Flow-Through Cell Electroporation Microchip Integrating

Sep 24, 2014 - ... appealing approach for high-throughput cell transfection, but cell viability of existing methods ... High cell viability microfluid...
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Flow-Through Cell Electroporation Microchip Integrating Dielectrophoretic Viable Cell Sorting Zewen Wei,† Xueming Li,‡,∥ Deyao Zhao,§ Hao Yan,‡ Zhiyuan Hu,*,† Zicai Liang,*,§ and Zhihong Li*,‡ †

National Center for Nanoscience and Technology, Beijing 100190, China National Key Laboratory of Science and Technology on Micro/Nano Fabrication, Institute of Microelectronics, Peking University, Beijing 100871, China § Institute of Molecular Medicine, Peking University, Beijing 100871, China ∥ Department of Microelectronics, Delft University of Technology, Delft 2628CT, The Netherlands ‡

S Supporting Information *

ABSTRACT: Microfluidics based continuous cell electroporation is an appealing approach for high-throughput cell transfection, but cell viability of existing methods is usually compromised by adverse electrical or hydrodynamic effects. Here we present the validation of a flow-through cell electroporation microchip, in which dielectrophoretic force was employed to sort viable cells. By integrating parallel electroporation electrodes and dielectrophoresis sorting electrodes together in a simple straight microfluidic channel, sufficient electrical pulses were applied for efficient electroporation, and a proper sinusoidal electrical field was subsequently utilized to exclude damaged cells by dielectrophoresis. Thus, the difficulties for seeking the fine balance between electrotransfection efficiency and cell viability were steered clear. After careful investigation and optimization of the DEP behaviors of electroporated cells, efficient electrotransfection of plasmid DNA was demonstrated in vulnerable neuron cells and several hard-to-transfect primary cell types with excellent cell viability. This microchip constitutes a novel way of continuous cell transfection to significantly improve the cell viability of existing methodologies.

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to the discontinuous operation and the trail-and-error process. The recent emerging microfluidic technologies opened new routes to create more efficient cell electroporation with higher throughput. Shrinking the spacing between electrodes to a few tens of micrometers significantly reduced the electroporation voltage to a few volts, thereby alleviating the harmful effects resulting from the high voltage.4 By introduction of welldesigned microfluidic channels, continuous cell electroporation was demonstrated on various cell types for large scale transfection in the order of millions cells.5 Despite all new

lectroporation is a promising nonviral technology for breaching cell membrane. Commercial bulk electroporation devices have been widely employed as a research tool at the cellular level for the delivery of various molecules,1 including oligo DNA, interference RNA, and molecular drugs. In conventional bulk electroporation devices, cells are treated with short, high-voltage pulses to create temporary pathways on the cell membrane to facilitate the uptake of molecules. An enhancement of the pulse amplitude would facilitate the uptake of more exogenous materials. However, the excessive pulse strength is often tied with the low viability induced by slow membrane recovery, loss of intracellular components, electrochemical damage, and Joule heating.2,3 In addition, the cell treatment speed of bulk electroporation devices was limited due © 2014 American Chemical Society

Received: June 23, 2014 Accepted: September 24, 2014 Published: September 24, 2014 10215

