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Representative Results The murine bone marrow-derived EC line STR-12 was grown on inserts with 5 ��m pores. The rate of EC growth was monitored under a microscope and when the EC were 100% confluent, the inserts were transferred into the wells in the lower compartment of the 3D device. Immediately before placing the inserts, the wells of the lower compartment were filled with culture medium alone (negative control) or with medium supplemented with stromal cell-derived factor-1 (SDF-1; 5 ng/ml and 50 ng/ml). Thereafter, the 3D Sitaxentan device was assembled and the chamber was filled with medium as described in the protocol. The test cells to be circulated in the upper compartment of the device were freshly harvested murine bone marrow cells (3.5 x 106 cells per chamber). A defined shear stress of 0.8 dyn/cm2 was applied by setting the peristaltic pump speed at 0.2 ml/min. The entire working system was then placed in the 5% CO2 incubator at 37 ��C and the cells were allowed to circulate and interact with the EC monolayer for 4 hr. At the end of that time, the circulating cells were collected, the chamber was disassembled, and the inserts were removed as described in the protocol. The transmigrated cells were harvested from the lower wells, washed, resuspended in fresh medium, and transferred to methylcellulose cultures supplemented with hematopoietic growth factors for colony-forming cell (CFC) assay (Figure 3). As expected, we found a significantly higher number of CFC had migrated across the EC monolayer to the wells containing 50 ng/ml SDF-1 than to wells containing 5 ng/ml SDF-1 or medium alone. As we described earlier, none of the current in vitro techniques available to study cell migration are capable of testing the effect of the local microenvironment on the ability of EC to support extravasation of migrating cells. To illustrate how this can be achieved with the 3D device, we examined extravasation of circulating hematopoietic cells across a layer of EC and a layer of bone marrow stromal cells. For this, a second (lower) insert containing a layer of stromal cells was juxtaposed to the upper insert containing the EC monolayer in the lower plate (Figure 4). The experiment was then performed as described above and the transmigrated cells were harvested from the wells and counted. The results demonstrated that insertion of an additional layer of stromal cells beneath the EC monolayer significantly increased the migration of hematopoietic cells toward SDF-1 (Figure 4). This finding is consistent with the notion that the local microenvironment contributes to the recruitment of circulating cells to the tissue. Figure 1. The 3D flow chamber device. A: The hermetically sealed 3D device consists of two compartments separated by a replaceable porous membrane (PM).