This work was funded by Natural Science Foundation of Zhejiang Province (LY22H020005), and National Natural Science Foundation of China (81873466).

All procedures involving animal subjects were approved by the Institutional Animal Care and Use Committee (IACUC) of Wenzhou Medical University (XMSQ2021-0057, July 19th, 2021). All reagents and consumables are listed in the Table of Materials.
1. Culture medium preparation
<ol>
	<li>10x M199 culture medium: Dissolve M199 powder to 10x concentration with 90 mL of deionized water and add 10 mL of fetal bovine serum (FBS), then pass through a 0.22 &#181;m filter. Store the medium at 4 &#176;C for up to 2 months.</li>
	<li>Complete endothelial culture medium: Add 50 mL of FBS, 5 mL of penicillin/streptomycin, and 5 mL of endothelial cell growth supplement to 460 mL of endothelial cell medium (ECM). Store the medium at 4 &#176;C for up to 1 month.</li>
</ol>
2. Vascular cell preparation
<ol>
	<li>Culture 1 x 105 vascular cells (primary cultured endothelial progenitor cells14, endothelial cells, or endothelial cell lines) with 8 mL of complete endothelial culture medium in 100 mm tissue culture dish at 37 &#176;C and 5% CO2 to 70% confluency.</li>
	<li>Remove the culture medium and rinse the dish 2x with 1x phosphate buffered saline (PBS) to remove unattached cells and debris. Remove PBS and add 3 mL of 0.25% trypsin containing 2.21 mM EDTA, and incubate at 37 &#176;C for 1 min.</li>
	<li>Neutralize the trypsin with 7 mL of complete endothelial culture medium, and gently rinse cells off the culture dish. Confirm cell detachment under a light microscope (40x magnification).</li>
	<li>Collect cell suspension in a 15 mL tube and centrifuge at 400 x g for 10 min. Remove supernatant and resuspend cells with 5 mL of complete endothelial culture medium.</li>
	<li>Count cells using a hemocytometer and move suspension containing 2 x 106 cells to a sterile 1.5 mL tube. Each plug contains 1.5 x 106 cells, additional 25% cells are used in case of waste. Each group includes at least 3 plugs. 
	NOTE: After trials it was found that 1.5 x 106 cells in 300 &#181;L of matrix gel led to the proper development of angiogenesis and was therefore chosen for experimentation.</li>
	<li>Centrifuge cell suspension at 400 x g for 5 min to pellet cells, and then remove supernatant.</li>
</ol>
3. Matrix gel preparation
<ol>
	<li>Pre-cool sterile 1.5 mL tubes containing cell pellet in a 4 &#176;C water bath. Pre-cool 30G 1 mL insulin syringes in a 4 &#176;C refrigerator.</li>
	<li>Completely thaw matrix gel in a 4 &#176;C water bath . Do not mix or vortex. Pre-cool 10x M199 and test reagent/drug-of-interest in a 4 &#176;C water bath.</li>
	<li>Mix pre-cooled matrix gel with 10x M199 containing 10% FBS and the reagent to be tested at a volume ratio of 8.8:1:0.2 to get a matrix gel and M199 mixture containing 1% FBS and the reagent.</li>
	<li>Resuspend cells with 400 &#181;L of the matrix gel mixture, mix gently to avoid forming bubbles. Keep the tube on ice until mice are prepared for injection. Keep the matrix gel mixture on ice all the time to avoid coagulation. 
	&#8203;NOTE: Here, 300 &#181;L of matrix gel mixture containing 1.5 x 106 cells was used to form gel plug. Prepare 25% extra gel according to 25% additional cells in step 2.</li>
</ol>
4. Mouse preparation
<ol>
	<li>Anesthetize 6-8 week old male Nu/Nu mouse (18-25 g) in the chamber of the animal anesthesia device with isoflurane (3% isoflurane in 100% oxygen at a flowrate of 1 L/min). After successful anaesthetization, confirmed by the absence of righting reflex and toe pinch reflex, move the mouse out of the chamber, move the mouse out of the chamber and put an anesthesia mask on the mouse and change the concentration of isoflurane to 1.5% in 100% oxygen at a flowrate of 1 L/min. 
	NOTE: Provide thermal support to the animal using a heating pad throughout the procedure.</li>
	<li>Use vet ointment on eyes to prevent dryness. Tape the limbs of the mice on the operation board in the prone position.</li>
</ol>
5. Matrix gel mixture injection
<ol>
	<li>Load 300 &#181;L of the matrix gel mixture into a 1 mL insulin syringe with a 30G needle. Avoid bubble formation. Quickly load the matrix gel (within 2 min) to avoid solidification during this time. Place the insulin syringe loaded with matrix gel on ice immediately.</li>
	<li>Clean the skin on the back of mice using 75% alcohol pads. Subcutaneously inject 300 &#181;L of matrix gel mixture into one side of the back of mice.</li>
	<li>Gently remove the needle from the injection site to prevent leakage of the matrix gel mixture. Check for a small hump at the injection site (Figure 1).</li>
	<li>Place the mouse on the heating pad for 2 min to let the matrix gel mixture coagulate and form plug.</li>
	<li>Repeat steps 5.2. to 5.4. to create plug on the other side of the back of the mouse. Mark the edges of the humps using a marker pen.</li>
	<li>Observe the mouse until it has regained sufficient consciousness to maintain sternal recumbency. Put the mouse in a separate cage until it is fully recovered. House the mice in SPF-class experimental animal laboratory at 22 &#177; 2 &#176;C with a 12 h light/dark cycle for 7 days.</li>
</ol>
6. Dextran-FITC injection through the tail vein
<ol>
	<li>After 7 days of matrix gel injection, resuspend 0.5 mg of dextran-FITC with 500 &#181;L of double distilled water (ddH2O) and then violently vortex to obtain the dextran-FITC solution at the final concentration of 1 &#181;g/&#181;L. 
	NOTE: Use dextran-FITC with molecular weight of ~150 kDa for angiogenesis assay, and use dextran-FITC with molecular weight of ~4 kDa for vascular permeability assay.</li>
	<li>Load 50 &#181;L of dextran-FITC solution into the 29G syringes.</li>
	<li>Fix the mouse on the tail vein injection instrument. Clean the tail with 75% alcohol cotton ball and gently inject 50 &#181;L of dextran-FITC through tail vein.</li>
	<li>Compress the injection site with cotton swab for 1 min to stanch bleeding, and then put the mice back in the cage for 30 min.</li>
	<li>Repeat steps 6.3. and 6.4. until all mice have received dextran-FITC injection.</li>
</ol>
7. Matrix gel plug collection
<ol>
	<li>Intraperitoneally inject 1% pentobarbital sodium in PBS (200 mg/kg body weight) and euthanize the mice with an IACUC approved procedure.</li>
	<li>Cut the skin along the marked border of the plug using surgical scissors and remove the skin above the matrix gel plug.</li>
	<li>Collect the plug and rinse in a small beaker with 1x PBS to wash away excess blood (Figure 2A). The color of plug can roughly delineate the degree of blood abundance and the content of neo-vessels (Figure 3A).</li>
</ol>
8. Embedding matrix gel plug and section preparation
<ol>
	<li>Cover the button of tissue embedding cassette with about 0.5 mL of tissue embedding gel, then immediately place matrix gel plug on the tissue embedding gel in the desired orientation as shown in Figure 2B. Then, embed the plug with additional tissue embedding gel.</li>
	<li>Put the cassettes into a -80 &#176;C freezer for 12 h to solidify.</li>
	<li>For thick section preparation, take out the solidified plug block and cut 12 &#181;m thick sections using the freezing microtome according to the instruction, and mount onto microscope slides.</li>
	<li>Slice 5-10 sections from each plug block.</li>
</ol>
9. Quantification of angiogenesis (Figure 3)
<ol>
	<li>Acquire fluorescence images from 5 independent fields of the section with an inverted fluorescence microscope at 488 nm wavelength under 10x magnification.</li>
	<li>Open the image file with Image J software (https://imagej.en.softonic.com/) and click the Angiogenesis Analyze button. Then, click Analyze HUVEC Phase Contrast button, and switch to Stat Results Table to gather information. Measure all 5 acquired fluorescence images for each section.</li>
	<li>Analyze the total length of neo-vessels from different groups to statistically evaluate the difference in angiogenesis between these groups. 
	&#8203;NOTE: Total master segments length indicates the total length of neo-vessels. The reagent that increases the length of neo-vessels is termed pro-angiogenic, while the reagent that decreases the length of neo-vessels is anti-angiogenic.</li>
</ol>
10. Quantification of vascular permeability (Figure 4)
<ol>
	<li>Acquire fluorescence images from 5 independent fields of the section with an inverted fluorescence microscope at 488 nm wavelength under 20x magnification.</li>
	<li>Open the image file with Image J software. Click the Freehand Selection button and circle the leakage area, then click on the Analyze menu and select the Measure option to get the area information of the leakage area. Use the ratio of leakage area and the area of image to indicate vascular permeability.</li>
	<li>Analyze the ratio of leakage area and area of the image from different groups to statistically evaluate the difference in vascular permeability between these groups.</li>
</ol>

Simplifying Angiogenesis Studies with a Modified Matrix Gel Plug Assay

<ol>
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The authors declare no conflict of interest.

