Flow-through electroporation of mammalian cells in decoupled flow streams using microcapillaries.
Ontology highlight
ABSTRACT:
SUBMITTER: Luo Y
PROVIDER: S-EPMC4032400 | biostudies-other | 2014 Sep
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Electroporation of in-vitro cultured cells is widely used in biological and medical areas to deliver molecules of interest inside cells. Since very high electric fields are required to electroporate the plasma membrane, depending on the geometry of the electrodes the required voltages can be very high and often critical to cell viability. Furthermore, in traditional electroporation configuration based on planar electrodes there is no a priori certain feedback about which cell has been targeted and delivered and the addition of fluorophores may be needed to gain this information. In this study we present a nanofabricated platform able to perform intracellular delivery of membrane-impermeable molecules by opening transient nanopores into the lipid membrane of adherent cells with high spatial precision and with the application of low voltages (1.5-2?V). This result is obtained by exploiting the tight seal that the cells present with 3D fluidic hollow gold-coated nanostructures that act as nanochannels and nanoelectrodes at the same time. The final soft-electroporation platform provides an accessible approach for controlled and selective drug delivery on ordered arrangements of cells.
| S-EPMC5561120 | biostudies-literature
Project description:The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.
| S-EPMC4331347 | biostudies-literature
Project description:Lentiviral vectors are beginning to emerge as a viable choice for human gene therapy. Here, we describe a method that combines the convenience of a suspension cell line with a scalable, nonchemically based, and GMP-compliant transfection technique known as flow electroporation (EP). Flow EP parameters for serum-free adapted HEK293FT cells were optimized to limit toxicity and maximize titers. Using a third generation, HIV-based, lentiviral vector system pseudotyped with the vesicular stomatitis glycoprotein envelope, both small- and large-volume transfections produced titers over 1×10(8) infectious units/mL. Therefore, an excellent option for implementing large-scale, clinical lentiviral productions is flow EP of suspension cell lines.
| S-EPMC3311912 | biostudies-literature
Project description:Nonviral methods have been explored as the replacement of viral systems for their low toxicity and immunogenicity. However, they have yet to reach levels competitive to their viral counterparts. In this paper, we combined physical and chemical methods to improve the performance of polyplex delivery of DNA and small interfering RNA. Specifically, gold nanoparticles (AuNPs) were used to carry polyplex (a chemical approach) while electroporation (a physical approach) was applied for fast and direct cytosolic delivery. In this hybrid approach, cationic polymer molecules condense and/or protect genetic probes as usual while AuNPs help fix polycations to reduce their cytotoxicity and promote the transfection efficiency of electroporation. AuNPs of various sizes were first coated with polyethylenimine, which were further conjugated with DNA plasmids or small interfering RNA molecules to form AuNPs-polyplex. The hybrid nanoparticles were then mixed with cells and introduced into cell cytosol by electroporation. The delivery efficiency was evaluated with both model anchor cells (i.e., NIH/3T3) and suspension cells (i.e., K562), together with their impact on cell viability. We found that AuNP-polyplex showed 1.5?2 folds improvement on the transfection efficiency with no significant increase of toxicity when compared to free plasmid delivery by electroporation alone. Such a combination of physical and chemical delivery concept may stimulate further exploration in the delivery of various therapeutic materials for both in vitro and in vivo applications.
| S-EPMC4696390 | biostudies-literature
Project description:Electroporation serves as a promising non-viral gene delivery approach, while its current configuration carries several drawbacks associated with high-voltage electrical pulses and heterogeneous treatment on individual cells. Here we developed a new micropillar array electroporation (MAE) platform to advance the electroporation-based delivery of DNA and RNA probes into mammalian cells. By introducing well-patterned micropillar array texture on the electrode surface, the number of pillars each cell faces varies with its plasma membrane surface area, despite their large population and random locations. In this way, cell size specific electroporation is conveniently carried out, contributing to a 2.5~3 fold increase on plasmid DNA transfection and an additional 10-55% transgene knockdown with siRNA probes, respectively. The delivery efficiency varies with the number and size of micropillars as well as their pattern density. As MAE works like many single cell electroporation are carried out in parallel, the electrophysiology response of individual cells is representative, which has potentials to facilitate the tedious, cell-specific protocol screening process in current bulk electroporation (i.e., electroporation to a large population of cells). Its success might promote the wide adoption of electroporation as a safe and effective non-viral gene delivery approach needed in many biological research and clinical treatments.
