A New Experimental System for the Extended Application of Cyclic Hydrostatic Pressure to Cell Culture

[+] Author and Article Information
Timothy M. Maul, Douglas W. Hamilton, Alejandro Nieponice, Lorenzo Soletti

Departments of Surgery and Bioengineering, and the McGowan Institute for Regenerative Medicine, University of Pittsburgh, 100 Technology Drive, Suite 200, Pittsburgh, PA 15219

David A. Vorp1

Departments of Surgery and Bioengineering, and the McGowan Institute for Regenerative Medicine, University of Pittsburgh, 100 Technology Drive, Suite 200, Pittsburgh, PA 15219VorpDA@upmc.edu


Corresponding author.

J Biomech Eng 129(1), 110-116 (Jun 23, 2006) (7 pages) doi:10.1115/1.2401190 History: Received September 27, 2005; Revised June 23, 2006

Mechanical forces have been shown to be important stimuli for the determination and maintenance of cellular phenotype and function. Many cells are constantly exposed in vivo to cyclic pressure, shear stress, and/or strain. Therefore, the ability to study the effects of these stimuli in vitro is important for understanding how they contribute to both normal and pathologic states. While there exist commercial as well as custom-built devices for the extended application of cyclic strain and shear stress, very few cyclic pressure systems have been reported to apply stimulation longer than 48h. However, pertinent responses of cells to mechanical stimulation may occur later than this. To address this limitation, we have designed a new cyclic hydrostatic pressure system based upon the following design variables: minimal size, stability of pressure and humidity, maximal accessibility, and versatility. Computational fluid dynamics (CFD) was utilized to predict the pressure and potential shear stress within the chamber during the first half of a 1.0Hz duty cycle. To biologically validate our system, we tested the response of bone marrow progenitor cells (BMPCs) from Sprague Dawley rats to a cyclic pressure stimulation of 12080mm Hg, 1.0Hz for 7days. Cellular morphology was measured using Scion Image, and cellular proliferation was measured by counting nuclei in ten fields of view. CFD results showed a constant pressure across the length of the chamber and no shear stress developed at the base of the chamber where the cells are cultured. BMPCs from Sprague Dawley rats demonstrated a significant change in morphology versus controls by reducing their size and adopting a more rounded morphology. Furthermore, these cells increased their proliferation under cyclic hydrostatic pressure. We have demonstrated that our system imparts a single mechanical stimulus of cyclic hydrostatic pressure and is capable of at least 7days of continuous operation without affecting cellular viability. Furthermore, we have shown for the first time that BMPCs respond to cyclic hydrostatic pressure by alterations in morphology and increased proliferation.

Copyright © 2007 by American Society of Mechanical Engineers
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Figure 1

Schematic and CFD model of the cyclic hydrostatic pressure system. (a) A constant pressure source (1) provides the bulk airflow that powers the cyclic pressure system. The pressure source consists of an in-house air pump and pure CO2 blended together (5% CO2, 20% O2, 75% N2). A heated passover circuit (2) humidifies the air to ∼100% before it passes through a resistor that controls the mean pressure (3). The air then passes through a sterile filter (4) before it enters the culture chamber (5) and builds a static pressure in the dead space above the culture media. Two stainless steel bars (6) deflect the incoming air and provide weight to stabilize the culture surface. An injection port (7) provides access for media sampling or chemical injection. The air then passes through a second sterile filter (8) on its way to a solenoid valve (9). When the valve is closed, pressure builds inside the system. When the valve is open, the pressure is released through a second resistor (10) that controls the diastolic pressure. Two pressure transducers (11) and their associated monitor (12) display the pressure inside the cyclic pressure chamber. A check valve (13), which has a cracking pressure of 250mm Hg, allows the system to release built-up pressure in the event the one of the filters becomes obstructed. The dashed box represents the interior of the incubator. (b) A three-dimensional model of the cyclic hydrostatic pressure chamber depicting the lid with the inlet, outlet, sampling ports, culture slides, o-ring groove, and stainless steel bars. (c) A 2-D CFD model of the pressure chamber using linear quadratic elements. Two boundary layers were created, one at the air/liquid interface (+), and the other at the base of the chamber where the cells would be located (∗). A sealed box (arrow) with an equivalent 2-D volume to the volume of the circuit between the chamber and the solenoid was used at the outlet. The x and y axes depict positional information (in meters) relevant to the velocity, pressure, and shear stress plots in subsequent figures.

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Figure 2

Pressure wave forms collected with the solenoid valve frequency set to (a) 0.5Hz, (b) 1.0Hz, and (c) 2.75Hz

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Figure 3

(a) Results for CFD analysis revealing a uniform pressure distribution across the length of the chamber. The dashed line indicates the position from which the velocity profile (c) was extracted. (b) Results for the pressure versus time versus position across the bottom of the cyclic pressure chamber indicate a smooth, continuous increase in pressure over the course of the simulation and along the length of the chamber. (c) A three-dimensional spatial velocity profile over time shows the development of parabolic flow in the air space, and that the velocity is zero in the liquid space (air-liquid interface located at y=6.3mm).

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Figure 4

Automated blood lab readings demonstrate similar values between the cyclic pressure (엯) and control experiments (∎) for (a) pH, (b) pCO2, and (c) HCO3−. (n=8). Dashed lines indicate the upper and lower limits for acceptable values for each measured variable. The stability in the HCO3− values indicates sufficient humidification. (d) pO2 values for pressure were elevated over controls throughout the experiments due to the increase in ambient pressure. These values, although statistically different (p<0.05), had similar FiO2 values when adjusted for ambient pressure and are not considered biologically significant.

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Figure 5

(a) Quantification of BMPC proliferation via cell counts revealed a 1.8-fold increase in cells exposed to cyclic pressure for 7days compared to controls. (b) BMPCs exposed to cyclic pressure demonstrated a significant reduction in their total area. (c) Shape index measurements indicated a more rounded morphology for BMPCs under cyclic pressure. (d) Coomassie blue stained images of pressure (left) and control (right) reflect the measured differences in morphology and proliferation that occurred upon stimulation with cyclic pressure. Images taken at 10× magnification.



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