Name: 2.5d model for ex vivo mechanical characterization of sprouting angiogenesis in living tissue
Uploaded: 2025-02-28T13:10:00.000+00:00
Duration: 10 min
Description: Sprouting angiogenesis is the formation of new blood vessels from pre-existing vasculature and is of great importance for physiological such as tissue ...

We thank the people from LifeTec for harvesting and transporting the porcine carotid arteries from the local slaughterhouse; Leon Hermans, Pim van den Bersselaar, and Adri&#224; Villacrosa Ribas (TU/e, ICMS) for the fruitful discussions on experimental procedures and mechanical characterization analysis. We gratefully acknowledge support by grants from the European Research Council (771168), the Netherlands Organization for Scientific Research (024.003.013), the Academy of Finland (307133, 316882, 330411 and 337531), and the &#197;bo Akademi University Foundation&#39;s Centers of Excellence in Cellular Mechanostasis (CellMech).

Porcine carotid arteries were used in this protocol. Porcine carotid arteries were harvested from Dutch Landrace hybrid pigs - aged 5-7 months and weight (alive) 80-120 kg - obtained from a local slaughterhouse. The protocols were compliant with the EC regulations 1069/2009 regarding slaughterhouse animal material for diagnosis and research as supervised by the Dutch Government (Dutch Ministry of Agriculture, Nature, and Food Quality) and were approved by the associated legal authorities of animal welfare (Food and Consumer Product Safety Authority). Ethical approval was not required since tissue was harvested from byproducts of already terminated animals. The time between death and tissue transport is 10-25 min, depending on the slaughterhouse.
NOTE: The Table of Materials summarizes details about the materials, equipment, and reagents used in this protocol. Protocols for 2D and 3D samples are described in the Supplementary File 1.
1. Preparation of 2D polyacrylamide (PAA) substrates
<ol>
	<li>In the fume hood, prepare bind-silane solution by mixing 12:1:1 absolute ethanol (for synthesis), acetic acid, and bind-silane at 4286 &#181;L, 357 &#181;L, and 357 &#181;L, respectively. Incubate 120 &#181;L/well bind-silane solution on a glass bottom 12 well plate for 1 h at room temperature. 
	CAUTION: Absolute ethanol is a highly flammable liquid and vapor (H225) and causes serious eye irritation (H319). Acetic acid is a flammable liquid and vapor (H226) and causes severe skin burns and eye damage (H314). Wear personal protective equipment and work in a fume hood.</li>
	<li>In the fume hood, wash the glass-bottom 12-well plate 3x with absolute ethanol (industrial) using a spray bottle. Discard the ethanol.</li>
	<li>Dry the glass bottom 12-well plate using nitrogen gas. If a white glace appears on top of the glass bottom, washing was not sufficient. Rewash the glass bottom 12 well plate.</li>
	<li>Prepare the PAA gel mixture according to the ratios in Table 1.
	<ol>
		<li>Add PBS to a microcentrifuge tube and resuspend 40% acrylamide, 2% bis-acrylamide, and fluorescent markers in the gel mixture. Vortex the solution right before gel preparation.</li>
		<li>As fast as possible, add 10% APS and TEMED to the solution and vortex after the addition of each element. Pipet a droplet of 11.5 &#181;L of gel mixture on the glass bottom of each well and gently place a 13 mm coverslip on top of the droplet.</li>
		<li>Gently tap and swirl the plate to evenly spread the gel mixture under the coverslip. If air bubbles appear, remove them by gently lifting the coverslip. Leave the gels to polymerize for 1 h at room temperature. Check polymerization using the remaining gel mixture in the tube. Polymerized PAA gels in the 12-well plate will display an inner halo. 
		CAUTION: Acrylamide is harmful if swallowed or if inhaled (H302 + H332), causes skin irritation (H315), may cause an allergic skin reaction (H317), causes serious eye irritation (H319), may cause genetic defects (H340), may cause cancer (H350), suspected of damaging fertility (H361f), and causes damage to organs through prolonged or repeated exposure if swallowed (H372). APS may intensify fire (oxidizer, H272), harmful if swallowed (H302), causes skin irritation (H315), may cause an allergic skin reaction (H317), causes serious eye irritation (H319), may cause allergy or asthma symptoms or breathing difficulties if inhaled (H334), and may cause respiratory irritation (H335). TEMED is a highly flammable liquid and vapor (H225), harmful if swallowed (H302), causes severe skin burns and eye damage (H314), and is toxic if inhaled (H331). Wear personal protective equipment.</li>
	</ol>
	</li>
	<li>After polymerization, add PBS to the well. Using tweezers and/or a bent needle, gently lift and remove the coverslip. Wash gels once in PBS.</li>
	<li>To facilitate collagen coating of the PAA gels, gels need to be functionalized using the crosslinker Sulfo-SANPAH. Add 75 &#181;L of 1 mg/mL Sulfo-SANPAH dissolved in ultrapure water to the PAA gel and incubate for 5 min under 365 nm UV light. 
	NOTE: Keep Sulfo-SANPAH protected from light and add ultrapure water right before UV incubation. A discoloration is visible from light red (before UV-light incubation) to dark red (after UV-light incubation). 
	CAUTION: Sulfo-SANPAH causes serious eye irritation (H319).</li>
	<li>In the biosafety cabinet, perform a quick sterile PBS wash of the Sulfo-SANPAH on the gels. Consequently, wash the functionalized PAA gels 2x in sterile PBS for 10 min. 
	NOTE: Starting from this point in the protocol, all steps will be performed under sterile conditions.</li>
	<li>In the biosafety cabinet, prepare a 0.1 mg/mL collagen type IV solution in PBS on ice. Pipet 50 &#181;L of 0.1 mg/mL collagen type IV droplet on top of the functionalized PAA gel and incubate overnight at 4 &#176;C.</li>
	<li>Wash the gels 2x in sterile PBS. Remove the PBS and leave the gels to dry for 5 min.</li>
	<li>Pipet a 50 &#181;L droplet of endothelial cell growth (ECG) medium on top of the gels and incubate for at least 1 h at 37 &#176;C and 5% CO2. 
	NOTE: Incubating the gels in the ECG medium improves cell and tissue attachments to the gels.</li>
</ol>
2. Preparation of modified Krebs solution for transport
NOTE: Prepare the modified Krebs solution fresh. In this protocol, the modified Krebs solution is prepared 1 day before tissue harvesting.
<ol>
