Microfabricated cantilevers for parallelized cell-cell adhesion measurements

Single-cell adhesion measured with atomic force microscopy (AFM) offers outstanding time and force resolution and allows the investigation of many important phenomena with unmatched precision. However, this technique suffers from serious practical limitations that hinder its effective application to a broader set of situations. Here we propose a different strategy based on the fabrication of large cantilevers and on the culture of the cells directly on them. Cantilevers are fabricated by standard micromachining, with an active area of 300 × 300 μm. A wedged structure is created so that the cantilever surface lies parallel to the substrate when mounted on an AFM system, so that the adhesion measurement probes the whole surface area at the same time. Thanks to the large area, cells can be seeded and grown on the cantilevers the day before the experi- ment, and let recover to optimal condition for the experiment. We used Human Embryonic Kidney cells, HEK 293A, to demonstrate the measurement of adhesion forces of up to 100 cells in parallel, and obtain a straightforward measurement of the average single cell adhesion energy. Our approach can improve significantly the cell-cell and cell-substrate adhesion statistics, reduce the experiment time and allow the investigation of the adhesion properties of cells that do not grow well in solution or on low adherent substrates, or that develop their characteristic features only after several hours or days of culture on a solid and adherent substrate.