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 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.
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 “dose” 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 “dosing” 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.
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.
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.
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.
4. Pump Sterilization
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.
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), maintains seal within the flow circuit. To ensure seal, spring clamps may be used to apply additional pressure on the gasket. Glass slides should be protected by polycarbonate sheet, cut to fit chamber area (Figure 6). Because flow velocity may additionally contribute to complications in maintaining seals, media may be altered through the addition of dextran to increase media viscosity. Through increased viscosity, fluid shear can be maintained at lower velocity. System response and integrity may be checked in the middle of the experiment simply by observing fluid levels and fluid color within the damping chamber and reservoir (Figure 3). The fluid level should maintain a constant value. Initial fluid level should be marked on both reservoir and damping chamber for checkup comparison. Adjustments can be made by the addition of media at the experiment initiation, or allowing entrance of air to damping chamber. Figure 4 illustrates a representative recording of flow velocity. Phenol red coloration should decrease over the course of the experiment. Upon completion of the experiment, media should be allowed to drain from the circuit, and stored at -80 °C freezer for future measure or use. Media may be analyzed for metabolite content or released cytokines, or used to monitor paracrine signaling to cellular responses by using it for cell culture. Cells may be imaged under phase contrast microscopy for morphology and confluency (Figure 5). Spindle like morphology is observed in ECs after HPF over 24 hr (Figure 7).
Figure 1. Flow circuit schematic. The flow meter labeled within the scheme is used to measure pulsatility index levels set by the damping chamber. Flow waveform should be measured with intended connection tube diameters to ensure system pressure remains physiologic. Please click here to view a larger version of this figure.
Figure 2. Flow chamber design and measurements. Outer channel is connected to vacuum. Center picture demonstrates clamping of the chamber. Picture at right illustrates gasket design. Please click here to view a larger version of this figure.
Figure 3. Flow Damping Chamber. Flow damping chamber schematic indicates intended flow direction and connection. Air release valve is opened, outlet valve is closed, and fluid fills the chamber to desired PI level. Upon opening of outlet valve and closing of air release valve, flow resumes and fluid levels within the chamber maintains levels. Please click here to view a larger version of this figure.
Figure 4. Sample flow waves with various pulsatility indices. High Pulsatility Flow (Left) and Low Pulsatility Flow (Right). Please click here to view a larger version of this figure.
Figure 5. Co-culture Flow Schematic. Co-culture shows pulsatile flow over endothelial cells, with smooth muscle cells in collagen gel. Cytokine release from cells may cross through the porous polycarbonate membrane. Please click here to view a larger version of this figure.
Figure 6. Endothelial Cell Morphology Changes with Flow Conditions. Confluent endothelial cell culture on functionalized polycarbonate membrane. Shown are before flow (left) and after flow (right). Flow is shown under differing conditions, static (no velocity), steady (constant velocity), high pulse (high pulsatility). Cells are stained with 2% crystal violet. Please click here to view a larger version of this figure.
Figure 7. Smooth Muscle Cell Protein Expression Changes with Flow Conditions. Smooth muscle cells expression of α-smooth muscle actin (SMA) and myosin heavy chain (SM-MHC) under differing conditions, static (no velocity), steady flow (constant velocity), and high pulse. Please click here to view a larger version of this figure.
