Dataset Information

A Flow-Through Cell Electroporation Device for Rapidly and Efficiently Transfecting Massive Amounts of Cells in vitro and ex vivo.

ABSTRACT: 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.

SUBMITTER: Zhao D

PROVIDER: S-EPMC4700452 | biostudies-other | 2016 Jan

REPOSITORIES: biostudies-other

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A Flow-Through Cell Electroporation Device for Rapidly and Efficiently Transfecting Massive Amounts of Cells in vitro and ex vivo.

Zhao Deyao D Huang Dong D Li Yang Y Wu Mengxi M Zhong Wenfeng W Cheng Qiang Q Wang Xiaoxia X Wu Yidi Y Zhou Xiao X Wei Zewen Z Li Zhihong Z Liang Zicai Z

Scientific reports 20160105

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 need ...[more]

PMID: 26728941

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Project description:A novel three-step combination of in vitro and ex vivo screening was established to massively screen host derived lactic acid bacteria (LAB) from the broiler chicken intestine with inhibitory activity against Escherichia coli. In a first step, a massive sample pool consisting of 7102 broiler-derived colonies from intestinal contents were established and sub-cultured. Supernatants thereof were incubated with an E. coli model strain to screen suitable isolates with inhibitory activity. A total of 76 isolates of interest were subsequently further studied based on either pH dependent or -independent activity in the second step of the assay. Here, in-depth growth inhibition of the E. coli model strain and the potential of isolates for lactic acid production as inhibitory substance were indexed for all isolates. Resulting scatter plots of both parameters revealed five isolates with exceptional inhibitory activity that were further studied under ex vivo condition in the third step of the assay. These isolates were taxonomically classified as strains of the species Lactobacillus agilis, Lactobacillus salivarius, and Pediococcus acidilactici. Samples from the broiler chicken intestine were inoculated with the Lactobacillus isolates and the E. coli model strain. After 8 and 24 h incubation, respectively, growth of the E. coli model strain was monitored by cultivation of the E. coli strain in antibiotic supplemented medium. By their superior inhibitory activity against the E. coli model strain, one L. agilis and one L. salivarius strain were selected and characterized for further application as probiotics in broiler chicken. Additionally, their antibiotic resistance patterns and resilience under gastric stress of isolates were also characterized. The results of this study demonstrate that the novel isolation procedure was able to efficiently and rapidly isolate and identify bacterial strains from a massive sample pool with inhibitory potential against specific types of bacteria (here E. coli). The introduction of the final ex vivo selection step additionally confirmed the inhibitory activity of the strains under conditions simulating the intestinal tract of the host. Furthermore, this method revealed a general potential for the isolation of antagonistic strains that active against other pathogenic bacteria with specific biomarker.

Project description:BackgroundThe dynamics of phosphatidylserine in the plasma membrane is a tightly regulated feature of eukaryotic cells. Phosphatidylserine (PS) is found preferentially in the inner leaflet of the plasma membrane. Disruption of this asymmetry leads to the exposure of phosphatidylserine on the cell surface and is associated with cell death, synaptic pruning, blood clotting and other cellular processes. Due to the role of phosphatidylserine in widespread cellular functions, an efficient phosphatidylserine probe is needed to study them. Currently, a few different phosphatidylserine labelling tools are available; however, these labels have unfavourable signal-to-noise ratios and are difficult to use in tissues due to limited permeability. Their application in living tissue requires injection procedures that damage the tissue and release damage-associated molecular patterns, which in turn stimulates phosphatidylserine exposure.MethodsFor this reason, we developed a novel genetically encoded phosphatidylserine probe based on the C2 domain of the lactadherin (MFG-E8) protein, suitable for labelling exposed phosphatidylserine in various research models. We tested the C2 probe specificity to phosphatidylserine on hybrid bilayer lipid membranes by observing surface plasmon resonance angle shift. Then, we analysed purified fused C2 proteins on different cell culture lines or engineered AAVs encoding C2 probes on tissue cultures after apoptosis induction. For in vivo experiments, neurotropic AAVs were intravenously injected into perinatal mice, and after 2 weeks, brain slices were collected to observe C2-SNAP expression.ResultsThe biophysical analysis revealed the high specificity of the C2 probe for phosphatidylserine. The fused recombinant C2 proteins were suitable for labelling phosphatidylserine on the surface of apoptotic cells in various cell lines. We engineered AAVs and validated them in organotypic brain tissue cultures for non-invasive delivery of the genetically encoded C2 probe and showed that these probes were expressed in the brain in vivo after intravenous AAV delivery to mice.ConclusionsWe have demonstrated that the developed genetically encoded PS biosensor can be utilised in a variety of assays as a two-component system of C2 and C2m2 fusion proteins. This system allows for precise quantification and PS visualisation at directly specified threshold levels, enabling the evaluation of PS exposure in both physiological and cell death processes.

Dataset Information

A Flow-Through Cell Electroporation Device for Rapidly and Efficiently Transfecting Massive Amounts of Cells in vitro and ex vivo.

Publications

A Flow-Through Cell Electroporation Device for Rapidly and Efficiently Transfecting Massive Amounts of Cells in vitro and ex vivo.

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