Physiologically, four major types of hepatic cells - the liver sinusoidal endothelial cells, Kupffer cells, hepatic stellate cells, and hepatocytes - reside inside liver sinusoids and interact with flowing peripheral cells under blood flow. It is hard to mimic an in vivo liver sinusoid due to its complex multiple cell-cell interactions, spatiotemporal construction, and mechanical microenvironment. Here we developed an in vitro liver sinusoid chip by integrating the four types of primary murine hepatic cells into two adjacent fluid channels separated by a porous permeable membrane, replicating liver's key structures and configurations. Each type of cells was identified with its respective markers, and the assembled chip presented the liver-specific unique morphology of fenestration. The flow field in the liver chip was quantitatively analyzed by computational fluid dynamics simulations and particle tracking visualization tests. Intriguingly, co-culture and shear flow enhance albumin secretion independently or cooperatively, while shear flow alone enhances HGF production and CYP450 metabolism. Under lipopolysaccharide (LPS) stimulations, the hepatic cell co-culture facilitated neutrophil recruitment in the liver chip. Thus, this 3D-configured in vitro liver chip integrates the two key factors of shear flow and the four types of primary hepatic cells to replicate key structures, hepatic functions, and primary immune responses and provides a new in vitro model to investigate the short-duration hepatic cellular interactions under a microenvironment mimicking the physiology of a liver.

Interactions between polymorphonuclear neutrophils (PMNs) and tumor cells have been reported to facilitate the adhesion and subsequent extravasation of tumor cells through the endothelium under blood flow, both of which are mediated by binding ␤ 2 -integrin to intercellular adhesion molecule 1 (ICAM-1). Here the adhesions between human WM9 metastatic melanoma cells, PMNs, and human pulmonary microvascular endothelial cells (HPMECs) were quantified by a gas-driven micropipette aspiration technique (GDMAT). Our data indicated that the cellular binding affinity of PMN-WM9 pair was 3.9-fold higher than that of the PMN-HPMEC pair. However, the effective binding affinities per molecular pair were comparable between the two cell pairs no matter whether WM9 cells or HPMECs were quiescent or cytokine-activated, indicating that the stronger adhesion between PMN-WM9 pair is mainly attrib- Tumor metastasis requires the detachment of malignant cells from primary tumor, invasion through blood/lymph vessels, transmigration across the endothelium, and finally, adhesion to the host cells (1). The adhesion and subsequent extravasation of tumor cells through the vascular endothelium are critical steps in this complex cascade (2). Malignant melanoma is the most deadly skin cancer with high metastatic potential (3). Although the binding of such adhesion molecules as P-selectin (4, 5), Lu-ECAM-1 (6), or integrin ␣ 4 ␤ 1 (VLA-4) (7) to their ligands has been found to support the adhesion of melanoma tumor cells (TCs) 3 with endothelial cells (ECs), the expression of the molecules seems insufficient to support the direct adhesion between TCs and ECs in blood flow (8). Rather, the presence of polymorphonuclear neutrophils (PMNs) potentially enhances TCs adhesion to the ECs and induces the subsequent extravasation under flow conditions (9 -11). A "two-step adhesion" hypothesis was introduced that involves initial PMN tethering on ECs followed by subsequent TCs being captured by tethered PMNs (10). Another line of in vitro evidence has indicated independently that TCs are able to aggregate with PMNs in a shear flow (12), which proposes an alternative hypothesis that TCs interact first with PMNs to form aggregates in a near-wall region of vascular endothelium prior to binding to EC through PMNs. Thus, it is physiologically important to elucidate how TCs, PMNs, and/or ECs interact with each other and what their underlying molecular mechanisms are.Regulations of PMN-EC or PMN-TC adhesion have been extensively studied at the cellular and molecular levels. Tethering and rolling of leukocytes on the endothelium are initiated by selectins and stabilized by ␤ 2 -integrins, which is followed by PECAM-1-induced transmigration through the extracellular matrix (13,14). Tumor cells are found to interact with PMNs under shear flow in a cone-plate viscometer (12,(15)(16)(17). Evidently, the interactions between ␤ 2 -integrin (LFA-1 or Mac-1) expressed on PMNs and intercellular adhesion molecule 1 (ICAM-1) expressed on TCs or ECs dominate both PMN-EC a...