Name: in vitro 3d cell-cultured arterial models for studying vascular drug targeting under flow
Uploaded: 2021-03-14T14:30:00.000+00:00
Duration: 7 min
Description: Watch this Scientific Journal Video about In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow at JoVE.com

This work was supported by the Israel Science Foundation (ISF grant # 902/18). Maria Khoury&#39;s scholarship was supported by The Baroness Ariane de Rothschild Women Doctoral Program.

NOTE: This protocol describes the fabrication of a 3D model of the carotid artery and can be applied to generate any other artery of interest by simply modifying the geometric parameters.
1. Design and fabrication of a 3D bifurcation of the human carotid artery model
<ol>
	<li>Choose images from patients or previously studied geometries of the human carotid artery bifurcation, and create a computer-aided design model of the mold that needs to be printed. 
	NOTE: The carotid artery bifurcation has one inlet and two outlets. It is important to design a 3D mold frame around the artery and temporary printing supports between the frame and the artery mold (Figure 2A-C).</li>
	<li>Print the geometries using a 3D printer. Cut the temporary printing supports, and use sandpaper to polish and smooth the molds, especially in the areas where the supports were cut. Rinse the sanded model with isopropyl alcohol to remove the plastic dust, and allow to completely dry in a chemical hood for 2-3 h. 
	NOTE: Here, the printed molds were made of clear resin v4 (Figure 2D,E).</li>
	<li>To easily dissolve the plastic, spray the molds with transparent lacquer inside a chemical hood, and allow to air-dry for 1 h. Repeat this 3x. 
	NOTE: Here, 2X Ultra Cover Clear Spray was used, but any other kind should be suitable as long as it is not wood lacquer. Confirm that there is no exposed plastic left because the plastic may react with the silicone and prevent it from properly solidifying. The quality of the sprayed surface will determine the quality of the surface of the final silicone model.</li>
	<li>Cut transparent rectangular slides/strips of smooth plastic of the same dimensions as the mold frame, and glue them using the lacquer to the frame on all sides and from one side of the frame, such that it will be sealed at the bottom and open at the top. Apply the lacquer using a paint brush inside a chemical hood, and allow the slides to completely dry for at least 24 h (Figure 2F).</li>
	<li>To prepare the silicone rubber mixture, add liquid silicone with its curing agent (mass ratio 1:10) in a plastic plate, and stir thoroughly&#160;to ensure complete mixing. For the carotid model, add 54 g of the silicone and 6 g of its curing agent.</li>
	<li>Cool the mixture for 15 min at 4 &#176;C, then degas it in a vacuum desiccator until all the air bubbles have been eliminated. Place the mold in the desiccator (with the open side facing up), slowly pour the silicone mixture into the mold, and again remove the air bubbles until the mixture is clear.</li>
	<li>Let the mold stand with the silicone overnight at room temperature (if possible, leave it in the desiccator without vacuum). If the mixture has not fully dried, incubate it at 60 &#176;C for another few hours.</li>
	<li>Once the mixture is fully dry, remove the transparent slides, and immerse the model in absolute acetone for 48 h in a chemical hood until the plastic is fully dissolved. Remove any plastic leftovers with a wooden stick. To evaporate the acetone trapped inside the model, incubate it at 60 &#176;C for at least 4 days before cell seeding.</li>
</ol>
2. Cell culture and seeding in models
<ol>
	<li>Prepare three connectors for the carotid model: one for the inlet and two for the outlets (see Table of Materials). Sterilize the model and the connectors by ultraviolet irradiation in a biological hood for 20 min.</li>
	<li>Coat the models with 4 mL of 100 &#181;g/mL fibronectin (in 1x phosphate-buffered saline, (PBS)) for 2 h at 37 &#176;C or overnight at 4 &#176;C. Inject the fibronectin solution into the model through the inlet using a 5 or 10 mL plastic syringe. Remove the fibronectin through the outlet, and wash the model with EC medium.</li>
	<li>Suspend 2.5 &#215; 106 cells/mL human umbilical vein endothelial cells (HUVECs, passage &#60; 6), and fill the model with 4 mL of the cell suspension using a 5 or 10 mL plastic syringe (Figure 3A). Place the model on a rotator inside an incubator (37 &#176;C) at a speed of 1 rpm for 48 h to ensure homogeneous seeding. Make sure the model is well-attached to the rotator (Figure 3B). Change the medium after 24 h inside a biological hood, and return to the rotator inside the incubator for another 24 h. 
	NOTE: After 24 h, the cells are seeded and can be imaged using a microscope.</li>
	<li>Remove the model from the rotator, and wash with 1x PBS using a 10 mL plastic syringe. To fix the cells, incubate the cells with 4 mL of 4% paraformaldehyde (PFA) to the model for 15 min inside a chemical hood, and then rinse 3x with PBS. Add 4 mL of PBS, and store at 4 &#176;C until the experiment (Figure 3C). Stain the cells inside the model using standard staining protocols (e.g., nuclear staining with 4&#8242;,6-diamidino-2-phenylindole (DAPI), Figure 3D).</li>
</ol>
3. Design of the perfusion system
<ol>