	<li>Sterilize a transport bottle by autoclaving. Fill a glass bottle with 90% (315 mL) of the total required transport volume (350 mL) of ultrapure water. Ensure water temperature is 15-20 &#176;C.</li>
	<li>While gently stirring the water using a stirring magnet, add 9.6 g/L (3.36 g) Krebs-Henseleit Buffer and stir until dissolved. Using a Pasteur pipet, rinse the weigh boat with a small volume of solution to include all traces of powder in the solution. Do not heat the solution.</li>
	<li>While stirring, add 0.373 g/L (130.55 mg) calcium chloride (CaCl2) to the solution and stir until dissolved. Rinse the weigh boat with a small volume of solution. 
	CAUTION: H319 causes serious eye irritation.</li>
	<li>While stirring, add 2.1 g/L (0.63 g) sodium bicarbonate (NaHCO3) to the solution and stir until dissolved. Rinse the weigh boat with a small volume of solution.</li>
	<li>While stirring, add 1 x 10-1 mM (13.15 mg) papaverine to the solution and stir until dissolved. Rinse the weigh boat with a small volume of solution. 
	NOTE: Papaverine is a smooth muscle relaxant used to prevent excessive vasoconstriction caused by cutting and handling the vessel during harvesting. 
	CAUTION: H301 is toxic if swallowed.</li>
	<li>In the fume hood, add 0.05 mM (1.2 &#181;L) 2-mercaptoethanol to the solution and stir. 
	NOTE: 2-mercaptoethanol is used to keep a low level of oxygen radicals. 
	CAUTION: 2-mercaptoethanol is toxic if swallowed or if inhaled (H301 + H331), fatal in contact with skin (H310), causes skin irritation (H315), may cause an allergic skin reaction (H317), causes serious eye damage (H318), suspected of damaging the unborn child (H361d), may cause damage to organs through prolonger or repeated exposure if swallowed (H373), and very toxic to aquatic life with long-lasting effects (H410). Wear personal protective equipment and work in a fume hood.</li>
	<li>While stirring, adjust pH to 7.2 by adding 1 N (1 M) HCl or 1 N (1 M) NaOH. The final pH aim is 7.4, but pH may rise 0.1-0.3 pH units during filtration. 
	CAUTION: HCl contains gas under pressure; it may explode if heated (H280), cause severe skin burns and eye damage (H314), and is toxic if inhaled (H331). Wear personal protective equipment.</li>
	<li>Continue working in a biosafety cabinet. Add 10% ultrapure water to bring the solution to the final volume.</li>
	<li>Sterilize the solution immediately by filtration using a membrane with a porosity of 0.22 &#181;m. Dispense the sterile solution directly in a sterile bottle. Add 7 mL of 2% Penicillin/Streptomyocin (P/S) to the solution. 
	NOTE: During transport, 2% P/S is added to the medium to remove all bacteria. During tissue culture, 1% P/S is added to the medium.</li>
	<li>Store the modified Krebs solution at 4 &#176;C until tissue harvesting.</li>
</ol>
3. Tissue harvesting
<ol>
	<li>Depending on the slaughterhouse, stun pigs through electrical shock or CO2. Subsequently, hang pigs from a hind limb, exsanguinated, and declared dead.</li>
	<li>Before entering the clean slaughtering process, scald pigs for hair removal, singe to remove the last hairs, and sterilize the outside of the carcass.</li>
	<li>Eviscerate the pigs by making a midline incision along the abdomen and carefully removing the internal organs. Depending on the slaughterhouse, the carotid artery was still attached to the carcass of the pig, or the carotid artery was already removed from the carcass with the pluck of thoracic organs.</li>
	<li>Using a sharp knife, harvest the carotid artery with some remaining surrounding tissue from the carcass or throat area tissue without touching the artery or applying mechanical strain.</li>
	<li>Place the tissue containing the carotid artery in the transport bottle containing sterile modified Krebs solution by briefly opening the bottle. Invert the bottle once to ensure that the entire tissue is covered in Krebs solution. Transport the tissues on ice to the laboratory. The transport takes approximately 30-45 min.</li>
</ol>
4. Tissue dissection
<ol>
	<li>Sterilize dissection equipment surgical tweezer (rough handling), epoxy-coated round tip tweezer (fine handling), scalpel, pincher, and coverslips by autoclaving.</li>
	<li>Prepare the biosafety cabinet before tissue harvesting/transport. Cover the dissection area with a surgical drape sheet. Mount dissection equipment (in sterile 50 mL tubes), surgical blades, and sterile coverslips. Fill two large Petri dishes with sterile PBS to prepare a dissection tray. Incubate the tissue culture in one small Petri dish with ECG medium at 37 &#176;C.</li>
	<li>After tissue transport, within the biosafety cabinet, transfer the carotid artery from the transport bottle (filled with modified Krebs solution) to a large Petri dish filled with sterile PBS using the surgical tweezer.</li>
	<li>Remove excessive tissue surrounding the carotid artery using the surgical tweezer and scalpel to create a clear view of the carotid artery.</li>
	<li>Remove 2-3 cm from both ends of the carotid artery to eliminate areas close to artery bifurcations by cutting with a scalpel. Remove arterial fascia surrounding the carotid artery using the fine round-tip tweezer. 
	NOTE: Renewing the scalpel blade helps cut the fascia more precisely.</li>
	<li>Transfer the carotid artery to a new large Petri dish filled with sterile PBS. Remove the remaining thin layer of arterial fascia as much as possible. 
	NOTE: The longer the carotid artery is in PBS, the more pieces of fascia tend to get loose. Removing fascia is important since it will obstruct vision during microscopy.</li>
	<li>Cut the clean carotid artery into rings approximately 2 mm in width. Transfer the carotid artery rings into the prewarmed small Petri dish filled with ECG medium and keep at 37 &#176;C.</li>
</ol>
5. Tissue seeding
NOTE: Tissue attachment was tested by adding no coverslip, a sterile untreated coverslip, or a sterile pluronic-treated coverslip (1% w/v pluronic in PBS, coverslips incubated overnight and washed in sterile ultrapure water before use) of different sizes on top of the arterial sheet after seeding on the PAA hydrogel.
<ol>
	<li>Remove ECG medium droplets from gels. Transfer a carotid artery ring to a clean medium Petri dish filled with sterile PBS.</li>
	<li>Using the round-tip tweezer and scalpel, cut the ring in half. Dissect half a ring into sheets of approximately 2 mm width to create arterial sheets with dimensions 2 x 2 mm. Mind the orientation of the endothelial side of the arterial sheet. Curvature and some remaining fascia of the arterial sheet may help determine this orientation. 
