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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Quantitative correlation of plaque localization with flow velocity profiles and wall shear stress.</a> Circulation Research. 53 (4), 502-514 (1983).</li><li>Chien, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+disturbed+flow+on+endothelial+cells.">Effects of disturbed flow on endothelial cells.</a> Annals of Biomedical Engineering. 36 (4), 554-562 (2008).</li><li>Malek, A. M., Alper, S. L., Izumo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+shear+stress+and+its+role+in+atherosclerosis.">Hemodynamic shear stress and its role in atherosclerosis.</a> JAMA. 282 (21), 2035-2042 (1999).</li><li>Glagov, S., Zarins, C., Giddens, D. P., Ku, D. 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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...

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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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

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

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 ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Targeting Neuronal Fiber Tracts for Deep Brain Stimulation Therapy Using Interactive, Patient-Specific Models

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for movement disorders and is being applied to a growing number of disorders. Computational modeling has been successfully used to predict the clinical effects of DBS; however, there is a need for novel modeling techniques to keep pace with the growing complexity of DBS devices. These models also need to generate predictions quickly and accurately. The goal of this project is to develop an image processing pipeline to incorporate structural magnetic resonance imaging (MRI) and diffusion weighted imaging (DWI) into an interactive, patient specific model to simulate the effects of DBS. A virtual DBS lead can be placed inside of the patient model, along with active contacts and stimulation settings, where changes in lead position or orientation generate a new finite element mesh and solution of the bioelectric field problem in near real-time, a timespan of approximately 10 seconds. This system also enables the simulation of multiple leads in close proximity to allow for current steering by varying anodes and cathodes on different leads. The techniques presented in this paper reduce the burden of generating and using computational models while providing meaningful feedback about the effects of electrode position, electrode design, and stimulation configurations to researchers or clinicians who may not be modeling experts.

The goal of this project is to develop an interactive, patient-specific modeling pipeline to simulate the effects of deep brain stimulation in near real-time and provide meaningful feedback as to how these devices influence neural activity in the brain.

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for ...

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 ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...