	<li>The perfusion system has two inlets and two outlet tubes. Merge the two inlets into a single 4 mm ID tube and again into two 6 mm ID tubes, which connect to the peristaltic pump. Merge the two 6 mm ID tubes coming out of the peristaltic pump into a single 4 mm tube, and connect it to an oscillation damper to eliminate any oscillations from the pump. Use a 250 mL narrow-mouthed bottle with a three-orifice lid as the damper.</li>
	<li>Connect one orifice to the inlet from the pump, close the second with a cork that is used for pressure venting in emergencies, and extend the third orifice (which is the outlet) to the bottom of the bottle.</li>
	<li>Connect the damper to the inlet of the cultured carotid model using the outlet tubing. Merge the two outlets of the model to one tubing, which will be the outlet of the system (All tubing are 4 mm ID).</li>
	<li>Split the outlet tubing to two outlet tubes (one for the closed circuit and the other for the waste container in the open circuit). Attach a plastic clamp to each tube. 
	NOTE: The combination of open/closed clamps will determine whether the system is in a closed or open circuit configuration. As shown in Figure 1, if clamps a and d are close while b and c are open, the system is a closed circuit; the reverse brings the system to open circuit configuration.</li>
	<li>Prepare three containers: one that can hold 300 mL fluid (for closed-circuit) and two others of 1 L each: one for washing and the other for waste (for open circuit).</li>
</ol>
4. Closed-circuit configuration: perfusion experiment and imaging
<ol>
	<li>Add 300 mL of PBS to the closed-circuit container, which is sufficient to fill the entire system, including the tubing and the model. Place one inlet tube and one outlet tube (open clamps b and c) inside the container.</li>
	<li>Fill the 1 L washing container with distilled water (for washing at the end of the experiment), and leave the other 1 L waste container empty. Place the other inlet and outlet tubing (close clamps a and d) in the washing and waste containers, respectively.</li>
	<li>Take the fixed cell cultured carotid model out of 4 &#176;C storage, and empty the PBS. Connect the inlet and outlets of the carotid as described in steps 3.3-3.4. Do not leave the model dry for a long time. Once the model is connected, activate the pump to perfuse fluid.</li>
	<li>Place the carotid model under the microscope. Open the tubing before the inlet and after the outlet of the carotid model. Set the peristaltic pump at 10 rpm, and turn it on. Increase the speed in increments of 5 rpm, every 4-5 min. Make sure there are no leakages.</li>
	<li>At 100 rpm, which equals the maximum flow rate of the physiological waveform of the human carotid artery (~400 mL/min), add fluorescent carboxylated polystyrene (PS) particles (2 &#181;m, at a concentration of 1.6 &#181;g/mL) to the 300 mL of PBS in the closed-circuit container. Image the region of interest every 10 s for 1.5 h (still single images or video as needed).</li>
</ol>
5. Open-circuit configuration: the washing step
<ol>
	<li>Open the clamps of the washing and waste tubes in the 1 L containers (clamps a and d), and immediately close the clamps of both the inlet and outlet tubes in the 300 mL container (clamps b and c) to change the system from a closed to open circuit configuration.</li>
	<li>Let most of the water flow from the washing container to the waste container at 100 rpm. Before it is completely transferred, press stop on the peristaltic pump, and close the tube clamps before the inlet and after the outlet of the carotid model.</li>
	<li>Using the appropriate filters, capture images of the model at the region of interest to show the deposition and adhesion of particles to the cells. Disconnect the carotid model. Carefully and slowly add 4 mL of PBS with a 10 mL syringe through the model&#39;s inlet.</li>
	<li>Connect a &#34;dummy model&#34;, (which is also a silicone rubber 3D carotid model used only for washing, without cultured cells) instead of the carotid model, and rinse the system. Add another 1 L of water, and wash the system again until all the water is transferred from the washing container to the waste. Turn off the peristaltic pump.</li>
</ol>
6. Data acquisition and analysis
<ol>
	<li>Acquire a digital movie of particle deposition at the region of interest with the images taken during the experiment, using a customized software code (see the Table of Materials).</li>
	<li>For mapping of the deposition of the particles along the model, tile multiple images to cover the examined region of interest (Figure 4A,B). 
	NOTE: A customized software code can be written to quantify the number of adhered particles at a site of interest (a sample file has been provided as Supplemental Information)17.</li>
</ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+behaviour+of+blood+cells+and+rigid+spheres+in+an+annular+vortex.">Flow behaviour of blood cells and rigid spheres in an annular vortex.</a> Philosophical transactions of the Royal Society of London. Series B, Biological. 279 (967), 413-445 (1977).</li><li>Goldsmith, H. L., Karino, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelets+in+a+region+of+disturbed+flow.">Platelets in a region of disturbed flow.</a> Transactions - American Society for Artificial Internal Organs. 23, 632-638 (1977).</li><li>Farcas, M. A., Rouleau, L., Fraser, R., Leask, R. 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The authors declare no conflicts of interest.

