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Endothelial Cell Culture Model for Replication of Physiological Profiles of Pressure, Flow, Stretch, and Shear Stress in Vitro Rosendo Estrada,† Guruprasad A. Giridharan,† Mai-Dung Nguyen,† Thomas J. Roussel,† Mostafa Shakeri,‡ Vahidreza Parichehreh,† Sumanth D. Prabhu,§ and Palaniappan Sethu*,† †
Department of Bioengineering and ‡Department of Mechanical Engineering, Speed School of Engineering, University of Louisville, Louisville, Kentucky 40208, United States § Institute of Molecular Cardiology, Department of Medicine, University of Louisville and Louisville VAMC, Louisville, Kentucky 40202, United States
bS Supporting Information ABSTRACT: The phenotype and function of vascular cells in vivo are influenced by complex mechanical signals generated by pulsatile hemodynamic loading. Physiologically relevant in vitro studies of vascular cells therefore require realistic environments where in vivo mechanical loading conditions can be accurately reproduced. To accomplish a realistic in vivo-like loading environment, we designed and fabricated an Endothelial Cell Culture Model (ECCM) to generate physiological pressure, stretch, and shear stress profiles associated with normal and pathological cardiac flow states. Cells within this system were cultured on a stretchable, thin (∼500 μm) planar membrane within a rectangular flow channel and subject to constant fluid flow. Under pressure, the thin planar membrane assumed a concave shape, representing a segment of the blood vessel wall. Pulsatility was introduced using a programmable pneumatically controlled collapsible chamber. Human aortic endothelial cells (HAECs) were cultured within this system under normal conditions and compared to HAECs cultured under static and “flow only” (13 dyn/cm2) control conditions using microscopy. Cells cultured within the ECCM were larger than both controls and assumed an ellipsoidal shape. In contrast to static control control cells, ECCMcultured cells exhibited alignment of cytoskeletal actin filaments and high and continuous expression levels of β-catenin indicating an in vivo-like phenotype. In conclusion, design, fabrication, testing, and validation of the ECCM for culture of ECs under realistic pressure, flow, strain, and shear loading seen in normal and pathological conditions was accomplished. The ECCM therefore is an enabling technology that allows for study of ECs under physiologically relevant biomechanical loading conditions in vitro.
T
he blood vessel is an active integrated organ consisting of endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts in a highly interactive signaling environment. The vasculature is capable of sensing both mechanical and biochemical signals and transducing these signals into intracellular cues to modulate tissue structure and function. The pulsatile flow of blood generates time-varying biomechanical forces in the form of pulsatile pressure, stretch, and shear stress that act on ECs (particularly arterial ECs) that form the innermost layer of the vessel. Pathological conditions affecting the cardiovascular system influence mechanical loading patterns and subject ECs to various types of biomechanical forces. ECs respond to these biomechanical force signals via conserved response mechanisms that include inflammation and tissue remodeling through events such as proliferation, cell migration, apoptosis, and cellcell and cellextracellular matrix (ECM) interactions that affect vascular tone and permeability. Normal large diameter arterial vessels experience average shear stress in the range of ∼1030 dyn/cm2 with peak values between 40 and 75 dyn/cm2. ECs cultured under these r 2011 American Chemical Society
conditions in vitro show an elongated and aligned phenotype, low EC turnover, reduced oxidative stress, low accumulation of low density lipoprotein (LDL), low DNA synthesis, and minimal expression of adhesion/inflammatory molecules.1,2 Low shear stress and disturbed oscillatory flow that typically occurs in regions of branching or bifurcation results in polygonal and randomly oriented phenotype, high EC turnover, increased accumulation of LDL, increased oxidative stress, increased DNA synthesis, and higher expression of proinflammatory adhesion molecules.1,2 Functionally, laminar and pulsatile laminar shear stress have been shown to cause vasodilatation via upregulation of nitric oxide synthase (NOS) and augmented levels of nitric oxide (NO)3 as well as increased production of Cu/Zn superoxide dismutase4 and dismutation of superoxide, which has been implicated in inducing endothelial dysfunction via several mechanisms including NO inactivation. Received: February 4, 2011 Accepted: March 1, 2011 Published: March 17, 2011 3170
dx.doi.org/10.1021/ac2002998 | Anal. Chem. 2011, 83, 3170–3177
Analytical Chemistry
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Table 1. Summary of Existing Systems Capable of Concomitant Stimulation of ECs with Pressure (Pr), Strain (ε), and Shear (τ) Loading model/year
system description
Aziz et al. vascular ECs cultured in flexible simulating device (VSD) (1994)
Pr ε τ
limitations
enabling features
(1) seeding, culture and imaging in a flexible tube
Enable assessment of changes due
rubber tubing and known pressure and flow applied
(2) nonrealistic (sinusoidal) pressure and flow waveforms
in two or more modes of stimulation similar to what occurs in disease
using a pump
(3) no demonstrations of waveforms
19
X
X X
associated with pathological conditions Zhao et al.8
ECs cultured in
(1) seeding, culture and imaging in
Strain synergistically modulate EC structure
(1995)
flexible silicone tubes
a flexible tube
and response to shear stress
driven using pump driven
(2) nonrealistic (sinusoidal) pressure
by a pulse generator
and flow waveforms
X
X X
(3) no demonstrations of waveforms associated with pathological conditions Peng et al.21
Servo controlled
Pulse Perfusion Model (2000)
X
X X
(1) seeding, culture and
(1) realistic mechanical pulsatile
feedback loop with
imaging in a flexible tube
forces on ECs
pump, resistor and
(2) significant retrograde flow,
(2) facilitate studies of phasic
adjustable flow
negative shear stress
shear, strain and pulsatile signal transduction.
