Cells are known to be the building blocks of life. Some of them prosper best alone, suspended, but most of them are integrated in a much larger 3D matrix such as tissue. These cells work together with nearby cells in the same tissue or those at the interface to adjacent, different, tissue.
This can be a natural interface as between tendons and bones, or an artificial one in the case of implants or biofilms. The forces leading cell-substrate and cell-cell interactions are vital for their structure and function, and their quantification is of interest to be aware of the mechanical strength of tissue and interfaces and related failures within.
Flex-FPM - The Standard Tool for Cell Adhesion
AFM has been used widely to learn about cell-substrate or cell-cell interactions at the single cell level [Helenius et al. (2008), Moreno-Encerrado et al. (2017)]. For this purpose, cells are normally immobilized chemically to a cantilever. Nevertheless, the chemical immobilization restricts the maximum available adhesion forces to a few hundred nanonewtons and it needs many cell experiments to get decisive results.
The restriction in force range is mostly difficult when studying cells after extended incubation times (hours to days), where forces may go beyond the micronewton range. This is mainly true during the study of confluent layers of cells. Here, the cell-cell interaction contributes adding up to the substrate adhesion that is usually studied.
Figure 1. Flex-FPM extending the FlexAFM functionality with FluidFM technology: Local sample manipulation using hollow cantilevers
Flex-FPM (Fig. 1) is a flexible instrument used to overcome these two main limitations. It was established at the ETH Zurich in the groups of Prof. Julia Vorholt and Dr. Tomaso Zambelli. By the use of FluidFM™ technology, the cell is attached to the cantilever by the use of negative pressure through a channel inside the cantilever. In comparison to chemical binding, higher forces can be achieved within a few seconds, reaching into the low micronewton range [Potthoff et al. (2012); Potthoff et al. (2014)].
The binding via aspiration is not just strong and fast, it is also reversible. Therefore, the same FluidFM™ probe can be used for multiple cells in a row. An outstanding number of over 200 different yeast cells were studied with a single cantilever in one day under varying environmental conditions [Potthoff et al. (2012)].
This number cannot be attained for mammalian cells. The throughput is still advanced than with chemical binding. Protocols have been set to clean the cantilever enzymatically with trypsin [Potthoff et al. (2014)] or chemically in a sodium hypochlorite solution [Jaatinen (2016)]. After cleaning, new cells can be aimed without the need for a new coating step.
Lately, FluidFM™ cell adhesion experiments were extended to study cell-cell interaction. This can either be the force between a cell (on the cantilever) and a cell below on a substrate (fig. 2 A) or between a cell and its surrounding cells in a confluent layer (fig. 2 B).
Figure 2. Cell-cell interactions (red springs) studied by aspiration of single cells to a hollow FluidFM™ probe (orange). A) Probing the force between a cell immobilized on the cantilever and a cell on the substrate. B) Picking a single cell from a confluent layer, probing cell-substrate (purple) and cell-cell (red) interactions.
Dr. Noa Cohen of Prof. Tanya Konry's group at Northeastern University in Boston studied cell-cell adhesion with a Flex-FPM system to collect additional insight into the progression of tumor and metastasis [Cohen et al. (2017)].
Figure 3 shows an optical image of the method illustrated in fig. 2 A that was used by Cohen for this study.
Figure 3. Optical image showing A) a single cell to be picked by a FluidFM probe B) the cell aspired to the cantilever and C) the FluidFM probe with aspired cell during a cell-cell adhesion measurement. Data courtesy of Tanya Konry group, Northeastern University, Boston USA.
Interactions among single MCF7 breast cancer cells on the cantilever with the different kinds of cells on the substrate were found to increase differently with incubation time. In these experiments, the reversible binding of cells permitted the different cell pairs to be studied with the same probe (fig. 4).
Figure 4. A) Typical force spectra between a MCF7 cell aspired to the cantilever and a non-cancerous, fibroblast (HS5) on the substrate at different contact times. B) Development of the force with contact time between the cells. Data courtesy of Tanya Konry group, Northeastern University, Boston USA.