Current approaches to study vascular targeting of particles fall short in replicating the physiological conditions present in the human body. Presented here is a protocol to fabricate 3D-reconstructed models of human arteries to study particle targeting to the ECs lining the artery under physiological flow applied using a customized perfusion system. When choosing the material for 3D printing, it is best to use a clear plastic to avoid pigment transfer to the silicone model, which should be as transparent as possible. In addition, it is important to choose a material that does not dissolve in acetone, but instead becomes soft and brittle and can subsequently easily be removed from the model.
The presented 3D models are made of silicone rubber, a transparent silicone, mixed with its curing agent. It is important to ensure that the mixture is always at room temperature or below, otherwise the crosslinking between the silicone and the curing agent will begin before the degassing and casting onto the molds. Although polydimethylsiloxane can also be used to fabricate such models (1:10 ratio with its crosslinker), silicone rubber is more durable. After immersing the model in acetone to dissolve the plastic, it is crucial to incubate it at 60 &#176;C for at least 4 days to ensure full evaporation of any acetone residue. If any acetone remains trapped inside the model, the cells will not grow properly. Changing the medium after 24 h and fixation of the cells after 48 h are the two steps that involve manual injection of fluid using a 10 mL syringe. It is therefore important to perfuse slowly, otherwise the cells might be washed out. 
The perfusion system has two inlets and two outlet tubes. Each tube has a plastic tube clamp for flow control. Most of the tubing system is comprised of 4 mm inner dimeter (ID) tubes, except for the tubes that are clamped in the pump, which are 6 mm ID tubes. The ID of the tubes clamped in the pump will determine the maximum flow rate that can be achieved in the system. This perfusion system can also generate a pulsatile waveform by superposing oscillations on the constant mean flow17 by connecting the outlet of the damper to an oscillator assembly, which superposes the oscillatory part of a desired waveform on the constant flow rate produced by the peristaltic pump. This configuration enables operation under oscillatory flow or under constant flow conditions when the oscillator is turned off.
In this paper, the perfusion system was customized based on experiments with the 3D human carotid artery bifurcation. Therefore, if other arterial models or other tubing are used, the amounts of fluids and flow rate may require adjustments. In such cases, the system and flow rate will have to be calibrated, while ensuring no cell detachment from the model walls. It is very important to gradually increase the flow rate in the peristaltic pump to guarantee that the cells are not washed away with the flow. Moreover, it is crucial to ensure that the entire system, including the tubing, the model, as well as the container are filled with fluids (e.g., in this case, it was filled with a total volume of 300 mL of fluid).In addition, before and after each experiment, the system should be washed with distilled water in an open-circuit configuration.
Blood can also be perfused into the models using the perfusion system17. In this case, extra caution must be exercised to prevent any leakage, especially if human blood is used. Moreover, the washing step is crucial as bleach must be perfused at the end of the experiment to ensure full wash out of the blood. After the bleach, water should be perfused as mentioned in the protocol. It is important to note that in this paper, carboxylated PS particles were used, which have a uniform composition and a narrow size distribution. Moreover, these particles adhere to the cells primarily through electrostatic interactions. However, other drug nanocarriers may be used, and specific targeting should be examined as well with ligand-labeled particles, e.g., to anti-intercellular adhesion molecule 1 and anti-platelet endothelial cell adhesion molecule 1, which will increase particle accumulation to ECs at the site of interest. 
In addition, in this protocol, ECs were fixed prior to connecting the models to the perfusion system and injection of the particles. The adhesion of particles to fixed cells represents the first stage in the binding process and therefore, experiments with live cells need to be performed, where internalization of particles may occur during later stages of the adhesion process. This protocol can be used to fabricate 3D arterial models for the study of drug carriers under physiological conditions. The outlined approach may assist in the study of delivery of agents under patient-specific conditions.

