The construction of an individually addressable cell array for selective patterning and electroporation

In basic cell biology research and drug discovery, it is important to rapidly introduce genes, proteins or drug compounds into cells without permanent damage. Here, we report a three dimensional SU-8 micro-well structure sandwiched with an indium tin oxide (ITO) electrode-covered slide from the top and an individually addressable array of microelectrodes on the bottom to allow parallel delivery of exogenous molecules into various cells in a spatially specific manner. A positive dielectrophoretic force was selectively applied by energizing appropriate electrodes to capture the dispersed cells at the bottom electrode, while the micro-wells were designed to confine cells in situ when the positive dielectrophoretic force is removed. The combination of spatial positive dielectrophoresis (pDEP) and micro-wells made it possible to construct cell microarrays with specific patterns. Once the cells become attached to the electrodes, different plasmids can be introduced sequentially for selective electroporation. The present cell arraying-assisted electroporation chip integrates a pDEP-assisted cell positioning function with selective electroporation to provide a simple and efficient method for gene transfer. This platform is ideal for high throughput screening of compounds in parallel and thus holds promise for applications in cellular and molecular research.     

Simulation and experimental demonstration of the electric field assisted electroporation microchip for in vitro gene delivery enhancement

Simulation and experimental demonstration of the in vitro gene delivery enhancement using electrostatic forces and electroporation (EP) microchips were conducted. Electroporation is a technique with which DNA molecules can be delivered into cells using electric field pulses. This study demonstrates that plasmid DNA can be attracted to the cell surfaces at the specific regions using an electrostatic force. Therefore, the DNA concentration on the cell surface is dramatically increased, which highly enhances the gene transfection efficiency compared to that without an attracting-electric field. The electrostatic force can be designed into specific regions, where the DNA plasmids are attracted to, to provide the region-targeting function. In this micro-device, the top electrode and the interdigitated electrodes provided the DNA attracting-electric field, and the interdigitated electrodes provided adequate electric fields for the electroporation process on the chip surface. Using the EP microchip, cells could be manipulated in situ without detachment if adherent cells were used for electroporation. Five different cells of two different types, primary cell and cell line, were successfully transfected under multi-pulse or single pulse electric field stimulation without applying an attracting-electric field. This study simulated and analyzed the electric field distributions at the DNA attracting and electroporation processes, and successfully demonstrated that the electrostatic force attracted DNA plasmids to specific regions and highly enhanced the gene delivery. In summary, this EP microchip should provide many potential applications for gene therapy.     

Efficient gene delivery in differentiated human embryonic stem cells

Human embryonic stem (hES) cells are capable of differentiating into pluralistic cell types, however, spontaneous differentiation generally gives rise to a limited number of specific differentiated cell types and a large degree of cell heterogeneity. In an effort to increase the efficiency of specified hES cell differentiation, we performed a series of transient transfection of hES cells with EGFP expression vectors driven by different promoter systems, including human cellular polypeptide chain elongation factor 1 alpha (hEF1alpha), human cytomegalo-virus, and chicken beta-actin. All these promoters were found to lead reporter gene expression in undifferentiated hES cells, but very few drug-selectable transfectants were obtained and failed to maintain stable expression of the transgene with either chemical or electroporation methods. In an attempt to increase transfection efficiency and obtain stable transgene expression, differentiated hES cells expressing both mesodermal and ectodermal markers were derived using a defined medium. Differentiated hES cells were electroporated with a hEF1alpha promoter-driven EGFP or human noggin expression vector. Using RT-PCR, immunocytochemistry and fluorescence microscopy, the differentiated hES cells transfected with foreign genes were confirmed to retain stable gene and protein expression during prolonged culture. These results may provide a new tool for introducing exogenous genes readily into hES cells, thereby facilitating more directed differentiation into specific and homogenous cell populations.     

