This research was supported by a grant (17172MFDS215) from Ministry of Food and Drug Safety, the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIP) (2017R1A2B4005463), and the Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education (2016R1A6A1A03007648).

Human ECFCs were isolated from human peripheral blood as described in a previous report27. Briefly, the mononuclear cell layer was separated from the whole blood using the Ficoll-Paque Plus, and cultured in the proper medium until the endothelial-like colonies were appeared. Colonies were collected and ECFCs were isolated using CD31-coated magnetic beads. MSCs were isolated from the adherent mononuclear cell (MNC) fraction of human adult bone marrow. The study protocol was approved by the institutional review board of Duksung Women&#8217;s University (IRB No. 2017-002-01).
1. Cell Culture
<ol>
	<li>Preparing ECFCs and MSCs medium and coat plates
	<ol>
		<li>Prepare endothelial cell growth medium MV2 (ECGM-MV2, except hydrocortisone) supplemented with 10% fetal bovine serum (FBS) and 1% glutamine-penicillin-streptomycin (GPS).</li>
		<li>Prepare mesenchymal stem cell growth medium-2 (MSCGM-2) containing 10% FBS and 1% GPS.</li>
		<li>For ECFC culture, coat the cell culture plates with 1% gelatin solution. To prepare 1% gelatin solution, dissolve 1 g of gelation powder in 500 mL of PBS with the magnetic stirrer, and sterilize with autoclave. Plates can be coated with 3 mL/60 mm, 5 mL/100 mm, or 15 mL/150 mm of 1% gelatin solution. Incubate coated plates for 15 min in a cell culture incubator (37 &#176;C and 5% CO2). After then, remove the remaining 1% gelatin solution by aspiration and wash the coated plates one time with PBS.</li>
	</ol>
	</li>
	<li>Growing ECFCs and MSCs
	<ol>
		<li>Seed 1 x 106 ECFCs on 1% gelatin-coated 150 mm plates, and grow using ECGM-MV2 (10% FBS, 1% GPS) in a cell culture incubator (37&#176;C and 5% CO2) to 80-90% confluency. Use ECFCs at passage numbers 7-10 to obtain consistent results.</li>
		<li>Seed 1 x 106 MSCs on non-coated 150 mm plates, and grow using MSCGM-2 in a cell culture incubator (37&#176;C and 5% CO2) to 80-90% confluency. Use MSCs at passage numbers 7-10 to obtain consistent results.</li>
	</ol>
	</li>
</ol>
2. Preparation of 1.2% w/v Methylcellulose Solution
<ol>
	<li>Measure 6 g of methyl cellulose in a 500 mL glass bottle and sterilize in an autoclave.</li>
	<li>Heat ECGM-MV2 (without FBS and GPS) at 60 &#176;C for 20 min, and add 250 mL of heated ECGM-MV2 to sterilized methyl cellulose powder. To maintain sterile conditions, carry out the process inside the flow hood.</li>
	<li>Add a sterilized magnetic stir bar and mix the solution for 20 min on the magnetic stirrer until the methyl cellulose is thoroughly wetted and evenly dispersed with no lumps. Add 250 mL of cold (4 &#176;C) ECGM-MV2 (without FBS and GPS) under the sterile condition and mix for an additional 10 min on the magnetic stirrer. Then, chill the solution in a refrigerator (4 &#176;C) overnight. 
	NOTE: Methylcellulose is a carbohydrate polymer that dissolve well in cool temperatures due to swelling and subsequent hydration.</li>
	<li>The next day, aliquot the solution in a 50 mL tube and centrifuge at 5,000 x g for 3 h at 4 &#176;C. Take the clear viscous supernatant solution and store at 4 &#176;C until use for up to 3-6 months. 
	NOTE: The sediment may contain residual cellulose fiber, so gently take the supernatant solution leaving behind at least 5 mL of volume. This volume can be adjusted according to experimental conditions.</li>
</ol>
3. Generation and Embedding of ECFCs-only, MSCs-only, and ECFCs-MSCs Spheroids
Day 1 
<ol>
	<li>Preparing ECFCs and MSCs cell suspension
	<ol>
		<li>Wash the 80-90% confluent ECFCs and MSCs by adding phosphate buffered saline (PBS) without calcium and magnesium, and aspirate.</li>
		<li>To detach ECFCs and MSCs from the culture plate, incubate them with 0.05% trypsin-EDTA for 3 min and 5 min in a cell culture incubator (37&#176;C and 5% CO2), respectively, in a cell culture incubator.</li>
		<li>Inactivate trypsin by adding DMEM medium containing 10% FBS and 1% GPS. Pipette the cells up and down to create a single cell suspension.</li>
		<li>Sediment the cells by centrifuging at 282 x g for 5 min at RT.</li>
		<li>Remove the supernatant, and re-suspend the cells in the respective medium.</li>
	</ol>
	</li>
	<li>Labeling ECFCs and MSCs with fluorescent dye 
	NOTE: Perform cell membrane labelling of ECFCs with PKH67 (green) and MSCs with PKH26 (red) fluorescent dye following the manufacturer&#8217;s instructions with slightly modifications as follows.
	<ol>
		<li>Wash the cells two times with serum free medium to remove FBS. 
