Improved visualization and quantitative analysis of drug effects using micropatterned cells.

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck (1-2). To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

The application of 3D micropatterning of agarose substrate for cell culture and in situ comet assays.

We report the fabrication of a 3D micropatterned agarose substrate that enables the culture of single or multiple cells. Patterning was performed on dried agarose using deep UV irradiation leading to 6-microm-deep micropatterns of 25-70 microm in diameter. Cell adhesion was facilitated by the specific grafting of ECM (extra cellular matrix) proteins such as fibronectin into the micropatterns. We show that the pattern size induced the adhesion of one or more cells, thus allowing precise control of the cell number used in the assay, and that cells proliferated similarly as in standard culture conditions. Moreover, cell polarity appeared well preserved on this substrate, so polarized cells like hepatoma HepaRG cells might maintain their differentiation status and act as primary human hepatocytes for hepatotoxicity testing. These 3D patterned culture slides have been successfully used for in situ comet assays and there is evidence that the genotoxic effects of sub-cytotoxic concentrations of drugs could be analyzed in a large number of single HeLa cells. Coupled with the parallel-based design of the 3D micropatterning, which allows automated image analysis, these results strongly indicate that this new cell array system is suitable for high-throughput cytotoxicity and genotoxicity screening applications.