Gene transfer and protein dynamics in stem cells using single cell electroporation in a microfluidic device

There is great interest in genetic modification of bone marrow-derived mesenchymal stem cells (MSC), not only for research purposes but also for use in (autologous) patient-derived-patient-used transplantations. A major drawback of bulk methods for genetic modifications of (stem) cells, like bulk-electroporation, is its limited yield of DNA transfection (typically then 10%). This is even more limited when cells are present at very low numbers, as is the case for stem cells. Here we present an alternative technology to transfect cells with high efficiency (>75%), based on single cell electroporation in a microfluidic device. In a first experiment we show that we can successfully transport propidium iodide (PI) into single mouse myoblastic C2C12 cells. Subsequently, we show the use of this microfluidic device to perform successful electroporation of single mouse myoblastic C2C12 cells and single human MSC with vector DNA encoding a green fluorescent-erk1 fusion protein (EGFP-ERK1 (MAPK3)). Finally, we performed electroporation in combination with live imaging of protein expression and dynamics in response to extracellular stimuli, by fibroblast growth factor (FGF-2). We observed nuclear translocation of EGFP-ERK1 in both cell types within 15 min after FGF-2 stimulation. Due to the successful and promising results, we predict that microfluidic devices can be used for highly efficient small-scale 'genetic modification' of cells, and biological experimentation, offering possibilities to study cellular processes at the single cell level. Future applications might be small-scale production of cells for therapeutic application under controlled conditions.     

Isotachophoresis for rapid transformation of Escherichia coli

A frequent limitation of electroporation (EP) and chemical transformation (CT) are the need of tedious and time-consuming procedures for inducing transformation competence, the substantial number of cells required, and the low transformation yields typically achieved. Here, we show a new and rapid electrokinetic method for transformation of small number of noncompetent Escherichia coli TOP10 cells (2–3 × 10⁵) at room temperature. Escherichia coli TOP10 cells and plasmid DNA are sequentially injected into a 50 μm ID capillary and focused into 11.5 nL by isotachophoresis (ITP) induced by application of high DC voltage (–16 kV). Through ITP, a large excess of plasmid DNA is brought in contact with the cell surface, with the contact time adjusted by application of a counter-pressure (1.3 psi) opposing the ITP movement. The transformation rate was more than 1000-fold higher compared to EP and CT at survival rates greater than 60%.     

Correlated atomic force microscopy and fluorescence lifetime imaging of live bacterial cells

We report on imaging living bacterial cells by using a correlated tapping-mode atomic force microscopy (AFM) and confocal fluorescence lifetime imaging microscopy (FLIM). For optimal imaging of Gram-negative Shewanella oneidensis MR-1 cells, we explored different methods of bacterial sample preparation, such as spreading the cells on poly-L-lysine coated surfaces or agarose gel coated surfaces. We have found that the agarose gel containing 99% ammonium acetate buffer can provide sufficient local aqueous environment for single bacterial cells. Furthermore, the cell surface topography can be characterized by tapping-mode in-air AFM imaging for the single bacterial cells that are partially embedded. Using in-air rather than under-water AFM imaging of the living cells significantly enhanced the contrast and signal-to-noise ratio of the AFM images. Near-field AFM-tip-enhanced fluorescence lifetime imaging (AFM-FLIM) holds high promise on obtaining fluorescence images beyond optical diffraction limited spatial resolution. We have previously demonstrated near-field AFM-FLIM imaging of polymer beads beyond diffraction limited spatial resolution. Here, as the first step of applying AFM-FLIM on imaging bacterial living cells, we demonstrated a correlated and consecutive AFM topographic imaging, fluorescence intensity imaging, and FLIM imaging of living bacterial cells to characterize cell polarity.     

Picoliter-volume aqueous droplets in oil: electrochemical detection and yeast cell electroporation

An electrochemical detection method was introduced for aqueous droplet analysis in oil phase of microfluidic devices. This method is based on the electrochemical signal difference between aqueous and oil. Applying a low alternating current (AC) voltage to a couple of Au microelectrodes, this method can offer size information and ion concentration range from 0.02 mmol/L to 1 mol/L of tens of picoliter to nanoliter aqueous droplets. Alternatively, applying a relative high AC voltage (18 Vpp) at a frequency of 1 kHz leads to electroporation of yeast cells encapsulated into picoliter droplets. We believe that this simple technique is useful for a number of aqueous droplet-based chemical and biological analyses as well as cell electroporation.     