		NOTE: Proteins and lipids in FBS reduces the effective dye concentration for labelling cells by binding.</li>
		<li>Count ECFCs and MSCs using a hemocytometer under the microscope, and transfer 3 x 106 ECFCs and 2 x 106 MSCs into 2 mL micro-tubes. 
		NOTE: These are the optimum cell densities for labelling cells with dyes. Use of a large number of cells causes poor and heterogeneous staining, whereas use of too few cells yields poor cells recovery.</li>
		<li>Centrifuge the tubes at 100 x g for 5 min at RT&#160;to obtain a cell pellet. Carefully aspirate the supernatant leaving no more than 15-25 &#181;L of residual volume.</li>
		<li>Re-suspend ECFCs and MSCs in 250 &#181;L of Diluent C from the dye kit. Gently pipette the cell suspension to ensure complete dispersion. 
		NOTE: Diluent C is a physiologic salt, and may reduce the staining efficiency of dye by forming micelles. Therefore, do not let cells stand in diluent C for long, and do not vortex the tubes.</li>
		<li>Prepare the staining solution for ECFCs by adding 5 &#181;L of PKH67 to 250 &#181;L of Diluent C (final concentration of 20 &#181;M) and for MSCs by adding 3 &#181;L of PKH26 to 250 &#181;L of Diluent C (final concentration of 12 &#181;M ). 
		NOTE: The final dye concentrations are different from the manufacturer&#8217; recommendations. High concentration would lead to cell clumping and cell toxicity. Low concentration would not be sufficient for staining.</li>
		<li>Add 250 &#181;L of ECFCs cell suspension to 250 &#181;L of PKH67 dye solution, and 250 &#181;L of MSCs to 250 &#181;L of PKH26 dye solution. Immediately mix the cells with dyes by pipetting up and down and incubate for 5 min at room temperature. For better results, cover the tube with aluminum foil and place on a shaker to ensure sufficient mixing during staining.</li>
		<li>Stop the staining process by adding 0.5 mL of FBS to the stained cell suspension, and then incubate for 1 min to allow FBS to bind excess unbound dye. Sediment the stained cells by centrifuging at 100 x g for 5 min. 
		NOTE: ECFCs and MSCs pellets are light yellow and pink, respectively, after staining.</li>
		<li>Carefully remove the supernatant and wash the cells two times with complete medium to remove excess dye. Centrifuge the tubes at 100 x g for 5 min and re-suspend the pellet in 2 mL of each complete medium.</li>
	</ol>
	</li>
	<li>Generating ECFC, MSC, or ECFC+MSC spheroids 
	NOTE: Apply the hanging drop technique to generate spheroids as described previously28. The steps for the preparation of spheroids are given below.
	<ol>
		<li>Count the stained ECFCs and MSCs.</li>
		<li>Suspend stained ECFCs, MSCs or ECFCs+MSCs in ECGM-MV2 containing 20% Methylcellulose solution and 5% FBS. Prepare at a cell density of 4 x 104 cells/mL for ECFCs or MSCs (25 &#181;L of cell suspension contains 1,000 cells). For two cell suspension of ECFCs and MSCs, prepare at a cell density of 2 x 104 cells/mL ECFCs and 0.4 x 104 cell/mL MSCs (25 &#181;L contains 500 ECFCs and 100 MSCs). 
		NOTE: Ratio of ECFCs to MSCs should not exceed 5:1. A larger number of MSCs could lead to excessive migration and irregular sprout morphology. A smaller number of MSCs could cause poor interaction between cells, yielding poor sprouting.</li>
		<li>Prepare a hydration unit by adding 15 mL of PBS into the bottom of a 150 mm culture plate.</li>
		<li>Transfer the cell suspension into the sterilized polystyrene rectangular reservoir for the use of multichannel pipette. Disperse the cells suspension evenly by pipetting.</li>
		<li>Deposit 25 &#181;L drops of cell suspension onto the cover of a 150 mm culture plate using a multi-channel pipette (approximately 100 drops/cover of the 150 mm plate). Invert the cover over a PBS-filled hydration unit and incubate in a cell culture incubator for 24 h. 
		NOTE: Methylcellulose solution provides a proper viscosity to the suspension solution. When the hanging drop method was performed without methylcellulose solution, drops were easily slipped down when the cover was inverted (Supplemental Figure 1). Furthermore, after overnight incubation, the spheroids were not perfectly formed without methylcellulose solution. This result indicate that proper viscosity by methylcellulose solution is necessary to form round shape of the spheroids.</li>
	</ol>
	</li>
</ol>
Day 2
<ol start="4">
	<li>Embeding spheroids in neutralized collagen gel
	<ol>
		<li>Warm FBS and ECGM-MV2 (without FBS and GPS) in a water bath (37 &#176;C). Place cold methylcellulose solution on a work bench to bring to room temperature.</li>
		<li>Place a 24-well plate in a cell culture incubator (37 &#176;C) for warming.</li>
