The authors wish to acknowledge funding sources, including AHA (13GRNT16990019 to W.T) and NHLBI (HL097246 and HL119371 to W.T.).

1. Silanization and Biomolecule Functionalization of Slide or Polycarbonate Membrane
Note: Many of the chemicals and solutions within this protocol have high evaporation rates (ethanol (EtOH), acetone, etc.). Other steps entail long incubation times for low evaporation rates. Paraffin film is recommended to seal containers. Caution: Many of the chemicals (including: sulfuric acid, acetone, (3-aminopropyl)triethoxysilane, glutaraldehyde, EtOH) are considered hazardous, or volatile. Consult material safety data sheet (MSDS) of each material for proper storage, handling, and disposal before use.
<ol>
	<li>Place new glass slide measuring 75 mm by 50 mm by 1 mm, in clean, glass slide staining dish without regard to orientation, ensuring full immersion. Use container for steps 1.1-1.12. 
	Note: For co-culture, include polycarbonate (PC) membrane with 0.4 micron pore size, cut to size of 75 mm by 50 mm, for all subsequent steps for PC functionalization and EC seeding.</li>
	<li>Immerse slide in sulfuric acid (20%), O/N. 
	Caution: Consult MSDS for sulfuric acid before use.</li>
	<li>Wash slide by immersing in deionized water (DI) for 5 min, three times, changing DI each time.</li>
	<li>Immerse slide in acetone for 30 min. 
	Caution: Consult MSDS for acetone before use.</li>
	<li>Immerse slide in 6% (3-aminopropyl)triethoxysilane / acetone solution O/N. 
	Caution: Consult MSDS for (3-aminopropyl)triethoxysilane and acetone before use.</li>
	<li>Immerse slide in acetone for 5 min, three times, changing acetone each time. 
	Caution: Consult MSDS for acetone before use.</li>
	<li>Wash slide by immersing in DI for 5 min, three times, changing DI each time.</li>
	<li>Immerse slide in 1.5% glutaraldehyde / DI solution for 60 min. 
	Caution: Consult MSDS for glutaraldehyde before use.</li>
	<li>Wash slide by immersing in DI for 5 min, two times, changing DI each time.</li>
	<li>Place slides into 70% ethanol (EtOH) for 30 min in sterile, laminar flow hood.</li>
	<li>Remove EtOH and allow remaining residue to evaporate. Immerse slides in sterile DI and drain in order to rehydrate the slides. 
	Note: Slides or PC membrane may be sealed in glass slide holder and stored at 4 &#176;C up to one week. Upon use for cell culture, repeat steps 1.10 and 1.11.</li>
	<li>Remove the slide from the slide holder and place it into a 100 mm by 100 mm square petri dish in the laminar flow hood. 
	Note: This container will be used for all remaining steps.
	<ol>
		<li>Prepare slide or PC for endothelial cell seeding for monoculture experiment.
		<ol>
			<li>Coat the functionalized slide or PC (step 1.11) with 1 ml fibronectin solution (25 &#956;g/ml), on one side, covering the approximate area exposed to cell culture chamber.</li>
			<li>Incubate slides or PC in sterile incubator set at 37 &#176;C for 1-2 hr.</li>
			<li>After incubation, aspirate the remaining solution using a glass aspirator pipette connected to a vacuum. 
			Note: Slides may now be stored O/N at 4 &#176;C for future use.</li>
		</ol>
		</li>
