The authors thank Sophie Kr&#252;ger and Gabriele Prinz for their excellent technical support. We thank Sebastian Maier for developing the ImageJ plugin to quantify spheroid sprouts and the Lighthouse Core Facility, Zentrum f&#252;r Translationale Zellforschung (ZTZ), Department of Medicine I, University Hospital Freiburg for the use of the IncuCyte system. The graphics were created with biorender.com. This work was supported by the Deutsche Forschungsgemeinschaft [Bu3135/3-1 + Bu3135/3-2 to F.B], the Medizinische Fakult&#228;t der Albert-Ludwigs- Universit&#228;t Freiburg [Berta-Ottenstein-Program for Clinician Scientists and Advanced Clinician Scientists to F.B.], the Else Kr&#246;ner-Fresenius-Stiftung [2021_EKEA.80 to F.B.] the German Cancer Consortium [CORTEX fellowship for Clinician Scientists to J.R.] and the Volker Homann Stiftung [to J.N.+F.B.] and the &#34;Freunde der Universit&#228;ts-Augenklinik Freiburg e.V.&#34; [to P.L.]

1. HUVEC cell culture
NOTE: Perform all following steps under sterile working conditions (sterile working bench).
<ol>
	<li>Expanding HUVECs
	<ol>
		<li>For both angiogenesis assays, use pooled human umbilical vein endothelial cells (HUVECs) or human microvascular endothelial cells (HMVECs).</li>
		<li>Cultivate the cells until they reach 90% confluence in an uncoated T75 flask with endothelial cell growth medium (EGM) before performing a split. Perform medium change 3 times a week. 
		NOTE: To expedite growth, the medium can be changed daily. Do not use HUVECs beyond the sixth passage. The study received the most consistent results running all experiments with HUVECs of the same passage.</li>
	</ol>
	</li>
	<li>Splitting HUVECs
	<ol>
		<li>When HUVECs reach the desired confluence in the T75 flask, rinse them once with 5 mL of phosphate-buffered saline (PBS), followed by incubating the cells with 0.5% trypsin for 2 min in the incubator. 
		&#8203;NOTE: Prolonged exposure to trypsin can damage HUVEC function.</li>
		<li>Gently detach the cells by tapping on the flask. Confirm successful detachment under an inverted phase contrast microscope.</li>
		<li>Add 8 mL of EGM, transfer the solution to a tube, and pellet the cells by centrifugation at 250 x g for 3 min.</li>
		<li>If cell counting is necessary, resuspend the cells in 5 mL of EGM and use a Neubauer chamber. Otherwise, add 40 mL of EGM and distribute it onto 4 fresh T75 cell culture flasks.</li>
	</ol>
	</li>
</ol>
2. Scratch wound migration assay
NOTE: The scratch wound migration assay requires a duration of 3 days for completion (Figure 1). Perform all the following steps under sterile working conditions (sterile working bench).
<ol>
	<li>Plating and starving of cells (day 1)
	<ol>
		<li>Using a specialized 96-well plate for live cell imaging, seed 20,000 HUVECs in 100 &#181;L of EGM per well. Allow the cells to settle for 6 h in the incubator. 
		NOTE: It is advisable to optimize cell numbers considering the varying speed of vascular endothelial cell growth between suppliers and laboratories. The objective is to achieve a perfect monolayer the following day before initiating the scratch. Overpopulated wells can alter endothelial cell behavior, e.g., by contact inhibition16, while an incomplete monolayer hampers the analysis drastically.</li>
		<li>Starve all wells overnight with 100 &#181;L of EBM per well supplemented with 2% FBS. 
		NOTE: Be careful not to damage the cell monolayer, especially when using a multichannel pipette.</li>
	</ol>
	</li>
	<li>Preparing stimulation solutions (day 2)
	<ol>
		<li>Dilute desired substances and controls in endothelial cell growth basal medium (EBM) supplemented with 2% FBS. For each well, use 100 &#181;L of solution. 
		NOTE: EBM supplemented with 2% FBS serves as a negative control, while recombinant human vascular endothelial growth factor (VEGF) at 25 ng/mL can be used as a positive control. Using 96-well plates, it is recommended that the experiments be designed with a minimum of technical replicates (4 wells with identical conditions), although it is suggested that 8 be used for optimal results. Strive to minimize potential edge or corner effects when planning the layout.</li>
	</ol>
	</li>
	<li>Creating the scratch (day 2)
	<ol>
		<li>Check cells under a microscope to verify a confluent cell monolayer and cell viability. Position the 96-well plate in a 96-well wound maker tool and generate the scratch by pressing down the lever of the device. Carefully lift off the lid of the wound maker tool before releasing the lever to prevent double scratching.</li>
		<li>Wash all wells twice using 200 &#181;L of EBM per well supplemented with 2% FBS. Confirm under a microscope that debris was successfully removed.</li>
	</ol>
	</li>
	<li>Cell stimulation (day 2)
	<ol>
		<li>Add 100 &#181;L of the prepared stimulation or control solutions per well.</li>
	</ol>
	</li>
	<li>Imaging (day 2-3)
	<ol>
		<li>Acquire images once per hour for a period of up to 24 h using an automated schedule provided by the live-cell imaging microscope. 