		<li>Cut the pointed 1 mL pipette tips 3-5 mm to aspirate spheroids and viscous suspensions, such as methylcellulose solution and type I collagen, comfortably and accurately.</li>
		<li>Prepare 3 mg/mL neutralized type I collagen gel on ice following the type I collagen gel manufacturer&#8217;s instruction. 
		NOTE: Neutralization should be performed on ice to avoid gelation of collagen at room temperature. For one well of a 24-well plate, 500 &#181;L of neutralized collagen is needed. Always prepare 1 mL extra volume of collagen. Collagen gel can be used up to 2-3 h after neutralization if kept on ice.</li>
		<li>Harvest the spheroids by rinsing the cover containing spheroids with 5 mL of PBS. Collect spheroid-suspended solution in a 50 mL conical tube. Re-rinse the cover with 5 mL of PBS to obtain the remaining spheroids. 
		NOTE: During harvest, closely observe to confirm the existence of spheroids. For more visual inspection, transfer about 50-100 &#181;L of spheroid-suspended solution on the slide glass, and check the round shape of the spheroids under the microscope.</li>
		<li>Sediment the spheroids by centrifuging at 282 x g for 5 min. Discard the supernatant carefully without disturbing the spheroids leaving behind not more than 100-200 &#181;L of residual volume.</li>
		<li>Gently tap on the wall of the tube so that spheroids are freely suspended in the remaining 100-200 &#181;L residual solution.</li>
		<li>Add ECGM-MV2 containing 5% FBS and 40% methylcellulose solution to the spheroid-containing tube. The volume added is determined based on approximately 100 spheroids/mL. Gently mix the spheroid suspension by pipetting with a blunt 1 mL pipette tip. 
		NOTE: Methylcellulose solution is widely used as a suspending agent that does not allow spheroids to sediment. FBS concentration should be 5%. Higher FBS concentration causes abnormal sprouting due to excessive growth factors. Lower FBS concentration cannot maintain healthy conditions for cells.</li>
		<li>Mix spheroid-suspension solution and neutralized-type I collagen gel (3 mg/mL) at a 1:1 ratio on ice. Use a blunt 1 mL pipette tip to avoid breaking of spheroids. 
		NOTE: 1 mL of the spheroids-suspending collagen gel solution is required for one well of a 24-well plate. Make an extra mixed suspension solution if possible.</li>
		<li>If the angiogenic properties of any agent(s) or chemical(s) need to be tested, transfer 1 mL of the spheroid-suspending collagen gel solution in a 1 mL micro-tube and add agent(s) or chemical(s) followed by gentle mixing with a blunt 1mL pipette tip. 
		NOTE:&#160;The volume of agent and chemical can sum to 200 &#956;L, which dilutes the type I collagen gel concentration from 1.5 mg/mL to 1.25 mg/mL, but does not affect type I collagen gel polymerization. Add same volume of vehicle to the control spheroids.</li>
		<li>Add the spheroid-suspending collagen gel solution into wells of a pre-warmed 24-well plate (0.9 mL/well) by pipetting, and then incubate for 30 min in a cell culture incubator for polymerization. 
		NOTE: During the spheroid embedding process, do not disturb the gel by agitating the plate.</li>
		<li>Cover the spheroid-suspending collagen gel by adding 100 &#181;L of ECGM-MV2 containing 2.5% FBS with/without VEGF. For ECFC-only spheroids, stimulate the cells with exogenous addition of VEGF (final concentration of 50 ng/mL) to form proper sprouts. ECFC+MSC spheroids do not require exogenous stimulation by any growth factors.</li>
		<li>Place the plate in a real-time cell recorder installed in a cell culture incubator (37 &#176;C and 5% CO2), and randomly focus on 5-10 spheroids (10x objective lens). Monitor the sprouting formation of each fluorescence-labeled spheroids every 1 h for 24 h without any disturbance.</li>
	</ol>
	</li>
</ol>
4. Quantitate Spheroid Sprouting
<ol>
	<li>Import the image files to ImageJ software to quantitate the number of sprouts and measure the length of each sprout. For co-culture spheroids, label ECFCs with green fluorescent dye before making spheroids. Then, measure the number and length of sprouts stained with green fluorescence (Supplemental Figure 2). Five randomly-selected spheroids were quantified per experimental group. 
	NOTE: Sprouts are collaboratively elongated structures formed by several ECFCs extending out of spheroids. Sprout length is measured as the length from the point of sprouts origin from the surface of the spheroids to the tip of sprouts.</li>
	<li>Express the values as means &#177; SEM of at least three independent experiments. Determine the statistically significant difference by one-way ANOVA for multiple comparisons or Student&#8217;s t-test for paired comparisons. 
	NOTE: P &#8804; 0.05 is considered statistically significant.</li>
</ol>