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advances brought by reduced electroporation voltage and precious control of electroporation process, cells treated by microfluidic electroporation devices still suffer from adverse environmental conditions, including pH variation,6 bubble accumulation,7 metal ion dissolution,8 excess heat generation,9 and hydrodynamic shear force.10 As a result, vulnerable cells could be harmed with various degrees, and the performance of microfluidic electroporation devices varies largely by cell types. Therefore, how to reach a compromise between the transfection efficiency and cell viability has become a key issue in the development of microfluidic electroporation systems. Since the proof-of-concept of microfluidic electroporation was first introduced in 2001,11 a variety of strategies have exploited particular channel geometries,12 electrode modifications,13 mechanical valves,14 microfluidic droplets,15 polyelectrolytic salt bridges,8 DC voltage16 and hydrodynamic focusing17 to improve the environmental conditions in microfluidic channel, as well as to decrease the applied voltage needed for efficient gene delivery. Our previous18 study also illustrated that isolating cells from unfavorable effects around the electrodes contributed efficient DNA and siRNA deliveries on various cells. However, harmful effects was remarkably reduced but not completely eliminated among all the above efforts. Moreover, ultrahigh electric field still needs to be applied on the cell membrane while seeking fine electroporation efficiency on some hard-to-transfect cells. Under that ultrahigh electrical field, unrecoverable cell membrane damage was sometimes unavoidable. While progress has been made in the field of flowthrough electroporation systems, fine transfection efficiency with excellent cell viability has yet to be realized, especially on those hard-to-transfect cells or delicate cells. For some frontier biological studies, such as the preparation or the screening of IPS (induced pluripotent stem) cells,19 cell colonies, in which most cells should be transfected, are required. Difficulties to achieve high transfection efficiency and excellent cell viability simultaneously prevented flowthrough electroporation to be an alternative to expensive and complicate viral-based methodologies. This study addresses this challenge with a novel strategy. We integrated two functions, cell electroporation and viable cell sorting, in a microfluidic chip monolithically. In the electroporation part, relatively high electric field was applied to acquire the highest possible electroporation efficiency; while in the dielectrophoretic cell sorting part, viable cells was then sorted out by dielectrophoresis (DEP). Eventually, viable cells with excellent transfection efficiency were collected. After exploring the behavior of the chip, we achieved efficient plasmid delivery with excellent viability on various cells.

Figure 1. Design of flow-through cell electroporation microchip integrating dielectrophoretic viable cell sorting. In the order of cell flow direction, the flow-through cell electroporation microchip is divided into three functional segments, including the electroporation segment (a), the concentration segment (b) and the DEP sorting segment (c). Green and red spots represent viable and nonviable cells, respectively. Flow direction, two inlets, and two outlets were marked on the figure. Outlet A, indicated by green arrow is for viable cells. Outlet B, indicated by red arrow is for nonviable cells. (d) Different forces exerted on viable cells and nonviable cells in DEP sorting segment and corresponding cell movements.

placed on the bottom of the microfluidic channel. The microfluidic channel was with 700 μm in width, 16 mm in length, and 30 μm in height. The electrodes were with 100 μm in width, 15 mm in length, and 500 μm in spacing. The whole electroporation segment maintained a uniform electrical field which was sufficient for cell electroporation (Supporting Information Figure S1). During the electroporation process, some cells became nonviable because of the inevitable cell membrane damage. The second segment was the concentration channel where another buffer flow was introduced to create a two-layer laminar flow (Figure 1b, Supporting Information Figure S2), and thereby pushed all the cells flowing along the lower side before they entered into the sorting segment. Both cell channel and buffer channel were 700 μm in width, so the total width of the concentration channel was 1.4 mm. The length of the concentration channel was 5 mm. Figure 1c shows the DEP sorting segment, including 30 pairs of parallel electrodes. The angle between the electrodes and the flow direction is 15 degrees. Both the width and the gap of the electrodes are 30 μm. The width, length and height of microfluidic channel in DEP sorting segment are 1.4 mm, 20 mm and 30 μm, respectively. As shown in Figure 1d, while passing through the sorting segment, the viable cells experienced both DEP force and hydrodynamic drag force, thus were deflected to the upper side by the resultant force. However, as to the nonviable cells, the cell membrane was damaged and thus permeable. The permittivity of the cytoplasm was balanced with buffer solution outside the cell membrane. Therefore, the DEP force exerting on the nonviable cells was very weak and the cells remained the original

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EXPERIMENTAL SECTION Design and Fabrication of the Microfluidic Chip. The schematic view of the microfluidic electroporation chip is shown in Figure 1. Distinct from single-functional devices, the proposed microchip integrated two functions, electroporation and viable cell sorting, in one microfluidic channel, thereby steered clear of the difficulties to maintain cell viability under unfavorable effects, such as high electrical field, hydrodynamic shear force, bubble accumulation, pH variations, and Joule heating. From a functional point of view, the chip was divided into three segments: electroporation, concentration, and sorting. First, cells were electroporated while flowing through the electroporation segment (Figure 1a), which consists of a microfluidic channel and a pair of parallel gold electrodes 10216

