(322d) An Orbital Shear Platform for in-Vitro Real-Time Endothelium Characterization

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].

12.0pt"> 

12.0pt">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-vitroplatform.

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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