| Cell-cell contact via VE-cadherin plays an important role in regulating numerous functions in endothelial cells, including proliferation. Using different experimental approaches to manipulate cell-cell contact, investigators have observed both inhibition and stimulation of proliferation. Attempts to reconcile these results were hampered by a technical inability to generate precisely defined multicellular aggregates. To address this issue and enable cell positioning for both cell biology and cell-based devices, we developed a novel approach to simultaneously position thousands of cells into defined groups using forces generated by dielectrophoresis (DEP). Our system, the Electronic Cell Arraying Device (eCAD) simultaneously traps thousands of cells on micropatterned electrodes. These 3-mum diameter electrodes, embedded within a flat culture surface, use electrical forces to attract, in general, one cell to each electrode. Fluid flow is then used to remove excess cells. The remaining cells, positioned in registration with the electrode array, adhere physiologically to the ECM-coated culture surface, and can be placed in standard culture conditions. The geometry of these patterned cells can be restricted by topological and/or chemical patterns on the culture surface. Analysis of proliferation, viability, and morphology indicated that cells are not harmed by the eCAD under appropriate conditions.;A combination of active patterning via DEP and control over cell-cell contact using microfabricated wells facilitated an investigation of whether the number of neighboring endothelial cells forming cell-cell contacts affected the proliferative response. Varying cell-cell contact resulted in a biphasic effect on proliferation: one contacting neighbor increased proliferation, while two or more neighboring cells partially inhibited this increase. We also observed that cell-cell contact increased the formation of actin stress fibers, suggesting the possibility that cell-cell contact regulated proliferation by altering cell mechanics. Expression of dominant negative RhoA (RhoN19) blocked the cell-cell contact mediated increase in stress fibers, and also abrogated the increase in proliferation with cell-cell contact. These findings concur with previous studies from our group in which the inhibition of myosin-generated cytoskeletal tension abrogated contact-mediated proliferation increases.;We next sought to determine whether it is tension across cell-cell contacts that is important to the contact-mediated proliferation changes, or whether cell-cell contacts cause an abrogation of intracellular tension which in turn regulates proliferation. To address this issue, we studied mixed pairs of cells, containing one cell expressing RhoN19 and one untreated cell. (Abstract shortened by UMI.). |