	NOTE: The size of the carotid artery rings might vary. Large rings yield approximately 6-8 sheets, while small rings yield 3-4 sheets. Large rings have enhanced attachment due to less curvature in the arterial sheet.</li>
	<li>Using the round tip tweezer, grab the arterial sheet at the back of the sheet (outside of the vessel wall) and place the sheet at the edge of the PAA gel with the endothelial inner lining facing the PAA substrate to avoid damaging the PAA substrate when placing the sheet.</li>
	<li>Using the tweezer or pincher, move the arterial sheet very gently to the center of the PAA hydrogel without touching the gel. Place the arterial sheet at the edge of the gel (the tissue might stick to the tweezer or pincher). 
	NOTE: Small remnants of arterial fascia at the outside of the vessel wall create an easy handle for grabbing the arterial sheet while the endothelial inner lining of the vessel wall is very smooth.</li>
	<li>Add 50 &#181;L of ECG medium on top of the arterial sheet placed on the PAA substrate. Ty to keep the droplet of ECG medium on top of the gel.</li>
	<li>Using the round-tipped tweezer, place a dry 13 mm coverslip on top of the arterial sheet on the PAA substrate in medium. Use the inner rim of the glass bottom to gently lower the coverslip until it touches the medium droplet and the medium spreads underneath it. 
	NOTE: The 13 mm coverslips are advantageous because they closely match the size of the inner wall of the glass bottom well plate. If you lower the coverslip too fast, the tissue will move to the edge of the coverslip.</li>
	<li>Leave the tissue to attach at 37 &#176;C and 5% CO2 for 5 h before adding 1 mL of ECG medium to each well. Place the arterial sheet on the PAA substrate at 37 &#176;C and 5% CO2 for 24 h.</li>
</ol>
6. Creation of the 2.5D model
<ol>
	<li>Autoclave a round-tip tweezer for coverslip removal. Prepare 1 M NaOH in ultrapure water, sterilize by filtration through a 0.22 &#181;m pore size filter, and store at 4 &#176;C. 
	NOTE: 1 M NaOH can be reused in future experiments.</li>
	<li>Using a sterile sharp needle with a bent tip, lift the 13 mm coverslip from the arterial sheet very gently and remove the coverslip. Use the inner ring of the glass bottom well as a support to lift and remove the coverslip. Any sideward movement increases the risk of detachment of the arterial sheet from the PAA substrate.</li>
	<li>In a sterile microcentrifuge tube, prepare the collagen type I mixture according to Table 2 on ice. Upon addition of collagen type I and NaOH to the solution, resuspend very well and invert the tube once to ensure that the mixture is homogeneously mixed. 
	NOTE: Upon addition of collagen type I to the mixture, the light pink-colored mixture turns colorless. Upon the addition of NaOH, the colorless mixture turns light pink.</li>
	<li>Remove the medium from the well using a vacuum suction system. Remove the medium surrounding the tissue as much as possible without touching the tissue. 
	NOTE: Too much medium surrounding the arterial sheet will prevent the collagen-type I gel from surrounding it.</li>
	<li>Add a 10 &#181;L droplet of collagen type I gel mixture on top of each arterial sheet and leave the collagen type I gel to polymerize for 1 h at 37 &#176;C and 5% CO2.</li>
	<li>Very gently, add 1 mL of pre-warmed ECG medium to each well and place the samples at 37 &#176;C and 5% CO2.</li>
</ol>
7. Live cell imaging
NOTE: Live cell imaging was performed using a Leica DMi8 or Nikon Ti2 Eclipse epi-fluorescence microscope equipped with thermal, CO2, and humidity control and controlled using Leica or NIS software. Adaptive focus control (Leica) and perfect focus system (Nikon) were used to maintain focus in time.
<ol>
	<li>After 24 h of culture in the 2.5D system, check for sprouting angiogenesis. Some arterial sheets already start to form cellular sprouts after 24 h of culture while other sheets need some more time to initiate endothelial sprouting.</li>
	<li>If sprouting angiogenesis has been initiated, refresh the ECG medium and place the 12-well plate in the stage holder within the 37 &#176;C pre-warmed incubation box of the microscope.</li>
	<li>Choose the objective of interest. Different objectives were used in this study, use a 10x objective to create a global overview of the formation of cellular sprouts and a 20x objective to perform TFM.</li>
	<li>Define channel(s) of interest. For TFM purposes, visualize the cellular sprouts using phase contrast and visualize the fluorescent markers (dark red) in the PAA substrate using a fluorescent channel at a wavelength of 660 nm.</li>
	<li>Select (multiple) region(s) of interest and&#160;find the focus plane. Turn on the focus system (adaptive focus control at Leica DMi8 or perfect focus system at Nikon Ti2 Eclipse) to ensure stable focus over timelapse imaging.</li>
	<li>Define the timelapse of interest by selecting a time interval and timelapse length. Different time intervals (5-20 min) and timelapse lengths (4-24 h) were used for different purposes in this study.</li>
	<li>For TFM purposes, after timelapse imaging, remove outgrowing cells by adding several droplets of 5% SDS in ultrapure water and wait several minutes. For each selected position, take a z-stack (a z-height of 2 &#956;m was defined with a step size of 0.2 &#956;m) of the fluorescent markers in the PAA substrate to obtain a relaxed state of the fluorescent markers as the reference image. 
	NOTE: Z-stack imaging is performed since cells might pull or push the fluorescent markers in the z-direction.</li>
</ol>
8. Traction force microscopy analysis
<ol>
	<li>Align and crop timelapse images relative to the best reference image for precise analysis.</li>
	<li>To measure displacements of fluorescent markers in the PAA hydrogel, perform Particle Image Velocimetry (PIV) between any timelapse image and the reference image using customized MatLab codes. Within the PIV analysis, divide the images into interrogation windows of 32 by 32 pixels with 0.5 overlap.</li>
	<li>Compute traction forces from the fluorescent markers&#39; displacements by Fourier transform-based traction microscopy of an infinite gel with a finite gel thickness using the Boussinesq equation27.</li>
</ol>