Transfer of foreign genes by electroporation and their expression in mammalian cells

Expression of foreign genes transferred into mammalian cells by electroporation has been studied. The pX1TK gene, pSV2Neo gene and pUCEJ oncogene have been introduced into MLTK- cells and NIH/3T3 cells, respectively. Stable transformation transient expression of TK gene by MLTK- cells as well as stable and malignant transformation of NIH/3T3 cells have been obtained. Transient expression frequency is about 80% and stable transformation frequency is about 10(-4). Integration of foreign genes into the cellular genome was verified with molecular hybridization. Tumor development was observed after inoculation of transformed cells into nude mice.     

TRANSFER OF FOREIGN GENES BY ELECTROPORATION AND THEIR EXPRESSION IN MAMMALIAN CELLS

Expression of foreign genes transferred into mammalian cells by electroporation has been studied. The pX1TK gene, pSV2Neo gene and pUCEJ oncogene have been introduced into MLTK-cells and NIH/3T3 cells, respectively. Stable transformation transient expression of TK gene by MLTK-cells as well as stable and malignant transformation of NIH/3T3 cells have been obtained. Transient expression frequency is about 80% and stable transformation frequency is about 10~(-4). Integration of foreign genes into the cellular genome was verified with molecular hybridization. Tumor development was observed after inoculation of transformed celts into nude mice.     

Bio-chip for spatially controlled transfection of nucleic acid payloads into cells in a culture

Transfection of siRNA and plasmid nucleic molecules to animal, microbial and plant cell cultures is a critical process in various research areas, including drug discovery, functional genomics and basic life science research. Till recent times, transfection of these exogenous molecules have been global in nature i.e. targeting all the cells in a culture and lacking capability to spatially confine the transfection to small populations of cells within a single culture. However, in emerging areas like high-throughput screening of large molecule libraries, there is a critical need to transfect multiple different molecules to locally specified regions of a single cell culture and monitor phenotypical changes in these different cell populations. In this study, we present a cell-based biochip that utilizes a microelectrode array to generate localized current density fields that induce electroporation to a targeted group of cells for site-specific transfection of exogenous molecules. More specifically, we optimize the transfection efficiency and viabilities for spatially controlled transfection of Alexa-Fluor-488 conjugated siRNA molecules into NIH3T3 fibroblast cell cultures. Optimal electroporation parameters are established at current density values ranging between 0.05-0.07 microA microm(-2) for high transfection efficiencies (>60%) while maintaining viability (>80%) on individual microelectrodes. Additionally, exogenous plasmid molecules are electroporated for site-specific GFP expression and monitored over 48 h in-situ. The microelectrode array technology reported here can therefore be potentially used for targeting specific cells in a culture with spatial precision and transfecting siRNA and plasmids. The microfabrication approach lends itself to significant high-throughput applications in drug-discovery research.     

Automatic medium exchange for micro-volume cell samples based on dielectrophoresis

Cell medium exchange is a crucial step for life science and medicine. However, conventional cell medium exchange methods, including centrifuging and filtering, show limited ability for micro-volume cell samples such as circulating tumor cell (CTC) and circulating fetal cell (CFC). In this paper, we proposed an automatic medium exchange method for micro-volume cell samples based on dielectrophoresis (DEP) in microfluidic chip. Fresh medium and cell suspension were introduced into the microfluidic channel as the laminar flow. Plane stair-shaped interdigital electrodes were employed to drive the cells from the cell suspension to fresh media directly by DEP force. Additionally, we characterized and optimized the cell medium exchange according to both the theory and experiments. In the end, we achieved a 96.9% harvest rate of medium exchange for 0.3 μL samples containing micro-volume cells. For implementing an automatic continuous cell medium exchange, the proposed method can be integrated into the automatic cell processing system conveniently. Furthermore, the proposed method is a great candidate in micro-volume cell analysis and processing, cell electroporation, single cell sequencing, and other scenarios.     