(3) no demonstrations of waveforms associated with pathological conditions Nakadate et al.20
System with
(1) nonrealistic (sinusoidal)
mimics physiological transmural pressure and
Pulsatile Perfusion
resistance an compliance
pressure and flow waveforms
shear stress in any size in vivo vessel
System (2008)
based on the 3 element
(2) no strain
X
X
Windkessel model
The effects of stretch on ECs and SMCs have been evaluated in vitro by culturing them on thin flexible substrates.5 Further, control over the direction and magnitude of stretch can be achieved by controlling the type of stretch via uniaxial,6 biaxial,7 and circumferential8 loading. ECs and SMCs in blood vessels are subject to uniaxial stretch (hoop stress) as a consequence of pulsatile pressure in a direction perpendicular to the flow of blood. Therefore, the immediate response of ECs to uniaxial stretch is directional alignment perpendicular to the direction of stretch9 (in the direction of flow in blood vessels) and stimulation of stretch-activated ion channels for Ca2þ ion transport.10 In vitro studies demonstrate that short-term stretch results in modulation of vessel tone through synthesis of superoxide11 known to play a role in vasoconstriction whereas prolonged exposure to stretch increases generation of vasodilators such as NO.12 Additionally, increased expression of endothelin 1 (ET1),12 a vasoconstrictor implicated in the progression of atherosclerosis, has been demonstrated in vitro. Unlike shear stress and stretch, relatively few in vitro studies have focused on the direct effects of pressure on EC structure and function.13 This can be attributed to the assumption that pulsatile pressure from blood flow causes the blood vessel to stretch and therefore the overall effect of pressure manifests itself primarily in the form of stretch. However, evaluation of pressure on ECs in vitro shows that pressure alone in the absence of stretch results in increased EC proliferation, cytoskeletal reorganization, and synthesis of ECM proteins.1416 Further, hydrostatic pressure indirectly affects cultured EC monolayer permeability via NO17 and Ca2þ signaling.18 Elevated hydrostatic pressure mediates an increase in Ca2þ transport into cultured ECs, which in turn reduces the permeability of the cultured EC monolayer.18
Most in vitro studies have limited investigations to evaluating the effects of isolated modes of mechanical stimulation. Despite the generation of large quantities of data regarding the EC stress response, which has led to significantly improved understanding of EC signaling mechanisms, the fact remains that the response of ECs to individual stimuli is very different from the simultaneous and coordinated stimulation sustained in vivo.8 Therefore, accurately mimicking the in vivo mechanical loading environment is essential to ensure relevance of the cellular events observed during in vitro studies. In reviewing literature, very few examples of systems that can accomplish concomitant stimulation with pressure, stretch, and shear stress have been demonstrated.8,1921 Each of these systems mimics two or more aspects of in vivo hemodynamic loading. However, each system is also associated with disadvantages. A summary of these systems along with their associated advantages and disadvantages are summarized in Table 1. To overcome these shortcomings, we have developed a new microfluidic “Endothelial Cell Culture Model” (ECCM) to culture ECs under realistic mechanical loading conditions and physiological pressure and flow patterns on a planar microscope compatible platform. The ECCM is based on the principles of existing mock flow loops designed to mimic a human circulatory system.2224 The platform itself consists of a rectangular cell culture channel. The cells are cultured on a suspended polymeric thin film inside the rectangular channel. The suspended thin film forms a concave shape inside the channel and represents a segment of the blood vessel wall. More importantly, the thin film in the channel stretches in response to pressure similar to a compliant blood vessel. This system uses easily adjustable analog controls including compliances, resistances, a collapsible pulsatile chamber, and a one way flow control valve to accurately 3171
dx.doi.org/10.1021/ac2002998 |Anal. Chem. 2011, 83, 3170–3177
Analytical Chemistry
Figure 1. (A) Schematic of the ECCM flow loop: (a) peristaltic pump, (b) pulmonary compliance, (c) pulmonary resistance, (d) collapsible chamber, (e) one-way valve, (f) inline flow sensor, (g) cell culture chamber, (h) aortic/systemic compliance, (i) inline pressure sensor, (j) aortic/systemic resistance, and (k) medium reservoir. (B) Picture of the actual setup, (C) schematic of the cell culture chamber, and (D) cross-section view of the cell culture chamber showing the thin membrane on which cells are cultured.
