Electrohydrodynamic cell concentration for biofabrication of agar-based 3D microtissues

We have developed a rapid and novel technique for concentrating cells into 3D clusters which are then embedded in an agar hydrogel. This method supports cell growth and proliferation with a steady nutrient supply through the porous hydrogel. For tissue engineering studies, the creation of cell clusters within a controlled size range is necessary to perform precise biological experiments and measure functions. Our precisely controlled cell clusters can be reproducibly formed for different sizes between 0.1 mm to 5 mm in diameter, with cell numbers up to 10(6) cells/cluster. The concentration of cells is achieved using a device we previously developed, that utilizes a combination of electrical and hydrodynamic phenomena. The results were obtained using an 80 mu L drop containing HeLa cancer cells placed over an interdigitated electrode array. The electrohydrodynamic phenomena was produced using a sinusoidal voltage (10 Vpk-pk and 50 kHz). This voltage application creates Joule heating resulting in a buoyancy driven convective flow pattern directed towards the center of the droplet. In addition, the same interdigitated electrode structure also induces dielectrophoretic (DEP) levitation of the suspended cells which prevents cell settling and non-specific adhesion. The dense clustering of cells is achieved within 10 minutes of AC voltage application. Furthermore, using staining we have demonstrated that cell viability was measured as 90% after the formation of agar-embedded cell clusters and 80% over 72 hours.