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Electroporation, Cell Sorting Procedures, and the Determination of the Performance. In electroporation, two inlets of the device were respectively connected to syringe pumps (Lange Instruments, China). The parallel electroporation electrodes were wired to a pulse generator (BTX Apparatus, ECM 830, USA). The DEP sorting electrodes were connected to a power amplifier (NF Corp., BA4825, Japan), which was controlled by a waveform generator (Agilent Technologies, 33250A, USA). The microfluidic channel was rinsed with alcohol before use to alleviate possible bubble gathering which could jam the channel in long-time processing. Cultured cells were harvested by trypsin treatment and resuspended to a density of 3 × 104 cells/μL in a modified hypo-osmolar buffer (5 mM KCl, 0.3 mM KH2PO4, 0.85 mM K2HPO4, 76 mM myo-inositol). Plasmid DNA was added to a final concentration of 20 μg/mL. The cells to be electroporated were first preloaded in a syringe and then pumped into the electroporation channel through the cell inlet, and buffer solution was pumped into the concentration channel through the buffer inlet. Electrical pulses for cell electroporation were applied to the cells passing between the electroporation electrodes. Sinusoidal electrical filed for DEP cell sorting was applied to the cells passing through the sorting electrodes. The frequency, amplitude, duration, and interval of the electrical pulses were individually optimized for different cell types. Normally, the processing time was 1 h. The syringe could be placed on a vibrator and gently shook at intervals to avoid possible cell precipitation. After electroporation and cell sorting, viable cells collected through the upper outlet were transferred into a 96-well cell culture plate, and a 200-μL culture medium was immediately added into each well. Nonviable cells were also collected and cultured as control. Twenty-four hours later, the number of GFP-expressing cells was counted in five randomly chosen fields, using ImageJ from NIH, under a fluorescence microscope (Olympus, IX71, Japan). The fluorescence threshold of GFP-expressing cells was manual determined by visually checking the transfected cells. Surviving cells were assessed by the propidium iodide (PI) exclusion assay. For each transfection, transfection rate (TR) was calculated by dividing the number of GFP-expressing cells by the number of living cells, while the cell survival rate (SR) was obtained by comparing the numbers of living cells in electroporated and unelectroporated samples to exclude the unavoidable natural cell death in the normal cell culture and proliferation process. The SR/TR for unsorted cells was estimated from an assay in which only the electroporation part (no sorting) was activated, while the SR/TR for viable and nonviable cells was estimated from another assay in which we activated both electroporation and sorting part. Cell Culture and Plasmids. The transfection efficiency of plasmid DNA was determined by pEGFP-C3 plasmid encoding an enhanced green fluorescent protein (Clontech, USA). Purification of plasmid DNA was performed using an EndoFree Plasmid Maxi Kit (Qiagen, Germany). HEK-293a, CHO, neuro-2a, and HSF cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Hyclone), 100 units/mL penicillin and 100 μg/mL streptomycin (Life Technologies, Gibco). HUVEC cells were grown in Endothelial Cell Medium (ECM) Kit (Sciencell, ECM 1001, USA). Cells were maintained at 37 °C in a 5% CO2 humidified incubator.

movement without deflection. In this way, electroporated cells with high viability could be collected through the upper outlet. The prototype of the flow-through electroporation device is shown in Figure 2. The fabrication process was previously

Figure 2. Prototype of flow-through cell electroporation microchip. The photo of the flow-through cell electroporation microchip and close-up of electroporation electrodes and DEP sorting electrodes. Flow direction, two inlets and two outlets were marked on the photo. Outlet A, indicated by green arrow is for viable cells. Outlet B, indicated by red arrow is for nonviable cells..

