where σm is the conductivity (S/m) of the cell suspending medium, and Cm is the capacitance of the plasma membrane. A cell of radius 10 μm and a typical Cm of 10 mF/m2, in a 100 mS/m solution, will thus exhibit a DEP crossover at ∼225 kHz. A viable cell will exhibit negative DEP (repulsion) at frequencies below ∼ fxo/4, and positive DEP (attraction) at frequencies above ∼4fxo. Following the procedure described previously by Menachary et al, the field gradient parameter (∇E2) of Equation 1 was determined to be ∼5 × 1011 V2/m3 at a radial distance of ∼40 μm from the electrode tip with an applied voltage of 1 V (rms) (7). With all other experimental factors remaining constant, this represents an enhanced DEP force compared to the DEP tweezer, without producing any noticeable electrical damage to the cells.
Cell viability is commonly identified through the use of chromogens and fluorophores. However, DEP can also distinguish between live and dead cells (1)(2). The plasma membrane of a dying cell loses its high resistance to passive ion leakage. This is manifested as an increase of the DEP crossover frequency defining where the polarizability parameter Re[CM] of Equation 1 changes from a negative to positive value as the field frequency is increased (2)(7). We used DEP to demonstrate that the single electrode could identify living and dead CHO cells within a population. In Figure 3, cells were first labeled with calcein-AM and ethidium homodimer-1 fluorophores to visually verify living and dead cells, respectively. Living cells displayed cell attraction at 10 MHz and repulsion from the electrode tip at 50 kHz, whereas dead cells where consistently repelled at both 10 and 50 kHz.
Lastly, we explored the use of single electrode DEP on smaller diameter, polarizable biological entities. Freshwater green algae, Eremosphaera viridis and growing lily pollen tubes were used for their pellucid unicellular bodies, which contain several chloroplasts and rapidly trafficking organelles, respectively. Both Eremosphaera and lily pollen tubes thrive in low conductivity media, making them amenable to DEP studies. In the following experiments, a single DEP electrode was positioned near either a single Eremosphaera cell (Figure 4A) or a growing lily pollen tube (Figure 4C). A signal of 100 kHz, 5 V (rms) was applied to both cells (Figure 4, B and D). The Eremosphaera were attracted to the electrode by positive DEP, indicating that the electric field penetrated the outer membrane and into the cell interior. The intracellular chloroplasts (Figure 4B) were also attracted towards the electrode tip. In the growing pollen tube, the flowing stream of organelles (Figure 4D) were repelled by negative DEP, greatly slowing the rate of cytoplasmic streaming within the growing tube. Both types of intracellular organelles exhibited strong redistributions in response to the DEP force. Upon removal of the voltage signal, the intracellular organelles evenly redistributed themselves in the Eremosphaera after approximately 1 hour, and cytoplasmic streaming recommenced within the lily pollen tube after seconds (data not shown).
The single electrode DEP design described provides a simpler and more cost-effective solution to small-scale cell manipulation over costly systems. The uncomplicated nature of its design and commercial availability provide significant advantages over previous single DEP electrodes. We have shown it is suitable for single cell manipulation, determining cell viability states, and redistributing intracellular organelles. These abilities may ultimately aid researchers interested in studying rare cells found within heterogeneous populations or exploring unknown functions of intracellular organelles. Moreover, use of our single electrode DEP design could be automated for higher throughput applications. The ability to spatially manipulate both cells and intracellular organelles opens doors toward studying multiple exciting and intriguing biological phenomena. For example, how are cell division, migration, and/or growth influenced by asymmetric organellular distribution? Its plasticity toward a wide range of cell types and efficiency make it amenable as a biological tool.
We thank R. Sanger for his assistance, W. Chowanadisai for providing the CHO cell line and R. R. Lew for initially providing the Eremosphaera. This research was funded by NIH-NCRR grant P41 RR001395 and supported by The Eugene and Millicent Bell Fellowship Fund in Tissue Engineering, the Hermann Foundation Research Development Fund Award, the Dennis and Alix Robinson Memorial, and the MBL Bell Center, grant GM092374. This paper is subject to the NIH Public Access Policy.
The authors declare no competing interests.
Address correspondence to David Graham, Eugene Bell Center for Regenerative Biology and Tissue Engineering, 7 MBL Street, Woods Hole, MA, USA. Email: [email protected]
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