The imperative for controlled mechanical stresses in unraveling cellular mechanisms of mechanotransduction

Abstract

Background: In vitro mechanotransduction studies are designed to elucidate cell behavior in response to a well-defined mechanical signal that is imparted to cultured cells, e.g. through fluid flow. Typically, flow rates are calculated based on a parallel plate flow assumption, to achieve a targeted cellular shear stress. This study evaluates the performance of specific flow/perfusion chambers in imparting the targeted stress at the cellular level.

Methods: To evaluate how well actual flow chambers meet their target stresses (set for 1 and 10 dyn/cm2 for this study) at a cellular level, computational models were developed to calculate flow velocity components and imparted shear stresses for a given pressure gradient. Computational predictions were validated with micro-particle image velocimetry (microPIV) experiments.

Results: Based on these computational and experimental studies, as few as 66% of cells seeded along the midplane of commonly implemented flow/perfusion chambers are subjected to stresses within +/-10% of the target stress. In addition, flow velocities and shear stresses imparted through fluid drag vary as a function of location within each chamber. Hence, not only a limited number of cells are exposed to target stress levels within each chamber, but also neighboring cells may experience different flow regimes. Finally, flow regimes are highly dependent on flow chamber geometry, resulting in significant variation in magnitudes and spatial distributions of stress between chambers.

Conclusion: The results of this study challenge the basic premise of in vitro mechanotransduction studies, i.e. that a controlled flow regime is applied to impart a defined mechanical stimulus to cells. These results also underscore the fact that data from studies in which different chambers are utilized can not be compared, even if the target stress regimes are comparable.

Figure 1

Schematic diagrams demonstrating characteristic dimensions of the flow chambers studied (not to scale).

Figure 2

Chamber 1 (Oligene) – computational model predictions are shown for the (A) velocity profile [m/s] at the center of the chamber (maximum velocity), (B) velocity within the region of interest, and (C) wall shear stress [dyn/cm²] within the region of interest for cell mechanotransduciton studies.

Figure 3

Chamber 1 (Oligene) – computational model predictions showing velocity profiles at midplane and wall shear stress profiles at the midplane and centerline of the chamber.

Figure 4

Chamber 2 (Bioptechs) – computational model predictions are shown for the velocity profile [m/s] at the center of the chamber (maximum velocity) and wall shear stress [dyn/cm²] along chamber surface, within the region of interest for cell mechanotransduciton studies.

Figure 5

Chamber 2 (Bioptechs) – computational model predictions showing velocity profiles at midplane and wall shear stress profiles at the centerline of the chamber.

Figure 6

Chamber 3 (Warner) – computational model predictions are shown for the velocity profile [m/s] at the center of the chamber (maximum velocity) and wall shear stress [dyn/cm²] along chamber surface, within the region of interest for cell mechanotransduciton studies.

Figure 7

Chamber 3 (Warner) – computational model predictions showing velocity profiles at midplane and wall shear stress profiles at the centerline of the chamber.

Figure 8

Case study based on computation model implementing geometry of Chamber 1 (Oligene) and including the cell monolayer on bottom surface. Looking from above, the shear stress [dyn/cm²] is mapped in region of interest for cell mechanotransduction studies.

Figure 9

Experimental measurement of velocity profiles and corresponding shear stresses for the case study implementing the geometry of Chamber 1 (Oligene). The shear stress profile is depicted along the surface of the cells and velocity profiles are shown (a) over a cell surface (shortened chamber height) and (b) between cell array (normalized height), in region of interest where a cell monolayer is cultivated within the chamber.

Figure 10

Experimental transverse velocity profile, of one focal plane, for the Oligene FCS chamber.

Figure 11

Experimental transverse velocity profile, of one focal plane, for the Bioptechs FCS2 chamber. Note: for this chamber, the area viewable for imaging differs from the gasket geometry; hence, the outer boundary indicates the area that is observable under the microscope.

Figure 12

Experimental transverse velocity profile, of one focal plane, for the Warner RC-30 HV chamber.