described20 and is shown schematically in Supporting Information Figure S3. A 4-in. Pyrex7740 (Corning Inc., USA) glass wafer was used as the substrate because of its high resistivity and transparency. Gold and chrome, respectively, served as functional and adhesive materials for both electroporation electrodes and DEP sorting electrodes. Gold was used because it has high conductivity, good chemical stability, and nontoxicity. The electrodes were sputtered and patterned on the glass substrate by simple one-mask lithography and wet etch. The thicknesses of the gold layer and chrome layers were 300 and 30 nm, respectively. The microfluidic channel was fabricated with polydimethylsiloxane (PDMS, Dow Corning, USA), which is a transparent silicone material commonly used for the manufacture of microfluidic structures. A silicon master mold was fabricated via wet etching. PDMS was cast into the mold and heated to 70 °C for 1 h before being peeled off and punched to create the inlet and outlet holes. After they were diced to the proper size and treated by oxygen plasma in a plasma cleaner (CAS Instruments, China), the PDMS chip and the glass/electrode substrate were aligned and bonded. Following that, they were baked at 120 °C for 1 h to achieve strong bonding. The proposed chip shares the same fabrication method with conventional planar-electrodes-based microfluidic electroporation chip. Therefore, employing cell sorting segment barely incurs extra cost. 10217

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Figure 3. DEP responses of different cell types. (a) DEP response of HEK-293a cells as frequencies increase from 50 kHz to 5 MHz. DEP force exerting on the viable cells is frequency-dependent, while the nonviable cells experienced only weak DEP force. The KCl concentration is 5 mM. (b) DEP responses of viable HEK-293a cells at different KCl concentrations as frequencies increase from 10 kHz to 5 MHz. (c) DEP responses of different types of viable cells as frequencies increase from 10 kHz to 5 MHz. The horizontal axes of (b) and (c) are logarithmically labeled. The voltage applied on sorting electrodes in panels a−c is 8 Vpp.

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RESULTS AND DISCUSSION Characterization of the DEP Responses. DEP force exerted on the cells can be described as21 FDEP = 2πεmR3Re(fCM )∇Erms 2

of KCl concentration in cell DEP responses of HEK-293a cells, in which the frequency of electrical field was set at 1.7 MHz. As shown in Figure 3b, from 0 to 10 mM, the higher the KCl concentration was, the higher the frequency needed to achieve strong positive DEP responses. While the KCl concentration increased to 15 mM, the DEP response stayed negative in the frequency range of 10 kHz to 5 MHz. Similar relationships between KCl concentration and DEP response on HSF and HUVEC cells were also observed (Supporting Information Figure S4). To get a balance between DEP sorting efficiency and electroporation efficiency, the KCl concentration was set at 5 mM in the subsequent assays. To balance the osmotic pressure of buffer, the concentration of myo-inositol was adjusted to 76 mM. Like electroporation, DEP sorting is a probe-free physical method. Theoretically, DEP sorting could be applied with no restriction on cell type. We then investigated the DEP responses of different kinds of viable cells with optimized buffer, as shown in Figure 3c. As expected, the DEP response versus frequency profiles of all kinds of cells are similar. All the cells experienced negative DEP force at low frequencies. Increasing the frequency from 100 kHz to 1 MHz resulted in a transition of DEP response from negative to positive. For each kind of cells, the positive DEP response reached its peak value while the frequency increased to the vicinity of 2 MHz. The results indicated that diverse cells could be sorted using the same chip and the similar electric setups. Optimization of DEP Cell Sorting. HEK-293a cells were used to determine the utility of DEP sorting electrodes in microfluidic channel. Figure 4a and b show the comparison of cell sorting results between unelectroporated cells and electroporated cells which were electrically stimulated by a commercial electroporation equipment (Eppendorf, Multiporator, Germany). Both electroporated and unelectroporated cells were stained by PI (propidium iodide) to indicate the dead cells. Cell solution was pumped into to the microchannel at a flow rate of 0.5 μL/min, and then pushed to the lower side by another buffer stream at a flow rate of 0.6 μL/min. In the DEP sorting segment, sinusoidal voltage with amplitude of 8 VPP and frequency of 1.7 MHz was applied on the electrodes. For unelectroporated cells (Figure 4a), almost all the cells remained alive and were driven to the upper side of the channel by the DEP force. Only a few dead cells (red spots) flew in the lower side. For electroporated cells (Figure 4b), some cells were inevitably harmed in various degrees under the electrical

