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. Video LinkThe video component of this article can be found at https://www.jove.com/video/2514/Protocol 1. Cell Preparation and Seeding on Micropatterns 1. Place 2 CYTOOchips in 2 independent wells of a 6 well plate ensuring that you read "CYTOO" in the lower right corner of the CYTOOchip.Micropatterns can be fluorescently labeled with Cy3 or Cy5 dyes.CYTOOChips used for this experiment have Cy3 fluorescently labeled micropatterns.CAUTION: Keep chips in the dark.2. Collect HeLa cells (~80% confluent) by trypsinization.Check under a microscope that cells are properly dispersed by the trypsin treatment.Collect and centrifuge cells at 300g for 4 min, then gently resuspend the cell pellet.Count cells and dilute to a concentration of 15,000 cells/ mL in completed DMEM/F12 culture media.3. Dispense 4 mL (60,000 cells) into each well containing a CYTOOchip. CAUTION:The plate should be moved as little as possible to avoid inducing any rotating movements in the medium as this will tend to concentrate cells at the center.4. Let the cells sediment for 10 min under the hood then move them to the cell incubator.After 10-20 min, cells will start to adhere to the micropatterns.After 10 min, check regularly under the microscope.5.As soon as cells have attached, change the cell medium and gently flush the coverslip surface using the following procedure: 6. Keeping the plate flat, gently aspirate the medium with a pipette from the side of the well.CAUTION: Never let the chip dry out, this means never aspirate off all the liquid but leave enough to cover the CYTOOchip at all times.7. Add 4 mL PBS and aspirate again first starting at the center of the chip then moving to the side of the well.Aspirate off the PBS as described without exposing the chip to air.Repeat 3-4 times.8. Check under the microscope for floating cells:• If a large number of floating cells remain, repeat the washing procedure.