Cells are said to be the building blocks of life. Most prosper best on their own, suspended, but some are included in a bigger 3D matrix like the tissue. These cells work mutually with nearby cells in the same tissue or those at the boundary to the neighboring, adjacent tissue. This can be a natural interface like those in the tendons and bones, or an artificial one, such as in the case of biofilms or implants.
The forces that govern cell-substrate and cell-cell interactions are essential for their structure and function, and their quantification is of interest to be attentive of the mechanical strength of tissue and interfaces and related failures contained in it.
Flex-FPM - The Standard Tool for Cell Adhesion
AFM has been commonly used in the study of cell-substrate or cell-cell interactions at the single cell level [Helenius et al. (2008), Moreno-Encerrado et al. (2017)]. For this intention, cells are usually immobilized chemically to a cantilever. On the other hand, the chemical immobilization gives limitation to the maximum obtainable adhesion forces to a few hundred nanonewtons and it has a required amount of cell experiments needed to get results that are decisive.
The limit in force range is generally difficult when studying cells after incubation times (hours to days) that are prolonged, where forces may go further than the micronewton range. This is primarily true during the study of confluent layers of cells. Here, the interaction between cells contributes, adding up to the substrate adhesion that is regularly studied.
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Figure 1. Flex-FPM extending the FlexAFM functionality with FluidFM technology: Local sample manipulation using hollow cantilevers
Flex-FPM (Fig. 1) is a flexible device used to defeat these two major limitations. It was first started at the ETH Zurich in the groups of Prof. Julia Vorholt and Dr. Tomaso Zambelli. Through the use of FluidFM™ technology, the cell is attached to the cantilever by utilizing negative pressure through a channel inside the cantilever. When compared to chemical binding, higher forces can be attained within a few seconds, reaching into the range of low micronewton [Potthoff et al. (2012); Potthoff et al. (2014)].
The binding via aspiration is reversible, adding to the fact that it is strong and fast. As a result, the same FluidFM™ probe can be utilized for several cells in a row. An exceptional number of over 200 different yeast cells were put into study with a single cantilever in one day under diverse environmental conditions [Potthoff et al. (2012)].
This number cannot be accomplished for mammalian cells. The throughput is still high in number compared with chemical binding. Protocols have been laid down to clean the cantilever enzymatically with trypsin [Potthoff et al. (2014)] or chemically in a sodium hypochlorite solution [Jaatinen (2016)]. After its cleanup, new cells can be expected with no need for a new coating step.
Cell-Cell Adhesion
FluidFM™ cell adhesion experiments were recently made more comprehensive and included the study of cell-cell interaction. This can be the force amid a cell (on the cantilever) and a cell below on a substrate (fig. 2 A). It can also be between a cell and its nearby cells in a confluent layer (fig. 2 B).
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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 made a study on cell-cell adhesion with a Flex-FPM system to gather other insights into the development of tumor and metastasis [Cohen et al. (2017)].
Figure 3 demonstrates an optical image of the method illustrated in fig. 2 A that Cohen utilized for this study.
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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.
Communications among single MCF7 breast cancer cells on the cantilever with the different varieties of cells on the substrate were found to grow in different ways depending on incubation time. During these experiments, the reversible binding of cells gave permission to the different cell pairs to be studied with the similar probe (fig. 4).
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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 largely studied the relationships between a cell and its neighbors in a confluent layer of cells (fig. 2 B) [Sancho et al. (2017)]. Fig. 5 illustrates the cantilever picking up a cell from a confluent layer (A) and the vacant space from where the cell was taken from. (B).
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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 found in the umbilical artery were found to utilize strong intercellular force (figures 6 A & B) that could be reduced widely by over expression of Muscle Segment Homeobox 1, to bring out the transition of endothelial-to-mesenchymal.
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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 transition is a procedure involved in cardiovascular development and disease. Corresponding to these adhesion experiments, the Flex-FPM system was also made use to carry out nano-indentation experiments by means of colloidal beads aimed to the cantilever.
Both of the said examples benefitted from FluidFM™ technology provided by the Flex-FPM solution. In the case of the confluent layer, the enormous forces of up to over 1.5 µN eradicate chemical binding to study cell-cell adhesion. In both cases, the reversible binding supplied the experiments with the vital speed-up to gain adequate statistics.
References
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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