Increasing throughput of AFM-based single cell adhesion measurements through multisubstrate surfaces

Cell adhesion is a complex process influenced by the specific adhesion molecules present on cell surfaces. These molecules vary across cell types and conditions, making adhesion substrate-specific. Prior research has shown that single-cell force spectroscopy is a valuable tool for measuring adhesion at the molecular level. However, the low throughput of this method limits its ability to test multiple substrates efficiently. This gap motivated the development of new tools to expand the scope of adhesion studies. Researchers needed a way to measure adhesion across multiple substrates simultaneously. No prior work had resolved the throughput issue in single-cell adhesion experiments. This limitation hindered the ability to compare adhesion patterns across diverse cell lines and substrates. The need for a high-throughput solution became increasingly apparent in the field.

The study aimed to address the low throughput of single-cell adhesion measurements using atomic force microscopy. The specific problem was the inability to test multiple substrates efficiently in one experiment. The motivation was to enable broader and more comprehensive adhesion studies. Researchers sought to develop a method that could measure adhesion to multiple substrates simultaneously. The goal was to improve the feasibility of experiments requiring multiple substrate comparisons. The study focused on creating a tool that would allow for high-throughput adhesion testing. This approach would allow for more detailed comparisons of adhesion behavior across cell lines. The researchers proposed that this tool could expand the utility of single-cell force spectroscopy.

Segmented polydimethylsiloxane (PDMS) masks were designed to allow simultaneous adhesion measurements on multiple substrates. These masks were fabricated to create distinct regions for each substrate. The masks were tested using four different cell lines: HeLa, prostate cancer, mouse kidney fibroblast, and MDCK. Three extracellular matrix proteins were selected for testing: fibronectin, collagen I, and laminin 332. Single-cell force spectroscopy was used to measure adhesion forces between cells and substrates. The adhesion of each cell line to each matrix protein was quantified and compared. The segmented masks enabled the measurement of adhesion to multiple substrates in a single experiment. This method improved the efficiency of adhesion testing compared to traditional single-substrate approaches.

The adhesion of each cell line to the three matrix proteins was found to be distinct. No two cell lines adhered equally to all three proteins. HeLa cells showed the highest adhesion to fibronectin compared to the other proteins. Prostate cancer cells adhered more strongly to collagen I than to fibronectin or laminin 332. Mouse kidney fibroblasts exhibited the strongest adhesion to laminin 332. MDCK cells adhered most strongly to fibronectin and collagen I. The segmented PDMS masks enabled the simultaneous testing of multiple substrates. This method significantly improved the throughput of single-cell adhesion experiments.

The segmented PDMS masks successfully improved the throughput of single-cell adhesion experiments. The masks allowed for the simultaneous measurement of adhesion to multiple substrates. The adhesion patterns of different cell lines to various matrix proteins were distinct. This finding suggests that cell adhesion is highly dependent on both cell type and substrate composition. The researchers propose that the masks can be used to expand the scope of adhesion studies. The masks are economical and versatile, making them suitable for a range of assays. The study demonstrated the feasibility of high-throughput adhesion testing using segmented substrates. The authors suggest that this approach could enhance the efficiency of future adhesion experiments.