Dr. Ana Sancho from the group Prof. Jürgen Groll's group at the University of Würzburg broadly studied the relations among a cell and its neighbors in a confluent layer of cells (fig. 2 B) [Sancho et al. (2017)]. Fig. 5 shows the cantilever picking up a cell from a confluent layer (A) and the vacant space from where the cell was detached (B).
Figure 5. Confluent layer of cells, where one is pulled out by FluidFM, adapted from: Sancho et al. (2017), Scientific Reports volume 7, 46152.
Human endothelial cells from the umbilical artery were found to make use of strong intercellular force (figures 6 A & B) that could be reduced extensively by over expression of Muscle Segment Homeobox 1, to bring about endothelial-to-mesenchymal transition.
Figure 6. A) Typical single cell force spectra of individual cells or cells in a confluent layer, depicting the increase in force by cell-cell interactions. B) Effect of MSX1 on the observed cell adhesion for individual cells and cells in a monolayer. Grey and black bars: control measurements on individual cells and monolayers, resp., pale and light blue MSX1 treated individual cells and cells in a monolayer, resp. Adapted from: Sancho et al. (2017), Scientific Reports volume 7, 46152.
This switch is a process involved in cardiovascular development and disease. Complementary to these adhesion experiments, the Flex-FPM system was also utilized to carry out nano-indentation experiments using colloidal beads aspired to the cantilever.
Both the examples strongly benefitted from FluidFM™ technology provided by the Flex-FPM solution. In the case of the confluent layer, the huge forces of up to over 1.5 µN eliminate chemical binding to study cell-cell adhesion. In both cases, the reversible binding provided the experiments with the essential speed-up to gain sufficient statistics.
Jonne Helenius, Carl-Philipp Heisenberg, Hermann E. Gaub, Daniel J. Muller Single-cell force spectroscopy Journal of Cell Science 2008 121: 1785-1791; doi:10.1242/jcs.030999
Alberto Moreno‐Cencerrado, Jagoba Iturri, Ilaria Pecorari, Maria D.M. Vivanco, Orfeo Sbaizero, José L. Toca‐Herrera Investigating cell‐substrate and cell–cell interactions by means of single‐cell‐probe force spectroscopy Microscopy Research & Technique 2017 80: 124-130; doi:10.1002/jemt.22706
Eva Potthoff, Orane Guillaume-Gentil, Dario Ossola, Jérôme Polesel-Maris, Salomé LeibundGut-Landmann, Tomaso Zambelli , Julia A. Vorholt Rapid and Serial Quantification of Adhesion Forces of Yeast and Mammalian Cells PLoS ONE 7(12): e52712; doi:10.1371/journal.pone.0052712
Eva Potthoff, Davide Franco, Valentina D’Alessandro, Christoph Starck, Volkmar Falk, Tomaso Zambelli, Julia A. Vorholt, Dimos Poulikakos, and Aldo Ferrari Toward a Rational Design of Surface Textures Promoting Endothelialization Nano Lett. 14, 2, 1069-1079; doi:10.1021/nl4047398
Leena Jaatinen Quantifying the effect of electric current on cell adhesion studied by single-cell force spectroscopy Biointerphases 11, 011004 (2016); doi:10.1116/1.4940214
Noa Cohen, Saheli Sarkar, Evangelia Hondroulis, Pooja Sabhachandan, Tania Konry Quantification of intercellular adhesion forces measured by fluid force microscopy Talanta Volume 174, 1 November 2017, Pages 409-413; doi:10.1016/j.talanta.2017.06.038
Ana Sancho, Ine Vandersmissen, Sander Craps, Aernout Luttun & Jürgen Groll A new strategy to measure intercellular adhesion forces in mature cell-cell contacts Scientific Reports volume 7, Article number: 46152 (2017); doi:10.1038/srep46152
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