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

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.