		NOTE: To ensure good image quality, scan every well immediately after transferring it into the incubator housing the live-cell imaging system. A 30 min acclimation time in the incubator helps to get a better picture quality. The microscope software is programmed to acquire one image per hour for each well at the midline of the defined scratch.</li>
	</ol>
	</li>
	<li>In the absence of a wound maker tool and a live cell imager, follow the steps described below.
	<ol>
		<li>Use a pipette tip in a 24-well plate to induce a scratch. Take images with a classic cell culture microscope at specific intervals. For this purpose, utilize a motorized microscope equipped with precise x and y tracking capabilities. This enables automatic positioning, ensuring consistent image capture at predefined locations. 
		NOTE: When manual imaging is necessary it is a viable option to only image T0 as well as one time point &#39;x&#39; hours following treatment. In the described protocol using HUVECs the time point T12 (12 h post-stimulation) was an attractive time point with a clear separation between negative and VEGF-stimulated positive control. However, running initial time course experiments with imaging every 2 h or 1 h is recommended to determine the optimal time point for the specific experimental settings in each lab.</li>
	</ol>
	</li>
	<li>Analysis (day 3)
	<ol>
		<li>The live-cell imaging microscope software may enable direct analysis of the acquired images. Define masks delineating the cell lawn, initial scratch, and relative wound density regions. Determine cell boundaries based on contrast differences. 
		NOTE: A segmentation adjustment of 1.6, a hole file of 500 &#181;m2, an adjusted size of 1 pixel, an area &#62;500 &#181;m, and an eccentricity &#62;0.6 were used for experiments run with HUVECs. Thorough visual vetting of the cell detection parameters relative to actual images is essential. With optimized configurations, the software calculates relative wound density (RWD) over time automatically and generates corresponding temporal graphs with the standard deviation (SD) of the technical replicates. This data can then be used for statistical analysis.</li>
	</ol>
	</li>
</ol>
3. RNA extraction with 2D cultivated cells
NOTE: Perform all the following steps until step 3.3 under sterile working conditions (sterile working bench).
<ol>
	<li>Plating cells (day 1)
	<ol>
		<li>Detach HUVECs per step 1.2 of the HUVEC cell culture protocol.</li>
		<li>Seed 50,000 cells per well in a 12-well plate with 2 mL of EGM per well.</li>
		<li>Change the medium after 6 h to EBM containing 2% FBS (starving media) for starving overnight.</li>
	</ol>
	</li>
	<li>Treatment of the cells (day 2)
	<ol>
		<li>Dilute agents of interest in EBM supplemented with 2% FBS. Replace starving media with the prepared dilution and incubate cells for the desired time in an incubator.</li>
	</ol>
	</li>
	<li>RNA extraction (day 2-3)
	<ol>
		<li>Lyse the cells with 750 &#181;L of Trizol per well and incubate for 10 min on an orbital shaker.</li>
		<li>Resuspend the samples with a 1000 &#181;L pipette a few times and then store them in specific low-binding RNA tubes (volume 1.5 mL). Store at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
4. Immunocytochemistry with 2D cultivated cells
NOTE: Perform all following steps until step 4.4 under sterile working conditions (sterile working bench).
<ol>
	<li>Preparation of coverslips
	<ol>
		<li>Incubate 100 coverslips with a diameter of 12 mm in 5% potassium hydroxide in methanol in a 50 mL tube for 30 min while shaking the applied solution. 
		NOTE: This is a very important step in deprotonating the surface of the coverslips and improving the conditions for coating and culture. At this point, ensuring that the coverslips are well distributed within the medium and don&#8217;t get dry and glued together is highly recommended.</li>
		<li>Wash the coverslips for 30 min 4 times with demineralized water.</li>
		<li>After washing, transfer them into 70% isopropyl solution for storage.</li>
	</ol>
	</li>
	<li>Coating of coverslips
	<ol>
		<li>Lean the coverslips individually against the wall of a square Petri dish to allow the evaporation of isopropyl alcohol.</li>
		<li>After 5 min transfer the coverslips into a 24 well plate (one coverslips per well). Apply 1 mL of a solution of 10 mg/mL collagen I in PBS to each well and incubate for 60 min at 37 &#176;C.</li>
		<li>Then, wash the coverslips 4-5 times for 5 min with PBS.&#160;</li>
	</ol>
	</li>
	<li>Cultivating cells
	<ol>
		<li>Detach HUVECs following the procedures outlined in step 1.2 of the HUVEC cell culture protocol.</li>
		<li>Seed 50,000 cells per well in a 24-well plate with 2 mL of EGM per well.</li>
		<li>After 6 h, change the medium to EBM containing 2% FBS to allow the cells to attach to the well and starve the cells overnight.</li>
