Name: in vitro 3d cell-cultured arterial models for studying vascular drug targeting under flow
Uploaded: 2021-03-14T14:30:00
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 bifur...

<ol>
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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamics+and+atherosclerosis.+Insights+and+perspectives+gained+from+studies+of+human+arteries.">Hemodynamics and atherosclerosis. Insights and perspectives gained from studies of human arteries.</a> Archives of Pathology &amp; Laboratory Medicine. 112 (10), 1018-1031 (1988).</li><li>Khoury, M., Epshtein, M., Zidan, H., Zukerman, H., Korin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+deposition+of+particles+in+reconstructed+models+of+human+arteries.">Mapping deposition of particles in reconstructed models of human arteries.</a> Journal of Controlled Release. 318, 78-85 (2020).</li></ol>

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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>&#160;Thermo-Fisher Scientific</td>
			<td>&#160;F8827</td>
			<td>&#160;Diameter = 2 &#181;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&#160;</td>
			<td>SolidWorks Corp.,&#160;Dassault Syst&#232;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></td>
			<td></td>
			<td></td>
			<td>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 flui...

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 ...

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