A flow through device for simultaneous dielectrophoretic cell trapping and AC electroporation

Finite element analysis was completed with COMSOL Multiphysics simultaneously using the electrostatics, creeping flow and particle tracing modules. A Dirichlet boundary condition with applied voltage was imposed at both electrodes. A no-slip condition was imposed at the electrode-glass interface and top channel surface which assumes simple Poiseuille flow. Infinite inlets and outlets were assumed at the two ends of the channel. Particles were introduced as constant time intervals. Here the influence of the electric double layer (EDL) on simulated electric fields was neglected as the associated capacitance will reduce the simulated field by less than 7%. Supplementary Section S5 describes in detail the determination of this capacitance value using electrochemical impedance spectroscopy (EIS).

The equivalent complex permittivities were calculated as outlined in Pethig (2017)19, for the HEK-293 cells using a double-shell model. Each cell component is assumed to contribute linearly to the effective permittivity and conductivity of the cell. A detailed explanation of the equations used for effective permittivity and CM factor determination is provided in the electronic supplementary information.

For simulation of the Clausius-Mosotti factor for dead cells, the assumption was made that plasma membrane conductance increased as the membrane degraded and was taken to be 100 S/m19. Because the formed PEG layer is porous and will exchange liquid with the surrounding medium, its dielectric properties were assumed to be equivalent to the 7% sucrose solution used in the described experiments.

A Fluigent pressure pump system (Fluigent, FlowEZ) is employed to maintain flow rate within the channel. The system regulates air pressure into a gas-impermeant fixed volume chamber containing the material to be flowed into the channel. A flow meter is placed in line with the fixed volume chamber and pressure pumps and is used as feedback by the pumps to regulate air pressure into the fixed volume chamber. Polyether ether ketone (PEEK) tubing with 200 um inner diameter and 700 μm outer diameter are used for fluid flow in the setup. 15 mL conical centrifuge tubes with a Fluiwell 1C-15 cap are used as the fixed volume reservoir. An optical image showing the full test setup is provided in Supplementary Figure S6.

A Tektronix AFG3251C arbitrary waveform generator is used to generate the simultaneous DEP/AC electroporation fields. The location and strength of the nDEP trap is verified experimentally using 9.9 μm polystyrene particles (ThermoFisher). Particles were suspended in a 7% sucrose solution (σ = 7.4 μS/cm) and introduced into the device at a constant flow rate under a 20VPP, 1 MHz applied signal. Once a particle was held, flow rate was steadily increased in 4 s intervals until the particle was no longer held against the flow rate. At this point, the hydrodynamic drag force was assumed to be equal to the DEP force acting on the particle. Using the equation for hydrodynamic drag force on a particle under shear given in Equation 5 17 the resulting DEP force is estimated. This equation is a modified form of Stoke’s law which accounts for particle-wall interaction. This method is preferred for estimation of the DEP force as the force will be altered by the presence of the particle close to the channel wall45. Here ηm is the viscosity of the medium, S is the shear rate and K is a coefficient accounting for the particle distance from the wall, taken to be 1.700517. S is calculated using Equation 6 after determining the equivalent linear flow rate, v, rate based on the channel cross section46. Here, dh is the hydraulic diameter used to estimate the effective diameter of the rectangular cross section given by Equation 7, where w and hc are the width and height of the channel, equal to 1 mm and 70 μm respectively47.

For plasmid transfection, cells grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum and penicillin/streptomycin were trypsinized and suspended in 7% sucrose solution at a density of 3 × 106 cells/mL. 13.47 μg of plasmid vector tagged with RFP and 10 μL of 4 mM Calcein AM was added to the cell solution. The mixture was introduced into the system under a 20 nL/min constant flow rate and plasmid transfection was carried out in the following sequence; (1) DEP trapping field of 4VPP was left on for 2 minutes to simultaneously immobilize and electroporate cells at trap locations. Visual confirmation was obtained via concurrent imaging with a Leica DMIRB epifluorescent microscope. (2) Flow rate was increased to 5 μL/min and cells were collected into 200 μL of DMEM with 10% fetal bovine serum and 20 mM HEPES with penicillin/streptomycin in a 1.5 mL microcentrifuge tube. The entire contents containing ≈10,000 cells was transferred to a 96-well plate for culture in a 37 ºC incubator. The process was repeated 3x for each of the test and control conditions. Cells were cultured overnight and imaging of a 440 μm × 330 μm region of the cultured well was completed to determine if transfection had occurred. A detailed description of the plasmid design is provided Supplementary Section S7.

Images are acquired using a Leica DMIRB epifluorescent microscope fitted with an AmScope MU300 CMOS microscope camera for image acquisition. A ≈577 nm ± 20 nm excitation filter is used for RFP imaging and a ≈470 nm ± 40 nm excitation filter is used for Calcein AM imaging.

All images were processed in ImageJ through separation of the red, green and blue channels with intensities normalized to background. The red channel is used for determination of FusionRed RFP intensity and the green channel is used for Calcein AM intensity. Cells locations were marked manually using the bright field image and viable cells were counted as those exhibiting Calcein AM fluorescence stronger than 5%. Transfected cells were marked as those exhibiting both RFP and Calcein AM intensity greater than 5%.

Partial support for this work was provided by NSF EEC-1444926 and by an Allen Discovery Center award from The Paul G. Allen Frontiers Group (12171). The authors gratefully acknowledge the support of the National Science Foundation for a Science and Technology Center on Emergent Behaviors of Integrated Cellular Systems (CBET-0939511). H.R.N. and S.S. would like to acknowledge the support of Office of Naval Research ONR N00014-16-1-2550 and N00014-12-1-0888. The authors would like to thank Dr. Qiaobing Xu and Caleb Neufeld for providing the propidium iodide cell stain and their discussions.

M.P., H.R.N. and S.S. conceived the experiment(s), M.P. and H.R.N. conducted the experiment(s), J.M. and M.L. designed the transfected plasmid and provided care of the HEK-293 cells. S.S. conceived the original idea, and supervised the project. All authors reviewed the manuscript.