| S-EPMC5141490 | biostudies-literature
Project description:Genetic modification of cell lines and primary cells is an expensive and cumbersome approach, often involving the use of viral vectors. Electroporation using square-wave generating devices, like Lonza's Nucleofector, is a widely used option, but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this methodology. In the present work, we show that our in-house developed buffers, termed Chicabuffers, can be efficiently used to electroporate cell lines and primary cells from murine and human origin. Using the Nucleofector II device, we electroporated 14 different cell lines and also primary cells, like mesenchymal stem cells and cord blood CD34+, providing optimized protocols for each of them. Moreover, when combined with sleeping beauty-based transposon system, long-term transgene expression could be achieved in all types of cells tested. Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. We also show that these buffers can be used in CRISPR-mediated editing of PDCD1 gene locus in 293T and human peripheral blood mononuclear cells. The optimized protocols reported in this study provide a suitable and cost-effective platform for the genetic modification of cells, facilitating the widespread adoption of this technology.
| S-EPMC5253374 | biostudies-literature
Project description:Isolation of cells and their transfection in a controlled manner is an integral step in cell biotechnology. Electric field approaches such as dielectrophoresis (DEP) offers a more viable method for targeted immobilization of cells without any labels. For transfection of cells to incorporate exogenous materials, electrical methods such as electroporation, are preferred over chemical and viral delivery methods since they minimally affect cell viability and can target many types. However prior approaches to both methods required multiple excitation sources, an AC source for DEP-based trapping and another DC source for electroporation. In this paper, we present a first of its kind flow through lab-on-chip platform using a single AC excitation source for combined trapping using negative dielectrophoresis (nDEP) and AC electroporation. Use of AC fields for electroporation eliminates the unwanted side effects of electrolysis or joule heating at electrodes compared to DC electroporation. Adjusting the flow rate and the electrical parameters of the incident AC field precisely controls the operation (trap, trap with electroporation and release). The platform has been validated through trapping and simultaneous transfection of HEK-293 embryonic kidney cells with a plasmid vector containing a fluorescent protein tag. Numerical scaling analysis is provided that indicates promise for individual cell trapping and electroporation using low voltage AC fields.
| S-EPMC6700080 | biostudies-other
Project description:Dense microcircuit reconstruction techniques have begun to provide ultrafine insight into the architecture of small-scale networks. However, identifying the totality of cells belonging to such neuronal modules, the "inputs" and "outputs," remains a major challenge. Here, we present the development of nanoengineered electroporation microelectrodes (NEMs) for comprehensive manipulation of a substantial volume of neuronal tissue. Combining finite element modeling and focused ion beam milling, NEMs permit substantially higher stimulation intensities compared to conventional glass capillaries, allowing for larger volumes configurable to the geometry of the target circuit. We apply NEMs to achieve near-complete labeling of the neuronal network associated with a genetically identified olfactory glomerulus. This allows us to detect sparse higher-order features of the wiring architecture that are inaccessible to statistical labeling approaches. Thus, NEM labeling provides crucial complementary information to dense circuit reconstruction techniques. Relying solely on targeting an electrode to the region of interest and passive biophysical properties largely common across cell types, this can easily be employed anywhere in the CNS.
| S-EPMC5766516 | biostudies-literature
Project description:Continuous cell electroporation is an appealing non-viral approach for genetically transfecting a large number of cells. Yet the traditional macro-scale devices suffer from the unsatisfactory transfection efficiency and/or cell viability due to their high voltage, while the emerging microfluidic electroporation devices is still limited by their low cell processing speed. Here we present a flow-through cell electroporation device integrating large-sized flow tube and small-spaced distributed needle electrode array. Relatively large flow tube enables high flow rate, simple flow characterization and low shear force, while well-organized needle array electrodes produce an even-distributed electric field with low voltage. Thus the difficulties for seeking the fine balance between high flow rate and low electroporation voltage were steered clear. Efficient in vitro electrotransfection of plasmid DNA was demonstrated in several hard-to-transfect cell lines. Furthermore, we also explored ex vivo electroporated mouse erythrocyte as the carrier of RNA. The strong ability of RNA loading and short exposure time of freshly isolated cells jointly ensured a high yield of valid carrier erythrocytes, which further successfully delivered RNA into targeted tissue. Both in vitro and ex vivo electrotransfection could be accomplished at high cell processing speed (20 million cells per minute) which remarkably outperforms previous devices.
| S-EPMC4700452 | biostudies-other
Project description:Manipulating gene expression in primary neurons has been a goal for many scientists for over 20 years. Vertebrate central nervous system neurons are classically difficult to transfect. Most lipid reagents are inefficient and toxic to the cells, and time-consuming methods such as viral infections are often required to obtain better efficiencies. We have developed an efficient method for the transfection of cerebellar granule neurons and hippocampal neurons with standard plasmid vectors. Using 96-well electroporation plates, square-wave pulses can introduce 96 different plasmids into neurons in a single step. The procedure results in greater than 20% transfection efficiencies and requires only simple solutions of nominal cost. In addition to enabling the rapid optimization of experimental protocols with multiple parameters, this procedure enables the use of high content screening methods to characterize neuronal phenotypes.
| S-EPMC2424126 | biostudies-literature