Versatile 2.5D Model for Analyzing Cellular Sprouting in Porcine Arteries

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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=Traction+force+microscopy+in+physics+and+biology.">Traction force microscopy in physics and biology.</a> Soft Matter. 10 (23), 4047-4055 (2014).</li><li>Trepat, X., 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=Physical+forces+during+collective+cell+migration.">Physical forces during collective cell migration.</a> Nat Phys. 5 (6), 426-430 (2009).</li><li>Santos-Oliveira, P., 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=The+force+at+the+tip+-+modelling+tension+and+proliferation+in+sprouting+angiogenesis.">The force at the tip - modelling tension and proliferation in sprouting angiogenesis.</a> PLoS Comput Biol. 11 (8), e1004436 (2015).</li><li>Boreddy, S. R., Sahu, R. P., Srivastava, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benzyl+isothiocyanate+suppresses+pancreatic+tumor+angiogenesis+and+invasion+by+inhibiting+HIF-&#945;/VEGF/Rho-GTPases:+Pivotal+role+of+STAT-3.">Benzyl isothiocyanate suppresses pancreatic tumor angiogenesis and invasion by inhibiting HIF-&#945;/VEGF/Rho-GTPases: Pivotal role of STAT-3.</a> PLoS One. 6 (10), 0025799 (2011).</li><li>Teng, R. J., Eis, A., Bakhutashvili, I., Arul, N., Konduri, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+superoxide+production+contributes+to+the+impaired+angiogenesis+of+fetal+pulmonary+arteries+with+in+utero+pulmonary+hypertension.">Increased superoxide production contributes to the impaired angiogenesis of fetal pulmonary arteries with in utero pulmonary hypertension.</a> Am J Physiol Lung Cell Mol Physiol. 297 (1), L184-L195 (2009).</li><li>Costa, G., 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=Asymmetric+division+coordinates+collective+cell+migration+in+angiogenesis.">Asymmetric division coordinates collective cell migration in angiogenesis.</a> Nature Cell Biol. 18 (12), 1292-1301 (2016).</li><li>Slater, K., Partridge, J., Nandivada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Corning tuning the elastic moduli of Corning Matrigel and collagen I 3D matrices by varying the protein concentration. , (2019).</li><li>Lee, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+chain+flexibility+on+cell+adhesion:+Semi-flexible+model-based+analysis+of+cell+adhesion+to+hydrogels.">Effect of chain flexibility on cell adhesion: Semi-flexible model-based analysis of cell adhesion to hydrogels.</a> Sci Rep. 9 (1), 2463 (2019).</li><li>Motte, S., Kaufman, L. 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V., Matsumoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cells+in+three-dimensional+collagen+matrix.">Imaging cells in three-dimensional collagen matrix.</a> Curr Prot Cell Biol. 10, Unit 10.18.1-Unit 10.18.20 (2010).</li><li>Labernadie, 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=A+mechanically+active+heterotypic+E-cadherin/N-cadherin+adhesion+enables+fibroblasts+to+drive+cancer+cell+invasion.">A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion.</a> Nat Cell Biol. 19 (3), 224-237 (2017).</li><li>Bazelli&#232;res, 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=Control+of+cell-cell+forces+and+collective+cell+dynamics+by+the+intercellular+adhesome.">Control of cell-cell forces and collective cell dynamics by the intercellular adhesome.</a> Nat Cell Biol. 17 (4), 409-420 (2015).</li><li>Uroz, M., 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=Traction+forces+at+the+cytokinetic+ring+regulate+cell+division+and+polyploidy+in+the+migrating+zebrafish+epicardium.">Traction forces at the cytokinetic ring regulate cell division and polyploidy in the migrating zebrafish epicardium.</a> Nat Mater. 18 (9), 1015-1023 (2019).</li><li>Dong, C., Nastaran, Z., Konstantopoulos, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomechanics in oncology. , (2018).</li><li>Schwarz, U. 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R., 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=Multidimensional+traction+force+microscopy+reveals+out-of-plane+rotational+moments+about+focal+adhesions.">Multidimensional traction force microscopy reveals out-of-plane rotational moments about focal adhesions.</a> Proc Natl Acad Sci U S A. 110 (3), 881-886 (2013).</li><li>Steinwachs, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+force+microscopy+of+cells+in+biopolymer+networks.">Three-dimensional force microscopy of cells in biopolymer networks.</a> Nat Meth. 13 (2), 171-176 (2016).</li><li>Franck, C., Maskarinec, S. A., Tirrell, D. 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The authors have no conflicts of interest to disclose.