<ol>
	<li>Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+life,+disease+and+medicine.">Angiogenesis in life, disease and medicine.</a> Nature. 438 (7070), 932-936 (2005).</li><li>Goodwin, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+assays+of+angiogenesis+for+assessment+of+angiogenic+and+anti-angiogenic+agents.">In vitro assays of angiogenesis for assessment of angiogenic and anti-angiogenic agents.</a> Microvascular research. 74 (2-3), 172-183 (2007).</li><li>Staton, C. A., Reed, M. W., Brown, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+analysis+of+current+in+vitro+and+in+vivo+angiogenesis+assays.">A critical analysis of current in vitro and in vivo angiogenesis assays.</a> International journal of experimental pathology. 90 (3), 195-221 (2009).</li><li>Lutolf, M. P., Gilbert, P. M., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+materials+to+direct+stem-cell+fate.">Designing materials to direct stem-cell fate.</a> Nature. 462 (7272), 433-441 (2009).</li><li>Fina, 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=Expression+of+the+CD34+gene+in+vascular+endothelial+cells.">Expression of the CD34 gene in vascular endothelial cells.</a> Blood. 75 (12), 2417-2426 (1990).</li><li>Delia, D., 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=CD34+expression+is+regulated+reciprocally+with+adhesion+molecules+in+vascular+endothelial+cells+in+vitro.">CD34 expression is regulated reciprocally with adhesion molecules in vascular endothelial cells in vitro.</a> Blood. 81 (4), 1001-1008 (1993).</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> Journal of Cell Science. 112 (19), 3249-3258 (1999).</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=Integration+of+endothelial+cells+in+multicellular+spheroids+prevents+apoptosis+and+induces+differentiation.">Integration of endothelial cells in multicellular spheroids prevents apoptosis and induces differentiation.</a> The Journal of cell biology. 143 (5), 1341-1352 (1998).</li><li>Gaengel, K., Genov&#233;, G., Armulik, 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=Endothelial-mural+cell+signaling+in+vascular+development+and+angiogenesis.">Endothelial-mural cell signaling in vascular development and angiogenesis.</a> Arteriosclerosis, thrombosis, and vascular biology. 29 (5), 630-638 (2009).</li><li>Hutmacher, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials+offer+cancer+research+the+third+dimension.">Biomaterials offer cancer research the third dimension.</a> Nature Materials. 9 (2), 90-93 (2010).</li><li>Korff, T., Kimmina, S., Martiny-Baron, G., 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=Blood+vessel+maturation+in+a+3-dimensional+spheroidal+coculture+model:+direct+contact+with+smooth+muscle+cells+regulates+endothelial+cell+quiescence+and+abrogates+VEGF+responsiveness.">Blood vessel maturation in a 3-dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness.</a> FASEB Journal. 15 (2), 447-457 (2001).</li><li>Gumkowski, F., Kaminska, G., Kaminski, M., Morrissey, L. W., Auerbach, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+mouse+vascular+endothelium.+In+vitro+studies+of+lymphatic,+large+blood+vessel+and+microvascular+endothelial+cells.">Heterogeneity of mouse vascular endothelium. In vitro studies of lymphatic, large blood vessel and microvascular endothelial cells.</a> Journal of Vascular Research. 24 (1-2), 11-23 (1987).</li><li>Asif, A. R., Oellerich, M., Armstrong, V. W., Hecker, M., Cattaruzza, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-786C+polymorphism+of+the+NOS-3+gene+and+the+endothelial+cell+response+to+fluid+shear+stress-a+proteome+analysis.">T-786C polymorphism of the NOS-3 gene and the endothelial cell response to fluid shear stress-a proteome analysis.</a> Journal of Proteome Research. 8 (6), 3161-3168 (2009).</li><li>Melero-Martin, J. 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=In+vivo+vasculogenic+potential+of+human+blood-derived+endothelial+progenitor+cells.">In vivo vasculogenic potential of human blood-derived endothelial progenitor cells.</a> Blood. 109 (11), 4761-4768 (2007).</li><li>Ingram, D. 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=Identification+of+a+novel+hierarchy+of+endothelial+progenitor+cells+using+human+peripheral+and+umbilical+cord+blood.">Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood.</a> Blood. 104 (9), 2752-2760 (2004).</li><li>Murasawa, S., Asahara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+progenitor+cells+for+vasculogenesis.">Endothelial progenitor cells for vasculogenesis.</a> Physiology (Bethesda). 20, 36-42 (2005).