Microfabricated cantilevers for parallelized cell-cell adhesion measurements

Abstract

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 experiment, 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.

Living Sample Viability Measurement Methods from Traditional Assays to Nanomotion

“…Controlling the location of samples allows for more flexibility in selecting optimal positions for reflection, resonance, and low noise [143]. The ink-jet printing immobilization process is monitored by a charge-coupled device (CCD) [163]. The ink-jet printing immobilization protocol is characterized by the fact that the location of cells or bacteria can be controlled.…”

“…A sample is limited by its size and by cantilever size. There is substantial potential for misinterpretation of data due to cell damage during the adhesion process [ 163 ]. Studies have demonstrated the effect of bacterial adhesion position distribution on the cantilever, and the effects increased when the adhesion positions were close to the cantilever’s free end [ 164 ].…”

“…The ink-jet technique enables the choice of immobilized samples’ positions on cantilevers, which enables the study of the effects of sample location on cantilever nanomotion behavior; see Figure 6 d. Controlling the location of samples allows for more flexibility in selecting optimal positions for reflection, resonance, and low noise [ 143 ]. The ink-jet printing immobilization process is monitored by a charge-coupled device (CCD) [ 163 ]. The ink-jet printing immobilization protocol is characterized by the fact that the location of cells or bacteria can be controlled.…”