In general, parallel plate flow chambers are designed to provide controlled flow of fluid across a cellular monolayer. Furthermore, flow profiles are generally controlled by the mass flow rate into the chamber and by the geometry of the channel through which the fluid flows. The geometry of the channel is typically defined by the surface of the substrate on which cells are seeded, bounded above by a compliant gasket and to the sides by the walls of the chamber and/or the gasket. We expand on this general design principle to create a chamber, and in effect a fluid geometry (via gasket design), that provides a physiologic and controlled mechanical stimulus to cells. We set two goals for the novel chamber, namely to construct a device that provides either single- or dual-flow regimes across the monolayer and to ensure controlled spatiotemporal distribution of shear stress on the cells through definition of appropriate gasket geometries. In addition, we placed particular attention on design for ease of use, as tests with commercial chambers had previously raised concerns with regard to leakage, bubble production, and lack of reproducibility of experimental conditions [20].
Chamber design
To use the dual flow modality, cells seeded within and/or on one or both sides of a permeable membrane, or cells seeded in a scaffold disc, or cells in situ (in a slice of tissue), are interposed between the two gaskets and flow is applied. For example, cells are seeded on a porous membrane (0.2 micron Anapore membrane, Nunc International, Denmark) that is interposed between two gaskets. The cell monolayer is interposed between two identical fluid layers, each with an independent inlet and outlet (Figure 1). The dual configuration provides relatively even flow across both surfaces of the monolayer, where the thin membrane (thickness is chosen by the user) between the regimes suspends the specimen in a physiologic manner. However, as mentioned previously, the membrane can be interchanged with a variety of other substrates, depending on the specific needs of the study, e.g. a layer in which cells are seeded or embedded can be placed between the gaskets or a slice of tissue with cells in situ can also be interposed between the gaskets. Furthermore, single-flow can also be achieved with a solid coverslip in place of the membrane.
Following the general flow design, the chamber itself consists of an upper and lower reservoir that is dictated by compressing rubber gaskets between two polycarbonate cases (Figure 2). The solid cases are fabricated such that the inlet and outlet channels are encased within the polycarbonate, in which the flow descends from the inlet channel, into the upper reservoir (formed by the geometry of the rubber gasket), and then exits via the polycarbonate outlet channel. Aside from the inlet/outlet channels, the flow regime, from the perspective of the cellular monolayer, is completely governed by the rubber gasket geometry as well as a glass coverslip above the monolayer which enables microscope visualization into the chamber. It should be noted that single-flow is achieved by the replacing the membrane with a glass coverslip, or by using a glass plug which completely "fills" the lower reservoir, decreasing the overall thickness of the chamber for ease of use when single-flow is desired.
Gasket design
Five different gasket geometries are created (Figure 3) to provide a variety of controlled stress fields to the cell monolayer, depending on the desired application,. Each gasket is compatible with the general parallel plate configuration; geometric differences control the specific spatial variation of shear stress imparted to the cell. In gasket I the geometry is designed to provide a constant shear stress to the majority of the cellular monolayer. In this case, the geometry contains zones of widening and tapering geometry that surround the parallel walls near the chamber midplane. In gasket II and III, the gasket geometry contains symmetric and asymmetric wide flow zones, whose walls taper immediately and linearly as they approach the outlet. Thus, the geometry provides specific gradients of stress to a monolayer. Finally, the last two gasket designs are designed to provide planar jet like flow, where zones with geometries that widen and taper abruptly define two different flow geometry lengths. Again, the geometry is created such that specific gradients of stress would be imparted to the cells, as opposed to the constant stress environment of gasket I.
Computational fluid dynamics
Computational models are created for each design to predict the fluid environment and imparted shear stress in each gasket. The inlet and outlet geometries do not change within the polycarbonate sections of the chamber. Hence, it is assumed that the entering and exiting flow are identical for each case and only the geometry within the rubber gaskets is modelled. Furthermore, the chamber design can accommodate a variety of gasket thicknesses. For the computational fluid dynamics (CFD) study, all gaskets are created with a thickness of 250 μm for comparison with previous predictions for commercial chambers [20]. In addition, gasket I is modelled for both 250 μm and 500 μm thicknesses to demonstrate flow variation attributable to gasket thickness. Gasket I is also modelled for single- and dual-flow scenarios, where the membrane is given a porosity of 50% and permeability of 10-16 m2. All other gaskets are modelled in single-flow mode. Inter-geometric variations in dual flow mode can be inferred from gasket I simulation results. In all cases, the cells are not included in the gasket fluid geometry; the effect of flow on cells has been reported previously [20]. For each flow model (gaskets I-V), the number of volumes were 89760, 117130, 117130, 179380, and 189180, respectively. The mesh density was increased until grid independence was achieved for each gasket (average finite volume = 10-13 m3).
The fluid environment is simulated for each model using a computational solver (CFD-ACE software package, Huntsville, AL) that calculates the velocity, pressure, and shear stress distribution. Flow is induced via a pressure gradient for each model to achieve a target wall shear stress at the exposed surface of the cell (at the interface with the flow channel's lower surface) is 1 dyn/cm2 [23–25, 7]. Also, no-slip conditions are enforced on the walls of each gasket. For all models, the continuity equation (1) and Navier-Stokes equations (2) are solved using a 2nd order upwind-discretization scheme in three dimensions, and the wall shear stress is calculated from the wall strain rate (3). ∇·u = 0 (1)
p(u·∇u) = -∇p + μ∇2
u (2)
(3)
where u is the velocity vector, ρ is the fluid density, p is the pressure, μ is the fluid viscosity, τ is shear stress, and is the strain rate. In all cases, the fluid is assumed to be similar to water and given a viscosity = 0.001 kg/ms and density = 1000 kg/m3. Flow is simulated for steady conditions with a local convergence criterion of 0.0001, where conservation of mass is used to validate each solution. It is important to note that, while simulations are calculated to achieve a target shear stress of 1 dyn/cm2, the results of these simulations can be scaled up for flow regimes in the laminar range (appropriate assumptions for flow regimes up to and beyond 20 dyn/cm2, i.e. regimes commonly applied for mechanotransduction research).
Chamber testing
In order to evaluate the chamber design in a realistic laboratory setting, bench-top testing is performed to determine ease of use and performance. During testing, both strengths and weaknesses are noted in comparison to the previously evaluated commercial chambers [20]. Ease of use testing consists of assembling the chamber for fluid/cell experiments, with emphasis placed on the choice of the materials and geometry for each design and how the chambers interface with standard laboratory equipment used in flow/cell studies (i.e. tubing, microscope, syringe pump, etc.). However, performance testing consists of actual flow through the assembled chamber, where problems associated with leaking, bubbling, visualization, etc. are the main areas of interest. In both modes, the novel design is compared with existing designs, providing a basis to evaluate improvement over the current state-of-the-art.