		<li>On the next day, dilute agents of interest in EBM supplemented with 2% FBS. Replace the starving solution in wells with the prepared dilution and incubate cells for the desired time in an incubator.</li>
	</ol>
	</li>
	<li>Fixation and blocking
	<ol>
		<li>Cultivate the cells for the desired time, then wash the cells with PBS for 5 min.</li>
		<li>Fix the cells with 2% paraformaldehyde (PFA) in PBS for 20 min at room temperature (RT).</li>
		<li>Wash with PBS 3-4 times for 5 min.</li>
		<li>Incubate samples with blocking buffer containing 5% normal goat serum (NGS), 0.1% Triton-X-100 in PBS for 1 h at RT. 
		NOTE: At this step, it is important to wash the samples to guarantee that PFA is removed completely. Choose the species of the serum based on the origin of the secondary antibody.</li>
	</ol>
	</li>
	<li>Staining
	<ol>
		<li>Incubate the samples overnight at 4 &#176;C in the blocking buffer with the primary antibody.</li>
		<li>On the next day, to remove any unbound primary antibody, wash the samples 3-4 times with PBS for 5 min each.</li>
		<li>Subsequently, incubate the samples for 1 h at RT with the corresponding secondary antibody and Phalloidin-FITC, diluted together in the blocking buffer.</li>
		<li>Wash an additional 3-4&#160;times.</li>
		<li>Invert the coverslips onto slides with a small droplet of DAPI-containing mounting medium, allow to dry in the dark, and seal with nail polish. Store samples in the dark at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
5. Spheroid sprouting assay
NOTE: The spheroid sprouting assay requires 3 days for completion (Figure 2). Perform all following steps until step 5.7 under sterile working conditions (sterile working bench).
<ol>
	<li>Generating spheroids in hanging drops (day 1)
	<ol>
		<li>Detach HUVECs following the procedures outlined in step 1.2 of the HUVEC cell culture protocol.</li>
		<li>Combine 200,000 cells with 8 mL of EGM and 2 mL of methocel stock solution, ensuring thorough mixing. Please refer to Supplementary File 1 for instructions on preparing the methocel stock solution.</li>
		<li>Dispense 25 &#181;L droplets of the solution onto the inverted inner surface of a large, squared Petri dish. Gently rotate the lid by 180&#176; and position it on the bottom of the cell culture vessel so that the droplets are hanging. Place the vessel in a cell culture incubator overnight.</li>
	</ol>
	</li>
	<li>Prepare the collagen gel (day 2)
	<ol>
		<li>Mix thoroughly 2.3 mL of rat tail collagen type I with 0.280 mL of 10x Medium 199 until the mixture achieves a consistently even yellow hue. Keep this mixture on ice throughout the entire process.</li>
	</ol>
	</li>
	<li>Titrate the collagen gel (day 2)
	<ol>
		<li>Titrate the mixture to achieve a physiological pH, indicated by an orange color, using NaOH (2 N).</li>
		<li>Add 50 &#181;L of HEPES buffer to the final product. 
		NOTE: To ensure an optimal dynamic range for the assay, it is imperative to approach as closely as possible to a physiological pH. Various batches of collagen may require different amounts of NaOH. Keep the mixture strictly on ice throughout the process and homogeneously mixed all the time.</li>
	</ol>
	</li>
	<li>Harvesting spheroids (day 2)
	<ol>
		<li>Rinse hanging drops from cell culture lids with 20 mL of PBS.</li>
		<li>Centrifuge the solution for 7 min at 250 x g. Discard the supernatant and resuspend the spheroids in 0.1 mL of FBS and 0.4 mL of EGM by gently tapping the tube.</li>
		<li>Add 2 mL of methocel stock solution and mix thoroughly. Use a pipette with a minimum capacity of 5 mL to avoid damaging the spheroids.</li>
	</ol>
	</li>
	<li>Adding spheroids to the 3D collagen matrix&#160;(day 2)
	<ol>
		<li>Add 2 mL of the prepared collagen mixture to the resuspended spheroids and mix thoroughly.</li>
		<li>Dispense 0.5 mL of the resulting mixture into the wells of a 24-well plate and incubate in a cell incubator for 30 min.</li>
	</ol>
	</li>
	<li>Stimulating spheroids in a 3D collagen matrix (day 2)
	<ol>
		<li>Prepare a dilution of desired substances in EBM. For each well, use 100 &#181;L of solution. 
		NOTE: EBM serves as a negative control, while vascular endothelial growth factor (VEGF) at 25 ng/mL (final concentration in 500 &#181;L of collagen matrix and 100 &#181;L EBM layer) can be used as a positive control.</li>
		<li>Incubate for the desired time. 
		NOTE: In a standard assay, 18-24 h is recommended, but timing needs to be carefully optimized in each lab.</li>
	</ol>
	</li>
	<li>Imaging spheroids (day 3)
	<ol>
		<li>Utilize an inverted microscope and imaging platform to capture images of all spheroids that are not in contact with the well rim, with each other, or show signs of damage. Maintain consistent settings and zoom levels across all conditions.</li>
	</ol>
	</li>
	<li>Analysis (day 3)
	<ol>
		<li>Utilize the measuring tool in ImageJ Fiji to ascertain the length of all sprouts from each spheroid in every image. 
		NOTE: Use the plugin provided in Supplementary File 2, which makes the analysis more efficient and generates an output .txt file.</li>
