RESUMEN
The electric cell-substrate impedance sensing (ECIS) is a well-established technique that allows for the real-time monitoring of cell cultures growing on gold-electrodes embedded in culture dishes. Its foundation lays on the insulating effect that cells present against the free-flow of electrons, as these passive electrical properties generate a characteristic complex impedance spectrum when a small-amplitude, non-invasive alternating current (AC) is provided through the electrodes, the living cells, and the culture media in the culture ware. In addition, it possesses the ability to create a wound that is highly confined to the electrode area by simply increasing the amplitude of the AC current in dependence of the pre-resistor strength for a defined pulse duration and at a specific frequency. Therefore, it represents a controlled and reproducible tool to carry out in vitro wound healing experiments. Accordingly, in this methods protocol, the use of the ECIS will be described in the context of the wound healing research: cardiac 3T3 fibroblasts will be wounded and their recovery dynamics analyzed based on the typical methodologies applied to the processing of ECIS data. In addition, cellular micromotions will be evaluated. Finally, fluorescence immunostaining of ECIS samples will be described in order to showcase the potential of the ECIS in combination with other well-established techniques to add further knowledge depth to the understanding of the complex wound healing dynamics.
Asunto(s)
Impedancia Eléctrica , Fibroblastos , Cicatrización de Heridas , Animales , Ratones , Fibroblastos/citología , Fibroblastos/metabolismo , Electrodos , Movimiento Celular , Técnicas de Cultivo de Célula/métodos , Técnicas Biosensibles/métodosRESUMEN
Shape, dynamics, and viscoelastic properties of eukaryotic cells are primarily governed by a thin, reversibly cross-linked actomyosin cortex located directly beneath the plasma membrane. We obtain time-dependent rheological responses of fibroblasts and MDCK II cells from deformation-relaxation curves using an atomic force microscope to access the dependence of cortex fluidity on prestress. We introduce a viscoelastic model that treats the cell as a composite shell and assumes that relaxation of the cortex follows a power law giving access to cortical prestress, area-compressibility modulus, and the power law exponent (fluidity). Cortex fluidity is modulated by interfering with myosin activity. We find that the power law exponent of the cell cortex decreases with increasing intrinsic prestress and area-compressibility modulus, in accordance with previous finding for isolated actin networks subject to external stress. Extrapolation to zero tension returns the theoretically predicted power law exponent for transiently cross-linked polymer networks. In contrast to the widely used Hertzian mechanics, our model provides viscoelastic parameters independent of indenter geometry and compression velocity.