HARRICK PLASMA

Living cell adhesion measured by force spectroscopy

Cell adhesion is the binding of a cell to another cell, extracellular matrix or a surface, using cell adhesion molecules. Many of these interactions involve transmembrane integrin receptors. Integrins cluster to provide dynamic links between extracellular and intracellular environments, by bi-directional signaling and stabilization of the focal adhesion points. The interactions involve complex couplings between cell biochemistry, structural mechanics, and surface bonding.
Adhesive interactions play a major role in the development of multicellular organisms, by guiding and anchoring cells into their appropriate locations. Adhesion is also important in the maintenance of the body: changes in the expression or function of cell adhesion molecules are implicated in all steps of tumor progression. Tumor cells are able to loosen their attachment to leave their original location and become lodged at distant tissues (metastasis). Implantology research and tissue engineering are directly focused on cell-surface interactions, since events leading to integration of an implant into bone and to long performance of the device take place at the interface formed between tissue and implant. Modifying the surface of an implant either by providing chemical and/or topographical cues encourages bone cell attachment. Cell adhesion is one way to increase osteointergration and to stabilize the implant. Tissue engineering aims to replace or restore the anatomic structure and function of damaged or missing tissue. Cell adhesion is a crucial parameter in the development of biodegradable scaffolds and cell sheet engineering techniques.
It is not easy to measure and quantify cell adhesion. Cell adhesion has been investigated using many techniques. The combination of cell biology with force spectroscopy provides a powerful tool for exploring the complexity of cell adhesion. The main objective of this work is to use force spectroscopy to quantify the long-term global adhesion between cells and surfaces and their response to modified surfaces. Atomic force microscopy-based force spectroscopy is capable of resolving individual cell binding events as well as global cell adhesion of living cells under physiological condition. It was used to study and quantify the adhesion of living cells to their growing substrate.
In order to determine if cell adhesion has to be studied independently of cell cycle or not, cell adhesion at different phases was measured. The adhesion of osteosarcoma cells to a glass surface was measured at different phases of the cell cycle. The cells were synchronized in three phases of the cell cycle: G1, S and G2M. Cells in these phases were compared with unsynchronized and native mitotic cells. Individual cells were attached to an atomic force microscope cantilever, brought into brief contact with the glass surface and, then pulled off again. The force-distance curves obtained allowed the work and maximum force of detachment as well as the number, amplitude, and position of discrete unbinding steps to be determined. The properties of the binding proteins present at the cell surface remained similar throughout the cell cycle, including mitosis. Therefore, next cell detachment experiments were allowed to be studied independently of cell cycle.
Long-term cell adhesion involves living adherent cells. The challenge was to find a method of attachment, between cells and atomic force microscope cantilevers, that allows their detachment from the surface. Fibronectin-coated cantilevers were used to detach individual immortalized fibroblasts from their growing substrate. The detachment of living adherent cells by force spectroscopy was tested on a number of chemically functionalized surfaces in order to validate the technique. The forces involved in the adhesion of fibroblasts were quantified. The cells were grown on glass surfaces as well as on surfaces used for cell sheet engineering: glass surfaces coated with polyelectrolyte multilayers (poly-L-lysine and hyaluronic acid) and thermally-responsive poly(N-isopropylacrylamide) brushes. Large differences in cellular adhesion were observed on polyelectrolyte coatings, depending on the number of polyelectrolyte bilayers. On poly(N-isopropylacrylamide)-grafted surfaces, changes of more than an order of magnitude were observed in cell adhesion above and below the lower critical solution temperature. Glass surfaces patterned with periodic poly(N-isopropylacrylamide) microdomains were also investigated. In this last case, it was shown that cellular adhesion could be reduced while keeping cellular morphology unchanged.
Finally, in order to investigate the potential and limitations of this technique, two important experimental parameters were altered: the topography and the cell type. Immortalized and primary fibroblasts were studied on flat and topographically structured quartz surfaces. Using a fibronectin-coated AFM cantilever, it was possible to detach a large proportion of the immortalized cells from the quartz surfaces. Their adhesion to the quartz surface, and the effects of topography on this adhesion, could be quantified. In contrast, few primary cells were detached under the same experimental conditions. A qualitative analysis of their behavior showed that immortalized fibroblasts adhered less strongly than primary fibroblasts to at least one quartz surface.
The potential and limitations of single cell force spectroscopy in the study of the adhesive properties of cells are discussed. Quantitative data of this nature should open the way for more rigorous investigation and comparison of the influence of different parameters on cell/substrate adhesion.

Weder, G.

University of Neuchâtel, Switzerland

2010

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