ibidi TIP

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Flow Assays

Impedance Measurements in Combination with the ibidi Pump System

Endothelial cells in vivo and in vitro respond to shear stress by activating intracellular signaling pathways, with morphological changes and gene expression. The shown experimental setup combines the ECIS System with the ibidi Pump System to more closely simulate the in vivo environment. Since endothelial cells are exposed to shear stress in their physiological environment, cell cultivation with a permanently circulating medium replicates the physiological conditions more accurately than static cultivation in open wells.


HUVECs (Human umbilical vein endothelial cells) cultivated under flow conditions for 340 hours. Resistance was measured using the ECIS System in conjunction with an ECIS Flow Array.

Laminar flow was added stepwise, after one day, during the proliferation phase (5/10/20 dyn/cm2). The cell layer’s response was a short increase of the resistance. The ongoing experiment was performed with 20 dyn/cm2 and showed a continuous decline in resistance over time. After 150 hours, shear stress was stopped, which resulted in a surprisingly clear positive slope of the resistance. Adding shear stress with 20 dyn/cm2 at 170 hours again resulted in a decline in the resistance. Stopping the flow brought the resistance back to a steady-state line.

The data shown indicates two different properties of the endothelial cell layer. In the short run (i.e., before confluence), the addition of shear stress leads to a non-permanent increase of the resistance. In the long run, a continuous laminar shear stress reduces the resistance of the cell layer. This reduction is reversible and can be neutralized by stopping the flow. These physiological differences are accompanied by strong morphological and biochemical changes in and within the cells. Cell-to-cell contacts, and the arrangement of the cells, will change after some days under flow conditions. The actin skeleton is well-organized, and the VE-cadherins will change from a diffused arrangement, to a well-organized one (see fluorescence images).

 


Appropriate Electrode Array for Flow Assays:

  • ECIS Flow Array1E and 10E

 

 


Optical Measurements

Experimental Example: Morphological Changes in Cell Cytoskeleton after Cultivation under Flow Conditions

Live cell imaging of HUVECs cultured under flow conditions (20 dyn / cm²) in μ-Slide I 0.4 Luer for 9 days. The cells were transduced with the adenoviral vector rAV CMV LifeAct-TagRFP 24 hours prior to running the experiment.

HUVECs cultivated and stained in a µ-Slide I 0.2 Luer for 7 days using a flow rate of 3.9 ml/min (20 dyn/cm2).
Blue: cell nucleus.
Green: VE-cadherins.
Red: actin filaments


Static conditions


Flow conditions