A single cell electroporation chip

Increasing the cell membrane's permeability can be accomplished via single cell electroporation. Polar substances that cannot otherwise permeate the plasma membrane (such as dyes, drugs, DNA, proteins, peptides, and amino acids) can thus be introduced into the cell. We developed a polymeric chip that can selectively immobilize and locally electroporate single cells. This easy-to-use chip focuses the electric field, eliminating the need to manipulate electrodes or glass pipettes. Moreover, this device allows parallel single cell electroporation. We demonstrate the effectiveness of our device design by electroporating HeLa cells using low applied voltages (< 1 V). We found the average transmembrane potential required for electroporation of HeLa cells to be 0.51 +/- 0.13 V. Membrane permeation is assessed electrically by measuring characteristic 'jumps' in current that correspond to drops in cell resistance, and microscopically by recording either the escape of cytoplasmic dye Calcein AM or the entrance of Trypan blue stain.     

Gene delivery by electroporation after dielectrophoretic positioning of cells in a non-uniform electric field

We report the use of dielectrophoresis (DEP) to position U-937 monocytes within a non-uniform electric field, prior to electroporation (EP) for gene delivery. DEP positioning and EP pulsing were both accomplished using a common set of inert planar electrodes, micro-fabricated on a glass substrate. A single-shell model of the cell's dielectric properties and finite-element modeling of the electric field distribution permitted us to predict the major features of cell positioning. The extent to which electric pulses increased the permeability of the cell membranes to fluorescent molecules and to pEGFPLuc DNA plasmids were found to depend on prior positioning. For a given set of pulse parameters, EP was either irreversible (resulting in cytolysis), reversible (leading to gene delivery), or not detectable, depending on where cells were positioned. Our results clearly demonstrate that position-dependent EP of cells in a non-uniform electric field can be controlled by DEP.     

Electroporation of cell membranes supporting penetration of photodynamic active macromolecular chromophore dextrans

The aim is to demonstrate that macromolecular chromophore dextrans (Cibacron-dextran) acting as photosensitizers can be transported easily into cancer cells by electroporation of their membranes (short electric pulses on cell suspension between electrodes). There are two possibilities, either:(A)irradiation starts with the electropulse-showed with easily penetrating thiopyronin-yielding nearly 100% dead cells;(B)irradiation starts after a resealing time of membrane pores during which macromolecular photosensitizers can penetrate into cells. In this way, fractions of Cibacron-dextran with molecular weights (Mw) 3300, 10,900 and 500,000 are now able to kill. This combination of bioelectrochemistry and photobiology will be suitable also for other biopolymers, connected with photodynamic active chromophores (e.g. chromopeptides) to transport them through cell walls and membranes into cells and tissues. The human cancer cells U-935 and K-562 (pulsed by 1.15 kV/cm field strength) additionally or synergistically reach high rates of necrotic cells (colored by trypan blue) by this combination.     

Anion Complexation and Transport by Isophthalamide and Dipicolinamide Derivatives: DNA Plasmid Transformation in E. coli

Tris-arenes based on either isophthalic acid or 2,6-dipicolinic acid have been known for more than a decade to bind anions. Recent studies have also demonstrated their ability to transport various ions through membranes. In this report, we demonstrate two important properties of these simple diamides. First, they transport plasmid DNA into Escherichia coli about 2-fold over controls, where the ampicillin resistance gene is expressed in the bacteria. These studies were done with plasmid DNA (～2.6 kilobase (kb)) in JM109 E. coli cells. Second, known methods do not typically transport large plasmids (>15 kb). We demonstrate here that transformation of large pVIB plasmids (i.e., >20 kb) were enhanced over water controls by ～10-fold. These results are in striking contrast to the normal decrease in transformation with increasing plasmid size.     

