1CYTOO Cell Architects, Grenoble, France, 2Centre Commun de Quantimétrie, Faculté de Médecine Rockefeller, Lyon, France
Degot, S., Auzan, M., Chapuis, V., Béghin, A., Chadeyras, A., Nelep, C., et al. Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells. J. Vis. Exp. (46), e2514, doi:10.3791/2514 (2010).
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.
1. Cell Preparation and Seeding on Micropatterns
2. Blebbistatin Treatment
3. Cell Fixation and Staining of Actin
4. Microscope Setup and Automated Image Acquisition
Numerous imaging software packages exist that control the microscope for fast automated image acquisition and display. This example uses Metamorph imaging software and automatic acquisitions are performed on a Nikon Eclipse Ti microscope equipped with a CCD Hamamatsu camera and an Intensilight mercury-fiber illuminator. Images are acquired at 20x magnification in three wavelengths corresponding to DAPI (nuclei), Cy-3 (micropatterns) and FITC-Phalloidin (actin). The number of micropatterns visualized per field will vary depending on the camera and objective setups. CYTOO micropatterns are positioned at defined intervals from each other (100 or 130 μm) across the chip greatly facilitating acquisition.
5. Automated Image Processing and Analysis
Many image processing and analysis software packages exist. The procedures described below outline image processing and analysis steps performed by 2 custom-made macros written for the open source program ImageJ. The first macro CellRef creates a Reference Cell, the second Hypotenuse measures rigidity/collapse of the cell membrane. Both are available upon request through www.cytoo.com.
6. Representative Results
Examples of what cells should look like on micropatterns immediately and several hours after the critical washing steps described in 1.6-1.8, are shown in Figure 5. After 15 min on CYTOOchips, round HeLa cells are observed at equal distances indicating that they have adhered to the fibronectin micropatterns (Figure 5a). Adhesion is complete when cells remain immobilized despite gentle shaking of the culture vessel. To minimize the number of micropatterns occupied by multiple cells, chips are then flushed with PBS to remove cells floating over the cytophobic areas. After several hours, cells that are fully stretched over the micropatterns will look like those shown in Figure 5b.
Typical single cell images obtained after incubation of cells with blebbistatin, fixation and staining of actin and nuclei are presented in Figure 6. After automated acquisition of multiple images and image processing using the ImageJ CellRef macro, a color-coded frequency map is generated that depicts the intensity of actin averaged over all cell images (Figure 6c and 6f). The comparison of the Reference Cell for nontreated and treated conditions shows the potential of this approach for easy observation of the phenotype under study and is especially useful for exploring cellular drug effects.
An example of one possible analysis of the effect of blebbistatin on cells is presented in Figure 7. The number of collapsed cells (ie. cells with a blank area existing under the theoretical hypotenuse) detected in 50 control cells and 50 cells treated with 5 μM blebbistatin as detected by the ImageJ Hypotenuse macro is shown. The increased number of collapsed cells in the blebbistatin treated population reflects relaxation of the stress fibers and hence tension at the membrane due to blebbistatin inhibition of actinomyosin contractile forces within the cell.
Figure 1. Procedure to create a new journal with Metamorph.
Figure 2. Flow chart of the automatic image processing procedure.
Figure 3. Filtering out of single cells.
Figure 4. Building a Reference Cell.
Figure 5. Cell seeding. A) Hela cells adhered to micropatterns after the flushing step (4x magnification) B) HeLa cells after full spreading on L micropatterns (10x magnification).
Figure 6. Comparison of nonpatterned and micropatterned cells in control and drug treated conditions. HeLa cells were seeded for 3 hours and treated with 5 μM blebbistatin for 1 hour (lower panel) or left untreated (upper panel). In control nonpatterned cells (a), actin assembles into multiple fibers which disassemble imperceptibly upon treatment with 5 μM blebbistatin (d). On L-micropatterns, control cells adopt a triangular shape (b) and develop a major actin fiber (stress fiber) between the 2 apices of the L. Compared to nonpatterned cells (d), blebbistatin induces a major phenotypic change on L-micropatterned cells (e). Direct visualization of drug effects and the comparison of control and treated conditions can be visualized rapidly with the Reference Cell presentation built from 20 cells. Similar to individual cells, a clear and intense stress fiber is present on the control Reference Cell (c), while blebbistatin treated cells collapse and the major stress fiber disappears (f).
Figure 7. Example of an analysis of the effects of blebbistatin on cells using the macro Hypothenuse. While 25% of control cells were collapsed, this increased 3 fold to 75% in the 5 μM blebbistatin treated cells reflecting the weakened actinomyosin contractile network (n=50 cells).
Choice of micropattern
While the effects of 5 μM blebbistatin are barely detectable on standard culture supports, efficient quantification is possible on micropatterns that concentrate actin into a single major stress fiber. This enhances visualization of the actin cytoskeleton aiding accurate quantification of blebbistatin effects on the cell. The choice of micropattern shape is critical in the design of the assay and is designed according to the phenotypic change to be highlighted in the study.
Micropatterns facilitate the visualization and quantification of drug effects, especially at low concentrations. The replacement of homogenous planar substrates with adhesive micropatterns for cell-based assays improves the accuracy, sensitivity and quality of data produced. In consequence, fewer cells are needed for analysis in order to achieve robust statistical outcomes 3. Adhesive micropatterns offer a real opportunity for improving functional studies and exploring phenotypic changes at lower drug concentrations in order to avoid inducing toxic or indirect drug effects. If adequate screening platforms are used, micropatterns can meet the demands of pharmaceutical companies for improving gene/drug screening applications. Some additional examples of recent publications that have exploited micropatterns for analyzing and quantifying cellular phenomenon are cited below 3-13.
All the authors except Anne Béghin are employees of CYTOO S.A, which produces reagents and instrumentation featured in this article.
|CYTOOchips 20x20 L-M-FN||CYTOO||10-114||CYTOOchips are available for adsorption of the protein of your choice|
|PBS 1x||GIBCO, by Life Technologies||14040-091|
|Counting chamber (Kova slide)||Prolabo||71287.01|
|DMEM/F-12 + Glutamax-I||GIBCO, by Life Technologies||25300-054|
|FBS (f tal bovine serum)||PAA Laboratories||31331-028|
|Penicillin & Streptomycin||PAA Laboratories||A15-101|
|Cytoskeleton Buffer 1X (CB)||Sigma-Aldrich||MES : M8250 KCl : I2636 MgCl : M2393 EGTA : E3889||10 mM MES pH 6.1, 138mM KCl,
|PFA 32 %||Electron Microscopy Sciences||15714|
|PFA 5%||Mix 10 mL of PFA 32% with 54 mL of CB 1.185X|
|PBS tablets||GIBCO, by Life Technologies||18912-014|
|Bovin serum albumin (BSA)||Sigma-Aldrich||A7806|
|Epifluorescent microscope||Nikon Instruments||Eclipse Ti|