(1)

And the complex Clausius−Mossotti function fCM = (εp − εm)/(εp + 2εm)

(2)

where εp and εm are the permittivity of the cell and buffer solution surrounding the cell, respectively. R is the diameter of the cell, and E is the electric field intensity. In our design, after experiencing electroporation, cells would spend about 2 s in the concentration segment before entering the sorting module. In this period, most survived cells finished its membrane resealing. For those cells with little chance to survive (nonviable cells), the cell membrane remained permeable, which means εp − εm ≈ 0 and DEP force exerted on nonviable cells is very weak. For viable cells, the frequency of the electric field determines whether the cells behave like insulator or dielectric sphere. HEK-293a (human embryonic kidney cells) was first employed for the characterization of DEP responses under different frequencies. As shown in Figure 3a, cell membrane blocked the electric field at low frequencies, thus the cells behaved like insulators and experienced negative DEP force. As frequency increased, the cell membrane began to polarize, lessening the negative DEP force. The DEP force was close to zero while the frequency was about 500 kHz. Cell membrane became electrictransparent at high frequencies, making the cell behave like a dielectric sphere with the frequency-dependent permittivity εp. The strength of DEP force was decides by εp as referred to eq 2. For HEK-293a cells, as the frequency increased, the DEP response of viable cells first increased, and then reached a plateau while the frequency were 1.4, 1.7, and 2 MHz. The DEP response finally reduced to zero as the frequency continually increased to 5 MHz. From our previous chip electroporation studies,18,22 buffer ionic strength is an important parameter determining the efficiency of cell electroporation. According to eq 1 and 2, buffer ionic strength also decides the buffer permittivity εm, and therefore affects the strength of DEP force. In our previously optimized buffer (25 mM KCl, 0.3 mM KH2PO4, 0.85 mM K2HPO4, 36 mM myo-inositol) for microfluidic electroporation,18 the concentration of KCl is the determining factor for the buffer ionic strength. Therefore, we studied the effects 10218

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viable cells versus the whole amount of viable cells. To maintain a constant width ratio between the cells flow and the buffer flow while entering the DEP segment, the velocity ratio between cell suspension and buffer flow was maintained at 1:1.2 for all experiments performed in the study (velocity of cell flow, 0.5 μL/min; buffer sheath flow, 0.6 μL/min). Figure 4c shows the tendency of sorting efficiency at frequencies ranging from 50 kHz to 4 MHz. Agreed with the DEP responses in Figure 3a, the best sorting result was obtained with the frequency ranging from 1.4 to 2 MHz while the strong positive DEP occurred. Figure 4d shows the influence of DEP voltage on the sorting efficiency, in which the frequency was set at the best fit value 1.7 MHz. According to eq 1, the DEP force is proportional to the applied voltage, thus the sorting efficiency increased with the voltage. However, under voltages higher than 20 V, some cells was trapped on the electrodes, and eventually damaged. Under moderate voltages (7, 8, 9, or 10 V), no significant reduce of cell viabilities between sorted and unsorted cells was observed. Therefore, a low voltage, 8 Vpp, was chosen for DEP sorting in order to promise high sorting efficiency and to prevent cells from damage. Figure 4e shows the relationship between sorting efficiency and flow velocity of cell suspension. Sorting efficiency decreased with the flow velocity. Although lower flow velocity promised better sorting efficiency, considering the cell treating speed, the flow rate of 0.5 μL/ min was used in the following assays. In fact, 0.5 μL/min was a relatively slow cell treating speed which limited the cell population in a certain time period. We actually tried several means to increase the cell flow speed. As the cell flow speeding up, to maintain a good sorting efficiency, the voltage should be enhanced for acquiring higher DEP force, or the number of the sorting electrodes should be increased for enabling longer sorting area. However, enhanced voltage would probably harm the cell viability, and increasing the electrodes number (or the channel length) might aggravate the channel jam and compromise the fabrication yield. Increasing the treating speed also required a longer electroporation electrode. Therefore, we employed the relatively low cell flow rate for the fine balance between sorting efficiency and cell viability. In our study, parallel utilizing multiple chips was found to be a feasible solution for increase the cell population. Taken together, these experiments indicated that flow velocity, strength and frequency of applied electrical field were crucial factors for DEP sorting efficiency. Under certain fluidic and electric conditions, the angle between sorting electrodes and flow direction could affect the relative direction of DEP force, and the resultant force exerted on cells. Figure 4f demonstrates the role of the angle of electrodes. With angles less than 15 degrees, the electrodes maintained a fine sorting efficiency. As the angles increased from 15 degrees to 30 degrees, the sorting efficiency dropped dramatically. On the other side, smaller angle required longer flow channel. Therefore, the angle of 15 degrees was considered as the optimal value. To sum up from Figure 4, the optimal sorting conditions were set at 1.7 MHz as the electric frequency, 8 Vpp as the voltage, 0.5 μL/min and 0.6 μL/min as the cell and buffer flow rate, and 15 degrees as the electrode angle for the following electrotransfection assays. Electrotransfection of Plasmid DNA. In the initial proof of concept assays, HEK-293a cells and plasmid DNA (pEGFPC3) were used as model system to determine whether the novel method did improve the cell viability while pursuing the high electrotransfection efficiency. The operating procedure is described in Electroporation, Cell Sorting Procedures, and the

