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.