Sprouting angiogenesis -&#160;the formation of new blood vessels -&#160;is a complex process regulated by both molecular and mechanical mechanisms. Whereas many 3D models have been developed over the past decades to study the molecular drivers (e.g., VEGF and Notch signaling) of sprouting angiogenesis, only little is known about cellular mechanics due to model limitations. Traction Force Microscopy (TFM) is a well-known technique for quantification of cellular forces in space and time, where 2D substrate deformations are converted into cellular tractions. Therefore, in this protocol, we describe a 2.5D ex vivo model, meaning that we locally provide the cells with a 3D environment while preserving the simplicity of a 2D model that allows for the quantification of traction forces during sprouting angiogenesis (Figure 1). To do so, we prepared and seeded a porcine arterial sheet (endothelial side down; Figure 2) on top of a collagen type IV coated polyacrylamide (PAA) hydrogel containing fluorescent markers. After the attachment of the arterial sheet using a 13 mm glass coverslip (Figure 3), we add a thin layer of collagen type I hydrogel that allows the formation of cellular sprouts (Figure 4). Using this model, we show that during cellular sprouting22,23,24,25, leader cells exert pulling forces (as was observed in literature19,20,21,22), but also that follower cells exert pushing forces (Figure 6). The resolution of the traction field obtained through our protocol allows for quantitative analyses of cellular kinematics and dynamics both in time and space that are typical of works resorting to traction force microscopy on compliant substrates37,38,39.
Moreover, we demonstrate the versatility of this 2.5D ex vivo model of sprouting angiogenesis by changing the mechanical cues of the microenvironment (Figure 5). While sprouting angiogenesis normally occurs at a physiological stiffness of 1 kPa35 - which can be mimicked by a 3D collagen type I hydrogel, tumor angiogenesis occurs in a stiffened microenvironment40 - which is beyond the stiffness range of conventional 3D collagen type I hydrogels. PAA substrate stiffness can easily be adjusted by changing the ratio of crosslinkers to generate a higher substrate stiffness. Using this model, we reveal that the onset of sprouting angiogenesis is stiffness-dependent. These substrates not only offer tunable stiffness but also enable systematic modulation of various other mechanical cues, e.g., matrix composition and density. In addition, this model allows us to study cellular mechanics while manipulating molecular regulators using conditioning of the medium (e.g., the effect of inhibition on Notch signaling on cellular mechanics) to understand the mechanobiological mechanisms of sprouting angiogenesis. This demonstrates the utility of this 2.5D ex vivo model of sprouting angiogenesis in a swatch of different microenvironments.
The model that we present makes use of conventional 2D TFM, which offers simpler analysis, higher spatial resolution, and easier implementation compared to 3D (viscoelastic) TFM, making it more accessible and cost-effective26,41. However, 3D (viscoelastic) TFM provides a more physiologically relevant environment by capturing traction forces in all three dimensions and accounting for the complex mechanical properties of the extracellular matrix, offering deeper insights into cell behavior in a more realistic context42,43,44,45. This effect of dimensionality also points towards a limitation of this 2.5D model. We use 2D TFM on the assumption that cells are migrating on a 2D substrate. While this is the case in this 2.5D model, cells are in a local 3D environment and thus adhere to the collagen type I gel layer and exert forces in this gel layer. The assumption that we adopted within this analysis is that the collagen type I gel layer is not mechanically coupled (no force transmission between these two hydrogels) to the PAA interface due to the order of magnitude difference in matrix stiffness, therefore, minimizing the effect of cellular forces on the collagen type I layer. This makes the force characterization using the 2.5D ex vivo model a simplified representation of the forces generated by the cells. In addition, this protocol requires precision and is extensive with several steps where samples can be lost, e.g. (i) cell visibility difficulties due to excessive tissue surrounding the arterial sheet (Figure 2), (ii) one out of three samples does not attach to the PAA substrate (Figure 3), (iii) not all samples will initiate the formation of cellular sprouts (Figure 5), (iv) cellular sprouts do not form at the PAA substrate, and (v) out-of-focus fluorescent markers when using thick arterial sheet on top of a low stiffness PAA hydrogel. Therefore, we optimized this method for a 12-well plate to ensure plenty of regions of interest to perform mechanical analysis of sprouting angiogenesis.
In conclusion, the presented approach for the simplified characterization of cellular traction forces of sprouting angiogenesis of a living porcine arterial sheet using a 2.5D model (Figure 7)&#160;can aid in creating more accurate and real-time insights into the mechanical interactions during angiogenesis within a native tissue context, facilitating the study of dynamic cellular processes with reduced complexity and improved reproducibility compared to fully 3D systems. This could enhance our understanding of how cells respond to mechanical cues in a more physiologically relevant environment while maintaining the analytical simplicity of 2D methods. This knowledge could advance the field of tissue engineering with the aim of creating blood vessels but also finding therapeutic drugs for the prevention of tumor angiogenesis with the aim to limit tumor growth and reduce metastasis.

