Figure 1. Darcy Flow Experimental Apparatus for the application of shear stress in 3D and Modified Boyden chamber invasion assay.

(A) The flow apparatus applied a constant hydrostatic pressure via a double reservoir system composed of a larger reservoir [A] feeding flow media into the smaller syringe reservoir [B] and a pressure release tube [C], both of which were fastened within a rubber seal [D] to the cell culture insert [E]. The pressure release tube [C] ensured that there were no fluctuations in pressure applied across the collagen/cell suspension [F]. Hydrostatic pressure [ΔP] drove media throughout the thickness of the gel [L] and exerted shear stress on the cell membranes. Flow media filtrate was collected in another reservoir [G]. (B) At the end of the flow period, the inserts containing the cell suspensions [E] were decoupled from the apparatus and the invasion assay ensued. During the migration period, 10 nM TGF-α directed cells to migrate through 8 µm pores towards the underside of the filter. At the end of the migration period, cells on the underside of the inserts were stained and migration rates were quantified.