In vivo, endothelial cells develop and differentiate under shear stress conditions. When starting cell-based assays that use endothelial cells, you should consider the possible influence of this mechanical force on cell morphology and physiology.
There is growing evidence that under in vitro conditions, the mechanical perturbations have a profound effect on the characteristics of the cell layer. In long-term experiments with HUVEC, three different phenotypes can be observed: a round flat cell after seeding, elongated cells after 1-2 days, and finally an oriented cobblestone appearance of a dense compact cell layer. The morphological changes are accompanied by physiological changes of the endothelial cell monolayer, which is measured by impedance monitoring.
Working Principle of Impedance Measurements
Cells are cultivated in channels with electrode arrays. Depending on the state of development of the endothelial cell monolayer, the gaps between the cells (influenced by the cell-cell contacts) change in size. These shifting gaps can be characterized by their change in conductivity when applying AC currents of different frequencies. The continuous red lines in the scheme represent the ion currents between the cells when applying the AC current.
Conductivity and Impedance Change of an Endothelial Cell Monolayer
Electrode with HUVEC under static conditions
Electrode with HUVEC under shear stress conditions
Static culture: After seeding, cells grow to confluence in 2 - 3 days. During this time, the conductivity is reduced, and the resistance rises to a plateau, which is then maintained under the subsequent static culturing over several days.
Flow culture: Applying shear stress reduces the conductivity and increases the impedance of the monolayer. Over a period of days the impedance of the endothelial monolayer decreases. The physiological properties of the cell monolayer are altered when compared to the static conditions.
Immunofluorescence Staining of Flow-Conditioned Endothelial Cells
Immunofluorescence stainings can easily be done in ibidi channel μ-Slides, subsequent to the performance of your specific experiment. When comparing the cultivation of HUVEC under static and flow conditions, the differences between static culture and flow conditioning are clearly visible.
Find detailed information about immunufluorescence staining in ibidi µ-Slides and µ-Dishes here.
Adherence Junctions (VE-Cadherins)
Flow-conditioned cells are elongated and show distinct stress fibers, whereas static culturing generates cells with a bigger surface and a chaotically structured actin skeleton. VE-Cadherins (adherence junction proteins of endothelial cells) are present in both conditions.
Red – F-Actin (Phalloidin-Alexa 633), Green – VE-Cadherin (VE-Cadherin (D87F2) XP, Rabbit mAb), Blue – Cell nuclei (DAPI)
Claudin-5, a tight junction protein, can be found at the cell-cell contact zone when the cells are flow-conditioned for five days. This shows that the impact of the mechanical shear stress is crucial for the differentiation of the cell layer.
Red – F-Actin (Phalloidin-Alexa 633) Green –Claudin-5 rabbit polyclonal AB (34-1600) Blue – Cell nuclei (DAPI)