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. Consu...

<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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+flow+co-culture+system+for+studying+mechanobiology+effects+of+pulse+flow+waves.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stiffening-Induced+High+Pulsatility+Flow+Activates+Endothelial+Inflammation+via+a+TLR2/NF-&#954;B+Pathway.">Stiffening-Induced High Pulsatility Flow Activates Endothelial Inflammation via a TLR2/NF-&#954;B Pathway.</a> PLoS ONE. 9 (7), e102195 (2014).</li><li>Wexler, L., 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=Coronary+Artery+Calcification:+Pathophysiology,+Epidemiology,+Imaging+Methods,+and+Clinical+Implications+A+Statement+for+Health+Professionals+From+the+American+Heart+Association.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+fibrosis+in+atherosclerosis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promoting+endothelial+recovery+and+reducing+neointimal+hyperplasia+using+sequential-like+release+of+acetylsalicylic+acid+and+paclitaxel-loaded+biodegradable+stents.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+Remodeling+in+Hypertension+Roles+of+Apoptosis+Inflammation,+and+Fibrosis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+carotid+artery+flow+velocities+after+stent+implantation:+a+fluid+dynamics+study+with+laser+Doppler+anemometry.">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. 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R., Tan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+pulsatility+flow+stimulates+smooth+muscle+cell+hypertrophy+and+contractile+protein+expression.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsatility+Index+of+Temporal+and+Renal+Arteries+as+an+Early+Finding+of+Arteriopathy+in+Diabetic+Patients..">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Flow+Patterns+on+the+Localization+and+Expression+of+VE-Cadherin+at+Vascular+Endothelial+Cell+Junctions:+In+vivo+and+in+vitro+Investigations.">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 border="0" cellpadding="0" cellspacing="0">
	<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)trethoxysilane</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 (70mm x 50mm)</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 &#956;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>NaHCO3</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 Figure 3.</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 Figure 2 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&#39;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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Research

JoVE Journal

Bioengineering

9.5K Görüntüleme.  University of Colorado at Boulder.  Bu protokol, hastalık patolojilerinde hücre yanıtını belirlenmesinde yardımcı olmak için, in vitro olarak fizyolojik veya patolojik kan akışını çoğaltır. Kan pompası alt bölmeyi sönümleme bir basınç katılmasıyla, damar boyunca kan akımı değinmeyecek edilebilir ve vasküler endotelyum, ya da taklit eden ko-kültür, bir tek tabaka üzerine koydu. 