Several approaches have recently been applied utilizing 3D models of human arteries1,2,3,4,5. These models replicate the physiological anatomy and environment of different arteries in the human body in vitro. However, they have been mainly used in cell biology studies. Current studies on vascular targeting of particles to the endothelium include in silico computational simulations6,7,8, in vitro microfluidic models9,10,11, and in vivo animal models12. Despite the insights they have provided, these experimental models have failed to accurately simulate the targeting process that occurs in human arteries, wherein blood flow and hemodynamics constitute dominant factors. For example, the study of particle targeting to atherosclerotic regions in the carotid artery bifurcation, which are known for their complex recirculation flow pattern and wall shear stress gradient, may impact the journey taken by the particles before they reach the endothelium13,14,15,16. Therefore, these studies must be performed under conditions that replicate the physiological environment, i.e., size, dimension, anatomy, and flow profile.
Recently, this research group fabricated 3D-reconstructed human arterial models to study the deposition and targeting of particles to the vasculature17. The models were based on geometrical 3D replicas of human blood vessels, which were then cultured with human ECs that subsequently lined their inner walls. In addition, when subjected to a perfusion system that produces physiological flow, the models accurately replicated physiological conditions. The perfusion system was designed to perfuse fluids at a constant flow rate, using a peristaltic pump in both closed and open-circuit configurations (Figure 1). The system can be used as a closed-circuit to map particle deposition and targeting to the cells seeded inside the carotid model. In addition, it can be used as an open circuit to wash out non-adherent particles at the end of the experiments and to clean and maintain the system. This paper presents protocols for the fabrication of 3D models of the human carotid bifurcation, design of the perfusion system, and mapping of the deposition of targeted particles inside the models.

