Paper BI-ThM5
Fabrication of Surface-Engineered 3D Micro-Well Arrays for High Resolution and High Throughput Analysis in a Single Cell Format

Thursday, October 18, 2007, 9:20 am, Room 609

In addition to substrate rigidity, matrix composition, and cell shape, dimensionality is now considered an important physical property of the cell microenvironment which directs cell behavior. However, available tools for the study of cell behavior in two-dimensional (2D) versus three-dimensional (3D) environments are difficult to compare, and no tools are available which provide 3D shape control of individual cells. Using microfabrication and replication techniques, we developed PDMS substrates for the culture of single cells in 3D arrays compatible with both high-resolution microscopy and high-throughput analysis. Cell adhesion was limited to within microwells by passivation of the flat upper surface through ‘wet-printing’ of a non-fouling polymer and backfilling of the wells with either specific adhesive proteins or lipid bilayers, the latter produced by exposing the oxidized PDMS wells to lipidic vesicles that spontaneously fused to form supported lipidic membranes. The surfaces were characterized at each fabrication stage by confocal laser scan microscopy (CLSM) in conjunction with labeled polymers and proteins, and the Quartz Crystal Microbalance (QCM-D) technique and FRAP to characterize bilayer formation on PDMS surfaces. Endothelial cells constrained within microwells were viable, although cell death was increased in very constrained microwells as has been reported for cells on flat substrates. In contrast to studies on 2D surfaces, actin stress fibers were present even within cells cultured in very constrained microwells, and in addition the cytoskeleton was 3D and not limited to the cell-substrate interface. These observations demonstrate that microwells can be used to produce microenvironments for large numbers of single cells with 3D shape control and can be added to a repertoire of tools which are ever more sought after for both fundamental biological studies as well as cell-based assays for drug development and screening. Future work is directed towards the study of (stem) cell differentiation and its dependence on surface (bio)chemistry, ligand mobility and substrate rigidity as well as the development of microwells with walls covered by cadherin-functionalized lipidic membranes simulating a microenvironment that is possibly closer to the one of cells in multicellular colonies and tissue.