Angiogenesis is the process of new blood vessel formation from pre-existing blood vessels. This process is essential during embryonic development, wound healing, and cancer progression, all of which are associated with biomechanical changes in the microenvironment1,2,3,4. At the onset of angiogenesis, hypoxic or injured tissues release vascular endothelial growth factor (VEGF) that will activate the endothelial cells of neighboring blood vessels to form endothelial sprouts - where two distinct leader and follower phenotypes are adopted through the molecular Notch signaling pathway5. Upon the formation of endothelial sprouts, a phenomenon known as sprouting angiogenesis, leader cells will degrade the surrounding extracellular matrix to collectively migrate towards the VEGF stimulus without losing cell-cell adhesions with the trailing follower cells6,7.
Over the past decades, there have been increasing numbers of sprouting angiogenesis assays described that investigate collective cell migration through various methodologies, each offering distinct benefits and limitations. These assays assess the coordinated movement of cell groups, such as endothelial cells, through 3D matrices, allowing for the study of cellular behaviors like sprouting, invasion, and collective migration in a controlled environment8,9,10. In vivo sprouting angiogenesis assays provide a comprehensive evaluation within a living organism, capturing intricate interactions, but are time-consuming, costly, prone to high variability, and difficult to quantify11,12. In vitro sprouting angiogenesis assays allow precise control over experimental conditions with high reproducibility and precise quantification but may not fully replicate in vivo complexities11,12,13. In contrast, ex vivo sprouting angiogenesis assays, of which the aortic ring assay is the most widely-performed model, use tissues outside the organism, preserving physiological relevance while avoiding in vivo complications14,15,16. Despite being technically challenging and sometimes struggling with tissue viability, ex vivo models offer a valuable balance between complexity and control, making them a promising approach for studying sprouting angiogenesis. While these models have been used extensively to study the molecular drivers of sprouting angiogenesis, the effect of mechanical cues and the mechanical behavior of cells remain poorly understood.
Multicellular migration during sprouting angiogenesis is highly dependent on cellular mechanics, as actomyosin-based contractile forces regulate endothelial cell invasion into the surrounding extracellular matrix17,18,19,20. Specifically, non-muscle myosin II motors, the major actin-based contractile machines within the cell21, have been observed to control cellular contractile forces during sprouting angiogenesis22,23. The leader cell is likely the predominant force-generating element of the sprout since deformations of the surrounding 3D extracellular matrix are significantly higher around the leader cell, specifically nearby actin-rich cellular protrusions23,24, compared to its followers22,23,25. Despite this growing evidence of the importance of cellular contractility in sprouting angiogenesis in 3D, a method for spatiotemporal mechanical characterization of cellular mechanics of sprouting angiogenesis is lacking.
The overall goal of this study is to develop a method that allows for the mechanical characterization of cellular migration during sprouting. By achieving spatiotemporal characterization of mechanical forces in a biologically relevant context, we aim to provide new insights into how cellular mechanics influence angiogenic sprout formation. To this end, we developed a 2.5D model system by creating a 2D polyacrylamide (PAA) hydrogel, seeding a carotid arterial sheet on it, and covering it with a thin layer of collagen type I gel to establish a localized 3D environment for the cells. Multicellular sprouts migrated out of the arterial sheet on the PAA-collagen gel interface. The advantage of this method compared to existing techniques is that the 2D PAA hydrogel allows analyses by traction force microscopy (TFM) - a well-known versatile technique where cells adhere to an elastic 2D substrate and will deform the substrate upon cellular traction forces26. These deformations can be captured, and cellular traction forces can be computed based on the mechanical properties of the substrate26. By adapting TFM for use in ex vivo living tissues, we aim to bridge the gap between in vitro control and in vivo relevance, providing a more comprehensive understanding of mechanical forces during angiogenesis.