<table><tbody><tr><td>3D printer</td><td>FormLabs</td><td>PKG-F2-REFURB</td><td></td></tr><tr><td>Acetone, absolute (AR grade)</td><td></td><td></td><td></td></tr><tr><td>Connectors</td><td>Nordson Medical</td><td>FTLL013-1</td><td>Female Luer</td></tr><tr><td></td><td></td><td>FTLL230-1</td><td>Female Luer</td></tr><tr><td></td><td></td><td>FTLL360-1</td><td>Female Luer</td></tr><tr><td></td><td></td><td>LP4-1</td><td>Male Luer Integral Lock</td></tr><tr><td>Damper</td><td>Thermo-Fisher Scientific</td><td>DS2127-0250</td><td>Nalgene Polycarbonate, Validation Bottle</td></tr><tr><td>Damper Cover</td><td>Thermo-Fisher Scientific</td><td>2162-0531</td><td>Nalgene Filling/Venting Closures</td></tr><tr><td>Elastosil Elastosil RT 601 A</td><td>Wacker</td><td>60003805</td><td></td></tr><tr><td>Elastosil RT 601 B</td><td>Wacker</td><td>60003817</td><td>The crosslinker</td></tr><tr><td>Endothelial Cell Media</td><td>ScienCell</td><td>1001</td><td></td></tr><tr><td>Fibrontectin</td><td>Sigma Aldrich</td><td>F0895-5mg</td><td></td></tr><tr><td>HUVEC</td><td>Lonza</td><td>CC-2519</td><td></td></tr><tr><td>Isopropyl alcohol, AR grade 99.5%</td><td></td><td></td><td>Remove plastic dust from the sanded model</td></tr><tr><td>Lacquer</td><td>Rust-Oleum</td><td></td><td>2X-Ultra cover Gloss Clear</td></tr><tr><td>Matlab</td><td>Mathworks</td><td></td><td>https://www.mathworks.com/products/matlab.html</td></tr><tr><td>Microscope</td><td>Nikon</td><td>SMZ25</td><td></td></tr><tr><td>Microscope Camera</td><td>Nikon</td><td>DS-Qi2</td><td></td></tr><tr><td>Peristaltic pump</td><td>Watson Marlow</td><td>530U IP31</td><td>With 2 pumpheads: 313D</td></tr><tr><td>Plastic tube clamp</td><td>Quickun</td><td>1-2240-stopvalve-2pcs</td><td></td></tr><tr><td>Polystyrene Particles</td><td>&amp;nbsp;Thermo-Fisher Scientific</td><td>&amp;nbsp;F8827</td><td>&amp;nbsp;Diameter = 2 &amp;micro;m</td></tr><tr><td>Printer resin</td><td>FormLabs</td><td>RS-F2-GPCL-04</td><td></td></tr><tr><td>Rotator</td><td>ELMI Ltd.</td><td></td><td>Intelli-Mixer RM-2</td></tr><tr><td>Solidworks&amp;nbsp;</td><td>SolidWorks Corp.,&amp;nbsp;Dassault Syst&amp;egrave;mes</td><td></td><td>https://www.solidworks.com/</td></tr><tr><td>Tubing</td><td>Watson Marlow</td><td>933.0064.016</td><td>Tubing for the pump: 6.4 mm ID</td></tr><tr><td colspan="4">All the other tubing: Silicon tubing: 4 mm ID</td></tr></tbody></table>

in vitro 3d cell-cultured arterial models for studying vascular drug targeting under flow

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of various important processes in the cardiovascular system. Recently, although several biological studies have been performed using such 3D models of human arteries, they have not been applied to study vascular targeting. This paper presents a new method to fabricate real-sized, reconstructed human arterial models using a 3D printing technique, line them with human endothelial cells (ECs), and study particle targeting under physiological flow. These models have the advantage of replicating the physiological size and conditions of blood vessels in the human body using low-cost components. This technique may serve as a new platform for studying and understanding drug targeting in the cardiovascular system and may improve the design of new injectable nanomedicines. Moreover, the presented approach may provide significant tools for the study of targeted delivery of different agents for cardiovascular diseases under patient-specific flow and physiological conditions.

