Decreasing the thresholds for electroporation by sensitizing cells with local cationic anesthetics and substances that decrease the surface negative electric charge

Grys, Maciej; Madeja, Zbigniew; Korohoda, W

doi:10.2478/s11658-013-0114-z

Cited by 7 publications

(8 citation statements)

References 36 publications

(49 reference statements)

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“…To preliminarily examine this hypothesis, lidocaine, a known ATPase ion channel inhibitor, was added to the electroporation buffer solutions (KCl and MgCl 2 at 500 μS/cm) at a final concentration of 10 mM [33][34][35] . Electric pulses of different applied energy were delivered, with cell viability assessed at 24 hours.…”

Section: Resultsmentioning

confidence: 99%

The effects of electroporation buffer composition on cell viability and electro-transfection efficiency

Sherba

Hogquist

Lin

et al. 2020

Sci Rep

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Electroporation is an electro-physical, non-viral approach to perform DNA, RNA, and protein transfections of cells. Upon application of an electric field, the cell membrane is compromised, allowing the delivery of exogenous materials into cells. Cell viability and electro-transfection efficiency (eTE) are dependent on various experimental factors, including pulse waveform, vector concentration, cell type/density, and electroporation buffer properties. In this work, the effects of buffer composition on cell viability and eTE were systematically explored for plasmid DNA encoding green fluorescent protein following electroporation of 3T3 fibroblasts. A HEPES-based buffer was used in conjunction with various salts and sugars to modulate conductivity and osmolality, respectively. Pulse applications were chosen to maintain constant applied electrical energy (J) or total charge flux (C/m 2). The energy of the pulse application primarily dictated cell viability, with Mg 2+-based buffers expanding the reversible electroporation range. The enhancement of viability with Mg 2+-based buffers led to the hypothesis that this enhancement is due to ATPase activation via re-establishing ionic homeostasis. We show preliminary evidence for this mechanism by demonstrating that the enhanced viability is eliminated by introducing lidocaine, an ATPase inhibitor. However, Mg 2+ also hinders eTE compared to K +-based buffers. Collectively, the results demonstrate that the rational selection of pulsing conditions and buffer compositions are critical for the design of electroporation protocols to maximize viability and eTE.

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Section: Resultsmentioning

confidence: 99%

The effects of electroporation buffer composition on cell viability and electro-transfection efficiency

Sherba

Hogquist

Lin

et al. 2020

Sci Rep

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“…Procaine and lidocaine also induce a variety of other effects upon cells besides changes in cell shape associated with changes in cytoskeleton organization. Changes in pinocytotic activity, in electric properties of cell surface as studied with cell electrophoresis, in sensitivity to electric fields used for cell electroporation and in cell movement have been described (NICOLSON et al 1976;STURROCK et al 1979;DICKSTEIN et al 1984;JOHNSON & DOWSE 1986;PIERZCHALSKA et al 1998;JOHNSON et al 2002;MICHALIK et al 2003;GRYS et al 2014). All these effects were, however, reversible and cells return faster to the original state than after treatment with trypsin.…”

Section: Resultsmentioning

confidence: 99%

“…Cell viability was tested using the fluorescein diacetate (FDA) and ethydium biomide (Eth Br) tests and a fluorescence microscope (Jenavert, Carl Zeiss Jena) as earlier described (GRYS et al 2014). In some experiments cell viability was additionally determined with the often used trypan blue exclusion test.…”

Section: Cell Viability Determinationmentioning

confidence: 99%

Efficacy of Local Anesthetics in Detachment of Normal 3T3 Mouse Fibroblasts and Prostate Cancer AT-2 Cells from Substrata, in Maintenance of Viable Cells in a Non-Adherent State, and in Preservation of Cell Surface Markers Detected with FlowSight Image Cytometry

Korohoda

Oczkowicz

Rolski

et al. 2015

folia biol (krakow)

