(335e) Introduction of Two Electrical Impedance Systems for the in-Vitro Characterization of Huvecs Undergoing Hydrodynamic Shear Stress

Velasco, V., University of Louisville
Gruenthal, M., University of Louisville
Berson, R. E., University of Louisville
Keynton, R., University of Louisville
Williams, S. J., University of Louisville
Kinetics of endothelial cells undergoing hydrodynamic shear stress are extremely dynamic and require a real-time evaluation. Electrical impedance systems have shown to have the ability to indicate numerous status factors of cellular monolayers in real-time, including cell viability [1], confluency [2], cell-cell integrity and morphology [3]â??[5]. Within this work, we introduce two electrical impedance chips for the in-vitro characterization of HUVECs undergoing hydrodynamic shear stress. Each system has a different fluid dynamic and structure environment that can cater to the scientist investigation interest and cell inoculation preference.

One system is a microfluidic platform where steady laminar shear stress is generated within a vessel-like structure. The microfluidic impedance platform consists of two microfabricated arrays of independently addressable gold electrode of various diameters (50 µm, 100 µm, and 200 µm), and a common large counter electrode on a borosilicate substrate. Different electrodes sizes were used to test and compare the behavior of different cell population sizes. The microfluidic chip also includes a two-channel design, such that one may contain cells, while the other remains without cells. This design feature helps to identify environmental variables affecting the endothelial cell data.

The other system, an orbital shear platform consists of a 35 mm diameter open-well formed by a PDMS mold. This design allows for more simplified and conventional cell inoculation methods. Unlike the steady laminar shear stress observed in the microfluidic platform, within the orbital shear platform, oscillatory hydrodynamic shear is reproduced. Oscillatory shear is more representative of the pulsatile nature of blood flow and also produces various shear values across the well dish, enabling simultaneous testing of different shear stress magnitudes on the same endothelial cell culture sample. Shear stress was induced by placing the platform on an orbital shaker at a rotation speed of 120 rpm. A computational model was used to determine the temporal and spatial resolution of wall shear stress for one orbit cycle [6]â??[8]. Similar to the microfluidic platform, the orbital shear platform consists of microfabricated arrays of 200 µm (in diameter) gold sensing electrodes and complimentary counter electrodes on a borosilicate substrate. Only 200 µm diameter electrode were incorporated into this design due to the complex fluid dynamics which benefit from more measurements of the same subpopulations size for reproducibility purposes. The chip contains 3 rows of electrodes which are spaced 120 degrees from each other. Electrodes are positioned at defined radial locations of 0, 2.5, 5.0, 7.5, 10.0, and 12.5 mm with respect to the center of the borosilicate chip. Within the rotating orbital well, cells experience a range of shear stress values and in differing directions at selected radial positions [6]. Depending on the electrode position, cells experienced shear values between 0.6-6.71 dyne/cm2.

Both systems have the ability to monitor cell adaptation to hydrodynamic shear stress in real-time via impedance spectroscopy measurements. Chips were inserted into commercially available card readers and connected to an in-house designed printed circuit board programmed to automatically record impedance spectra from a high-precision impedance analyzer (HP4294a) at specified time intervals. Human umbilical vein endothelial cells (HUVECs) were used as an initial endothelium model and cultured within the chips. The collected impedance spectra were fitted to an equivalent circuit model. The individual electrical elements are extracted and used as representative variables for the cellular monolayer and microenvironment behavior. In both systems despite the different fluid dynamic environments, results indicate that the onset of shear stress (acute flow) generates an increase in the cell monolayer resistance, which is followed by a decrease. This is all accompanied by the re-orientation and elongation of cells.


[1] X. Zhang, F. Li, A. N. Nordin, J. Tarbell, and I. Voiculescu, â??Toxicity studies using mammalian cells and impedance spectroscopy method,â? Sens. Bio-Sensing Res., vol. 3, pp. 112â??121, 2015.

[2] S. Michaelis, R. Robelek, and J. Wegener, â??Studying Cellâ??Surface Interactions In Vitro: A Survey of Experimental Approaches and Techniques,â? in Tissue engineering III: cell - surface interactions for tissue culture techniques., vol. 126, 2012, pp. 33â??66.

[3] J. Seebach, P. Dieterich, and F. Luo, â??Endothelial barrier function under laminar fluid shear stress,â? Lab. â?¦, vol. 80, no. 12, pp. 1819â??31, Dec. 2000.

[4] J. Seebach, G. Donnert, R. Kronstein, S. Werth, B. Wojciak-Stothard, D. Falzarano, C. Mrowietz, S. W. Hell, and H. J. Schnittler, â??Regulation of endothelial barrier function during flow-induced conversion to an arterial phenotype,â? Cardiovasc. Res., vol. 75, pp. 596â??607, 2007.

[5] S. Arndt, J. Seebach, K. Psathaki, H. J. Galla, and J. Wegener, â??Bioelectrical impedance assay to monitor changes in cell shape during apoptosis,â? Biosens. Bioelectron., vol. 19, pp. 583â??594, 2004.

[6] J. M. D. Thomas, A. Chakraborty, M. K. Sharp, and R. E. Berson, â??Spatial and temporal resolution of shear in an orbiting petri dish,â? Biotechnol. Prog., vol. 27, pp. 460â??465, 2011.

[7] A. Chakraborty, S. Chakraborty, V. R. Jala, B. Haribabu, M. K. Sharp, and R. E. Berson, â??Effects of biaxial oscillatory shear stress on endothelial cell proliferation and morphology,â? Biotechnol. Bioeng., vol. 109, no. 3, pp. 695â??707, 2012.

[8] R. E. Berson, M. R. Purcell, and M. K. Sharp, â??Computationally Determined Shear on Cells Grown in Orbiting Culture Dishes,â? in ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY: OXYGEN TRANSPORT TO TISSUE XXIX, 2008, pp. 189â??198.