This paper presents a new protocol to map the deposition of particles inside real-sized 3D human artery models, which may provide a new platform for drug delivery research. Using a 3D printing technique, a model of the human carotid bifurcation artery was fabricated (Figure 2). The model was made of silicone rubber and seeded with human ECs (Figure 3). Importantly, this protocol enabled the replication of physiological conditions, especially with respect to fluid dynamics. A perfusion system was designed to infuse particles to the carotid bifurcation under constant flow at the magnitude of the physiological waveform characteristic of the carotid. Figure 1 presents the perfusion system, which consists of the peristaltic pump, an oscillation damper, the cultured bifurcation model, tubing, and fluid containers.
To map the deposition and adhesion of the perfused particles, the arterial model was imaged under a stereomicroscope, both at the end of the experiment and after washing (step 5.3). The images were captured using 2x objective and tiled together to form a whole image of the model. Then, the number of adhered particles was calculated using a customized software code. To examine the formation of the recirculation pattern at the bifurcation, 10 &#181;m fluorescent glass beads were infused into the model. Figure 4A shows the recirculation, which suggests that the conditions inside the model mimic physiological conditions.
To map the deposition of particles inside the model, 2 &#181;m fluorescent carboxylated PS particles were infused, and their adhesion to the ECs was imaged (Figure 4B,C). These particles adhered differently at various regions along the model-more adhesion was observed out of the recirculation area, where wall shear stress is high. These results have been previously discussed to show that the adhesion of particles is a function of the model&#39;s geometry, particle surface characteristics, and shear stress17. These deposition maps are relatively simple and may be quickly obtained for screening drug carriers&#39; affinity and targeting under physiological conditions in patient-specific models.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62279/62279fig01.jpg" /> 
Figure 1: The perfusion system. A perfusion system was designed to perfuse fluids under constant flow. It is comprised of (1) a peristaltic pump, (2) an oscillation damper, (3) the cultured 3D arterial model, and three glass containers: two with a 1 L capacity (4 and 6) and a third that can hold 300 mL fluid (5). The system can operate in two configurations: (i) an open circuit, in which clamps a+d are open and b+c are closed, or (ii) a closed circuit, in which clamps a+d are closed and b+c are open. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62279/62279fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62279/62279fig02.jpg" /> 
Figure 2:&#160;Fabrication process of a 3D carotid artery bifurcation model. (A-C) Human carotid bifurcation, the mold frame around the artery, and temporary printing supports were designed. (D, E) The geometries were printed using a 3D printer. (F) The temporary printing supports were cut, and the model was sanded and sprayed with lacquer. Then, transparent rectangular slides were glued to the frame from all sides. Silicone rubber was cast when the glue was dry. Abbreviation: 3D = three-dimensional. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62279/62279fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62279/62279fig03.jpg" /> 
Figure 3: Seeding of ECs inside 3D models of the carotid artery. (A) Real-sized 3D model of the human carotid bifurcation made of silicone rubber. The model was cultured with human ECs and filled with cell medium. (B)&#160;The cultured model was placed on a rotator at 37 &#176;C for 48 h. (C) Images of the cultured ECs inside the 3D model in brightfield and (D) with DAPI for nuclear staining in blue. Scale bars = 10 &#181;m. Abbreviations: ECs = endothelial cells; DAPI = 4&#8242;,6-diamidino-2-phenylindole; 3D = three-dimensional. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62279/62279fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62279/62279fig04.jpg" /> 
Figure 4: Perfusing and mapping the adhesion of particles. (A) Streak-line image of streamlines and recirculation (dashed rectangle) generated upon perfusion of 10 &#181;m fluorescent glass particles at a constant flow of 400 mL/min through the model. (B) Deposition map of the 2 &#181;m fluorescent carboxylated PS particles (in red) inside the 3D-cultured model. Scale bar = 2 mm. (C) Adhesion of the particles (in red) to the cultured ECs (in blue-DAPI) inside the model at a 10x magnification. Scale bar = 10 &#181;m. Abbreviations: PS = polystyrene; 3D = three-dimensional; ECs = endothelial cells; DAPI = 4&#8242;,6-diamidino-2-phenylindole. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62279/62279fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Information: <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62279/JoVE_Particle_Counting_MK.m" target="_blank">Please click here to download&#160;this File.</a>

Watch this Scientific Journal Video about In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow at JoVE.com

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

Here, we present a new protocol to study and map the targeted deposition of drug carriers to endothelial cells in fabricated, real-sized, three-dimensional human artery models under physiological flow. The presented method may serve as a new platform for targeting drug carriers within the vascular system.