We present a reliable and convenient method for the quantitative evaluation of angiogenesis in vivo without staining. In this protocol, vascular cells were mixed with matrix gel in the presence of pro-angiogenic or anti-angiogenic reagents, and then subcutaneously injected into the back of Nu/Nu mice to form gel plug (Figure 1). After 7 days of gel plug formation, dextran-FITC was intravenously injected and circulated for 30 min. The gel plug was collected and embedded with tissue e...

Angiogenesis, the process of formation of neo-vessels from pre-existing vessels, plays a critical role in many physiological and pathological processes, such as embryonic development, wound healing, atherosclerosis, tumor development, etc.1,2,3,4,5. This dynamic process involves several steps, including the degradation of the matrix, vascular cell proliferation, migration and self-organization to form tubular structures and the stabilization of the neo-vessels6. Promoting angiogenesis has been demonstrated to be critical in the treatment of myocardial infarction, stroke and other kinds of ischemic diseases7 while inhibiting angiogenesis has been considered a promising strategy in the treatment of cancers8 and rheumatoid diseases9. Angiogenesis has been considered an organizing principle for drug discovery10. Thus, the construction of a reliable and convenient method to assess the extent of angiogenesis is critical for mechanical research or drug discovery in angiogenesis-dependent diseases.
Several in vitro and in vivo models have been developed to evaluate angiogenesis11. Among these, two-dimensional (2-D) models, like matrix gel tube formation assay12, cannot form functional tubular structures. The animal models, such as the hind limb ischemia model13,14, can reproduce the angiogenesis process but are complex and require a laser speckle blood flow imaging system. 3D models of vascular morphogenesis, like matrix gel plug assay, provide a simple platform that can mimic the process of angiogenesis in vivo15, but the detection of angiogenesis requires immunohistochemistry or immunofluorescence staining16,17,18, which are variable and poorly visualized.
Here, we describe a protocol for a modified matrix gel plug assay where vascular cells were mixed with matrix gel and subcutaneously injected into the back of mice to form a plug. In the plug, vascular cells need to degrade the matrix, proliferate, migrate, and self-organize to finally form functional vessels with blood flow in the internal environment. Thereafter, fluorescent-labeled dextran is injected via the tail vein, to flow through the plug, and the label is visualized to indicate neo-vessels. The content of angiogenesis can be quantitatively evaluated by the length of the vessels. This method can form functional vessels that cannot be produced in 2-D angiogenesis models12, and does not need complex stain process as in ordinary matrix gel plug assay11. It also does not require expensive specific instruments like laser speckle blood flow imaging system in hind limb ischemia model13,14,19. This method is versatile, low-cost, quantifiable, and easy to perform, and can be used to determine the pro- or anti-angiogenic capability of drugs or be used in mechanical research involved in angiogenesis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adhesion Microscope Slides</td>
			<td>CITOTEST</td>
			<td>188105</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia System</td>
			<td>RWD</td>
			<td>R640-S1</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counter</td>
			<td>Invitrogen</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryoslicer</td>
			<td>Thermo Fisher</td>
			<td>CryoStar NX50</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrans-FITC-150kDa</td>
			<td>WEIHUA BIO</td>
			<td>WH007N07</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrans-FITC-4kDa</td>
			<td>WEIHUA BIO</td>
			<td>WH007N0705</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding Cassettes</td>
			<td>CITOTEST</td>
			<td>80203-0007</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Medium</td>
			<td>ScienCell</td>
			<td>35809</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Growth Supplements</td>
			<td>ScienCell</td>
			<td>1025</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10100147C</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast Growth Factor 1</td>
			<td>AtaGenix</td>
			<td>9043p-082318-A01</td>
			<td>FGF1</td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>Nikon</td>
			<td>ECLIPSE Ni</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>Boruida</td>
			<td>30-50-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Syringe</td>
			<td>BD</td>
			<td>300841</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD</td>
			<td>R510-22-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory&#160;Balance</td>
			<td>Sartorius</td>
			<td>BSA124S-CW</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356234</td>
			<td>Matrix gel</td>
		</tr>
		<tr>
			<td>Medium 199 powder</td>
			<td>Gibco</td>
			<td>31100-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtubes</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimal Cutting Temperature (OCT) Compound</td>
			<td>SUKURA</td>
			<td>4583</td>
			<td>Tissue embedding gel</td>
		</tr>
		<tr>
			<td>Palmitate Acid</td>
			<td>KunChuang</td>
			<td>KC001</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Liquid</td>
			<td>Solarbio</td>
			<td>P1400</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline</td>
			<td>Solarbio</td>
			<td>P1022</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Instruments</td>
			<td>RWD</td>
			<td>RWD</td>
			<td></td>
		</tr>
		<tr>
			<td>Tail Vein Injection Instrument</td>
			<td>KEW BASIS</td>
			<td>KW-XXY</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA Solution</td>
			<td>Solarbio</td>
			<td>T1320</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Low Temperature Freezer</td>
			<td>eppendorf</td>
			<td>U410</td>
			<td></td>
		</tr>
		<tr>
			<td>Vascular Endothelial Growth Factor</td>
			<td>CHAMOT</td>
			<td>CM058-5HP</td>
			<td>VEGF</td>
		</tr>
	</tbody>
</table>