		<li>Follow 1.12.2 steps only for preparing EC/SMC co-culture. Prepare and seed smooth muscle cell (SMC) culture for co-culture experiment.
		<ol>
			<li>Place silicone gasket on top of the slide (step 1.11), with dimensions of 75 mm by 50 mm outer diameter, inner diameter of 50 mm by 30 mm, thickness of 0.5 mm, and perforations which align with flow chamber vacuum.</li>
			<li>Prepare 1 ml SMC in 10% FBS/DMEM suspension. Neutralize 1 ml solution of 2 mg/ml collagen type-I with 7% NaHCO3 and 0.1 M NaOH solution to pH of 7.4, and mix with SMC suspension with final cell density of 2 x 106 cells/ml.</li>
			<li>Spread SMC solution within gasket and incubate slides at 37 &#176;C and 5% CO2 for 30 min.</li>
			<li>After incubation, cover the slide with 25 ml 0.1% fetal bovine serum (FBS) mixed in Dulbecco&#8217;s Modified Eagle Medium (DMEM) for 72 hr.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Culture endothelial cells (ECs) on glass slide/polycarbonate membrane.
	<ol>
		<li>Seed 1 ml ECs in 10% FBS/DMEM, with initial concentration of 6.0 x 105 cells/ml, on fibronectin surfaces of glass slide or polycarbonate membrane.</li>
		<li>Incubate cells in an incubator at 37 &#176;C, 5% CO2, and 10% FBS for 120 min.</li>
		<li>Cover slides containing cells with additional 10% FBS/DMEM, and place in incubator in 37 &#176;C, 5% CO2 O/N, until 70%-80% confluent.</li>
	</ol>
	</li>
</ol>
2. Determination of Fluid Viscosity and Volumetric Flow Rate
Note: Rotating viscometers are sensitive equipment, and the viscometer user manual should be consulted before calibrating, zeroing, or performing measurements.
<ol>
	<li>Determine desired fluid shear (&#964;) from literature of the experiment&#8217;s targeted vasculature.</li>
	<li>Measure 1% FBS/DMEM viscosity (&#956;), using a rotational viscometer.</li>
	<li>Determine required volumetric flow rate (Q) from Poiseuille equation: 
	<img alt="Equation 1" src="/files/ftp_upload/53224/53224eq1.jpg" />, 
	where &#964; = fluid shear, &#956; = viscosity, Q = vol. flow rate, w = chamber width, and h = chamber height).</li>
	<li>Set pump to flow rate derived in step 2.3, and use for all subsequent steps.</li>
</ol>
3. Determination of Pulsatility Index
Note: All connection ports within the system should be connected with appropriately sized lock-ring-to-barb, or female luer-to-barb connections. Connecting PVC tubing may then be connected to barb fittings, and circuit completed.
<ol>
	<li>Connect flow circuit as in Figure 2, with damping chamber (Figure 3) and ultrasonic flow meter in correct flow direction. 
	Note: Ultrasonic flow meter is a sensitive piece of equipment, and the user manual should be consulted before use.</li>
	<li>Fill flow circuit and reservoir with DI water, by ensuring reservoir outlet tube (to pump) is submerged within reservoir volume. Visualize flow waveform using a flow meter.</li>
	<li>Open air release valve on the damping chamber to change fluid/air volume ratio. Close air release valve at different fluid level intervals and calculate pulsatility index (PI = (VMax - VMin) / VMean) by using flow waveform peak (VMax), trough (VMin), and mean (VMean). Record PI values in notebook.</li>