</li><li>Rafii, S., Lyden, D., Benezra, R., Hattori, K., Heissig, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+and+haematopoietic+stem+cells:+novel+targets+for+anti-angiogenesis+therapy.">Vascular and haematopoietic stem cells: novel targets for anti-angiogenesis therapy.</a> Nature Review Cancer. 2 (11), 826-835 (2002).</li><li>Shi, S., Gronthos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+niche+of+postnatal+mesenchymal+stem+cells+in+human+bone+marrow+and+dental+pulp.">Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp.</a> Journal of Bone and Mineral Research. 18 (4), 696-704 (2003).</li><li>Crisan, 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=A+perivascular+origin+for+mesenchymal+stem+cells+in+multiple+human+organs.">A perivascular origin for mesenchymal stem cells in multiple human organs.</a> Cell Stem Cell. 3 (3), 301-313 (2008).</li><li>Lee, H., Kang, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+tube+formation+assay+using+human+endothelial+colony+forming+cells+for+in+vitro+evaluation+of+angiogenesis.">Advanced tube formation assay using human endothelial colony forming cells for in vitro evaluation of angiogenesis.</a> Korean Journal of Physiology &amp; Pharmacology. 22 (6), 705-712 (2018).</li><li>Melero-Martin, J. 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=Engineering+robust+and+functional+vascular+networks+in+vivo+with+human+adult+and+cord+blood-derived+progenitor+cells.">Engineering robust and functional vascular networks in vivo with human adult and cord blood-derived progenitor cells.</a> Circulation Research. 103 (2), 194-202 (2008).</li><li>Kang, K. T., Allen, P., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineered+human+vascular+networks+transplanted+into+secondary+mice+reconnect+with+the+host+vasculature+and+re-establish+perfusion.">Bioengineered human vascular networks transplanted into secondary mice reconnect with the host vasculature and re-establish perfusion.</a> Blood. 118 (25), 6718-6721 (2011).</li><li>Kang, K. T., Coggins, M., Xiao, C., Rosenzweig, A., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+vasculogenic+cells+form+functional+blood+vessels+and+mitigate+adverse+remodeling+after+ischemia+reperfusion+injury+in+rats.">Human vasculogenic cells form functional blood vessels and mitigate adverse remodeling after ischemia reperfusion injury in rats.</a> Angiogenesis. 16 (4), 773-784 (2013).</li><li>Kang, K. T., Lin, R. Z., Kuppermann, D., Melero-Martin, J. M., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+colony+forming+cells+and+mesenchymal+progenitor+cells+form+blood+vessels+and+increase+blood+flow+in+ischemic+muscle.">Endothelial colony forming cells and mesenchymal progenitor cells form blood vessels and increase blood flow in ischemic muscle.</a> Scientific Reports. 7 (1), 770 (2017).</li><li>Paupert, J., Sounni, N. E., Noel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphangiogenesis+in+post-natal+tissue+remodeling:+lymphatic+endothelial+cell+connection+with+its+environment.">Lymphangiogenesis in post-natal tissue remodeling: lymphatic endothelial cell connection with its environment.</a> Molecular Aspects of Medicine. 32 (2), 146-158 (2011).</li><li>Lu, P., Weaver, V. M., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix:+a+dynamic+niche+in+cancer+progression.">The extracellular matrix: a dynamic niche in cancer progression.</a> Journal of Cell Biology. 196 (4), 395-406 (2012).</li><li>Melero-Martin, J. M., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+13.+An+in+vivo+experimental+model+for+postnatal+vasculogenesis.">Chapter 13. An in vivo experimental model for postnatal vasculogenesis.</a> Methods in Enzymology. 445, 303-329 (2008).</li><li>Laib, A. 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=Spheroid-based+human+endothelial+cell+microvessel+formation+in+vivo.">Spheroid-based human endothelial cell microvessel formation in vivo.</a> Nature Protocols. 4 (8), 1202-1215 (2009).</li><li>Leuning, D. 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=The+cytokine+secretion+profile+of+mesenchymal+stromal+cells+is+determined+by+surface+structure+of+the+microenvironment.">The cytokine secretion profile of mesenchymal stromal cells is determined by surface structure of the microenvironment.</a> Scientific Reports. 8 (1), 7716 (2018).</li><li>Shah, S., Kang, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-Cell+Spheroid+Angiogenesis+Assay+System+Using+Both+Endothelial+Colony+Forming+Cells+and+Mesenchymal+Stem+Cells.">Two-Cell Spheroid Angiogenesis Assay System Using Both Endothelial Colony Forming Cells and Mesenchymal Stem Cells.</a> Biomolecules &amp; Therapeutics (Seoul). 26 (5), 474-480 (2018).</li></ol>