<table><tbody><tr><td>2% bis-acrylamide</td><td></td><td>Bio-Rad</td><td>1610143</td></tr><tr><td>2-mercaptoethanol</td><td></td><td>Merck Life Science</td><td>60-24-2</td></tr><tr><td>3-(Trimethoxysilyl)propyl methacrylate</td><td>Bind-Silane</td><td>Sigma-Aldrich</td><td>440159-100ML</td></tr><tr><td>40% acrylamide</td><td></td><td>Bio-Rad</td><td>1610140</td></tr><tr><td>Aboslute ethanol (for analysis)</td><td></td><td>VWR International</td><td>1.00983.1000</td></tr><tr><td>Absolute ethanol (industrial)</td><td></td><td>VWR International</td><td>83813.41</td></tr><tr><td>Acetic acid, glacial 100%</td><td></td><td>Merck</td><td>1000562500</td></tr><tr><td>Ammonium persulfate</td><td>APS</td><td>Bio-Rad</td><td>7727-54-0</td></tr><tr><td>antibody (primary) - calponin</td><td></td><td>abcam</td><td>ab46794</td></tr><tr><td>antibody (primary) - CD31</td><td></td><td>Serotec</td><td>MCA1746</td></tr><tr><td>antibody (primary) - &amp;alpha;-smooth muscle actin</td><td>&amp;alpha;SMA</td><td>Dako</td><td>M0851</td></tr><tr><td>antibody (secondary) - goat-anti-mouse-IgG1 Alexa 488</td><td></td><td>Molecular Probes</td><td>A21121</td></tr><tr><td>antibody (secondary) - goat-anti-mouse-IgG2a Alexa 555</td><td></td><td>Molecular Probes</td><td>A21137</td></tr><tr><td>antibody (secondary) - goat-anti-rabbit-IgG Alexa 555</td><td></td><td>Molecular Probes</td><td>A21428</td></tr><tr><td>Autoclave</td><td></td><td>Astell</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>CaCl2</td><td>Calbiochem</td><td>208291-250GM</td></tr><tr><td>Collagen type I, rat-tail</td><td></td><td>Corning</td><td>354236</td></tr><tr><td>Collagen type IV, human placenta</td><td></td><td>Merck Life Science</td><td>C5533-5MG</td></tr><tr><td>Endothelial Cell Growth Medium</td><td>ECG medium</td><td>Promocell</td><td>C-22111</td></tr><tr><td>Expoxy-coated round tip tweezer</td><td>fine tweezer</td><td>Rubis Pinzette</td><td>E78144-2A</td></tr><tr><td>Fluorescent marker, dark red</td><td></td><td>Invitrogen</td><td>F8807</td></tr><tr><td>Glass coverslips, &amp;Oslash;13 mm, #1</td><td></td><td>Epredia</td><td>CB00130RA120MNZ0</td></tr><tr><td>Glass coverslips, &amp;Oslash;13 mm, #1.5</td><td></td><td>Epredia</td><td>CB00120RAC20MNZ0</td></tr><tr><td>Hydrochloride acid, 25%</td><td>HCl</td><td>Merck</td><td>1.100316.1000</td></tr><tr><td>Krebs-Henseleit buffer</td><td></td><td>Sigma-Aldrich</td><td>K3753</td></tr><tr><td>Microscope, Leica Application Suite X software, version 3.5.7.23225</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Microscope, Leica DMi8 epifluorescent microscope</td><td></td><td>Leica Microsystems</td><td></td></tr><tr><td>Microscope, Nikon Ti2 Eclipse</td><td></td><td>Nikon</td><td></td></tr><tr><td>Microscope, NIS-Elements AR software</td><td></td><td>Nikon</td><td></td></tr><tr><td>N,N,N&#039;,N&#039;-tetramethylethane-1,2-diamine</td><td>TEMED</td><td>Merck Life Science</td><td>110-18-9</td></tr><tr><td>Nalgene bottle</td><td></td><td>Thermo Scientific</td><td>2187-0016</td></tr><tr><td>Needle, 21Gx1&quot;</td><td></td><td>Henke Sass Wolf</td><td>HK4710008025</td></tr><tr><td>Normal serum, goat</td><td></td><td>Gibco</td><td>10098792</td></tr><tr><td>Papaverine hydrochloride</td><td></td><td>Sigma</td><td>61-25-6</td></tr><tr><td>Penicillin/Streptomyocin (10 000 U/mL)</td><td>P/S</td><td>Gibco</td><td>15140163</td></tr><tr><td>Petri-dish, large (145x20mm)</td><td></td><td>Greiner Bio-one</td><td>639160</td></tr><tr><td>Petri-dish, small (60x15mm)</td><td></td><td>Greiner Bio-one</td><td>628160</td></tr><tr><td>Phosphate Buffered Saline</td><td>PBS</td><td>Sigma</td><td>P4417</td></tr><tr><td>Pluronic F-127</td><td></td><td>Merck Life Science</td><td>P2443-250G</td></tr><tr><td>Puncture needle, sharp closed tip</td><td></td><td>unknown</td><td></td></tr><tr><td>Scalpel, no. 4</td><td></td><td>Swann-Morton</td><td></td></tr><tr><td>Sodium hydrogen carbonate</td><td>NaHCO3</td><td>VWR International</td><td>144-55-8</td></tr><tr><td>sulfosuccinimidyl 6-(4&#039;-azido-2&#039;-nitrophenylamino)hexanoate</td><td>Sulfo-SANPAH</td><td>Thermo Scientific</td><td>22589</td></tr><tr><td>Surgical blade, no. 20</td><td></td><td>Swann-Morton</td><td></td></tr><tr><td>Surgical drape sheet</td><td></td><td>Foliodrape</td><td>2775001</td></tr><tr><td>Surgical tweezer</td><td></td><td>Lettix</td><td>400024</td></tr><tr><td>Triton X-100</td><td></td><td>Merck</td><td>9036-19-5</td></tr><tr><td>UV lamp</td><td></td><td>Analytik Jena</td><td>95-0042-13&amp;nbsp;</td></tr><tr><td>well plate, 96-well, F-bottom</td><td></td><td>Greiner Bio-one</td><td>655180</td></tr><tr><td>well plate, glass bottom 12-well</td><td></td><td>MatTek</td><td>P12G-0-14-F</td></tr></tbody></table>