mimic hemodynamic waveforms associated with normal and pathological conditions. Culture of cells on a planar surface simplifies cell seeding using standard cell culture techniques and imaging using confocal microscopy. This paper describes the design, construction, operation, and validation of the ECCM to culture ECs under conditions of (patho)physiological mechanical loading. With appropriate modifications, this system is also capable of coculture with SMCs and compatible with our previously developed microfluidic cardiac cell culture model (μCCCM),25 which was developed to culture cardiomyocytes under conditions of pressure and stretch similar to that observed in the left ventricle and can possibly be integrated downstream of cultured cardiomyocytes.
’ MATERIALS AND METHODS ECCM Components. The ECCM consists of a peristaltic pump to induce and manipulate flow, a cell culture chamber with a compliant thin membrane that mimics a vessel wall, a pneumatically driven pulsatile chamber, a one-way valve, two tunable flow resistance elements to adjust preload and afterload, and two tunable compliance elements that represent arterial and venous compliance (Figure 1). Fabrication of Cell Culture Chamber. The cell culture chamber is a rectangular channel with a compliant thin membrane that serves as the floor of the channel. The chamber was fabricated using standard soft lithography techniques as detailed in the Supporting Information. Pressure and Fluid Flow Measurement. Flow and pressure measurements were made upstream of the cell culture chamber inlet and downstream of the outlet, respectively. Flow measurements were collected real time using an inline, transit time flow
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probe (Transonics, Ithaca, NY). Pressure measurements were accomplished using an inline pressure sensor (Validyne, San Fransisco, CA). Signal conditioning was accomplished using transducer amplifiers (Ectron, San Diego, CA) and transit-time flow meters (Transonics, Ithaca, NY) and other peripheral conditioners integrated in an instrumentation system compliant with Good Laboratory Practice (GLP) guidelines. Signal conditioned data were low pass filtered at 60 Hz, analog to digitally converted (AT-MIO-16E-10 and LabVIEW, National Instruments, Austin, TX) at a sampling rate of 500 Hz, and stored on a personal computer for postprocessing and analysis (Strain Measurement and Shear Stress Estimation. See Supporting Information). Human Aortic Endothelial Cell Culture. HAECs cells (Invitrogen, Carlsbad, CA) were used for all experiments. Cells were cultured using supplier recommended culture medium for 24 h and then maintained under static conditions (control) or laminar flow or under perfusion and pulsatile stretch. (For details see the Supporting Information). Cell Size Measurement and Immunofluorescence Microscopy. HAECs were fixed with 4% paraformaldehyde in 1 PBS for 20 min, washed two times with wash buffer (1 PBS containing 0.05% Tween-20 (Fisher Scientific, Fair Lawn, NJ)), and permeabilized with 0.5% Triton X-100 (Fisher Scientific, Fair Lawn, NJ) for 2 min at room temperature. Then, cells were washed two times with wash buffer, blocked with 1% BSA in freshly prepared 1 PBS for 30 min and incubated with primary antibody antihuman mouse β-catenin (1:50; Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 1 h. Cells were washed three times with wash buffer for 5 min each time and incubated at room temperature with the second antibody, FITC-conjugated goat antimouse (1:100; Millipore, Billerica, MA). After 1 h, cells were washed three times with wash buffer. For negative controls, the same procedure was performed without adding the primary antibody. For the detection of F-actin, cells were washed two times with 1% BSA in freshly prepared 1 PBS for 30 min and incubated at room temperature for 1 h with TRITC-conjugated phalloidin (1:100; Millipore, Billerica, MA). Light Diagnostics mounting fluid (Millipore, Billerica, MA) was added to the cells, and cells were examined using a Nikon Eclipse A1 confocal microscopy system (Nikon Instruments, Melville, NY). Cell size was estimated using phase contrast microscopy in combination with fluorescent βcatenin staining. Both phase contrast and fluorescence images obtained at 40 magnification were overlaid to distinguish and map cell boundaries and analyzed using Metamorph software (Molecular Devices, Sunnyvale, CA) to obtain the area of a cell. Measurements were made of 10 cells in each sample, and the area was averaged. Results represent means ( standard error of means (SEM) for N = 10. Student’s t test (p-value
