18 (2), 153-174 (1986).</li><li>Armulik, A., Genove, G., Betsholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes:+developmental,+physiological,+and+pathological+perspectives,+problems,+and+promises.">Pericytes: developmental, physiological, and pathological perspectives, problems, and promises.</a> Dev Cell. 21 (2), 193-215 (2011).</li><li>Tattersall, I. W., 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=In+vitro+modeling+of+endothelial+interaction+with+macrophages+and+pericytes+demonstrates+Notch+signaling+function+in+the+vascular+microenvironment.">In vitro modeling of endothelial interaction with macrophages and pericytes demonstrates Notch signaling function in the vascular microenvironment.</a> Angiogenesis. 19 (2), 201-215 (2016).</li><li>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=Normalization+of+tumor+vasculature:+an+emerging+concept+in+antiangiogenic+therapy.">Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy.</a> Science. 307 (5706), 58-62 (2005).</li><li>Tahergorabi, Z., Khazaei, 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+review+on+angiogenesis+and+its+assays.">A review on angiogenesis and its assays.</a> Iran J Basic Med Sci. 15 (6), 1110-1126 (2012).</li><li>Popson, S. A., 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=Interferon-induced+transmembrane+protein+1+regulates+endothelial+lumen+formation+during+angiogenesis.">Interferon-induced transmembrane protein 1 regulates endothelial lumen formation during angiogenesis.</a> Arterioscler Thromb Vasc Biol. 34 (5), 1011-1019 (2014).</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 (7070), 937-945 (2005).</li><li>Newman, A. C., Nakatsu, M. N., Chou, W., Gershon, P. D., Hughes, C. 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The authors have nothing to disclose.

This protocol presents a method for characterizing the complex stages and heterotypic cellular interactions of sprouting angiogenesis by enabling the researcher to employ genetic and imaging approaches to conduct thorough mechanistic investigations. When performing the assay, it is essential that efficient endothelial coating of the beads takes place during the bead agitation steps. Poor endothelial coating will be made evident, if the beads do not appear to have a golf ball-like rough surface the next morning prior to g...

Angiogenesis is vital to appropriate embryogenesis and wound healing, and it also plays key roles in numerous diseases including cancer progression1 and coronary artery disease.2,3 Having a better understanding of how angiogenesis occurs during normal development, and how it is reactivated in pathologic contexts, is critical for the development of novel, effective therapeutics. Faithful in vitro models that recapitulate the important stages and cell types involved in angiogenesis in vivo are needed to allow researchers to better characterize the molecular mechanisms driving angiogenesis and make novel discoveries in endothelial regulation.
Nakatsu and Hughes have optimized a sprouting bead assay that they have demonstrated undergoes the many known stages of sprouting angiogenesis.4,5 The purpose of the method presented here is to build upon the assay optimized by Nakatsu and Hughes by incorporating perictyes into the assay, so that the paracrine and juxtracrine roles of mural cells in endothelial cell sprouting can be incorporated in novel angiogenesis studies. Pericytes are mural cells that are defined by their role as cells that maintain close physical contact with endothelial cells due to their being embedded in the vascular basement membrane.6 Pericytes and endothelial cells engage in complex cross-talk via signaling pathways including Notch signaling, Ang-Tie2, PDGFR&#946;, TGFR&#946;, and many others.7,8 Mouse models deficient in these signaling pathways demonstrate poor pericyte coverage of developing vasculature in embryogenesis, leading to poor vascular remodeling and dysfunctional vasculature.7 In addition, the role of pericytes in pathologic angiogenesis is important but oftentimes under-appreciated. For example, a unique feature of tumor vasculature is that the vessels are more immature, leaky, and dysfunctional due to poor pericyte coverage.9 It has been proposed that the presence or absence of pericytes dramatically impacts the phenotype of tumor blood vessels and is an important mediator of responses to antiangiogenic and antitumor therapies.9 Thus, including the role of pericytes in in vitro assays is key to more completely capture the important mechanisms of endothelial regulation.
Although there are many in vitro and ex vivo assays currently employed to study angiogenesis, there are shortcomings to consider in each. Some, such as endothelial proliferation and endothelial migration assays, are overly simplified and focus on one endothelial function in an isolated setting on tissue culture plastic.10 Other assays occur in a more 3 dimensional (3D) setting, such as the Matrigel tube formation assay,10 but these assays are still oversimplified and focus more on the ability of endothelial cells to migrate and form de novo vascular structures, as opposed to sprouting from pre-existing vasculature. Furthermore, none of these assays incorporate mural cell types. There are ex vivo models such as the ring aorta assay that do incorporate pericytes present in the host organ, but genetic manipulation of these models is much more challenging due to the necessity of generating knockout or transgenic mouse models of the pathways of interest. The bead sprouting assay is ideal because it models endothelial sprouting, proliferation, migration, and even anastomosis and lumen formation in a 3D matrix.4 The assay faithfully allows for mechanistic assessment of the many different stages of sprouting, while still allowing for direct genetic modification of either the endothelial cells or pericytes in a more controlled setting. The fibrin clots containing the sprouting beads can be easily fixed, stained, and imaged at different stages of sprouting; these sprouts can also be placed in a live imaging chamber to perform real-time imaging of sprouting. The methodology presented here is ideal for studying basic mechanisms of angiogenesis through in depth phenotyping and thorough analysis of the pathways activated during angiogenesis.