Figure 4. Optimization of DEP sorting parameters. To evaluate the effects of DEP sorting parameters on sorting efficiency, HEK-293a cells were electroporated by a commercial equipment (Eppendorf, Multiporator, Germany) and then injected into DEP sorting segment with buffer flow. Nonviable cells were indicated by PI staining (red spots). (a) The majority of unelectroporated cells maintained its viability, deflecting to the top. (b) Part of electroporated cells (red spots) became nonviable and maintained their flow path in the bottom, while other viable cells were directed to the top. The reason for the slight mismatch of DIC and PI image was the cell drifting during the image capture period. The sorting efficiency was presented as a function of electric field frequency (c), voltage applied on sorting electrodes (d), cell flow velocity (e), and the angle between sorting electrodes and cell flow direction (f). The number of cells was counted by ImageJ from NIH. The sorting efficiency was estimated by the deflected viable cells versus the whole amount of viable cells. All the data in panels c−f are the average of three independent assays. Each data was shown as the mean ± standard deviation..

stimulation. The membrane of fatally damaged cell became permanently permeable, being stained to red by PI. In the DEP sorting segment, cells with low viability (red spots) experienced weak DEP force and flew along the lower side of the channel without deflection, while cells remained viable were forced to the upper side. To optimize the cell sorting performance of this device, effects of varying frequency, voltage, flow velocity, as well as electrode angle, were determined, in terms of sorting efficiency. The sorting efficiency was estimated by the number of deflected 10219

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Figure 5. Electrotransfection of plasmid DNA. To determine the utility of the integrated sorting electrodes in improving cell viability, pEGFP plasmid was transfected into different cells types. Twenty-four hours after electroporation, the viable cells (a), the nonviable cells (b), and the unsorted cells (c) as control were fluorescently imaged. The green spots represent GFP-expressing cells while the red spots represent dead cells stained by PI. The number of GFP-expressing cells was counted in five randomly chosen fields by ImageJ from NIH; the number of living cells was determined by PI exclusion. (d) Quantitative analysis of transfection rate (TR) and survival rate (SR) of unsorted, viable, and nonviable HEK-293a cells. (e) Quantitative analysis of TR and SR of Neuro-2a cells. (f) Quantitative analysis of TR and SR of HUVEC cells with both low electroporation voltage and high electroporation voltage. (g) TR and SR of HUVEC cells using commercial cuvette-based electroporation equipment (Multiporator, Eppendorf, Germany), the voltage was 1200 V. (h) Quantitative analysis of TR and SR of HSF cells. (i) TR and SR of HSF cells using commercial batch-mode electroporation equipment (Multiporator, Eppendorf, Germany); the voltage was 1600 V. Transfection rate (TR) was estimated by the number of transfected cells versus living cells; survival rate (SR) was estimated by the number of living cells in treated and unelectroporated samples. All the data in panels d−i are the average of three independent assays. Each data was shown as the mean ± standard deviation. Individually optimized microfluidic electroporation parameters: HEK-293a, pulse strength 60 V, pulse duration 0.1 ms and pulse interval 2 s; Neuro-2a, 90 V, 0.1 ms and 2 s; HUVEC cells with low voltage 70 V, 0.1 ms and 2 s; HUVEC cells with high voltage 100 V, 0.2 ms and 2 s; HSF cells with low voltage 90 V, 0.1 ms, 2 s; HSF cells with high voltage 120 V, 0.5 ms, 1 s.

