Biomedical Engineering Reference
In-Depth Information
for this particular pair of cells and substrate. A quantitative indicator that has been
used to describe the strength of cell-substrate interactions is the centrifugal force
necessary to detach 50% of the initial cell population. The centrifugation assay is,
however, a low-throughput assay as only a single force can be applied per
experiment. Moreover, in some cases the strength of cellular adhesion may exceed
the forces that can be applied in these assays with reasonable effort.
Channavajjala et al. [ 41 ] applied the centrifugation assay to quantify the
adhesion strength of tumor cells to immobilized HIV-1 Tat-protein, containing the
amino acid sequence RGD, in comparison to other specific ECM proteins such as
fibronectin and vitronectin. HIV-1 Tat was shown to mediate cell adhesion, but,
unlike the ECM proteins, the interaction between cells and the surface-immobi-
lized protein was mechanically weak.
4.2.2 Hydrodynamic Flow Experiment
In hydrodynamic flow experiments the cell-covered surface is placed in a laminar
flow channel and the cells are challenged with increasing flow velocities of the
fluid. As in the centrifugation assay, the flow of liquid generates mechanical forces
on the cell body tangential to the surface, which may lead to detachment (Fig. 8 b).
Most frequently a parallel-plate flow apparatus is used. Here the opposing side of
the channel is a parallel plate that moves with a preset velocity. A laminar shear
flow is generated over the cell surface by viscous coupling of the liquid. In order to
guarantee laminar flow the gap height between the parallel plates has to be small
compared to the length of the flow path. In a parallel-plate flow chamber the shear
stress is constant and depends on the flow rate and the gap between the two plates.
Thus, the applied shear stress can be easily adjusted by altering one of these two
parameters. After exposing the cells to laminar flow for a definite period of time,
the number of adherent cells is counted and compared to the number before the
onset of flow, providing the fraction of adherent cells that were capable of resisting
a given laminar shear stress.
Using a laminar flow assay Xiao and Truskey [ 44 ] analyzed the adhesion
strength of endothelial cells grown on a glass substrate that had been pre-coated
with linear or cyclic RGD peptides as well as fibronectin. The critical shear stress,
defined as the shear stress required to detach 50% of the cells from the coated
substrate surface, was determined to be (59 ± 13) dyne/cm 2 for cyclic and (39 ±
4) dyne/cm 2 for linear RGD peptide. The value for fibronectin-coated surfaces was
lower than those for the peptide coatings.
By mounting the flow chamber on the stage of an inverted phase-contrast
microscope and using time-lapse video microscopy, the dynamics of cell
detachment can be monitored simultaneously. However, a major limitation of
these flow systems is that the detachment forces are usually non-uniform along
the cell surface and cannot be calculated without simplifying assumptions about
cell shape.
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