modified in vivo matrix gel plug assay for angiogenesis studies

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized equipment, or are hard to perform for subsequent quantitative analysis. Here we present a modified matrix gel plug assay to evaluate angiogenesis in vivo. In this protocol, vascular cells were mixed with matrix gel in the presence or absence of pro-angiogenic or anti-angiogenic reagents, and then subcutaneously injected into the back of recipient mice. After 7 days, phosphate buffer saline containing dextran-FITC is injected via the tail vein and circulated in vessels for 30 min. Matrix gel plugs are collected and embedded with tissue embedding gel, then 12 &#181;m sections are cut for fluorescence detection without staining. In this assay, dextran-FITC with high molecular weight (~150,000 Da) can be used to indicate functional vessels for detecting their length, while dextran-FITC with low molecular weight (~4,400 Da) can be used to indicate the permeability of neo-vessels. In conclusion, this protocol can provide a reliable and convenient method for the quantitative study of angiogenesis in vivo.

Author Spotlight: Investigating Angiogenesis and Vessel Permeability Through a Modified Matrix Gel Plug Assay

Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies

Our research deals with angiogenesis, the process of formation of new vessels from preexisting vessels. It plays a critical role in many physiological and pathological processes, such as embryonic development or curing atherosclerosis, tumor development, etc. The construction of a reliable model to evaluate the extent of angiogenesis is critical for myocardial research or the discovery of medicines for angiogenesis-depend diseases.However, most of the existing models are complex, expensive, and require specialized equipment. Here, we present a modified matrix gel plug assay that can be used to investigate angiogenesis and vessel permeability without staining in a simple, more intuitive, as well as efficient manner.

Figure 1 is the flowchart depicting how to prepare the mixture of matrix gel, vascular cells, culture medium and reagent. The mixture was then subcutaneously injected into the back of Nu/Nu mice and heated using a heating pad to accelerate its coagulation to finally form gel plug.
Figure 2A is the flowchart to indicate vessels with fluorescent labeled dextran. Fluorescent labeled dextran was injected via the tail vein and circle for 3...

Watch this Scientific Journal Video about Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies at JoVE.com

The method presented here can evaluate the effect of reagents on angiogenesis or vascular permeability in vivo without staining. The method uses dextran-FITC injection via the tail vein to visualize neo-vessels or vascular leakage.

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized ...

modified-in-vivo-matrix-gel-plug-assay-for-angiogenesis-studies

Research

JoVE Journal

Biology

Preparing Vascular Cells for In Vivo Matrix Gel Plug Assay

Begin by preparing the complete endothelial culture medium using 460 milliliters of endothelial cell medium, and adding into it, 50 milliliters of FBS, five milliliters of penicillin streptomycin, and five milliliters of endothelial cell growth supplement. The prepared media can be stored at four degrees celsius for a month. Next, in a 100 millimeter tissue culture dish containing 0.1 million vascular cells at eight milliliters of the prepared complete endothelial culture medium and culture the cells at 37 degrees Celsius and 5%carbon dioxide until 70%co-fluency is achieved.Then, remove the culture medium from the dish and rinse the cells twice with PBS to eliminate unattached cells and debris. After removing the PBS, add three milliliters of 0.25%trypsin, containing 2.21 millimolar EDTA to the cells and incubate the dish at 37 degrees Celsius for one minute. Verify cell detachment under a light microscope at 40 times magnification.Neutralize the trypsin with seven milliliters of complete endothelial culture medium and gently rinse the cells off the culture dish. Centrifuge the cell suspension after collecting it in a 15 milliliter tube at 400G for 10 minutes. Re-suspend the cells in five milliliters of complete endothelial culture medium after removing the supernatant, then count the cells using a hemo-cytometer, and transfer a calculated volume of suspension containing 2 million cells to a sterile 1.5 milliliter tube.Pellet the cells by centrifuging the suspension at 400G for five minutes before removing the supernatant. The vascular cells are now ready for mixing with the matrix gel for matrix gel plug assay.