Determination of the Performance section. To determine the utility of the integrated sorting electrodes in improving cell viability, a pair of simple but popular parallel electrodes was employed for electroporation. The electric parameters (pulse strength 60 V, pulse duration 0.1 ms and pulse interval 2 s) for electroporation were optimized according to our previous work.18 Other parameters were set at the optimized number in Optimization of DEP Cell Sorting section. After electroporation and sorting, cells collected through outlet A in Figures 1 and 2 were defined as viable cells, while the other cells from outlet B were defined as nonviable cells. As control, unsorted cells refer to those electroporated cells flowing through the unpowered sorting segment. As expected, the optical and corresponding fluorescent images clearly showed the differences of plasmid transfection efficiency and cell viability between viable cells (Figure 5a), nonviable cells (Figure 5b), and unsorted cells (Figure 5c). ImageJ from NIH was employed for the quantitative analyses of transfection rate (TR) and cell survival rate (SR) in five independent assays (Figure 5d). For unsorted cells, the TR was around 80%, while the SR was no more than 50%. For viable cells, the SR was remarkably improved to 90%, while the TR remained high

(75%). In addition, both decreased TR (30%) and poor SR (less than 5%) of nonviable cells hinted an efficient sorting process in which the vast majority of unharmed cells were deflected to upper side of the channel and collected as viable cells. To evaluate the flexibility of the microfluidic chip in cell electrotransfection, pEGFP plasmid was electroporated into a range of cell types representing different levels of difficulty in gene delivery. In contrast to easy-to-transfect HEK-293a cells, we included the following: Neuro-2a cells, a neuroblastoma cell line; HUVEC cells, a primary human umbilicalvein endothelial cell and HSF cells, a primary human skin fibroblast cell. All the three types of cells mentioned above are known to be difficult to transfect by chemical approaches such as Lipofectamine 2000. Figure 5e shows the electrotransfection of Neuro-2a cells, a vulnerable neuron cell line that plays an important role in studying neural signal transmission. The electric parameters (pulse strength 90 V, pulse duration 0.1 ms, and pulse interval 2 s) were optimized according to our previous work.18 Compared with unsorted cells, sorted viable neuro-2a cells exhibited a remarkably enhanced SR (from about 30% to more than 80%) while maintaining the similar TR (about 65%). 10220

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CONCLUSION Microfluidic electroporation has been demonstrated to be a viable method for continuous electrotransfection of large scale of cells, but the unsatisfactory cell viability induced by adverse electric or hydrodynamic effects has limited the application of this useful technology on many important but vulnerable cell types, such as neuron cells or primary cells. In the present study, we demonstrated a flow-through electroporation microchip by implementing dielectrophoretic viable cell sorting in a conventional microfluidic electroporation chip based on parallel electrodes. Using standard microfabrication technology, a proof of concept device was fabricated. After carefully investigating and optimizing the DEP behaviors of electroporated cells in microfluidic channel, four cell types, including difficult-totransfect primary cells and vulnerable neuron cells, were efficiently electrotransfected with excellent cell viability. Compared with previously reported methods to improve the viability of electroporated cells by precisely adjusting electric and hydrodynamic parameters, this new method was found to have the following prominent features: (i) Because it benefited from steering clear of the difficulties of maintaining cell viability under unfavorable effects or excessive electric field, relatively high electric fields could be applied for efficient electroporation of many difficult-to-transfect cells; (ii) The efficient sorting of viable cells also ensures an acceptable viability of those vulnerable cell types. Furthermore, another clear advantage of the work is that the design and fabrication process of the simple DEP sorting electrodes are both fully compatible with existing microfluidic electroporation devices based on planar electrodes. Therefore, the presented sorting method opens new opportunities for the further improvement of cell viability in other state-of-art designs, and barely increases the cost.