<table><tbody><tr><td>Sterile Pipette tips</td><td>VWR</td><td></td><td></td></tr><tr><td>Pipettors</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Complete EGM2 Media Bullet Kit</td><td>Lonza</td><td>CC-3162</td><td>HUVEC Media</td></tr><tr><td>MEM</td><td>Gibco</td><td>11095114</td><td>10T1/2 Media</td></tr><tr><td>DMEM</td><td>Gibco</td><td>11965118</td><td>NHLF Media</td></tr><tr><td>Tissue culture-grade PBS</td><td>Gibco</td><td>14190-144</td><td>Magnesium and calcium free</td></tr><tr><td>Accutase</td><td>Life Technologies</td><td>A1110501</td><td>For lifting HUVEC</td></tr><tr><td>Trypsin</td><td>Life Technologies</td><td>15050065</td><td>For lifting 10T 1/2 and NHLF</td></tr><tr><td>Customs siRNAs</td><td>Sigma</td><td></td><td></td></tr><tr><td>Lipofectamine RNAiMax</td><td>Life Technologies</td><td>13778150</td><td></td></tr><tr><td>HUVEC</td><td>Lonza</td><td>C2517A</td><td></td></tr><tr><td>10T 1/2</td><td>ATCC</td><td></td><td></td></tr><tr><td>NHLF</td><td>ATCC</td><td></td><td></td></tr><tr><td>Cytodex 3 microcarrier beads</td><td>Sigma</td><td>C3275</td><td></td></tr><tr><td>Tissue culture-coated 6 and 10 cm plates</td><td>Corning</td><td></td><td></td></tr><tr><td>Fibrinogen from bovine plasma</td><td>Sigma</td><td>F8630</td><td></td></tr><tr><td>Thrombin</td><td>Sigma</td><td>t9549</td><td></td></tr><tr><td>Aprotinin</td><td>Sigma</td><td>a3428</td><td></td></tr><tr><td>Falcon Round-Bottom Tubes</td><td>Corning</td><td></td><td></td></tr><tr><td>Tissue culture incubator and hood</td><td></td><td></td><td></td></tr><tr><td>24-well glass bottom plates</td><td>MatTek</td><td>P24G1.513F</td><td>Glass-bottom plates are needed only if the sprouts are going to be imaged. If not, tissue culture plastic is also acceptable.</td></tr><tr><td>Sterile Filtration Device</td><td></td><td></td><td></td></tr></tbody></table>

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 allows for a tight association of the two cell types in vitro, and the presence of the pericytes complements the occurrence of sprouting (Figure 1A, B). The protocol also enables effective silencing (e.g. via RNA interference) of a gene of interest in a cell type of interest (such as VEGFA specifically in endothelial cells or PDGFRβ in pericytes)7,12 d...

Watch this Scientific Journal Video about Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay at JoVE.com

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

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 ...

incorporating-pericytes-into-an-endothelial-cell-bead-sprouting-assay

Research

JoVE Journal

Developmental Biology

8.0K Views.  University of North Carolina at Chapel Hill. Dit protocol presenteert een kraal roman in vitro assay dat meer modellen op de juiste wijze het proces van in vivo angiogenese kiemen door pericytes op te nemen. Deze wijziging maakt het mogelijk de bepaling van de kraal te recapituleren meer getrouw de heterotypic cellulaire interacties tussen endotheliale cellen en muurschildering cellen die essentieel zijn voor angiogenese.

Video: Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

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

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.

Endothelial progenitors derived from induced pluripotent stem cells (iPSC-EPs) have the potential to revolutionize cardiovascular disease treatments and to enable the creation of more faithful cardiovascular disease models. Herein, the encapsulation of iPSC-EPs in three-dimensional (3D) collagen microenvironments and a quantitative analysis of these cells&#8217; vasculogenic potential are described.

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...

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 ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Bioengineering

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

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 ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

Medicine

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

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

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

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

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

Study of Angiogenesis Using Scratch Wound Migration and Spheroid Sprouting Assays

Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A detailed understanding of the underlying molecular mechanisms and reliable screening models are essential for targeting diseases effectively and developing new therapeutic options. Several in vitro assays have been developed to model angiogenesis, capitalizing on the opportunities a controlled environment provides to elucidate angiogenic drivers at a molecular level and screen for therapeutic targets.
This study presents workflows for investigating angiogenesis in vitro using human umbilical vein endothelial cells (HUVECs). We detail a scratch wound migration assay utilizing a live cell imaging system measuring endothelial cell migration in a 2D setting and the spheroid sprouting assay assessing endothelial cell sprouting in a 3D setting provided by a collagen matrix. Additionally, we outline strategies for sample preparation to enable further molecular analyses such as transcriptomics, particularly in the 3D setting, including RNA extraction as well as immunocytochemistry. Altogether, this framework offers scientists a reliable and versatile toolset to pursue their scientific inquiries in in vitro angiogenesis assays.

Author Spotlight: Investigating Angiogenesis Through Challenges and Innovations in Assay Development

This study presents the two-dimensional (2D) scratch wound migration assay and the three-dimensional (3D) spheroid sprouting assay, along with their respective downstream analysis methods, including RNA extraction and immunocytochemistry, as suitable assays to study angiogenesis in vitro.

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A ...

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 ...

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

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