The authors have nothing to disclose.

The present study introduce an improved in vitro angiogenesis assay system utilizing co-culture spheroids formed by two human vascular cell progenitors, ECFCs and MSCs. Co-culture spheroid system can mimic in vivo vascular sprout formation, which is accomplished by interaction and incorporation between endothelial cells and pericytes. Compared to other in vitro angiogenesis assays that reflect only ECs-mediated angiogenesis, this co-culture assay system is more representative of the multistep cascade of physiologic angio...

Approximately 500 million people worldwide are expected to benefit from angiogenesis-modulating therapy for vascular malformation-associated diseases such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis1. Thus, the development of drugs that control angiogenesis has become an important research area in the pharmaceutical industry. During the drug development process, in vivo animal study is necessary to explore the effects of drug candidates on physiologic functions and systemic interactions between organs. However, ethical and cost issues have increased the concerns regarding animal experiments2. Therefore, improved in vitro assay systems are needed to obtain more accurate and predictable data leading to the better decision-making before animal experiments. Current in vitro angiogenesis assays usually measure proliferation, invasion, migration, or tubular structure formation of endothelial cells (ECs) seeded in two-dimensional (2D) culture plates3. These 2D angiogenesis assays are quick, simple, quantitative, and cost-effective, and have significantly contributed to the discovery of angiogenesis-modulating drugs. However, several issues remain to be improved.
Such 2D in vitro assay systems cannot reflect complex multi-step events of angiogenesis that occurs in in vivo physiologic conditions, leading to inaccurate results that cause discrepancies between in vitro assay data and clinical trial outcomes4. 2D culture conditions also induce the change of cellular phenotypes. For example, after proliferation in 2D culture plates, ECs have a weak cellular phenotype as manifested by reduced expression of CD34 and several signals that govern cellular responses5,6. To overcome the limitations of 2D culture-based angiogenesis assay systems, three-dimensional (3D) spheroid angiogenesis assay systems have been developed. Sprouting followed by tubular structure formation from spheroids formed by ECs reflect in vivo neo-vascularization processes7,8. Thus, the 3D spheroid angiogenesis assay has been considered an effective assay system for screening potential pro- or anti-angiogenesis drugs.
Most 3D spheroid angiogenesis assays utilize only ECs, mainly human umbilical vein endothelial cells (HUVECs) or human dermal microvascular endothelial cells (HDMECs) to focus on the cellular response of ECs during angiogenesis. However, blood capillaries are composed of two cell types: ECs and pericytes. Elaborating bi-directional interaction between ECs and pericytes is critical for proper vascular integrity and function. Several diseases, such as hereditary stroke, diabetic retinopathy, and venous malformation, are associated with altered pericyte density or decreased pericyte attachment to the endothelium9. Pericytes are also known as a key element of the angiogenic process. Pericytes are recruited to stabilize newly-formed vessel structures by ECs. In this regard, mono-culture spheroid angiogenesis assay does not incorporate pericytes7,10. Therefore, co-culture spheroids formed by ECs and pericytes may provide a valuable approach to more closely mimic physiologic angiogenic events.
The present study aimed to develop a 3D co-culture spheroid angiogenesis assay with a combination of human endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) to more closely reflect in vivo angiogenesis. Co-culture spheroid system as an in vitro representation assembly of a normal blood vessel was first established by Korff et al. in 200111. They combined HUVECs and human umbilical artery smooth muscle cells (HUSMCs), and demonstrated that co-culture of two mature vascular cells decreased the sprouting potential. Mature ECs (HUVECs) are known to progressively lose their ability to proliferate and differentiate, which negatively affects their angiogenesis responses12,13. Mature perivascular cells (HUSMCs) can cause endothelial cell inactivation through the abrogation of the vascular endothelial growth factor (VEGF) responsiveness11. The main difference between Korff&#8217;s and our co-culture spheroid system is the cell types used. We applied two vascular precursors, ECFCs and MSCs, to establish a proper angiogenesis assay system to screen and investigate pro-or-anti-angiogenic agents. ECFCs are the precursor of ECs. ECFCs have robust proliferation capacity compared with mature ECs14, which enable to overcome the limitation of ECs. ECFCs contribute to new vessel formation in many post-natal pathophysiologic conditions15,16,17. MSCs are pluripotent stem cells that have the capacity to differentiate into pericytes, thereby contributing to angiogenesis18,19.
In previous reports, ECFCs and MSCs showed synergistic effects on in vitro tube formation20, in vivo neo-vascularization21,22, and improved reperfusion of ischemic tissues23,24. In the present study, ECFCs and MSCs were used to form co-culture spheroids and embedded in type I collagen gel to reflect an in vivo 3D environment. Collagen is considered as a major constituents of the extracellular matrix (ECM) surrounding ECs25. The ECM plays a critical role in regulating cell behavior26. The assay protocol proposed here can be easily and quickly carried out within two days using common laboratory techniques. For effective cell tracking during the sprouting process, each cell type can be fluorescently labeled and monitored using a real-time cell recorder. The newly-established 3D co-culture spheroid angiogenesis assay system is designed to increase sensitivity for evaluating potential angiogenic modulators and to provide predictable information in advance of in vivo study.