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 and advanced techniques, consult the literature.2 Critical in culturing cells under fluid shear over extended periods are: 1) maintain a sealed flow circuit, excepting the reservoir, and 2) prevent contamination throughout the experiment. Sealing of the flow circuit is essential in maintaining media volume and correct PI levels, with introduction of unintended air affecting desired flow parameters. Additional air in the system may partially obstruct flow within the stream, disturb flow path and enhance the compliance of the overall system. While volumetric flow rates would be maintained, decreased cross-sectional area from this partial obstruction would lead to localized increases in velocity, and surface discontinuities. Both flow velocity variation and surface discontinuities would contribute to flow destabilization, and possibly turbulence. Lastly, any trapped air into the pressurized flow system would act as a compressible damping of the flow, thereby decreasing the PI from its desired level. As the flow circuit is of constant volume, trapped air also would cause leakage of media which can adversely impact cells, increasing metabolite concentration over time. Blood pump sealing and function are reliant on temperature, therefore the placement of the pump in the incubator may introduce sealing complications. Both increased presence of air within the flow circuit and loss of media through pump sealing or malfunction could increase variability of the cell environment, and lead to inaccurate results. Prevention of contamination during the experiment is important in all cell culture. Sealing of the flow circuit must again be stressed to prevent entrance of contaminants as well. Flow media provides many of the nutrients and sugars necessary for microorganism growth, therefore cleaning of the circuit immediately after use is necessary. Additionally, an opaque pump cover, reduces effectiveness of UV radiation in sterilization of the overall pump. This, coupled with the incompatibility of the acrylic piston with ethanol, requires full sterilization of the circuit using hydrogen peroxide (H2O2). Prevention of contamination growth when the system is not in use, use of sterilizing solution, and maintaining a sterile environment all may introduce complications either by allowing contamination or sterilizing cell culture. Limitations of this technique include the inability to properly model the complex flow wave in physiologic blood flow. While the flow system does offer a close approximation, different aspects of the complex waveform seen in physiologic flow may not be controlled or changed. Also, reversing shear stress in reciprocating flow is a complication seen in some arterial disease, and may not by replicated with this flow system. Additionally, long-term study of cell response could be more complicated and challenging, mainly due to the performance of the pump. Reducing the downstream resistance is essential to maintain the integrity of flow system and pumping efficiency. Lastly, the above system uses glass slides and polycarbonate membranes, which do not physiologically model the vasculature, and does not consider mechanical signaling resulting from arterial stretch or basal membrane rigidity. Previously, one study has used a pulsatile system capable of reproducing pulsatile flow using syringe pumps12. Efficacy of the system in reproducing the high PI could be affected by limitations on the pump speed and sealing. The method of damping chamber is additionally simpler in its application, not requiring pump programming. Future applications of the flow system would allow for mechanical signaling as a result of arterial stretch by incorporation of elastic tubing to replace the flow chamber and slide. Through this improvement, mismatched compliance and luminal surface discontinuities may be further studied.
The authors have nothing to disclose.
The authors wish to acknowledge funding sources, including AHA (13GRNT16990019 to W.T) and NHLBI (HL097246 and HL119371 to W.T.).
Acetone | Sigma-Aldrich | 34850 | |
Sulfuric Acid | Sigma-Aldrich | 320501 | |
(3-Aminopropyl)trethoxysilane | Sigma-Aldrich | 440140 | |
Glutaraldehyde Solution | Sigma-Aldrich | G5882 | |
Ethanol | Sigma-Aldrich | 459844 | |
Glass Slide (70mm x 50mm) | Sigma-Aldrich | CLS294775X50 | |
Polycarbonate Membrane | Millipore Corp. | HTTP09030 | |
Silicone Gasket | Grace Bio-Labs | RD 475464 | |
Fibronectin (25 μg/mL) | Sigma-Aldrich | F1141 | |
Collagen Type-I | Sigma-Aldrich | C3867 | |
NaHCO3 | Fluka | 36486 | |
NaOH | Sigma-Aldrich | S5881 | |
Damping Chamber | This chamber is custom made, and may be requested using the engineering drawing of Figure 3. | ||
Blood Pump | Harvard Apparatus | 529552 | |
Poly-Vinyl Carbonate Tubing | US Plastic | 65066, 65063, 65062 | Various sizes may be required |
Luer Connections | Nordson Medical | Various | Various sizes will be required, and a number of parts should be purchased for replacement use. |
Culture Chamber | Machined in-house | Custom | 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 |
Square Petri Dish | Cole-Parmer | EW-14007-10 | |
Glass Slide Holder | Capitol Scientific | WHE-900303 | |
Fetal Bovine Serum | Mediatech, Inc. | 35-010-CV | |
Dulbecco's Modified Eagle Medium | Mediatech, Inc. | 10-013-CV | |
Flow Meter | Sonotec, GmbH | Sonoflow co.55/060 | |
Sylgard Elastomer Kit | Sigma-Aldrich | 761036-5EA | |
14 G Steel Cannula | General Laboratory Supply | S8365-1 |