		<li>Import the data into a spreadsheet, R, or any other preferred software, and use the average cumulative length of all sprouts per spheroid as the readout for each condition. 
		NOTE: Results from different gels cannot be combined in the form of absolute sprouting lengths. Data from each treatment group need to be normalized to the EBM or VEGF control group (relative sprouting length) before data can be merged.</li>
	</ol>
	</li>
</ol>
6. RNA extraction with 3D cultivated cells
<ol>
	<li>Spheroid sprouting assay
	<ol>
		<li>Perform the spheroid sprouting assay as described in section 5.</li>
	</ol>
	</li>
	<li>Lysis of collagen gel
	<ol>
		<li>Wash spheroid gels with warm PBS for 5 min.</li>
		<li>Cut the spheroid gels into quarters and transfer all 4 quarters into a 50 mL tube. Use a scalpel for the mechanical dissection of the gels.</li>
		<li>Treat gel samples with lysis solution (containing 2 mg/mL Collagenase D in PBS) and incubate for 45 min at 37 &#176;C on a shaker.</li>
		<li>Wash gels gently with PBS supplemented by 2 mM EDTA to stop the reaction of the lysis buffer.</li>
	</ol>
	</li>
	<li>RNA extraction
	<ol>
		<li>Resuspend the lysed gels in 750 &#181;L of Trizol and incubate for 10 min to guarantee sufficient RNA extraction as described in step 3.3.</li>
		<li>Resuspend the samples with a 1000 &#181;L pipette a few times and transfer samples to specific low-binding RNA tubes (volume 1.5 mL). Store samples at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
7. Immunocytochemistry of spheroids in a 3D collagen matrix
<ol>
	<li>Spheroid sprouting assay
	<ol>
		<li>Perform the spheroid sprouting assay as described in section 5.</li>
	</ol>
	</li>
	<li>Fixation and blocking
	<ol>
		<li>Fix the gels with 4% PFA in PBS for 1 h. 
		&#8203;NOTE: To ensure good fixation of the cells in the gel, gels need to be detached carefully on the sides of the wells with a scalpel and tweezers following rinsing with PBS.</li>
		<li>Wash gels gently 3-4 times with PBS and then fully detach them from the surface of the wells and transfer them into new 24-well plates.</li>
		<li>Incubate gels for 1 h at RT with the blocking solution (containing 5% NGS and 0.1% Triton-X-100 in PBS) on an orbital shaker.</li>
	</ol>
	</li>
	<li>Staining
	<ol>
		<li>Incubate the samples overnight at 4 &#176;C on an orbital shaker with the primary antibody diluted in blocking buffer. 
		NOTE: Turning the gels during this incubation did not improve staining quality.</li>
		<li>The next day, wash the gels gently 4-5 times with PBS for 5 min each.</li>
		<li>Incubate the corresponding secondary antibody, diluted with Phalloidin-FITC in blocking buffer overnight at 4 &#176;C on an orbital shaker.</li>
		<li>Conduct 4-5 additional washing steps with PBS.</li>
		<li>Transfer the gels onto microscope slides and mount them with two drops of DAPI-containing mounting medium on a coverslip placed onto each gel. Allow the samples to dry before sealing with nail polish and store them in the dark at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Microscopy
	<ol>
		<li>Image all samples on a confocal microscope. Use the 20x objective and focus on the region of interest. Acquire images as Z-stacks, as well as individual focal plane images. 
		NOTE: Acquiring Z-stack and focal plane images at multiple sections throughout the 3D sample is essential to capture the complete representation, especially for thick 3D samples.</li>
	</ol>
	</li>
</ol>
8. Human retinal microvascular endothelial cells (HRMVECs)
NOTE: All the described steps can also be performed with microvascular endothelial cells, e.g., human retinal microvascular endothelial cells (HRMVECs). In that case, the medium needs to be switched to a specific microvascular endothelial cell medium containing 10% fetal bovine serum (FBS) for cultivation as well as specific steps in each assay. HRMVEC-specific differences to the assay protocols are outlined below:
<ol>
	<li>Scratch wound assay
	<ol>
		<li>Cultivate cells in 10% FBS microvascular endothelial cell medium instead of EGM.</li>
		<li>Seed 20,000 cells/well.</li>
		<li>Do not starve the cells overnight.</li>
		<li>After performing the scratch, change the medium to basal endothelial cell medium containing 5% FBS instead of EBM with 2% FBS.</li>
	</ol>
	</li>
	<li>Immunocytochemistry of 2D cultivated HRMVECs
	<ol>
		<li>Cultivate the cells in 10% microvascular endothelial cell medium instead of EGM.</li>
		<li>Do not starve the cells overnight.</li>
		<li>Change the medium right before treatment to EBM + 5% FBS instead of EBM+2% FBS.</li>
	</ol>
	</li>
	<li>Spheroid sprouting assay
	<ol>
		<li>Seed the cells as hanging drops with microvascular endothelial cell medium containing 10% FBS instead of EGM.</li>
	</ol>
	</li>
	<li>Immunocytochemistry of spheroids in a 3D collagen matrix
	<ol>
		<li>Seed the cells as hanging drops with microvascular endothelial cell medium containing 10% FBS instead of EGM.</li>
	</ol>
	</li>
</ol>