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Cells from substrata, in maintenance of viable cells in a non-adherent state, and in preservation of cell surface markers detected with FlowSight image cytometry. Folia Biologica (Kraków) 63: 249-255. The local anesthetics procaine, lidocaine and tetracaine permit the reversible detachment of viable cells and their passaging or preservation in a non-adherent state in the absence of proteolytic enzymes. The effects of these anesthetics, dissolved in various media, on cell viability, cell detachment from substrata and preservation of cells in a non-adherent state, were compared using the AT-2 line of rat prostate carcinoma cells of moderate malignancy and the 3T3 mouse fibroblast cell line. It was found that all three local anesthetics can induce cell rounding followed by detachment of over 95% of viable cells in both lines in Ca 2+ /Mg 2+-free PBS. Tetracaine in 1 mM concentration was the most effective in induction of fast cell detachment. However, procaine and lidocaine in 16 mM concentrations were found to be optimal for preservation of cells in a non-adherent state and for the maintenance of cell viability for at least 2 h. The tested anesthetics also cause cell rounding and detachment when present in various cell culture media but these processes occurred much more slowly and less efficiently than in Ca 2+ /Mg 2+-free PBS. Normal 3T3 mouse fibroblasts after detachment and passaging undertake growth reaching the same saturation density in cultures after detachment with procaine or lidocaine as after passaging using trypsin solution. The results suggest that the application of local anesthetics can be a very simple and effective technique for cell passaging in tissue cultures. This technique might decrease side-effects and cell injury caused by trypsinization or cell scraping. The preservation of cells in suspension in a non-adherent state may facilitate analysis of cell surface properties and fractionation of cell mixtures. Avoiding the use of trypsin allows for the preservation of cell surface proteins ICAM, CXCR4, and HCAM analyzed with FlowSight® image flow cytometry.

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“…All experiments were performed with normal HSFs and 3 cancer cell lines, including human melanoma A375 cells and 2 rat prostate cancer cell lines from the Dunning R-3327 system: Highly malignant Mat-LyLu and moderately malignant AT-2 (1,30,31). The cells were plated in 6-well Falcon culture plates at a density of 20 000 cells per well, 24 h prior to the addition of 9-AAA or 5-FU (Sigma-Aldrich, St. Louis, MO, USA).…”

Section: Cell Culturesmentioning

confidence: 99%

“…Nevertheless, when normal and cancer cells were left to grow in the presence of 9-AAA for 2-5 days, the rat prostate cancer cells and human melanoma cells died, whereas normal human skin fibroblasts (HSFs) continued to grow (1).…”

Section: Introductionmentioning

confidence: 99%

9-AAA inhibits growth and induces apoptosis in human melanoma A375 and rat prostate adenocarcinoma AT-2 and Mat-LyLu cell lines but does not affect the growth and viability of normal fibroblasts

et al. 2016

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Abstract. The present study found that, similarly to 5-f luorouracil, low concentrations (1-10 µM) of 9-aminoacridine (9-AAA) inhibited the growth of the two rat prostate cancer AT-2 and Mat-LyLu cell lines and the human melanoma A375 cell line. However, at the same concentrations, 9-AAA had no effect on the growth and apoptosis of normal human skin fibroblasts (HSFs). The differences between the cellular responses of the AT-2 and Mat-LyLu cell lines, which differ in malignancy, were found to be relatively small compared with the differences between normal HSFs and the cancer cell lines. Visible effects on the cell growth and survival of tumor cell lines were observed after 24-48 h of treatment with 9-AAA, and increased over time. The inhibition of cancer cell growth was found to be due to the gradually increasing number of cells dying by apoptosis, which was observed using two methods, direct counting and FlowSight analysis. Simultaneously, cell motile activity decreased to the same degree in cancer and normal cells within the first 8 h of incubation in the presence of 9-AAA. The results presented in the current study suggest that short-lasting tests for potential anticancer substances can be insufficient; which may result in cell type-dependent differences in the responses of cells to tested compounds that act with a delay being overlooked. The observed differences in responses between normal human fibroblasts and cancer cells to 9-AAA show the requirement for additional studies to be performed simultaneously on differently reacting cancer and normal cells, to determine the molecular mechanisms responsible for these differences.

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Decreasing the thresholds for electroporation by sensitizing cells with local cationic anesthetics and substances that decrease the surface negative electric charge

Cited by 7 publications

References 36 publications

The effects of electroporation buffer composition on cell viability and electro-transfection efficiency

The effects of electroporation buffer composition on cell viability and electro-transfection efficiency

Efficacy of Local Anesthetics in Detachment of Normal 3T3 Mouse Fibroblasts and Prostate Cancer AT-2 Cells from Substrata, in Maintenance of Viable Cells in a Non-Adherent State, and in Preservation of Cell Surface Markers Detected with FlowSight Image Cytometry

9-AAA inhibits growth and induces apoptosis in human melanoma A375 and rat prostate adenocarcinoma AT-2 and Mat-LyLu cell lines but does not affect the growth and viability of normal fibroblasts

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