Mouse myoblastic cells (C2C12)
C2C12 (ATCC) cells were cultured in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 100 IU ml–1 penicillin, 100 mg ml–1 streptomycin, 2 mM L-glutamine, 250 mg ml–1 fungizone and 1% sodium pyruvate. Cells were harvested by incubation with 0.025% (w/v) trypsin (Invitrogen).
Mesenchymal stem cells (MSCs)
Mesenchymal stem cell growth medium consisted of MEM (Invitrogen), 10% FBS (Cambrex), 1 ng ml–1 bFGF (Invitrogen), 100 µg ml–1 penicillin, 100 IU ml–1 streptomycin (Invitrogen), and 0.4 mmol ml–1 ascorbic acid (Sigma). Cells were harvested by incubation with 0.25% (w/v) trypsin (Invitrogen). Cells were used up to passage number 6 (approximately 40 passage doublings)
For electroporation
Cells were harvested using 0.25% trypsin. Trypsin was inactivated by addition of complete growth medium, followed by centrifuging. Cells (C2C12 or MSC) were resuspended in electroporation buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.68 mM KCl, and 1.7 mM MgCl2, 25 mM glucose, pH 7.4) at a concentration of 0.5×106 cells ml–1.
Generation of an EGFP-ERK1 fusion construct
ERK1 DNA was amplified using PCR with Pfu Polymerase (Promega). The PCR primers were directed against the start and stopcodon and included specific restriction sites for easy isolation. The PCR products were cloned into PCR2.1 using a TOPO TA cloning kit (Invitrogen). ERK1 was isolated from PCR2.1 by digestion with XhoI and BamHI and ligated into the XhoI and BamHI sites of pEGFP-N1 (Clontech).
Instrumentation
The microfluidic device was mounted onto an X-Y-Z translation stage on an inverted wide fluorescence microscope (Leica DM IRM, Leica Microsystems, Wetzlar, GmbH, Germany). The microscope system is equipped with a mercury lamp, 20×, 40×, 50×, 63× objectives, and a fluorescence filter set (BP 480/40, LP 515). In addition, a computer-controlled CCD camera (Leica DFC300 FX) was mounted on the microscope for image recording. For the electroporation signal, a custom-made Labview (National Instruments) application controls a function generator card (NI5041, National Instruments) and a data acquisition card (NI PCI-6221, National Instruments), which were connected to the electrodes of the device via an especially home-designed holder.
Cell imaging and correction for photobleaching
Track images were taken every 5 min with 488 nm excitation and a LP 515 emission filter. Fluorescence intensity changes in either the cytoplasm or nucleus of the cells was determined using ImageJ software.13 For correction of photobleaching, it is assumed that over the time of the experiment, the total fluorescent intensity in the cell is constant. In order to determine the corrected intensity, the mean intensity is calculated inside a mask of the whole cell (I0) and inside a region of interest, defined manually, both in the cytoplasm and in the nucleus of the cell upon their position over time. Subsequently, each pixel in each frame was multiplied by the factor (I0/It), where I0 is the original intensity (t = 0) and It is the current frame intensity. For time zero (t = 0 min) the correction factor is 1, but as the image bleaches, the correction factor gets larger. This correction factor was applied to the track images for the nuclear translocation of EGFP-ERK1, as well as to the graphs of the fluorescence intensity versus time.
Electric field distribution
The electric field distribution in the cell trapping device has been modeled using finite element modeling software (Femlab 3.0, Comsol, Sweden) for a 2D situation. The DC conductive medium model was used for a static state situation, which solves Gauss' differential equation. The boundary conditions are considered as being electrically insulated, with the two electrodes being 1 V and grounded. The subdomain has a conductivity of 0.1 S m–1.
Results
Electroporation of C2C12 cells
In order to introduce DNA into cells, we determined the electrical parameters for electroporation of mammalian cells. Since human adult stem cells are relatively hard to isolate and cannot be kept in culture indefinitely, we used a C2C12 mouse myoblastic cell line that is similar in size and has some of the properties of the MSC.
The voltage (E-field) and pulse length needed for cell membrane permeabilization of individual C2C12 cells that are trapped in the cell trap device were determined by using propidium iodide (PI) as membrane integrity indicator. In a series of experiments, pulses with different amplitudes and/or pulse lengths were applied to individual cells and the fluorescence intensity of the cell was measured one minute after each single pulse. At the start of each experiment, trapped cells were not red fluorescent, indicating that the cell membrane was intact. First the minimum voltage for successful PI uptake was determined for a pulse length of 6 ms. Electrical pulses of 6 ms with increasing amplitude from 1 to 3 V (steps of 0.5 V) and an interval pulse of 1 min were applied to the cells (Fig. 2a–b and supplementary Fig. S1). Subsequently, for the lowest voltage for electroporation of C2C12 cells (2 V), the effect of the pulse length on PI uptake was studied (supplementary Fig. S2). In order to test DNA uptake under the defined parameters, a DNA construct encoding green fluorescent protein (EGFP-N1) was electroporated into C2C12 cells. Twenty-four hours after electroporation, EGFP expression was visible in all cells as green fluorescent staining (Fig. 2d and Table 1). The cells also showed healthy morphology observed under light microscopy images. This healthy morphology was maintained at least 120 h post-transfection (after which the cells had divided one time). Thus, a potential of 2 V for at least 6 ms results in single cell electroporation of all C2C12 cells. This electroporation procedure resulted for the first time in GFP transfection and expression into single C2C12 cells and was used to transfect plasmids into MSCs.