This study investigates the processes of cell membrane puncturing and the factors influencing these processes through force spectroscopy experiments. A custom-designed tall and sharp tip was fabricated on a commercially available microcantilever using two-photon polymerization (2PP). Force spectroscopy curves were measured at several locations on the cell while the tip was inserted inside the cell. A cell membrane insertion event, identified as a ”force drop” in force spectroscopy curves, is often hardly visible or does not occur. In this study, the effects of probe tip diameter, cantilever spring constant, insertion velocity, and cell height of mouse preosteoblast cells were systematically analysed for their influence on how often the force drops occur and how clearly they are visible. Data processing algorithms were employed to process thousands of force spectroscopy curves, enabling large sample statistical analysis using multiple linear regression. The results demonstrate that selecting cells with maximum height and size increases force drop occurrence from 10% to 40%. Furthermore, while decreasing the tip diameter from 2 to 0.7 µm did not affect the occurrence rate, it increased force drop visibility from ∼250 to ∼600 pico-Newton (pN). Additionally, cantilevers with a spring constant of 0.2 N/m achieved higher force drop occurrence (40%) and visibility (∼300 pN), compared to more flexible cantilevers with a spring constant of 0.02 N/m (20% occurrence, ∼40 pN visibility). These findings provide valuable insights for optimizing experimental parameters in cell membrane mechanics research.

Cancer metastasis, the spread of cancer cells from a primary tumor to distant organs, is the leading cause of cancer-related deaths. During metastasis, cancer cells undergo significant changes in their mechanical properties, including alterations in cell elasticity. Cancer cells generally exhibit higher elasticity than normal cells, a key feature that may contribute to their ability to migrate and invade other tissues. Although extensive research has focused on cell-substrate interactions, these studies do not fully replicate the physiological environment, where cells are frequently in direct contact with each other. In this study, Atomic Force Microscopy (AFM) in force spectroscopy mode, utilizing a micro-sized cantilever with a hemispherical tip, was used to quantify the Young’s modulus (E) of MCF-7 breast cancer cells in two configurations: cell-substrate and cell-cell. The resulting force-indentation curves were analyzed and fitted to the Hertz contact model to determine the Young’s modulus. The results showed that the Young’s modulus in the cell-cell configuration was Ecc = 205.8 ± 50.88 Pa, while in the cell-substrate configuration, it was Ecs = 187.95 ± 78.26 Pa. These findings suggest that MCF-7 cells are slightly less elastic in the cell-cell configuration. This challenges the expectation of a more significant difference between the two configurations and highlights the importance of considering biological variability and experimental conditions when interpreting cell mechanical properties.