	<li>Mark resulting fluid level and PI on the damping chamber, using a felt tipped, permanent ink pen for future use. Determine desired PI levels from pathology literature11.</li>
	<li>Silicon Tube Formation- Optional, more advanced method of controlling pulsatility
	<ol>
		<li>Mix the silicone elastomer base and curing agent at different ratios (e.g., a base-to-crosslinker ratio from 10:1 to 36:1) for varied targeted elastic moduli, as illustrated previously.2</li>
		<li>Fabricate silicone tubes by repeated dip-cure process, briefly dipping steel cannula (14 G) into silicone prepolymer mixture with a predetermined base-to-crosslinker ratio.</li>
		<li>Place the prepolymer-coated cannula into an oven set at 60 &#176;C for 4 hr to cure the polymer coating on the cannula, switching the direction in the middle of curing process.</li>
		<li>Repeat the dipping process with the same silicone elastomer mixture and place back in the oven for additional 4 hr, which results in ~0.3 mm thick tubes.</li>
		<li>Remove the ultrathin silicone tube from the stainless steel cannula.</li>
		<li>Connect tubes to flow circuit instead of damping chamber, and test PI for each as in 3.3.</li>
	</ol>
	</li>
</ol>
4. Pump Sterilization
<ol>
	<li>Immerse the flow chamber (Figure 2), PVC tubing, and gaskets in 10% hydrogen peroxide (H2O2). Wash with sterile DI before step 4.</li>
	<li>Place the blood pump on a spare incubator shelf. 
	Note: Incubator shelves should be stainless steel, and be able to support the weight of the entire flow circuit.</li>
	<li>Fill the flow circuit and reservoir with 10% H2O2 by ensuring reservoir outlet tube (to pump) is submerged within reservoir volume. Circulate H2O2 by pumping through circuit for 20 min in the laminar flow hood with UV light on.</li>
	<li>Aspirate all H2O2 from tubing, and wash with sterile PBS by pumping through flow circuit.</li>
</ol>
5. Flow Chamber Assembly
Note: The flow chamber consists of an acrylic, custom made plate, with vacuum ports and inlet and outlet flow ports (see Figure 2). Chamber assembly consists of placing the flow chamber and gaskets on top of culture slides, properly aligned, and is described below.
<ol>
	<li>Warm up 1% FBS/DMEM in a 37 &#176;C water bath and prepare flow chamber.
	<ol>
		<li>Take EC slide out of media (from step 1.13) and place gasket on top with cell side up.</li>
		<li>(Optional) Take PC membrane out of media (from step 1.13) and place gasket on top with cell side up. Place co-culture slide with seeded SMC (step 1.12.2) underneath PC membrane.</li>
	</ol>
	</li>
	<li>Align vacuum channel of the flow chamber with the holes in the gasket (Figure 2).</li>
	<li>Attach vacuum tube to vacuum ports (Figure 2) ensuring no media leaks to vacuum.</li>
	<li>Place polycarbonate sheet underneath glass slide and clamp flow chamber assembly with spring clamps, one on each side of flow chamber (Figures 2 and 5).</li>
	<li>Place flow system in incubator at 37 &#176;C and 5% CO2, and fill circuit with media by pumping from filled reservoir at low pulsatility. Maintain this flow for 4 hr for preconditioning cells.</li>
	<li>Adjust fluid volume in damping chamber to marked PI level, and culture for desired time.</li>
</ol>