2.5d model for ex vivo mechanical characterization of sprouting angiogenesis in living tissue

Sprouting angiogenesis is the formation of new blood vessels from pre-existing vasculature and is of great importance for physiological such as tissue growth and repair and pathological processes, including cancer and metastasis. The multistep process of sprouting angiogenesis is a molecularly and mechanically driven process. It consists of induction of cellular sprout by vascular endothelial growth factor, leader/follower cell selection through Notch signaling, directed migration of endothelial cells, and vessel fusion and stabilization. A variety of sprouting angiogenesis models have been developed over the years to better understand the underlying mechanisms of cellular sprouting. Despite advancements in understanding the molecular drivers of sprouting angiogenesis, the role of mechanical cues and the mechanical driver of sprouting angiogenesis remains underexplored due to limitations in existing models. In this study, we designed a 2.5D ex vivo model that enables us to mechanically characterize cellular sprouting from a porcine carotid artery using traction force microscopy. The model identifies distinct force patterns within the sprout, where leader cells exert pulling forces and follower cells exert pushing forces on the matrix. The model's versatility allows for the manipulation of both chemical and mechanical cues, such as matrix stiffness, enhancing its relevance to various microenvironments. Here, we demonstrate that the onset of sprouting angiogenesis is stiffness-dependent. The presented 2.5D model for quantifying cellular traction forces in sprouting angiogenesis offers a simplified yet physiologically relevant method, enhancing our understanding of cellular responses to mechanical cues, which could advance tissue engineering and therapeutic strategies against tumor angiogenesis.

2.5D Model for Ex Vivo Mechanical Characterization of Sprouting Angiogenesis in Living Tissue

Sprouting angiogenesis, fundamental for development and disease, involves complex molecular and mechanical processes. We present a versatile 2.5D ex vivo model that analyzes cellular sprouting from porcine carotid arteries, revealing stiffness-dependent angiogenesis and distinct leader-follower cell mechanics. This model aids in advancing tissue engineering strategies and cancer therapy approaches.

Sprouting angiogenesis is the formation of new blood vessels from pre-existing vasculature and is of great importance for physiological such as tissue ...

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JoVE Journal

Biology

188 Views.  Eindhoven University of Technology. Het kwantificeren van de celmechanica die het ontkiemen van angiogenese in 3D-levende weefsels aanstuurt, is nog steeds moeilijk, en toch is het noodzakelijk om vooruitgang te blijven boeken op het gebied van regeneratieve geneeskunde. Dit protocol maakt de taak toegankelijker door de toegankelijkheid van kwantitatieve 2D-methoden te combineren met de representativiteit van 3D-weefsels. Bestaande methoden voor de kwantificering van cellulaire krachten tijdens...

Video: 2.5D-model voor ex vivo mechanische karakterisering van kiemende angiogenese in levend weefsel

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

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

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

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

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

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

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

Ex vivo Mechanical Loading of Tendon

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37&deg;C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.

A new in vitro system for simultaneously loading four tendons in culture is described.

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune cells, and the coordination with lymphatic vessels and nerves. The multi-cell, multi-system interactions necessitate the investigation of angiogenesis in a physiologically relevant environment. Thus, while the use of in vitro cell-culture models have provided mechanistic insights, a common critique is that they do not recapitulate the complexity associated with a microvascular network. The objective of this protocol is to demonstrate the ability to make time-lapse comparisons of intact microvascular networks before and after angiogenesis stimulation in cultured rat mesentery tissues. Cultured tissues contain microvascular networks that maintain their hierarchy. Immunohistochemical labeling confirms the presence of endothelial cells, smooth muscle cells, pericytes, blood vessels and lymphatic vessels. In addition, labeling tissues with BSI-lectin enables time-lapse comparison of local network regions before and after serum or growth factor stimulation characterized by increased capillary sprouting and vessel density. In comparison to common cell culture models, this method provides a tool for endothelial cell lineage studies and tissue specific angiogenic drug evaluation in physiologically relevant microvascular networks.

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis.

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune ...

Developmental 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 ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Bioengineering

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 ...

Microfluidic Model to Mimic Initial Event of Neovascularization

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the ...

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 ...

Medicine

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 ...

2.5D Model for Ex Vivo Mechanical Characterization of Sprouting Angiogenesis in Living Tissue

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