Study of Angiogenesis Using Scratch Wound Migration and Spheroid Sprouting Assays

<ol>
	<li>Risau, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+angiogenesis.">Mechanisms of angiogenesis.</a> Nature. 386 (6626), 671-674 (1997).</li><li>Gariano, R. F., Gardner, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+angiogenesis+in+development+and+disease.">Retinal angiogenesis in development and disease.</a> Nature. 438 (7070), 960-966 (2005).</li><li>Mancuso, M. R., Kuhnert, F., Kuo, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+angiogenesis+of+the+central+nervous+system.">Developmental angiogenesis of the central nervous system.</a> Lymphat Res Biol. 6 (3-4), 173-180 (2008).</li><li>Tonnesen, M. G., Feng, X., Clark, R. A. <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+wound+healing.">Angiogenesis in wound healing.</a> J Investig Dermatol Symp Proc. 5 (1), 40-46 (2000).</li><li>Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+angiogenesis+in+tumor+growth+and+metastasis.">Role of angiogenesis in tumor growth and metastasis.</a> Semin Oncol. 29, 15-18 (2002).</li><li>Crawford, T. N., Alfaro, D. V., Kerrison, J. B., Jablon, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetic+retinopathy+and+angiogenesis.">Diabetic retinopathy and angiogenesis.</a> Curr Diabetes Rev. 5 (1), 8-13 (2009).</li><li>Ng, E. W., Adamis, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+angiogenesis,+the+underlying+disorder+in+neovascular+age-related+macular+degeneration.">Targeting angiogenesis, the underlying disorder in neovascular age-related macular degeneration.</a> Can J Ophthalmol. 40 (3), 352-368 (2005).</li><li>Blanco, R., Gerhardt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vegf+and+notch+in+tip+and+stalk+cell+selection.">Vegf and notch in tip and stalk cell selection.</a> Cold Spring Harb Perspect Med. 3 (1), 006569 (2013).</li><li>Hellstr&#246;m, M., Kal&#233;n, M., Lindahl, P., Abramsson, A., 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=Role+of+PDGF-B+and+PDGFR-&#946;+in+recruitment+of+vascular+smooth+muscle+cells+and+pericytes+during+embryonic+blood+vessel+formation+in+the+mouse.">Role of PDGF-B and PDGFR-&#946; in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse.</a> Development. 126 (14), 3047-3055 (1999).</li><li>Nowak-Sliwinska, 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=Consensus+guidelines+for+the+use+and+interpretation+of+angiogenesis+assays.">Consensus guidelines for the use and interpretation of angiogenesis assays.</a> Angiogenesis. 21 (3), 425-532 (2018).</li><li>Korff, T., Augustin, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensional+forces+in+fibrillar+extracellular+matrices+control+directional+capillary+sprouting.">Tensional forces in fibrillar extracellular matrices control directional capillary sprouting.</a> J Cell Sci. 112, 3249-3258 (1999).</li><li>Rapp, 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=2D+and+3D+in+vitro+angiogenesis+assays+highlight+different+aspects+of+angiogenesis.">2D and 3D in vitro angiogenesis assays highlight different aspects of angiogenesis.</a> Biochim Biophys Acta Mol Basis Dis. 1870 (3), 167028 (2024).</li><li>Rapp, 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=STAT3+signaling+induced+by+the+IL-6+family+of+cytokines+modulates+angiogenesis.">STAT3 signaling induced by the IL-6 family of cytokines modulates angiogenesis.</a> J Cell Sci. 136 (1), 260182 (2023).</li><li>Heiss, 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=Endothelial+cell+spheroids+as+a+versatile+tool+to+study+angiogenesis+in+vitro.">Endothelial cell spheroids as a versatile tool to study angiogenesis in vitro.</a> FASEB J. 29 (7), 3076-3084 (2015).</li><li>Rapp, 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=Oncostatin+m+reduces+pathological+neovascularization+in+the+retina+through+m&#252;ller+cell+activation.">Oncostatin m reduces pathological neovascularization in the retina through m&#252;ller cell activation.</a> Invest Ophthalmol Vis Sci. 65 (1), 22 (2024).</li><li>Fagotto, F., Gumbiner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+contact-dependent+signaling.">Cell contact-dependent signaling.</a> Dev Biol. 180 (2), 445-454 (1996).</li><li>Rapp, 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=Addressing+bias+in+manual+segmentation+of+spheroid+sprouting+assays+with+U-Net.">Addressing bias in manual segmentation of spheroid sprouting assays with U-Net.</a> Mol Vis. 29, 197-205 (2023).</li><li>Boyden, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemotactic+effect+of+mixtures+of+antibody+and+antigen+on+polymorphonuclear+leucocytes.">The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes.</a> J Exp Med. 115 (3), 453-466 (1962).</li><li>Guy, J. B., 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=Evaluation+of+the+cell+invasion+and+migration+process:+A+comparison+of+the+video+microscope-based+scratch+wound+assay+and+the+Boyden+chamber+assay.">Evaluation of the cell invasion and migration process: A comparison of the video microscope-based scratch wound assay and the Boyden chamber assay.</a> J Vis Exp. (129), e56337 (2017).</li><li>Decicco-Skinner, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+tube+formation+assay+for+the+in+vitro+study+of+angiogenesis.">Endothelial cell tube formation assay for the in vitro study of angiogenesis.</a> J Vis Exp. (91), e51312 (2014).</li><li>Vernon, R. B., Angello, J. C., Iruela-Arispe, M. L., Lane, T. F., Sage, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reorganization+of+basement+membrane+matrices+by+cellular+traction+promotes+the+formation+of+cellular+networks+in+vitro.">Reorganization of basement membrane matrices by cellular traction promotes the formation of cellular networks in vitro.</a> Lab Invest. 66 (5), 536-547 (1992).</li><li>Craig, L. E., Spelman, J. P., Strandberg, J. D., Zink, M. C. <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+from+diverse+tissues+exhibit+differences+in+growth+and+morphology.">Endothelial cells from diverse tissues exhibit differences in growth and morphology.</a> Microvasc Res. 55 (1), 65-76 (1998).</li><li>M&#252;ller, A. M., Hermanns, M. I., Cronen, C., Kirkpatrick, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+adhesion+molecule+expression+in+cultured+human+macro-and+microvascular+endothelial+cells.">Comparative study of adhesion molecule expression in cultured human macro-and microvascular endothelial cells.</a> Exp Mol Pathol. 73 (3), 171-180 (2002).</li><li>Chiew, G. G. Y., Wei, N., Sultania, S., Lim, S., Luo, K. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineered+three-dimensional+co-culture+of+cancer+cells+and+endothelial+cells:+A+model+system+for+dual+analysis+of+tumor+growth+and+angiogenesis.">Bioengineered three-dimensional co-culture of cancer cells and endothelial cells: A model system for dual analysis of tumor growth and angiogenesis.</a> Biotechnol Bioeng. 114 (8), 1865-1877 (2017).</li><li>Smith, L. 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=Oxygen-induced+retinopathy+in+the+mouse.">Oxygen-induced retinopathy in the mouse.</a> Invest Ophthalmol Vis Sci. 35 (1), 101-111 (1994).</li><li>Lambert, V., 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=Laser-induced+choroidal+neovascularization+model+to+study+age-related+macular+degeneration+in+mice.">Laser-induced choroidal neovascularization model to study age-related macular degeneration in mice.</a> Nat Protoc. 8 (11), 2197-2211 (2013).</li><li>Kastana, 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=Matrigel+plug+assay+for+in+vivo+evaluation+of+angiogenesis.">Matrigel plug assay for in vivo evaluation of angiogenesis.</a> Methods Mol Biol. 1952, 219-232 (2019).</li></ol>

