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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S., Hughes, T. J., Decuzzi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+deposition+patterns+for+nanoparticles+in+an+inflamed+patient-specific+arterial+tree.">Vascular deposition patterns for nanoparticles in an inflamed patient-specific arterial tree.</a> Biomechanics and Modeling in Mechanobiology. 13 (3), 585-597 (2014).</li><li>Charoenphol, P., Huang, R. B., Eniola-Adefeso, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+role+of+size+and+hemodynamics+in+the+efficacy+of+vascular-targeted+spherical+drug+carriers.">Potential role of size and hemodynamics in the efficacy of vascular-targeted spherical drug carriers.</a> Biomaterials. 31 (6), 1392-1402 (2010).</li><li>Ta, H. T., Truong, N. P., Whittaker, A. K., Davis, T. P., Peter, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+particle+size,+shape,+density+and+flow+characteristics+on+particle+margination+to+vascular+walls+in+cardiovascular+diseases.">The effects of particle size, shape, density and flow characteristics on particle margination to vascular walls in cardiovascular diseases.</a> Expert Opinion on Drug Delivery. 15 (1), 33-45 (2018).</li><li>Cooley, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+particle+size+and+shape+on+their+margination+and+wall-adhesion:+implications+in+drug+delivery+vehicle+design+across+nano-to-micro+scale.">Influence of particle size and shape on their margination and wall-adhesion: implications in drug delivery vehicle design across nano-to-micro scale.</a> Nanoscale. 10 (32), 15350-15364 (2018).</li><li>Jiang, X. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantum+dot+interactions+and+flow+effects+in+angiogenic+zebrafish+(Danio+rerio)+vessels+and+human+endothelial+cells.">Quantum dot interactions and flow effects in angiogenic zebrafish (Danio rerio) vessels and human endothelial cells.</a> Nanomedicine: Nanotechnology, Biology, and Medicine. 13 (3), 999-1010 (2017).</li><li>Zarins, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carotid+bifurcation+atherosclerosis.+Quantitative+correlation+of+plaque+localization+with+flow+velocity+profiles+and+wall+shear+stress.">Carotid bifurcation atherosclerosis. 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. 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 ...

The present protocol may serve as a new platform for the study and development of drug carriers to target disease sites within the human vascular system. The main advantage of our technique is that it allows the study of drug carriers'accumulation inside human-replicated 3d arterial model. This is done under physiological conditions, including blood flow.This approach can be used to optimize drug carriers for the diagnosis and treatment of a wide range of vascular diseases, including a atherosclerotic arteries. Begin by selecting images from patients or previously studied geometries of the human carotid artery bifurcation and using the images to create a computer-aided design model of the mold. Use a 3d printer to print the design, remove the temporary printing supports and rinse the model with acetone before using sandpaper to polish and smooth the molds, especially the areas from which the supports were cut.After sanding, rinse the model with isopropyl alcohol to remove any plastic dust and allow the model to dry in a chemical hood for two to three hours. To facilitate easy disillusion of the plastic, spray the model with transparent lacquer three times, allowing the lacquer to air-dry for one hour between applications. After the last application, use a paint brush and lacquer to glue transparent, rectangular strips of smooth plastic to each side of the frame, such that the model will be sealed at the bottom and open at the top.After drying, place the mold in a desiccator and slowly poor freshly-prepared silicone rubber solution into the opening. Remove air bubbles until the mixture is clear and leave the mold inside the desiccator overnight. When the mixture is fully dry, remove the transparent slides and immerse the model in absolute acetone for 48 hours in a chemical hood until the plastic is fully dissolved.To evaporate the trapped acetone before cell seeding, incubate the model for at least four days at 60 degrees Celsius. To seed the model with cells, first sterilize the model and two inlet and outlet connectors by ultraviolet light or radiation. After 20 minutes, using a syringe to coat the model with four milliliters of 100 microgram per milliliter fibronectin for two hours at 37 degrees Celsius.At the end of the incubation, remove the fibronectin solution through the outlet and wash the model with endothelial cell medium. Use a syringe to fill the rinsed model with four milliliters of a 2.5 times 10 to the sixth endothelial cells per milliliter of endothelial cell medium cell suspension and secure the model to a rotator inside a cell culture incubator 48 hours at one revolution per minute, to ensure homogenous distribution of the cells. At the end of the incubation, use a 10 milliliter plastic syringe to wash the model with PBS.Fix the cells for 15 minutes, with four milliliters of 4%paraformaldehyde, then wash the model three times with PBS as demonstrated before storing the model at four degrees Celsius in four milliliters of fresh PBS. Before beginning an experiment, use the outlet tubing to connect an oscillation damper connected to a peristaltic pump to the inlet of the cultured carotid model and use a piece of tubing to merge the two outlets. Then split the outlet tubing to two outlet tubes and add a plastic clamp to each tube.To set up a closed circuit configuration, add 300 milliliters of PBS to a closed circuit container and place one inlet and one outlet tube inside the PBS filled container with the B and C clamps open, respectively, then place the other inlet tube into a one-liter washing container filled with distilled water, and the other outlet tube into an empty one-liter waste container with the A and D clamps closed, respectively. Place the carotid model under a stereo microscope, set the pump to a starting flow rate of 10 revolutions per minute, with five revolution per minute increment increases every four to five minutes. When the flow rate reaches 100 revolutions per minute, add 1.6 micrograms of fluorescent carboxylated polystyrene particles per milliliter of PBS to the closed circuit container and image the region of interest every 10 seconds for one and a half hours.To set up an open circuit configuration, open the A and D clamps and immediately close the B and C clamps. Let most of the water flow from the washing container to the waste container at 100 revolutions per minute. Before the water has been completely transferred, stop the pump and close the tube clamps before the inlet and after the outlet of the carotid model.Using the appropriate filters, capture images of the model at the region of interest to show the deposition and adhesion of the particles to the cells. Upon completion of the experiment, use a customized software code to analyze the images captured at the region of interest, enter the name of the image and set the estimated threshold. Then run the code.Inspect the resulting image and the counted number of particles in the image. In this representative analysis, photo micrographs of cultured endothelial cells seeded in a 3d carotid artery model can be observed by brightfield and fluorescence confocal microscopic imaging. Fluorescent 10 micron diameter glass beads, seeded into the 3d model exhibited a recirculation pattern, suggesting a successful mimicking of physiological conditions within the model.Imaging of the particle deposition revealed that a higher level of adhesion of the particles to the cells was observed outside of the recirculation area where the wall shear stress was high. Following this protocol, functionalized particles with specific binding kinetics and a core culture of cells within different human artery models may be explored to improve the model and to study specific formulations. This technique provides a new platform for studying the vascular targeting of drug carriers.We have recently used it to show how the additivity of particles can be tailored to target different diseased vessel regions.

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Research

JoVE Journal

Bioengineering

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

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 ...

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 ...