<table><tbody><tr><td>0.05% Trypsin EDTA (1x)</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>Bevacizumab</td><td>Roche</td><td>NA</td><td>Commercial name: Avastin</td></tr><tr><td>Dulbecco Modified Eagle Medium</td><td>Gibco</td><td>11885-084</td><td>DMEM</td></tr><tr><td>Dulbeco&#039;s Phosphate buffered saline (10x)</td><td>Gibco</td><td>21600-010</td><td>PBS (10x)</td></tr><tr><td>Dulbeco&#039;s Phosphate buffered saline (1x)</td><td>Corning</td><td>21-031-CVR</td><td>PBS (1x)</td></tr><tr><td>Endothelial cell Growth medium MV2 kit</td><td>Promocell</td><td>C-22121</td><td>ECGM-MV2</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Atlas</td><td>FP-0500A</td><td>FBS</td></tr><tr><td>Gelatin</td><td>BD Sciences</td><td>214340</td><td></td></tr><tr><td>L-Glutamine&amp;ndash;Penicillin&amp;ndash;Streptomycin</td><td>Gibco</td><td>10378-016</td><td>GPS</td></tr><tr><td>Mesenchymal stem cell growth medium-2</td><td>Promocell</td><td>C-28009</td><td>MSCGM-2</td></tr><tr><td>Methyl cellulose</td><td>Sigma-Aldrich</td><td>M0512</td><td></td></tr><tr><td>PKH26 Fluorescent Cell Linker Kits</td><td>Sigma-Aldrich</td><td>MINI26</td><td>PKH26</td></tr><tr><td>PKH67 Fluorescent Cell Linker Kits</td><td>Sigma-Aldrich</td><td>MINI67</td><td>PKH67</td></tr><tr><td>Sodium Hydroxide</td><td>Sigma-Aldrich</td><td>S5881</td><td></td></tr><tr><td>Type I collagen gel</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>Vascular endothelial growth factor A</td><td>R&amp;amp;D</td><td>293-VE-010</td><td>VEGF</td></tr></tbody></table>