We next targeted to difficult-to-transfect primary cells. Although cultured cells serve as important models for understanding basic biological processes, primary cells are physiologically closer to the real biological systems and have greater potential in biomedical studies. However, beaching the membrane of primary cells usually relied on more intensive electrical field which is very likely to aggravate the cell mortality. On the other side, the expression of DNA and further biological studies often required excellent cell viabilities. As a result, successful transfection of important primary cells, such as HUVEC and HSF, has never been reported with conventional microfluidic electroporation systems. Figure 5f shows the electrotransfection of HUVEC cells. First, we used the optimized electrical parameters (pulse strength 70 V, pulse duration 0.1 ms and pulse interval 2 s) from our previous chip electroporation study.22 It showed that the TR and SR for unsorted cells were 30% and 50%, while the numbers for sorted viable cells were 50% and 85%. To pursue higher TR, we employed a stronger electric field (100 V, 0.2 ms, 2 s). The TR for unsorted cells was increased to 75%, however the SR was dramatically dropped to less than 20%, which is unacceptable. Using DEP sorting, the SR was enhanced to more than 80% while the TR was maintained to 75%. It is worthwhile to note that the DEP sorting process in fact concentrated the viable cells right after the electroporation. For some important biological applications, such as IPS (induced pluripotent stem) cells production, increased density of viable cells and transfected cells not only facilitated a good culture condition, improving the cell proliferating, but also significantly reduced the time cost for screening the IPS cell clones. Figure 5h shows the electrotransfection of HSF cells which was frequently used in producing IPS cells. In conventional parallel-electrode device, the electric parameters could only be set at a low voltage (90 V, 0.1 ms and 2 s) for a high SR (60%), compromising the TR to less than 10%. Under this low voltage, even employing DEP sorting could barely improve the TR, leaving the enhancement of SR to 80% meaningless. After trailand-error assays, a more intensive electric field (120 V, 0.5 ms, 1 s) was employed to improve the TR to about 30%, three times higher than the number with low voltage. Despite the SR of unsorted cells was down to less than 20%, a satisfactory SR of 80% was still achieved on sorted viable cells. A commercial cuvette-based electroporation equipment (Multiporator, Eppendorf, Germany) and corresponding buffer kit were employed to electrotransfect pEGFP into both HUVEC and HSF cells for comparison (Figure 5g and i). According trialand-error test of the voltage, which was in fact the only adjustable parameter, the electroporation voltage for HUVEC and HSF were set to 1200 and 1600 V, respectively, for the best balance between SR and TR. The results demonstrated that our sorting strategy helped increasing both transfection efficiency and cell viability. To validate the reliability of the microfluidic chip, a prolonged test using HEK-293a cells was performed continuously over the course of 2 h. Consistent cell transfection efficiency and viability were observed, revealing that this chip can stably function over a long time course. Taken together, the optimal and consistent electrotransfection indicates that DEP sorting was effective on improving the cell viability, and in addition, allowing us to consider a more intensive electric field for some difficult-to-transfect cells.

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ASSOCIATED CONTENT

S Supporting Information *

Additional materials as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Fax 86-10-82545643. *E-mail: [email protected]. Fax: +86-10-62769862. *E-mail: [email protected]. Fax: 86-10-62751789. Author Contributions

Z.W. and X.L. contributed equally to the work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 61204118, 61176111 and 81273422), the National High Technology Research and Development Program of China (863) (No. 2012AA022501), and National Drug Program of China (No. 2011ZX09102-01112, 2012ZX09102301-006).

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