The authors declare no conflict of interest in this project.

In this report, we presented a spectrum of techniques with functional and molecular readouts to study angiogenesis in vitro. 
The migration assay represents a well-established technique used across all fields of wet laboratory work. We chose the commercially available live-cell imaging approach to take advantage of the 96-well format suitable for screening and dose-response experiments, the standardized and reproducible wound size created by the WoundMaker tool, the opportunity to ob...

Angiogenesis, which refers to the formation of new blood vessels from pre-existing ones1, is a crucial process during physiological development and pathologic conditions. It is indispensable for providing energy to highly metabolically active tissues such as the retina2 or the developing central nervous system3 and during the healing of damaged tissue4. Abnormal angiogenesis, on the other hand, is the basis for numerous diseases. Solid tumors, such as colorectal cancer or non-small cell lung cancer, facilitate their growth and the necessary energy supply by promoting angiogenesis5. Apart from cancer, neovascular diseases of the eye like diabetic retinopathy or age-related macular degeneration, which represent leading causes of blindness in the developed world, result from aberrant vessel growth6,7. A detailed understanding of the underlying pathomechanism is crucial to comprehend how physiological angiogenesis can be enhanced, for instance, in wound healing while better controlling pathological conditions such as vasoproliferative eye diseases.
On a cellular level, vascular endothelial cells are activated by various signaling molecules in angiogenesis, initiating cell proliferation and migration8. The cells subsequently organize into a hierarchy, with non-proliferating tip cells forming filopodia at the leading edge of the developing vessel branch8. Alongside, fast-proliferating stalk cells trail the tip cells, contributing to the formation of the emerging vessel. Subsequently, other cell types, such as pericytes or smooth muscle cells, are recruited to further stabilize the nascent branch9.
To explore molecular processes at the vascular endothelial cell level, numerous in vitro protocols have been developed and recently reviewed10. These assays typically fall into two categories: more simplistic but scalable 2D approaches and more elaborate 3D protocols. In a recent project, we conducted a comprehensive comparative analysis between the 2D scratch wound migration assay and the 3D spheroid sprouting assay11 to assess the extent of their differences and their ability to model various aspects of angiogenesis12. 
While both offer the advantages of being reliable and easily implementable, on a molecular level, the 3D spheroid sprouting assay was favorable in addressing key aspects of angiogenesis compared to in vivo data, such as metabolic switches or cell-matrix interactions. Since in vitro angiogenesis assays are used to evaluate the angiomodulatory potential of signaling pathways13 and to screen for therapeutic agents, transferability of in vitro results to in vivo settings is essential. Furthermore, the opportunity for omics-based analyses on the RNA and protein levels to characterize the molecular changes in response to targeted modulation of angiogenic processes under controlled conditions remains an important benefit compared to in vivo settings14,15.
In this publication, we present key assays for exploring angiogenesis-related questions through the utilization of a live-cell imaging migration assay and a spheroid sprouting assay, including subsequent molecular analyses like RNA sequencing for transcriptomic analysis and immunohistochemistry on the protein level.

