Defining the Critical Shear Stress Range in Long Term ... · definition of shear stress parameters...
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cells in focus Claudin-5 (Alexa 555) DAPI ibidi GmbH, Martinsried, Germany +49 (0) 89 520 4617 - 0 www.ibidi.de Conclusion Our results highlight the importance of the exact definition of shear stress parameters since only a range between >0.7 and <30 dyn/cm² seems to mimic physiological flow conditions for HUVECs. Defining the Critical Shear Stress Range in Long Term HUVEC Cell Culture Christina Fey 1 , Anita Reiser 2 , Helga Wagner 3 , Armin Bieser 3 , Roman Zantl 3 1 Technische Hochschule Nürnberg Georg Simon Ohm; 2 Friedrich-Alexander-Universität Erlangen-Nürnberg; 3 ibidi GmbH, Martinsried Cell Orientation Elongation Capacity at 64,000 Hz Resistance at 4,000 Hz Axis Ratio* sin (angle) Long term flow conditioning using the ibidi Pump System • Flow conditioning of HUVEC (P3/P4) with ibidi Pump System in Collagen IV- coated µ-Slides I Luer over 96 hours • Flow measurements at 0.7, 5, 10 and 30 dyn/cm² • Immunofluorescence staining: Claudin-5 (tight junctions) • Impedance measurements using ECIS ZΘ (Applied Biophysics) 0° 180° 90° sin (angle) 1 0 RFU* *Major axis/minor axis *Mean Fluorescence Intensity: Cell−Cell Contact/Cytoplasm Norm. Capacity Norm. Resistance Intensity Claudin-5 Cell Physiology Cell-cell Contacts 0.7 dyn/cm² 0.7 dyn/cm² 0.7 dyn/cm² 0h 50h 100h 0.4 0.6 0.8 1.0 1.2 Flow Control 0h 3h 24h 48h 72h 96h 0.6 0.8 1.0 0h 3h 24h 48h 72h 96h 1.0 2.0 3.0 4.0 0.5 1.0 1.5 2.0 2.5 0h 3h 24h 48h 72h 96h 1 1.4 1.8 2.2 2.6 0h 50h 100h 5 dyn/cm² 5 dyn/cm² 5 dyn/cm² **** **** **** **** **** 0h 3h 24h 48h 72h 96h 0.6 0.8 1.0 * * * * *** 0h 3h 24h 48h 72h 96h 1.0 2.0 3.0 4.0 * **** ** * * 0.5 1.0 1.5 2.0 2.5 0h 3h 24h 48h 72h 96h 0h 50h 100h 0.4 0.6 0.8 1.0 1.2 1 1.4 1.8 2.2 2.6 0h 50h 100h 10 dyn/cm² 10 dyn/cm² 10 dyn/cm² **** **** **** **** **** 0h 3h 24h 48h 72h 96h 0.6 0.8 1.0 ** 0h 3h 24h 48h 72h 96h 1.0 2.0 3.0 4.0 **** **** **** **** 0.5 1.0 1.5 2.0 2.5 0h 3h 24h 48h 72h 96h 0h 50h 100h 0.4 0.6 0.8 1.0 1.2 1 1.4 1.8 2.2 2.6 0h 50h 100h 30 dyn/cm² 30 dyn/cm² 30 dyn/cm² **** **** **** **** **** 0h 3h 24h 48h 72h 96h 0.6 0.8 1.0 **** *** 0h 3h 24h 48h 72h 96h 1.0 2.0 3.0 4.0 * **** *** ** 0.5 1.0 1.5 2.0 2.5 0h 3h 24h 48h 72h 96h 0h 50h 100h 0.4 0.6 0.8 1.0 1.2 1 1.4 1.8 2.2 2.6 0h 50h 100h Flow Control Flow Control Results Low shear stress of 0.7 dyn/cm² had no effect on tested parameters and correlates to steady state observations. Morphology • Cell orientation was significantly changed at higher shear stress compared to 0.7 dyn/cm². • Elongation of cells decreases after 24 hours shear stress dependend. Cell Physiology • According to the tested parameters, the shear stress of 30 dyn/cm² proved to be far from phy- siological conditions. We observed a leakage of the cell layer indicated by the sharp and ....inhomogeneous increase of C and the distur- ....bed optical coherence of the cell layer. Cell-cell Contact Protein • The tight junctions were down-regulated under shear stress >0.7 dyn/cm². Introduction Long term in vitro experiments of HUVEC under shear stress displayed a dynamic modification in cell morphology and cell-cell contacts leading to a dramatic change in the physiological behavior of the cell monolayer. Up to now it is unknown which shear range is responsible for the observed changes in cell behavior. Therefore we investigated shear stress values between 0.7 and 30 dyn/cm² over 96 hours, regarding the physiological properties and morphological characteristics of the cell layer. In parallel to the long term impedance measurements, a cell-cell contact protein of tight junctions was counterstained with immunofluorescence. Morphology
Transcript of Defining the Critical Shear Stress Range in Long Term ... · definition of shear stress parameters...
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®cells in focus
Cla
ud
in-5
(Ale
xa
55
5)
DA
PI
ibidi GmbH, Martinsried, Germany +49 (0) 89 520 4617 - 0 www.ibidi.de
ConclusionOur results highlight the importance of the exact
definition of shear stress parameters since only a
range between >0.7 and 0.7 dyn/cm².
IntroductionLong term in vitro experiments of HUVEC under shear stress displayed a dynamic modification in cell morphology and cell-cell contacts leading
to a dramatic change in the physiological behavior of the cell monolayer.
Up to now it is unknown which shear range is responsible for the observed changes in cell behavior. Therefore we investigated shear stress
values between 0.7 and 30 dyn/cm² over 96 hours, regarding the physiological properties and morphological characteristics of the cell layer.
In parallel to the long term impedance measurements, a cell-cell contact protein of tight junctions was counterstained with
immunofluorescence.
Morphology