<ol><li>Scott-Drechsel, D., Su, Z., Hunter, K., Li, M., Shandas, R., Tan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+flow+co-culture+system+for+studying+mechanobiology+effects+of+pulse+flow+waves%5BTitle%5D)+AND+%22Cytotechnology%22%5BJournal%5D)">A new flow co-culture system for studying mechanobiology effects of pulse flow waves.</a> Cytotechnology. 64 (6), 649-666 (2012).</li>
<li>Tan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stiffening-Induced+High+Pulsatility+Flow+Activates+Endothelial+Inflammation+via+a+TLR2%2FNF-%CE%BAB+Pathway%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Stiffening-Induced High Pulsatility Flow Activates Endothelial Inflammation via a TLR2/NF-κB Pathway.</a> PLoS ONE. 9 (7), e102195(2014).</li>
<li>Wexler, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coronary+Artery+Calcification%3A+Pathophysiology%2C+Epidemiology%2C+Imaging+Methods%2C+and+Clinical+Implications+A+Statement+for+Health+Professionals+From+the+American+Heart+Association%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Coronary Artery Calcification: Pathophysiology, Epidemiology, Imaging Methods, and Clinical Implications A Statement for Health Professionals From the American Heart Association.</a> Circulation. 94 (5), 1175-1192 (1996).</li>
<li>Lan, T. -H., Huang, X. -Q., Tan, H. -M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vascular+fibrosis+in+atherosclerosis%5BTitle%5D)+AND+%22Cardiovasc+Pathol%22%5BJournal%5D)">Vascular fibrosis in atherosclerosis.</a> Cardiovasc Pathol. 22 (5), 401-407 (2013).</li>
<li>Lee, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Promoting+endothelial+recovery+and+reducing+neointimal+hyperplasia+using+sequential-like+release+of+acetylsalicylic+acid+and+paclitaxel-loaded+biodegradable+stents%5BTitle%5D)+AND+%22Int+J+Nanomedicine%22%5BJournal%5D)">Promoting endothelial recovery and reducing neointimal hyperplasia using sequential-like release of acetylsalicylic acid and paclitaxel-loaded biodegradable stents.</a> Int J Nanomedicine. 9, 4117-4133 (2014).</li>
<li>Intengan, H. D., Schiffrin, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vascular+Remodeling+in+Hypertension+Roles+of+Apoptosis+Inflammation%2C+and+Fibrosis%5BTitle%5D)+AND+%22Hypertension%22%5BJournal%5D)">Vascular Remodeling in Hypertension Roles of Apoptosis Inflammation, and Fibrosis.</a> Hypertension. 38 (3), 581-587 (2001).</li>
<li>Greil, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changes+in+carotid+artery+flow+velocities+after+stent+implantation%3A+a+fluid+dynamics+study+with+laser+Doppler+anemometry%5BTitle%5D)+AND+%22J+Endovasc+Ther%22%5BJournal%5D)">Changes in carotid artery flow velocities after stent implantation: a fluid dynamics study with laser Doppler anemometry.</a> J Endovasc Ther. 10 (2), 275-284 (2003).</li>
<li>Egorova, A. D., van der Heiden, K., Poelmann, R. E., Hierck, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Primary+cilia+as+biomechanical+sensors+in+regulating+endothelial+function%5BTitle%5D)+AND+%22Differentiation%22%5BJournal%5D)">Primary cilia as biomechanical sensors in regulating endothelial function.</a> Differentiation. 83 (2), S56-S61 (2012).</li>
<li>Liu, S. Q., Goldman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+blood+shear+stress+in+the+regulation+of+vascular+smooth+muscle+cell+migration%5BTitle%5D)+AND+%22IEEE+T+Bio-Med+Eng%22%5BJournal%5D)">Role of blood shear stress in the regulation of vascular smooth muscle cell migration.</a> IEEE T Bio-Med Eng. 48 (4), 474-483 (2001).</li>
<li>Scott, D., Tan, Y., Shandas, R., Stenmark, K. R., Tan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+pulsatility+flow+stimulates+smooth+muscle+cell+hypertrophy+and+contractile+protein+expression%5BTitle%5D)+AND+%22AJP%3A+Lung+C%22%5BJournal%5D)">High pulsatility flow stimulates smooth muscle cell hypertrophy and contractile protein expression.</a> AJP: Lung C. 304 (1), L70-L81 (2013).</li>
<li>Panaritis, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pulsatility+Index+of+Temporal+and+Renal+Arteries+as+an+Early+Finding+of+Arteriopathy+in+Diabetic+Patients.%5BTitle%5D)+AND+%22Ann+Vasc+Surg%22%5BJournal%5D)">Pulsatility Index of Temporal and Renal Arteries as an Early Finding of Arteriopathy in Diabetic Patients.</a> Ann Vasc Surg. 19 (1), 80-83 (2005).</li>
<li>Miao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+Flow+Patterns+on+the+Localization+and+Expression+of+VE-Cadherin+at+Vascular+Endothelial+Cell+Junctions%3A+In+vivo+and+in+vitro+Investigations%5BTitle%5D)+AND+%22J+Vasc+Res%22%5BJournal%5D)">Effects of Flow Patterns on the Localization and Expression of VE-Cadherin at Vascular Endothelial Cell Junctions: In vivo and in vitro Investigations.</a> J Vasc Res. 42 (1), 77-89 (2005).</li>
</ol>