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.

Comparison experiments were performed using mono-culture spheroids (ECFCs-only) and co-culture spheroids (ECFCs+MSCs) to examine whether MSCs play a considerable role in ECFCs-mediated angiogenesis. Sprouting formation from each spheroid was monitored for 24 h by a real-time cell recorder that could capture the progression of angiogenic sprouting from spheroids. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from individual spheroids. Five ra...

Watch this Scientific Journal Video about Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells at JoVE.com

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

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 ...

three-dimensional-angiogenesis-assay-system-using-co-culture

Research

JoVE Journal

Developmental Biology

9.8K Views.  Duksung Women's University. Dit 3D co-cultuur sferoïde systeem is ontworpen om fysiologische angiogenese na te bootsen. Het zal effectief zijn voor de evaluatie van potentiële angiogene modulatoren en biedt voorspelbare informatie voorafgaand aan in vivo studies. Dit systeem maakt gebruik van co-cultuur sferoïden gevormd door twee vasculaire voorloper cellen en kan worden gebruikt om cellulaire interacties te onderzoeken, ontkiemen, en de buisvormige rijping van fysiologische angiogenese.Als we patiëntencelle...

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

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

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

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

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

Biology

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 ...

Cancer Research

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

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

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

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

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

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

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

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

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

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

Bioengineering

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

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 ...

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...

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 ...

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

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