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of ...

in-vitro-3d-cell-cultured-arterial-models-for-studying-vascular-drug

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

3.1K Views.  Technion - IIT. Here, we present a new protocol to study and map the targeted deposition of drug carriers to endothelial cells in fabricated, real-sized, three-dimensional human artery models under physiological flow. The presented method may serve as a new platform for targeting drug carriers within the vascular system. 

Video: In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

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

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

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

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

Developmental Biology

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Immunology and Infection

Micropatterning and Assembly of 3D Microvessels

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

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

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

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

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

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

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

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

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

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

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

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

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current in vitro screening models that have sufficient throughput only have limited physiological relevance, which hinders the translation of findings from in vitro to in vivo. On the other hand, microfluidic cell culture platforms have shown unparalleled physiological relevancy in vitro, but often lack the required throughput, scalability and standardization. We demonstrate a robust platform to study angiogenesis of endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) in a physiological relevant cellular microenvironment, including perfusion and gradients. The iPSC-ECs are cultured as 40 perfused 3D microvessels against a patterned collagen-1 scaffold. Upon the application of a gradient of angiogenic factors, important hallmarks of angiogenesis can be studied, including the differentiation into tip- and stalk cell and the formation of perfusable lumen. Perfusion with fluorescent tracer dyes enables the study of permeability during and after anastomosis of the angiogenic sprouts. In conclusion, this method shows the feasibility of iPSC-derived ECs in a standardized and scalable 3D angiogenic assay that combines physiological relevant culture conditions in a platform that has the required robustness and scalability to be integrated within the drug screening infrastructure.

This method describes the culture of iPSC-derived endothelial cells as 40 perfused 3D microvessels in a standardized microfluidic platform. This platform enables the study of gradient-driven angiogenic sprouting in 3D, including anastomosis and stabilization of the angiogenic sprouts in a scalable and high-throughput manner.

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current ...

Studying Tissue Rigidity and Shear Stress Effects on Endothelial Cells

In Vitro Model Integrating Substrate Stiffness and Flow to Study Endothelial Cell Responses

We present an innovative in vitro model aimed at investigating the combined effects of tissue rigidity and shear stress on endothelial cell (EC) function, which are crucial for understanding vascular health and the onset of diseases such as atherosclerosis. Traditionally, studies have explored the impacts of shear stress and substrate stiffness on ECs, independently. However, this integrated system combines these factors to provide a more precise simulation of the mechanical environment of the vasculature. The objective is to examine EC mechanotransduction across various tissue stiffness levels and flow conditions using human ECs. We detail the protocol for synthesizing gelatin methacrylate (GelMA) hydrogels with tunable stiffness and seeding them with ECs to achieve confluency. Additionally, we describe the design and assembly of a cost-effective flow chamber, supplemented by computational fluid dynamics simulations, to generate physiological flow conditions characterized by laminar flow and appropriate shear stress levels. The protocol also incorporates fluorescence labeling for confocal microscopy, enabling the assessment of EC responses to both tissue compliance and flow conditions. By subjecting cultured ECs to multiple integrated mechanical stimuli, this model enables comprehensive investigations into how factors such as hypertension and aging may affect EC function and EC-mediated vascular diseases. The insights gained from these investigations will be instrumental in elucidating the mechanisms underlying vascular diseases and in developing effective treatment strategies.

We synthesized and characterized a tunable gelatin-based substrate for culturing vascular endothelial cells (ECs) under relevant vascular flow conditions. This biomimetic surface replicates both physiological and pathological conditions, enabling the study of mechanical forces on EC behavior and advancing our understanding of vascular health and disease mechanisms.

We present an innovative in vitro model aimed at investigating the combined effects of tissue rigidity and shear stress on endothelial cell (EC) function, ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

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 ...

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

Summary

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Chapters in this video

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

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

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

Micropatterning and Assembly of 3D Microvessels

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

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

In Vitro Model Integrating Substrate Stiffness and Flow to Study Endothelial Cell Responses

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca²⁺]_iand Nitric Oxide Production

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing