Highly controlled electrofusion of individually selected cells in dielectrophoretic field cages.

The prospect of novel therapeutic approaches has renewed the current interest in the fusion of rare cells, like stem cells or primary immune cells. While conventional techniques are only capable of mass fusion, lab-on-a-chip systems often still lack an acceptable method for making the cells available after processing. Here, we present a microfluidic approach for electrofusion on the single-cell level that offers high control over the cells both before and after fusion. For cell pairing and fusion, we employed dielectrophoresis and AC voltage pulses, respectively. Each cell has been characterized and selected before they were paired, fused and released from the fluidic system for subsequent analysis and cultivation. The successful experimental evaluation of our system was further corroborated by numerical simulations. We obtained fusion efficiencies of more than 30% for individual pairs of mouse myeloma and B cell blasts and showed the proliferating ability of the hybrid cells 3 d after fusion. Since aggregates of more than two cells can be fused, the technique could also be developed further for generating giant cells for low-noise electrophysiology in the context of semi-automated pharmaceutical screening procedures.

Gravitation-driven stress-reduced cell handling.

We present a simple lab-on-chip device for handling small samples of delicate cells, e.g. stem cells. It uses a combination of sedimentation and dielectrophoresis. The transport of cells is driven by gravitation. Dielectrophoresis uses radio-frequency electric fields for generating particle-selective forces dependent on size and polarisability. Electrodes along the channels hold particles and/or cells in a defined position and deflect them towards different outlets. The absence of external pumping and the integration of injection and sampling ports allow the processing of tiny sample volumes. Various functions are demonstrated, such as contact-free cell trapping and cell/particle sorting. Pairs of human cells and antibody-coated beads, as they are formed for T cell activation, are separated from unbound beads. The cells experience only low stress levels compared with the stress levels in dielectrophoresis systems, where transport depends on external pumping. Our device is a versatile yet simple tool that finds applications in cellular biotechnology, in particular when an economic solution is required.

Accumulation and trapping of hepatitis A virus particles by electrohydrodynamic flow and dielectrophoresis.

Hepatitis A virus particles (d = 27 nm) were successfully accumulated and trapped in a microfluidic system by means of a combination of electrohydrodynamic flow and dielectrophoretic forces. Electric fields were generated in a field cage consisting of eight microelectrodes. In addition, high medium conductance (0.3 S/m) resulted in sufficient Joule heating and the corresponding spatial variation of temperature, density, and permittivity to induce electrohydrodynamic flow in the vicinity of the field cage. Flow vortices transport particles toward the center of the field cage, where dielectrophoretic forces cause permanent entrapment and particle aggregation. Spatial distribution of temperature, density, and permittivity as well as resulting flow patterns were modeled numerically and are in good agreement with experimental results. This accumulation scheme might be applicable to sample concentration enhancement in biosensor applications.

Electrophoresis in gel channels.

We describe a novel approach to generate dynamic pH gradients suited to fractionate or purify samples of biomolecules or particles such as proteins and viruses in tiny volumes. The method combines diffusion and electromigration between micro-scaled channels embedded in hydrogel. For the used geometry and in accordance with numerical calculations the gel-channel system reaches a tuneable, steady-state pH gradient after a few minutes. For quantification of experimentally generated pH-profiles, the concentration independent extinction ratio of phenol red at two wavelengths is used. The proposed electrophoretic flow-cell is simple and flexible since no Immobilines are required to establish the pH gradient.

Microdevices for manipulation and accumulation of micro- and nanoparticles by dielectrophoresis.

Microfluidic devices with three-dimensional (3-D) arrays of microelectrodes embedded in microchannels have been developed to study dielectrophoretic forces acting on synthetic micro- and nanoparticles. In particular, so-called deflector structures were used to separate particles according to their size and to enable accumulation of a fraction of interest into a small sample volume for further analysis. Particle velocity within the microchannels was measured by video microscopy and the hydrodynamic friction forces exerted on deflected particles were determined according to Stokes law. These results lead to an absolute measure of the dielectrophoretic forces and allowed for a quantitative test of the underlying theory. In summary, the influence of channel height, particle size, buffer composition, electric field, strength and frequency on the dielectrophoretic force and the effectiveness of dielectrophoretic deflection structures were determined. For this purpose, microfluidic devices have been developed comprising pairs of electrodes extending into fluid channels on both top and bottom side of the microfluidic channels. Electrodes were aligned under angles varying from 0 to 75 degrees with respect to the direction of flow. Devices with channel height varying between 5 and 50 microm were manufactured. Fabrication involved a dedicated bonding technology using a mask aligner and UV-curing adhesive. Particles with radius ranging from 250 nm to 12 microm were injected into the channels using aqueous buffer solutions.