2Cellular Dynamics Program, Marine Biological Laboratory, Woods Hole, MA, USA
3Institute for Integrated Micro and Nano Systems, School of Engineering, The University of Edinburgh, Edinburgh, UK
The selection, isolation, and accurate positioning of single cells in three dimensions are increasingly desirable in many areas of cell biology and tissue engineering. We describe the application of a simple and low cost dielectrophoretic device for picking out and relocating single target cells. The device consists of a single metal electrode and an AC signal generator. It does not require microfabrication technologies or sophisticated electronics. The dielectrophoretic manipulator also discriminates between live and dead cells and is capable of redistributing intracellular organelles.
Dielectrophoresis (DEP) is a proven technique for characterizing and manipulating cells by imposing forces through applied AC electrical field gradients. This technique, which can be used on diverse cell types and multicellular organisms, has been largely worked on in physics and engineering laboratories and has received limited attention as a potentially useful tool for biologists (1-3). Most studies utilizing DEP use sophisticated planar DEP microelectrode arrays coupled to microfluidics systems for large-scale separation of thousands of cells (4-6). These devices have shown significant advances with the advent of photolithography-based microfabrication technologies and new polymers, however they are not suitable for small-scale separations of rare cells within a heterogeneous population and are not commercially available. Similarly, cell sorting flow cytometers are optimized for high-throughput applications but are not appropriate for all cell types and often require large quantities of sample material that not all applications have the fortune of. Additionally, these systems are costly and often require a trained technician for proper operation. Other cell manipulation technologies, including optical tweezers, enable single cell manipulation but also require highly specialized and expensive instrumentation.
We have previously reported a DEP electrode designed to pick out and relocate single target cells from a cell culture (7). The electrode took the form of two electrochemically etched gold wires insulated from each other except for a short region near the electrode tips, which formed the working ends of the DEP “tweezer” design single electrode. Consistent fabrication of this design was difficult, and great care was required in preventing both cell damage arising from contact of cells with electrode tips, as well as damage to the tips themselves. To date, single DEP electrodes have been handmade, making them inherently prone to manufacturing inconsistencies. Fabrication of these designs are technically difficult and require instrumentation not standard to most labs, such as capillary pullers, metal etching equipment, and sputter coaters (7-10). Despite these difficulties, single electrode DEP designs offer advantages over planar designs in single cell manipulation and in small-scale separations of primarily rare cell types. However, both configurations utilizing DEP as a technique have found limited practical use in biological research, in part to technical limitations.
Here, we report the characterization and implementation of an improved DEP electrode. Our design is comprised of a single, commercially available microelectrode and requires only a micromanipulator, microscope, and AC signal generator for use. Unlike prior single and planar DEP electrodes, our design is of simpler composition and commercially available, enabling greater consistency and ease of use. Furthermore, our single electrode design provides the ability to select rare cells from a heterogeneous population and applies exacting positive or negative DEP forces to finite regions of a cell. Our design is capacitively coupled to ground, negating the need for a direct ground in the liquid medium and thus aiding in overall experimental setup. In this report, we demonstrate how this electrode is capable of small-scale separations of single cells and demonstrate its ability to assess viability states without the use of chromogens. Lastly, we demonstrate a novel application of single electrode DEP in the spatial manipulation of intracellular organelles.
Materials and methods
Clonal cell lines CHO (gifted by W. Chowanadisai, UC Davis, USA), HeLa (CCL-2; ATCC, Manassas, VA, USA),and HeLa mCherry-H2B (gifted by Daniel Gerlich, ETH Zurich) were cultured in medium containing Dulbecco's modified Eagle medium (Gibco, Invitrogen, Carlsbad, CA, USA) containing high glucose and sodium bicarbonate, supplemented with 10% FBS (Gibco) and 100 mg/mL penicillin/streptomycin (Invitrogen). All cell lines were grown at 37°C, in 5% CO2. Eremosphaera viridis (no. LB 2600; UTEX, USA) were grown as previously described (11). Lily pollen grains were germinated in a low calcium Dickinson's medium [5 mM MES, 1.27 mM Ca(NO3)2, 1 mM KNO3, 0.16 mM H3BO3, 5% (w/v) sucrose, pH 5.5] at ambient temperature.
Cell viability labeling was performed by first trypsinizing cells and washing twice in 1× Hank's balanced salt solution (HBSS; no. 14025-092; Gibco) containing calcium and magnesium. Cells were incubated in 2 µM calcein-AM (AnaSpec, Fremont, CA, USA) and 4 μM ethidium homodimer-1 (AnaSpec) for 20 minutes at room temperature. Serial dilutions were performed to acquire a cell density suitable for cell separation. For cell separations of fluorescent and nonfluorescent cells, the above procedure was followed for loading calcein-AM. Both loaded and nonloaded populations were then mixed together.
Scanning electron microscopy was performed on noncoated microelectrodes with a Zeiss Supra 40 VP microscope (Carl Zeiss, Munich, Germany) at an accelerating voltage of 2 KV. All light and fluorescent microscopy was performed on a Zeiss Axiovert 40 CFL (Carl Zeiss).
DEP on cells
Prior to use, mammalian cells were detached from the bottom of the culture dish with trypsin treatment and washed twice in 1× HBSS. Cells were then transferred to a low conductivity medium after two rinses with 0.1× HBSS (conductivity of 114 mS/m) supplemented with 2.25 g/50 mL sucrose (CAS: 57-50-1; Fisher Scientific, Pittsburgh, PA, USA) to attain an osmolarity of ∼300 mOsm. The conductivity of the 0.1× HBSS solution was 114 mS/m. A 1% agarose-0.1× HBSS cushion was prepared in 35 × 10-mm Petri dishes (Falcon 35 1008; Becton Dickinson, Franklin Lakes, NJ, USA) to prevent cell adhesion to the bottom of the dish and to prevent collision of the electrode tip with the bottom of the dish. Serial dilutions of cells were performed to acquire a cell density suitable for cell separations. After dilutions and washing in 0.1× HBSS-sucrose, cells were transferred to the agarose bottom dish for DEP experimentation. E. viridis and lily pollen were directly transferred from growth media and −80°C storage, respectively, to low calcium Dickinson's medium for experimentation. A 1% agarose bottom cushion containing low calcium Dickinson's medium was used for preparations of these cell types. The DEP single electrode (tip resistance of 1 MΩ stainless steel microelectrode, no. SS30031.0A10; MicroProbes for Life Science, Gaithersburg, MD, USA) was positioned by use of a mounted micromanipulator (Narishige, Tokyo, Japan). A signal generator (model 4045; B&K Precision, Yorba Linda, CA, USA) was used for all sinusoidal wave forms. An aluminum foil ground was made by cutting a 10 cm2 piece of aluminum and placing a small hole (∼0.5 cm diameter) in the center (to enable microscopy). A 5 cm piece of silver wire with a 1 mm diameter (7440-22-4; Alfa Aesar, Ward Hill, MA, USA) was secured to the aluminum foil and to a grounded alligator clip. The foil ground was taped in place to the microscope stage.
Results and discussion
The DEP cell manipulator described here consists of a single, commercially available metal microelectrode. Although this design generates larger DEP forces at the surface of the electrode than our previous design, cell damage is avoided due to a thin porous metal oxide coating on the microelectrode tip. The oxide coating appears to shield direct exposure of a cell to the energized electrode end, reducing exposure to undesirable AC field exposure effects (12-14). A scanning electron micrograph of a 1 MΩ electrode is shown in Figure 1A. The electrode body is coated with a thin layer of Parylene and the exposed stainless steel tip has a metal-oxide coating.