Ring-Shaped Micropatterning Cell Chirality Assay: An In Vitro Technique to Determine Multicellular Chirality Based on Cell Alignment on Ring-Shaped Micropatterns

To determine in vitro mammalian cell chirality - the left-right bias of cells to align in a leftward or rightward orientation under confining conditions - begin with custom-fabricated gold-coated slides in a media-containing multi-well plate. The slides comprise micropatterns of ring-shaped islands of cell adhesion-promoting fibronectin, backfilled with cell adhesion-resistant self-assembled monolayer.

Next, seed an appropriate volume of mammalian cell suspension onto the slides and incubate. The cells adsorb onto the fibronectin-containing ring micropatterns. The surrounding self-assembled monolayer prevents non-specific cell attachment outside the ring boundaries.

Replace the media, removing any unattached cells, with fresh media. Add an actin-interfering drug to one set of slides and incubate.

The adhered cells proliferate, spreading and assuming the shape of the underlying fibronectin islands, whilst reorganizing their actin fibers in response to the spatial cues, which confine the cell monolayer, promoting biased alignment.

Now, treat the slides with paraformaldehyde for cell fixation. Under phase-contrast microscopy, the cells on the ring micropatterns appear as dark regions with bright contours aligned according to their preferential bias. 

Using software, calculate each cell's angle of alignment as the deviation from the ring's circumferential direction. Average to determine the mammalian cell chirality. Actin-interfering drug-treated cells may exhibit reverse chirality.  

Before seeding the cells, warm the media and trypsin in a water bath, tempered at 37 degrees Celsius.

For better cell attachment, soak the patterned slide in a 12-well plate containing culture media, and warm it at 37 degrees Celsius in the incubator. Once the cells are trypsinized, neutralize the cells with FBS-containing media. Pellet down the cells at 100 x g for 3 minutes, and then, resuspend the cell pellet in fresh media. Count the cells, and dilute the cell suspension to achieve the concentration of 200,000 cells per milliliter.

Add 0.5 milliliters of the cell suspension to each well containing one gold slide. Gently shake the plate a few times for uniform cell seeding, before incubating for 15 minutes for cell attachment. After 15 minutes, check the cell attachment under a microscope, and if required, incubate for some more time.

Once the cells are attached, aspirate out the media containing unattached cells from each well, and add 1 milliliter of fresh culture media. Culture the cells in the incubator for 24 hours and check confluency to determine if chirality has formed.

When the required confluency is achieved, fix the cells by removing the culture media. After rinsing with PBS once, add 4% paraformaldehyde solution to the slide, and incubate at room temperature for 15 minutes, before rinsing again with PBS three times.

To acquire the images, use a phase contrast microscope with camera functionality, and capture each ring on the slide at high resolution.

For chirality characterization, download the MATLAB code files. Add the code folder, and subfolders to the MATLAB path and open the "ROI_selection.m" file. In line 4, change the directory to the desired data folder.

Change the image size in line 14, with the first two figures representing the inner circle size of the ring, while the other two representing the outer.

To determine the region of interest or ROI in the phase contrast images, execute the MATLAB code "ROI_selection.m" m by clicking the Run button. Manually drag the selection square to fit the ring, then, double click on the image to confirm the selection.

After selecting ROI from all the images in the folder, a ".mat" file will be generated to store the ROI information for each image. Then, open the "Analysis_batch.m" file and change the directory of the folder as demonstrated before. Click on the Run button to execute the code "Analysis_batch.m" to determine the chirality of multiple cellular ring patterns.

A "datatoexcel.txt" file will be generated, containing circular statistics for each ring, as well as the numbers of clockwise, non-chiral, and anticlockwise rings.