The authors have nothing to disclose.

This protocol describes a method of reproducing pulsatile flow in vitro, and may be instrumental first step in determining contribution of flow conditions to disease pathologies. Previous studies using this protocol have found flow conditions contribute to vascular inflammatory response.1,10 Additionally, this protocol is intended for experienced laboratories. As such, neither in depth fluid mechanics, nor biochemical analysis is described herein. For more advanced fluid dynamics,1</su...

Morbidity rates for cardiovascular diseases are the largest in America, with many resulting from unhealthy vasculature. Healthy arteries consist of elastic tissue, with soft luminal surface coated with an endothelial cell (EC) monolayer. Arterial flow may be modeled as an oscillating wave function with positive mean flow rate. The pulsatility index (PI) is the quotient of oscillation magnitude and mean flow (PI = (Max. - Min.) / Mean),1 and has been modeled in vitro with variable vessel elasticity.2 Arterial elasticity is important in storage of flow energy from heart contractions, dilating under systolic pressure, and plays a significant role in modulating blood flow PI. Because the heart maintains a consistent, pulsatile, volumetric flow, arterial expansion increases cross-sectional area, enhancing flow stability by reducing flow velocity, shear stress, and PI. Frequently, unhealthy arteries present changes to elasticity or compliance, displaying stiffening from vascular remodeling, scar tissue or calcification3,4. Additionally, other vascular disorders, such as neointimal hyperplasia (NIH),5 aneurysm and hypertension6 and vascular fibrosis4, may constrict vessel diameter. However, current drug treatment and device treatment of vascular diseases often neglect the importance of vessel wall compliance or blood flow dynamics in vascular disease which is often complicated by changes in vessel morphology and properties. Neither balloon angioplasty nor stenting answer the complication of wall elasticity7. Therefore, in vitro modeling of blood flows resulting from arterial disease and treatments is important in investigating disease pathologies and future efficacy of treatment. Herein, we describe a method of replicating physiological and pathological blood flow designed to determine cell response in vascular disease pathologies. Fluid flow causes shear stress at the vessel wall, which is an important mechanical signal in vessel health, affecting all cells within the vasculature. Several mechanical sensors on the vascular endothelium for fluid shear have been identified, including primary cilium shown in recent studies for endothelial mechanosensing8. Endothelial cell activity and morphology are affected by flow velocity, direction, and pulsatility. Additionally, smooth muscle cell (SMC) migration can be affected by mechano-signals of low velocity flow through interstitial fluid9, and can also be through the paracrine signaling from endothelial cells through their response to flow and mechano-transduction of flow signals via cytokine release10. The &#8220;dose&#8221; dependence of mean shear, PI, and paracrine signaling may also be interdependent. To this end, the determination of vascular cell response to fluid shear with varied &#8220;dosing&#8221; in monolayer culture or co-culture in vitro could provide mechanistic insights into vascular remodeling and improve disease and treatment prediction. The flow system used in this experiment consists of a blood pump, an upstream flow damping air reservoir, a downstream flow meter only used during experimental setup, a downstream cell culture, parallel-plate flow chamber, and media reservoir. Control of vascular flow variables such as mean flow rate, beats per minute, and PI may be achieved through controlling flow rate, pulse frequency, and introduction of pressure damping. Pulsatile blood pumps are available with variable stroke displacement, at controlled stroke frequency, relating directly to mean volumetric flow rate, and pulse frequency. Introduction of an air reservoir within the flow circuit allows for pressure damping, reducing flow oscillation magnitude. Media is an incompressible fluid, while air within the damping chamber is compressible, allowing excess pressure from the flow wave to be absorbed by air compression. The air to media ratio allows for control over how much damping occurs. A custom cell culture flow chamber 75 mm in length by 50 mm in width was created from acrylic. Flow enters through the inlet port, and expands through the inlet manifold, providing consistent flow across the entirety of the flow chamber. Similar flow and structures are present at chamber outlet. Cells are seeded onto functionalized slides, and subsequently attached to the flow chamber. This allows for large populations, easily retrieved after the study. Co-culture experiments may use a porous polycarbonate membrane to eliminate cell-to-cell contact between cultures while allowing cytokine/flow transport. This system has previously been used to model high PI flow and its effect on endothelial monolayer culture and EC/SMC co-culture1,10, to investigate cell response to pathologically high PI disease. By describing the protocol used to model these flow conditions, we hope to aid others in determining flow signal contribution to cell response.