<table><tbody><tr><td>10x Medium 199</td><td>Sigma-Aldrich</td><td>M0650</td><td></td></tr><tr><td>Alexa Fluor 647-conjugated AffiniPure F(ab)&amp;lsquo;2-Fragment</td><td>Jackson IR&amp;nbsp;</td><td>115-606-072</td><td></td></tr><tr><td>Axio Vert. A1</td><td>Zeiss</td><td></td><td></td></tr><tr><td>CapturePro 2.10.0.1</td><td>JENOTIK Optical Systems</td><td></td><td></td></tr><tr><td>Collagen Type 1 rat tail</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>Collagenase D</td><td>Roche&amp;nbsp;</td><td>11088858001</td><td></td></tr><tr><td>Endothelial Cell Basal Medium</td><td>Lonza</td><td>CC-3156</td><td>EBM</td></tr><tr><td>Endothelial Cell Growth Medium</td><td>Lonza</td><td>CC-3162</td><td>EGM</td></tr><tr><td>Ethylenediaminetetraacetic acid&amp;nbsp;</td><td>Serva</td><td>11290.02</td><td>EDTA</td></tr><tr><td>Fetal bovine serum</td><td>Bio&amp;amp;SELL</td><td>S 0615</td><td>FBS</td></tr><tr><td>Human Umbilical Vein Endothelial Cells, pooled</td><td>Lonza</td><td>C2519A</td><td>HUVEC</td></tr><tr><td>IncuCyte ImageLock 96-well plates&amp;nbsp;</td><td>Sartorius</td><td>4379</td><td></td></tr><tr><td>Incucyte S3 Live-Cell Analysis System</td><td>Sartorius</td><td></td><td></td></tr><tr><td>Methocel</td><td>Sigma</td><td>m-0512</td><td></td></tr><tr><td>Microvascular Endothelial Cell Medium with 10% FBS</td><td>PB-MH-100-4090-GFP</td><td>PELOBiotech</td><td></td></tr><tr><td>NaOH</td><td>Carl Roth</td><td>P031.2</td><td></td></tr><tr><td>Phalloidin-Fluorescein Isothiocyanate Labeled (0.5 mg/mL Methanol)</td><td>Sigma-Aldrich</td><td>P5282-.1MG</td><td>Phalloidin-FITC</td></tr><tr><td>Phosphate-buffered saline</td><td>Thermo Fisher Scientfic</td><td>14190-094</td><td>PBS</td></tr><tr><td>Primary Human Retinal Microvascular Endothelial Cells</td><td>Cell Systems</td><td>ACBRI 181</td><td></td></tr><tr><td>ProLong Glass Antifade Mountant with NucBlue</td><td>Invitrogen by ThermoFisher Scientific</td><td>2260939</td><td></td></tr><tr><td>QIAzol Lysis Reagent</td><td>QIAGEN</td><td>79306</td><td></td></tr><tr><td>recombinant human Vascular Endothelial Growth Factor</td><td>PeproTech</td><td>100-20</td><td>VEGF</td></tr><tr><td>Squared petri dish</td><td>Greiner</td><td>688102</td><td></td></tr><tr><td>Trizol</td><td>Qiagen</td><td>79306</td><td></td></tr><tr><td>Trypsin</td><td>PAN-Biotec</td><td>P10-024100</td><td></td></tr><tr><td>VEGF-R2 (monoclonal)</td><td>ThermoFisher Scientific Inc.</td><td>B.309.4</td><td></td></tr><tr><td>WoundMaker</td><td>Sartorius</td><td>4493</td><td></td></tr></tbody></table>

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.

For the migration assay, it is crucial to thoroughly examine the images captured at the t = 0 h time point to ensure the presence of a fully formed cell monolayer is accurately detected by the system (Figure 1B). Additionally, the clarity and straightness of the scratch border should be confirmed (Figure 1B). The cell-free area ought to be largely free of debris. At the end of the assay, a group stimulated with, for example, 25 ng/mL VEGF as a positive control s...

