Electro-mechanical manipulation of mammalian cells in suspension

The purpose of this study has been to describe the development and demonstration of a microfabricated platform for mechanical characterization of individual living mammalian cells in suspension. The technique uses electrical polarization forces to trap and stretch cells in time-varying, non-uniform fringing electric fields. This work was motivated by the apparent under-utilization of electrical stresses for the mechanical characterization of live cells, and the methods described here permitted mechanical characterization of diverse (previously uncharacterized) mammalian cell-types.

Mechanical characterization of cells is usually achieved by probing local structures near the cell-surface, and very few techniques apply uniaxial stresses to whole individual cells. Most mammalian cells adopt a relatively simple (spherical) geometry when they are suspended in a liquid (aqueous) medium. This simplifies cell-manipulation and permits relatively straightforward interpretation of mechanical data. Mammalian cells are increasingly being used outside of their natural environments, for example within microfluidic devices, which requires precise cell-manipulation protocols. Present miniaturization trends within experimental biotechnology are producing new tools for the precise manipulation of individual living cells, and electric fields feature prominently within this context. Electric fields exert forces on cells without the requirement of mechanical contact between cells and device structures and can therefore be described as “tractor beams”, which can move, trap, or deform electrically polarisable objects such as biological cells.

Mechanical characterization of cells by electro-deformation (ED) will be described within the larger context of cell electro-manipulations. To better understand the behaviour of cells in electric fields, we used dielectrophoresis (DEP) to position human monocytes (U937) within a non-uniform electric field prior to electro-poration (EP) for gene delivery. DEP positioning and EP pulsing were both accomplished using a common set of inert planar electrodes, micro-fabricated on a glass substrate. A single-shell model of the cell's dielectric properties and finite-element modeling of the electric field distribution permitted us to predict the major features of experimentally observed cell positioning. The extent to which electric pulses increased the permeability of the cell-membranes to florescent molecules and to pEGFP-Luc DNA plasmids were found to depend on prior positioning. For a given set of pulse parameters, EP was either irreversible (resulting in cytolysis), reversible (leading to gene delivery), or not detectable, depending on where cells were positioned. Our results clearly demonstrate that position-dependent EP of cells in a non-uniform electric field can be controlled by DEP.

The same planar microelectrodes used for DEP and EP were then used to measure mechanical properties of individual mammalian cells in suspension by deforming the cells in time-varying, non-uniform electric fields. Electrical stresses generated by the planar microelectrodes were used to trap and stretch cells, while (ED) was observed using optical microscopy. Two distinct cell-types were compared after fitting strain data with a three-parameter “standard linear solid” (SLS) model of viscoelasticity, and with a two-parameter power-law (PL) method. Chinese hamster ovary (CHO) cells were found to be approximately twice as stiff as U937 human promonocytes, and CHO cells displayed an elastic behaviour with full recovery of initial shape, while U937 strain data bore witness to plastic deformation.

We then extended these measurements to include two additional cell-types (L929 and HEK293); confocal immuno-fluorescent microscopy was used for visualization and semi-quantitative analysis of the cell-cytoskeleton (CSK) for all cell-types. We treated U937 cells with microfilament (MF)- and intermediate-filament (IF)- disrupting drugs, latrunculin-A (Lat-A), and acrylamide (ACR), respectively, to assess their effects on the CSK and on the mechanical properties of that cell-type. The measured viscoelastic properties of individually deformed cells depended on cortical actin (CA) thickness and were significantly affected by Lat-A treatments. U937 and HEK293 cells had thin CA and were more easily deformed than CHO and L929 cells, which were stiffer and had thicker CA layers.

The results presented in this thesis demonstrate that electrical stresses generated by micro-fabricated electrodes permit mechanical characterization of distinct mammalian cell-types, and therefore accomplish the main objective of this work.