In this project, we will design and test an experimental system for measuring adherent cell elastic properties. Changes in cell elasticity have been proposed as a marker for correlation with various human diseases: cancer cells, for example, are able to modify both their morphology and the substrate strain field depending on aggressiveness and stiffness. An accurate measurement of cell elasticity is therefore crucial for understanding cell response to mechanical loading. In the proposed system the cells to be tested will be seeded in a silicone chamber devised with an elastic modulus close to that of the cells itself, and uniaxially stretched by a linear actuator. A high resolution camera mounted on a 40X inverted microscope will be employed to acquire a picture of the substrate underlying the cells before and after the stretching. The system will be designed to automatically compensate for the motion imposed to the chamber along the stretching direction and the unavoidable small movements along the vertical axis by using two additional motors controlled by a custom made software. The reference and deformed pictures will then be in focus, and analyzed with ImageJ software to return the substrate strain field. The maximum strain occurring in the substrate beneath each cell is a parameter linearly related to cell stiffness. All the tests needed to develop the system will be performed by using osteoblast-like cells. The sensitivity of the system will be evaluated using a cell model with artificially increased stiffness and one with artificially decreased stiffness. Atomic Force Microscopy measurements of cell stiffness will yield to a calibration of the proposed system. Finally, the use of conductive gels will be tested to device a new approach for the measurement of cell stiffness, by correlating the change in resistance with the applied force, to devise a new low cost and independent tool for better clarifying the mechanisms by which cells sense mechanical stimuli.
In the last years, the measurement of cell elasticity has become of high interest, since it has been proposed as a new marker for correlation with various human diseases. In addition to the most standard technique, the atomic force microscopy, a huge number of methodologies have been devised to measure cell stiffness, but new insights are still needed. In fact, all these approaches have technical issues, being invasive, or very expensive, or specific for some cell types, or dependent on a hard data analysis. Recently, a new technique based on the measurement of substrate deformation beneath and surrounding the cell was proposed. The main advantage of this technique is that it is completely non-invasive, since it takes advantage of the adhesion forces already existing between the cell and the substrate. However, it has to be noted that it allows only for measuring the average cell stiffness.
On the basis of this idea, we will develop a new system to measure the maximum strain underlying each single cell, to subsequently measure cell elasticity. The proposed research is innovative for the following reasons:
- the proposed system will be low-cost, versatile and independent. A software will be realized to allow the cell to be tested to be always centered and properly focused, by synchronizing three linear motors. Two motors will be in charge of stretching and centering the cell along the longitudinal direction, while the other one will automatically compensate the small movements along the vertical axis. Such a device could be therefore easily moved to be used with any kind of inverted microscope. Moreover, silicone based membranes with different elasticity will be realized to test cells having different stiffness (e.g. cells from bone or skeletal muscle tissue).
- the technique sensitivity will be assessed to precisely understand whether it could be used to discriminate between different cell types or between control and damaged cells. To this aim we will use a model of artificially hardened cells and one of artificially softened cells, also with reference to cells originating from tissues with different stiffness (e.g. cells from bone and skeletal muscle tissue);
- the use of conductive gels (e.g. Master Bond) will be tested for the first time to further refine the relationship between the maximum strain beneath the cell and the cell elasticity. The changes in resistance occurring during cell stretching will be correlated to the applied force through a proper calibration, allowing for measuring cell stiffness. The technique will be verified by comparison with the measurements performed with the gold standard technique, the atomic force microscopy.
Therefore the proposed study will have a direct impact on the field of mechanotransduction, and will contribute to elucidate fundamental issues related to the measurement of cell elasticity through the use of new non-invasive techniques.