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.

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 ...

investigating-angiogenesis-on-functional-molecular-level-leveraging

Research

JoVE Journal

Medicine

1.1K Views.  University of Freiburg. Angiogenese is een kenmerkend proces dat betrokken is bij de embryologie en bij de ontwikkeling van ziekten, waaronder tumorgroei en vasoproliferatieve oogziekte. In dit project willen we een uitgebreid kader van in vitro technieken presenteren die kunnen worden gebruikt om angiogenese op moleculair niveau te bestuderen in een zeer gecontroleerde setting, en om te screenen op mogelijke therapeutische opties. Het vertalen in vitro naar in vivo en uiteindelijk ...

Video: Auteur in de schijnwerpers: onderzoek naar angiogenese door middel van uitdagingen en innovaties in de ontwikkeling van assay

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an &#34;in-incubator&#34; video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

Cancer Research

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human central nervous system (CNS). However, both the cytological origin and the evolutionary process of HBs (including neovascularization) remain controversial, and anti-angiogenesis for VHL-HBs, based on classic HB angiogenesis, have produced disappointing results in clinical trials. One major obstacle to the successful clinical translation of anti-vascular treatment is the lack of a thorough understanding of neovascularization in this vascular tumor. In this article, we present a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in HBs, as well as its role in HBs. With this procedure, researchers can accurately understand the complexity of HB neovascularization and identify the function of this common form of angiogenesis in HBs. These protocols can be used to evaluate the most promising anti-vascular therapy for tumors, which has high translational potential either for tumors treatment or for aiding in the optimization of the anti-angiogenic treatment for HBs in future translations. The results highlight the complexity of HB neovascularization and suggest that this common form angiogenesis is only a complementary mechanism in HB neovascularization.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into changes in clinical standards used for treating wounds and decreasing morbidity and mortality for patients. Wound healing is a complex process that requires strategic cell and tissue interaction and function. One of the many critically important functions of wound healing is individual and collective cellular migration. Upon injury, various cells from the blood, surrounding connective, and epithelial tissues rapidly migrate to the wound site by way of chemical and/or physical stimuli. This migration response can largely dictate the outcomes and success of a healing wound. Understanding this specific cellular function is important for translational medicine that can lead to improved wound healing outcomes. Here, we describe a protocol used to better understand cellular migration as it pertains to wound healing, and how changes to the cellular environment can significantly alter this process. In this example study, dermal fibroblasts were grown in media supplemented with fetal bovine serum (FBS) as monolayer cultures in tissue culture flasks. Cells were aseptically transferred into tissue culture treated 12-well plates and grown to 100% confluence. Upon reaching confluence, the cells in the monolayer were vertically scratched using a p200 pipet tip. Arsenic diluted in culture media supplemented with FBS was added to individual wells at environmentally relevant doses ranging 0.1-10 &#956;M. Images were captured every 4 hours (h) over a 24 h period using an inverted light microscope to observe cellular migration (wound closure). Images were individually analyzed using image analysis software, and percent wound closure was calculated. Results demonstrate that arsenic slows down wound healing. This technique provides a rapid and inexpensive first screen for evaluation of the effects of contaminants on wound healing.

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

This study focuses on an in vitro model of wound healing (scratch assay) as a mechanism for determining how environmental contaminants such as arsenic influence cellular migration. The results demonstrate that this in vitro assay provides rapid and early indications of changes to cellular migration prior to in vivo experimentation.

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into ...

Environment

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 ...

Developmental Biology

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Bioengineering

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

A Cost Effective and Adaptable Scratch Migration Assay

Cell migration is a key component in both physiological and pathological events. Normal cell migration is required for essential functions such as development and mounting an immune response. When a defect or alteration occurs with the cell migration process, it can have detrimental outcomes (i.e., cancer metastasis, wound healing, and scar formation). Due to the importance of cell migration, it is necessary to have access to a cell migration assay that is affordable, adaptable, and repeatable. Utilizing the common scratch migration assay, we have developed a new approach to analyzing cell migration that uses general laboratory equipment. The method described uses visual markers that allow for recapturing specific areas of interest without the use of time-lapse microscopy. In addition, it provides flexibility in the experimental design, ranging from altering the migration matrix substrate to the addition of pharmacological modifiers. Furthermore, this protocol outlines a way to account for the area of cell migration, which is not considered by several methods when examining cell migration. This new approach offers a scratch migration assay to a larger audience and will provide greater opportunity for researchers to examine the physiological and pathophysiological impact of cell migration.

We present a cost-effective method to the scratch migration assay that provides a new approach for determining cell migration without the use of equipment-intensive methods. While fibroblasts were used in this protocol, it can be adapted and utilized to study additional cell types and influences on cell migration.

Cell migration is a key component in both physiological and pathological events. Normal cell migration is required for essential functions such as ...

Biology

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 ...

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

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