386800 An Orbital Shear Platform for in-Vitro Real-Time Endothelium Characterization

Tuesday, November 18, 2014: 1:45 PM
Marquis Ballroom C (Marriott Marquis Atlanta)
Vanessa Velasco1, Mark Gruenthal2, Stuart J. Williams1, Jonathan M. D. Thomas3, R. Eric Berson3 and Robert Keynton4, (1)Mechanical Engineering, University of Louisville, Louisville, KY, (2)Mechanical Engineering, University of Louisville , Louisville, KY, (3)Chemical Engineering, University of Louisville, Louisville, KY, (4)Department of Bioengineering, University of Louisville, Louisville, KY

Introduction

        Atherosclerosis is the buildup of plaque in lesions in the inner most lining of arteries, known as the endothelium. As plaque hardens, the arteries become narrow and limit blood flow leading to the onset of cardio disease, such as congestive heart failure, stroke and hypertension. Studies have shown that the development of atherosclerosis is linked to changes in the endothelium function [1-2]. Shear stress can control endothelium and in some cases leading to increased endothelium permeability which in turn can lead to lesions where plaques forms [3-4].

Electrical impedance measurements can be used to monitor the permeability and cell movement of adherent cells, such as endothelial cells [5]. In this work, we present an impedance based in-vitro platform used to monitor the behavior of cultured Human Umbilical Vein Endothelial Cells (HUVECs) under fluid-dynamic oscillatory shear.

Material and methods

The platform (Figure 1) consists of a microfabricated array of 200 µm (in diameter) gold sensing electrodes and complimentary counter electrodes on a borosilicate substrate. The chip contains 3 rows of electrodes which are spaced 120 degrees from each other. Electrodes are positioned at defined radial locations with respect to the center of the borosilicate chip.  A PDMS mold forms a 35 mm diameter well. Shear stress is induced by placing the platform on an orbital shaker (Figure 2). Within the rotating orbital well, cells experience a range of shear stress values and in differing directions at different radial positions [6].

Figure 1:  (Left) An image of the completed chip including a 35 mm PDMS well.  (Right) A microscopic image of a sensing electrode (200 µm in diameter).

Figure 2: Illustration of the orbital shear in-vitro platform and its experimental setup.

                                                                                                                  

For this work, HUVECs were seeded and allowed to adhere in the platform overnight in static condition. Impedance spectra (Agilent 4294A) were acquired one hour prior to the onset of shear and then 60 hours after initiating shear. Cells were monitored at radial positions 0, 3, 9, and 15 mm. The orbital shaker was set to 120 rpm and cells were exposed to ~0-10 dyne/cm2 shear. Numerical simulations for this case are demonstrated in Figure 3.

Figure 3: Numerical simulation of the shear magnitude at different radial position for a rotating cell culture well at 120 rpm, 0.95 cm orbital radius, and 2.0 mm fluid height.

Results and Conclusions

Acquired impedance measurements (Figure 4) indicate that at radial locations of 9 mm and 15 mm, impedance initially increased after initiating shear before reaching a plateau and subsequently decreasing. A previous publication suggests that the original increase is the result of cells flattening when fluid flow begins. While the subsequent decrease, demonstrates how cell junctions begin to weaken in order to align themselves in the direction of flow [7].  However, cells near the center at radial positions 0 mm and 3 mm were not uniformly oriented, similar to those observed in unsteady flow. Hence, an insignificant increase in impedance is observed which is indicative of a permeable endothelium.

The in-vitro platform presented within here is unlike other common in-vitro platforms based on microchannels because this device subjects cells to both unsteady and steady hydrodynamic shear simultaneously. Shear stress can be easily modulated by changing the rotational velocity and/or liquid volume, and the device requires very simple cell seeding and maintenance procedures. It also provides to acquire real time data, avoiding the need of lengthy fluorescent cell staining and microscope imaging.

Figure 4: Normalized impedance measurements of HUVECs under orbital shear at various radial locations within the in-vitro platform.

REFERENCES

  1. Davies, P.F. and S.C. Tripathi, Mechanical-stress mechanisms and the cell – an  endothelial paradigm. Circulation Research, 1993. 72(2): p. 239-245.
  2. Ross, R., The pathogenesis of atherosclerosis - a perspective for the 1990s. Nature, 1993. 362(6423): p. 801-809.
  3. Keynton, R.S., et al., The effect of graft caliber upon wall shear within in vivo distal vascular anastomoses. J Biomech Engr., Feb. 1999, vol. 121, p. 79-87.
  4.  Davies, P.F., et al., Turbulent fluid shear stress induces vascular endothelial cell turnover in vitro. Proc. of the National Academy of Sciences, 1986. 83(7): p. 2114-2117.
  5. Giaever, I. and C.R. Keese, Micromotion of mammalian-cells measured electrically. Proceedings of the National Academy of Sciences of the United States of America, 1991. 88(17): p. 7896-7900.
  6. Chakraborty, A., et al., Effects of biaxial oscillatory shear stress on endothelial cell proliferation and morphology. Biotechnol Bioeng, 2012. 109(3): p. 695-707.
  7. DePaola, N., et al., Electrical impedance of cultured endothelium under fluid flow. Ann Biomed Eng, 2001. 29(8): p. 648-56.

 


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