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Microfluidic Irreversible Electroporation-A Versatile Tool to Extract Intracellular Contents of Bacteria and Yeast.

ABSTRACT: Exploring the dynamic behavior of cellular metabolism requires a standard laboratory method that guarantees rapid sampling and extraction of the cellular content. We propose a versatile sampling technique applicable to cells with different cell wall and cell membrane properties. The technique is based on irreversible electroporation with simultaneous quenching and extraction by using a microfluidic device. By application of electric pulses in the millisecond range, permanent lethal pores are formed in the cell membrane of Escherichia coli and Saccharomyces cerevisiae, facilitating the release of the cellular contents; here demonstrated by the measurement of glucose-6-phosphate and the activity of the enzyme glucose-6-phosphate dehydrogenase. The successful application of this device was demonstrated by pulsed electric field treatment in a flow-through configuration of the microfluidic chip in combination with sampling, inactivation, and extraction of the intracellular content in a few seconds. Minimum electric field strengths of 10 kV/cm for E. coli and 7.5 kV/cm for yeast S. cerevisiae were required for successful cell lysis. The results are discussed in the context of applications in industrial biotechnology, where metabolomics analyses are important.

SUBMITTER: Rockenbach A

PROVIDER: S-EPMC6835232 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Microfluidic Irreversible Electroporation-A Versatile Tool to Extract Intracellular Contents of Bacteria and Yeast.

Rockenbach Alexander A Sudarsan Suresh S Berens Judith J Kosubek Michael M Lazar Jaroslav J Demling Philipp P Hanke René R Mennicken Philip P Ebert Birgitta E BE Blank Lars M LM Schnakenberg Uwe U

Metabolites 20190930 10

Exploring the dynamic behavior of cellular metabolism requires a standard laboratory method that guarantees rapid sampling and extraction of the cellular content. We propose a versatile sampling technique applicable to cells with different cell wall and cell membrane properties. The technique is based on irreversible electroporation with simultaneous quenching and extraction by using a microfluidic device. By application of electric pulses in the millisecond range, permanent lethal pores are for ...[more]

PMID: 31574935

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Project description:BackgroundHigh-frequency irreversible electroporation (H-FIRE) is a novel, next-generation nanoknife technology with the advantage of relieving irreversible electroporation (IRE)-induced muscle contractions. However, the difference between IRE and H-FIRE with distinct ablation parameters was not clearly defined. This study aimed to compare the efficacy of the two treatments in vivo.MethodsTen Bama miniature swine were divided into two group: five in the 1-day group and five in the 7-day group. The efficacy of IRE and H-FIRE ablation was compared by volume transfer constant (Krans), rate constant (Kep) and extravascular extracellular volume fraction (Ve) value of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), size of the ablation zone, and histologic analysis. Each animal underwent the IRE and H-FIRE. Temperatures of the electrodes were measured during ablation. DCE-MRI images were obtained 1, 4, and 7 days after ablation in the 7-day group. All animals in the two groups were euthanized 1 day or 7 days after ablation, and subsequently, IRE and H-FIRE treated liver tissues were collected for histological examination. Student's t test or Mann-Whitney U test was applied for comparing any two groups. One-way analysis of variance (ANOVA) test and Welch's ANOVA test followed by Holm-Sidak's multiple comparisons test, one-way ANOVA with repeated measures followed by Bonferroni test, or Kruskal-Wallis H test followed by Dunn's multiple comparison test was used for multiple group comparisons and post hoc analyses. Pearson correlation coefficient test was conducted to analyze the relationship between two variables.ResultsHigher Ve was seen in IRE zone than in H-FIRE zone (0.14 ± 0.02 vs. 0.08 ± 0.05, t = 2.408, P = 0.043) on day 4, but no significant difference was seen in Ktrans or Kep between IRE and H-FIRE zones at all time points (all P > 0.05). For IRE zone, the greatest Ktrans was seen on day 7, which was significantly higher than that on day 1 (P = 0.033). The ablation zone size of H-FIRE was significantly larger than IRE 1 day (4.74 ± 0.88 cm2vs. 3.20 ± 0.77 cm2, t = 3.241, P = 0.009) and 4 days (2.22 ± 0.83 cm2vs. 1.30 ± 0.50 cm2, t = 2.343, P = 0.041) after treatment. Apoptotic index (0.05 ± 0.02 vs. 0.73 ± 0.06 vs. 0.68 ± 0.07, F = 241.300, P < 0.001) and heat shock protein 70 (HSP70) (0.03 ± 0.01 vs. 0.46 ± 0.09 vs. and 0.42 ± 0.07, F = 64.490, P < 0.001) were significantly different between the untreated, IRE and H-FIRE zones, but no significant difference was seen in apoptotic index or HSP70 between IRE and H-FIRE zone (both P > 0.05). Electrode temperature variations were not significantly different between the two zones (18.00 ± 3.77°C vs. 16.20 ± 7.45°C, t = 0.682, P = 0.504). The Ktrans value (r = 0.940, P = 0.017) and the Kep value (r = 0.895, P = 0.040) of the H-FIRE zone were positively correlated with the number of hepatocytes in the ablation zone.ConclusionsH-FIRE showed a comparable ablation effect to IRE. DCE-MRI has the potential to monitor the changes of H-FIRE ablation zone.

Dataset Information

Microfluidic Irreversible Electroporation-A Versatile Tool to Extract Intracellular Contents of Bacteria and Yeast.

Publications

Microfluidic Irreversible Electroporation-A Versatile Tool to Extract Intracellular Contents of Bacteria and Yeast.

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