Preparing Matrix Gel with Vascular Cells for In Vivo Matrix Gel Plug Assay

To begin, prepare 10 times M199 culture medium by dissolving M199 powder to 10 times concentration in 90 milliliters of deionized water, followed by the addition of 10 milliliters of FBS. Filter the solution through a 0.22 micron filter. The filtered medium can be stored at four degrees Celsius for up to two months.For preparing the matrix gel with vascular cells, use about 2 million vascular cells. Cool the sterile 1.5 milliliter tube containing the appropriately prepared cell pellet in an ice bath. Thaw the matrix gel completely in an ice bath without mixing or vortexing it.Similarly, cool the prepared 10 times M199 culture media along with the test reagent or drug of interest to four degrees Celsius. Next, maintaining a volume ratio of 8.8 to 1 to 0.2, combine the pre-cooled matrix gel with the pre-cooled 10 times M199 media containing 10%FBS, and the pre-cooled test reagent to produce the matrix gel mixture. Finally, re-suspend the pre-cooled cells in 400 microliters of the prepared matrix gel mixture by mixing gently to avoid forming bubbles.Keep the tube containing the cells in matrix gel on ice until the mice are ready for the matrix gel injection.

Matrix Gel Mixture Injection into Mouse for Matrix Gel Plug Assay

To begin, place the 30 gauge one milliliter insulin syringes in a four degrees Celsius refrigerator for cooling. Keep the freshly prepared matrix gel mix containing vascular cells on ice until the mouse is ready for the injection. Next, place the properly anesthetized mouse on the operating board in a prone position and secure its limbs onto the board using tape.Load a pre cooled insulin syringe with 300 microliters of the matrix gel mixture, avoiding the formation of bubbles. Disinfect the skin on the back of the mouse using 75%alcohol pads, then subcutaneously inject 300 microliters of the matrix gel mixture in the syringe into one side of the animal's back. Gently remove the needle from the injection site to prevent any leakage of the gel mixture.Ensure the formation of a small hump at the injection site. Position the mouse on a heating pad for two minutes to allow the matrix gel mixture to coagulate and form a plug. Repeat the demonstrated steps to create a plug on the other side of the mouse's back.Mark the edges of the hump using a marker pen. House the animal in a specific pathogen free experimental animal laboratory at 20 to 24 degrees with 12 hour light and 12 hour dark cycles for seven days.

Dextran-FITC Injection and Matrix Gel Plug Collection from Mouse for Matrix Gel Plug Assay

To inject the mouse with Dextran, FITC seven days post the matrix gel injection, RESUSPEND 0.5 milligrams of Dextran, FITC in 500 microliters of double distilled water. Thoroughly vortex it to obtain a one microgram per microliter Dextran, FITC solution. Fill a 29 gauge syringe with 50 microliters of the Dextran FITC solution.Secure the mouse on the tail vein injection instrument, sanitize the tail with a 75%alcohol soaked cotton ball before carefully injecting 50 microliters of the Dextran FITC solution in the syringe into the tail vein. Apply pressure to the injection site with the cotton swab for one minute to staunch bleeding. Return the mouse to its cage and let it relax for 30 minutes before collecting the matrix gel plug.To collect the gel plug, cut the skin of the euthanized mouse along the marked border of the plug, using surgical scissors. And remove the skin above the matrix gel plug. Collect the plug and rinse it in a beaker with phosphate buffered saline to wash off any excess blood.Next, cover the bottom of the tissue embedding cassette with about 0.5 milliliters of tissue embedding gel. Then swiftly place the matrix gel plug on the tissue embedding gel in the planned orientation. Embed the plug with additional tissue embedding gel.Solidify the cassettes at minus 80 degrees Celsius for 12 hours. After removing the solidified plug block, cut 12 micron thick sections using a freezing microtome. Then mount the sections onto microscope slides.

538 Views.  Pingyang Affiliated Hospital of Wenzhou Medical University.  The method presented here can evaluate the effect of reagents on angiogenesis or vascular permeability in vivo without staining. The method uses dextran-FITC injection via the tail vein to visualize neo-vessels or vascular leakage.

Simplifying Angiogenesis Studies with a Modified Matrix Gel Plug Assay

Summary