A platform method for plasmid isoforms analysis by capillary gel electrophoresis

In the production of novel biological products, plasmids are often engineered into delivery vectors for target genes, which can be used directly as vaccines or as intermediate products for gene/cell therapy. Plasmid DNA exists in several topological forms such as supercoiled, linear, and open circular. As supercoiled plasmid shows the highest efficiency in transfecting eukaryotic cells, the content of supercoiled plasmids becomes an important indicator of plasmid quality. CGE is an effective analysis method for separating different topological structures of plasmids. For the purpose of providing plasmid manufacturers and regulatory agencies with an efficient and readily used tool for monitoring the quality of plasmids, this article identifies the optimal separation and detection conditions of CGE, presents a platform-based plasmid analytical method, and uses plasmid of different sizes to verify the feasibility of this method. In terms of detector, the LIF detector has obvious advantages over the ultraviolet detector in sensitivity and resolution. Using the optimal CE condition (10× gel buffer), baseline separation of different topological forms and impurities can be achieved for different plasmid sizes (5.9, 7.8, 15.4 kb). In addition, 6.5 kb plasmid was used to compare the different separation technologies such as CGE-LIF, ion exchange chromatography and agarose gel electrophoresis. The result shows that CGE-LIF can provide better resolution and quantitation accuracy than ion exchange chromatography and agarose gel electrophoresis. CGE-LIF, as a quick and convenient method to separate and quantify plasmids, has the advantages of high sensitivity, high resolution, and high quantitative accuracy. Therefore, it is ideal for analysis of plasmids with different sizes, and it can also be used as a platform method for manufacturers and regulatory agencies to monitor the purity and stability of plasmids.     

Efficiency of DNA Transfection of Rat Heart Myoblast Cells H9c2(2-1) by Either Polyethyleneimine or Electroporation

Expression of exogenous DNA in vitro is significantly affected by the particular transfection method utilized. In this study, we evaluated the efficiency of two transfection methods, chemically mediated polyethyleneimine (PEI) treatment and physically mediated electroporation, on a rat heart myoblast cell line, H9c2(2-1). After PEI transfection of pPgk-1/EGFP into H9c2(2-1) cells, EGFP expression could be easily detected by fluorospectrometer after 48 h (210 ± 12 RFU) and continued to increase after 72 h (243 ± 14 RFU). However, when H9c2(2-1) cells were transfected by electroporation (200 V, 500 μF, and one pulse), low level EGFP expression was observed after 48 h (49 ± 4 RFU) or 72 h (21 ± 14 RFU). In contrast, the easily transfectable control CHO-K1 cell line displayed a stronger EGFP expression than the H9c2(2-1) cells either by PEI or electroporation transfection. When transfection efficiencies were assayed by flow cytometry after 72 h, 13.6 ± 2.2% of PEI and 10.1 ± 1.5% of electroporation (250 V, 500 μF, and two pulses) transfected cells of H9c2(2-1) expressed EGFP, and PEI-transfected cells appeared to be less damaged (viability 93.6%) as compared to electroporation-transfected cells (39.5%). However, both PEI and electroporation (580 V, 50 Ω, and 50 μF) were effective for transfection of CHO-K1 with a higher efficiency, cell viability, and EGFP expression than H9c2(2-1). Our results indicate that the transfection efficiency of different methods varies among cell lines and that PEI is more efficient than electropolation for transfection of H9c2(2-1) whereas both PEI and electroporation are suitable for CHO-K1 transfection.     

Influence of the physiological state on the electric field mediated transformation efficiency of intact mycobacterial cells

The sensitivity of Mycobacterium fortuitum at various growth stages to electric field pulses has been investigated in order to transform the cells by electroporation. In contrast to older cells, early- and mid-exponential growing cells are very field sensitive and, simultaneously, transformable to a relatively high degree. At an electric field strength E = 7 kV/cm and a pulse duration τ ≈ 13 ms (one pulse) an optimum transformation rate of about 2 × 10⁴ transformants/μg DNA was observed. On the basis of the different fast change of transformation rate and field sensitivity during cell growth we suppose a remarkable influence of biochemical changes in the cell wall composition on the efficiency of the electric field mediated transformation.     

TRANSFORMATION OF BALB 3T3 CELLS EXPOSED TO A GERMICIDAL UV LAMP AND A SUNLAMP

Abstract— The effect of germicidal UV and sunlamp exposure on direct and simian virus-40 (SV-40) transformation of Balb 3T3 cells was studied. Transformation was determined by the ability of transformed cells to grow as clones in agar. Radiation from these lamps enhanced direct transformation, and enhanced viral transformation to approximately the same degree. Enhanced transformation was seen with exposures of light that caused no measurable cell killing, which suggests that the induction of new transformants is involved rather than the selection of pre-existing transformants. Induction is also suggested by post-irradiation growth kinetics experiments.