<table><tbody><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>34850</td><td></td></tr><tr><td>Sulfuric Acid</td><td>Sigma-Aldrich</td><td>320501</td><td></td></tr><tr><td>(3-Aminopropyl)triethoxysilane</td><td>Sigma-Aldrich</td><td>440140</td><td></td></tr><tr><td>Glutaraldehyde Solution</td><td>Sigma-Aldrich</td><td>G5882</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>459844</td><td></td></tr><tr><td>Glass Slide (70&amp;#160;mm x 50&amp;#160;mm)</td><td>Sigma-Aldrich</td><td>CLS294775X50</td><td></td></tr><tr><td>Polycarbonate Membrane</td><td>Millipore Corp.</td><td>HTTP09030</td><td></td></tr><tr><td>Silicone Gasket</td><td>Grace Bio-Labs</td><td>RD 475464</td><td></td></tr><tr><td>Fibronectin (25 &amp;mu;g/ml)</td><td>Sigma-Aldrich</td><td>F1141</td><td></td></tr><tr><td>Collagen Type-I</td><td>Sigma-Aldrich</td><td>C3867</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fluka</td><td>36486</td><td></td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>S5881</td><td></td></tr><tr><td>Damping Chamber</td><td></td><td></td><td>This chamber is custom made, and may be requested using the engineering drawing of &lt;strong&gt;Figure 3&lt;/strong&gt;.</td></tr><tr><td>Blood Pump</td><td>Harvard Apparatus</td><td>529552</td><td></td></tr><tr><td>Poly-Vinyl Carbonate Tubing</td><td>US Plastic</td><td>65066, 65063, 65062</td><td>Various sizes may be required</td></tr><tr><td>Luer Connections</td><td>Nordson Medical</td><td>Various</td><td>Various sizes will be required, and a number of parts should be purchased for replacement use.</td></tr><tr><td>Culture Chamber</td><td>Machined in-house</td><td>Custom</td><td>Acrylic may be purchased in sheets and machined for intended use. The engineering drawing shown in &lt;strong&gt;Figure 2&lt;/strong&gt; may be used to recreate this chamber.</td></tr><tr><td>Square Petri Dish</td><td>Cole-Parmer</td><td>EW-14007-10</td><td></td></tr><tr><td>Glass Slide Holder</td><td>Capitol Scientific</td><td>WHE-900303</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Mediatech, Inc.</td><td>35-010-CV</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Mediatech, Inc.</td><td>10-013-CV</td><td></td></tr><tr><td>Flow Meter</td><td>Sonotec, GmbH</td><td>Sonoflow co.55/060</td><td></td></tr><tr><td>Sylgard Elastomer Kit</td><td>Sigma-Aldrich</td><td>761036-5EA</td><td></td></tr><tr><td>14 G Steel Cannula</td><td>General Laboratory Supply</td><td>S8365-1</td><td></td></tr></tbody></table>

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.

Maintenance of flow conditions is reliant on correct assembly of the flow circuit (Figure 1). Tubing diameter is an important selection in assembly, with larger diameters reducing flow resistance and subsequent pressure drop before and after the culture chamber. To ensure intended pressure and flow velocity, assemble the system with flow meter before experiment with intended tubing. Alignment of culture chamber vacuum channel (Figure 2), with perforations on silicon gasket (not pictured)...

Watch this Scientific Journal Video about In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling at JoVE.com

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.

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 ...

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Bioengineering

9.6K Views.  University of Colorado at Boulder.  Dit protocol repliceert fysiologische of pathologische bloedstroom in vitro te helpen bij het ​​bepalen celrespons ziekte pathologieën. Door de invoering van een druk rustkamer stroomafwaarts van een bloed pomp, kan de bloedstroom over het vaatstelsel worden gerecapituleerd en opgelegd aan een monolaag van vasculaire endotheel of mimetische co-cultuur. 

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

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

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

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

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

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

Developmental Biology

Micropatterning and Assembly of 3D Microvessels

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

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

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

Differentiation and Mechanical Stimulation of hiPSC-Derived Endothelial Cells

In Vitro Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides transporting oxygen and nutrients to ensure fetal growth, fetal circulation controls many crucial developmental events taking place within the endothelial layer through mechanical cues. Biomechanical signals induce blood vessel structural changes, establish arteriovenous specification, and control the development of hematopoietic stem cells. The inaccessibility of the developing tissues limits the understanding of the role of circulation in early human development; therefore, in vitro models are pivotal tools for the study of vessel mechanobiology. This paper describes a protocol to differentiate endothelial cells from human induced pluripotent stem cells and their subsequent seeding into a fluidic device to study their response to mechanical cues. This approach allows for long-term culture of endothelial cells under mechanical stimulation followed by&#160;retrieval of the endothelial cells for phenotypical and functional characterization. The in vitro model established here will be instrumental to elucidate&#160;the intracellular molecular mechanisms that transduce the signaling mediated by mechanical cues, which ultimately orchestrate vessel development during human fetal life.

Author Spotlight: Studying hiPSC-Derived Endothelial Cells Cultured Under Fluidic-Mediated Mechanical Stimulation 

Described here is a simple workflow to differentiate endothelial cells from human pluripotent stem cells followed by a detailed protocol for their mechanical stimulation. This allows for the study of the developmental mechanobiology of endothelial cells. This approach is compatible with downstream assays of live cells collected from the culture chip after mechanical stimulation.

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides ...

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

Immunology and Infection

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 ...

Microfluidic Model to Mimic Initial Event of Neovascularization

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the ...

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.

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.

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

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...

Studying Tissue Rigidity and Shear Stress Effects on Endothelial Cells

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

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

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

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

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

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