Real-time monitoring of cytotoxic effects of electroporation on breast and colon cancer cell lines

Cvetković, Danijela; Živanović, Marko; Milutinović, Milena; Djukic, Tijana; Radović, Miloš; Cvetković, Aleksandra; Filipović, Nenad; Zdravković, Nebojša

doi:10.1016/j.bioelechem.2016.10.005

Cited by 20 publications

(19 citation statements)

References 27 publications

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“…Thus, Fig. 2 B provides the rationale that we used AC voltages of 40 kHz for pulsing, as reported earlier 9 , 10 .…”

Section: Resultsmentioning

confidence: 84%

“…Alternatively, real-time techniques have been described, which are mainly based on video-microscopy reporting on the uptake of extracellular material or the release of cytoplasmic molecules 7 . In addition to imaging, electrochemical approaches have been used to monitor membrane integrity of single cells during pulsing 8 , the barrier function of cell layers before and after in situ electroporation 9 , the effect of suspension electroporation on post-pulse cell layer formation 10 or the electrical properties of a tissue 11 . Whereas these techniques were essentially applied to monitor the invasiveness of an electric pulse, none of them was used to record the cell response to the presence of bioactive molecules that had been delivered to the cytoplasm by means of pulsing.…”

Section: Introductionmentioning

confidence: 99%

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Impedance analysis of adherent cells after in situ electroporation-mediated delivery of bioactive proteins, DNA and nanoparticles in µL-volumes

Stolwijk

Wegener

2020

Sci Rep

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Specific intracellular manipulation of animal cells is a persistent goal in experimental cell biology. Such manipulations allow precise and targeted interference with signaling cascades, metabolic pathways, or bi-molecular interactions for subsequent tracking of functional consequences. However, most biomolecules capable of molecular recognition are membrane impermeable. The ability to introduce these molecules into the cytoplasm and then to apply appropriate readouts to monitor the corresponding cell response could prove to be an important research tool. This study describes such an experimental approach combining in situ electroporation (ISE) as a means to efficiently deliver biomolecules to the cytoplasm with an impedance-based, time-resolved analysis of cell status using electric cell-substrate impedance sensing (ECIS). In this approach, gold-film electrodes, deposited on the bottom of regular culture dishes, are used for both electroporation and monitoring. The design of the electrode layout and measurement chamber allows working with sample volumes as small as 10 µL. A miniaturized setup for combined electroporation and impedance sensing (µISE-ECIS) was applied to load different adherent cells with bioactive macromolecules including enzymes, antibodies, nucleic acids and quantum dot nanoparticles. The cell response after loading the cytoplasm with RNase A or cytochrome c (in the presence or absence of caspase inhibitors) was tracked by non-invasive impedance readings in real-time.

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“…Thus, Fig. 2 B provides the rationale that we used AC voltages of 40 kHz for pulsing, as reported earlier 9 , 10 .…”

Section: Resultsmentioning

confidence: 84%

Section: Introductionmentioning

confidence: 99%

Impedance analysis of adherent cells after in situ electroporation-mediated delivery of bioactive proteins, DNA and nanoparticles in µL-volumes

Stolwijk

Wegener

2020

Sci Rep

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“…Further efforts to reduce cytotoxicity have also explored the effect of incorporating chemical modifications on both the Cas9 mRNA 20 as well as the sgRNA, 7,21,22 which has proven to be an effective means to reduce immune detection, improve stability, and reduce cytotoxicity during this process. In addition, electroporation, 23 delivery of exogenous mRNA, 8 and AAV transduction 24 have been shown to also elicit cytotoxicity in a variety of cell lines and primary patientderived cells. It is currently unclear which elements of the protocol are most responsible for this toxicity, much less which specific genes and pathways may be involved in this response.…”

Section: Introductionmentioning

confidence: 99%

Global Transcriptional Response to CRISPR/Cas9-AAV6-Based Genome Editing in CD34+ Hematopoietic Stem and Progenitor Cells

et al. 2018

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Genome-editing technologies are currently being translated to the clinic. However, cellular effects of the editing machinery have yet to be fully elucidated. Here, we performed global microarray-based gene expression measurements on human CD34 hematopoietic stem and progenitor cells that underwent editing. We probed effects of the entire editing process as well as each component individually, including electroporation, Cas9 (mRNA or protein) with chemically modified sgRNA, and AAV6 transduction. We identified differentially expressed genes relative to control treatments, which displayed enrichment for particular biological processes. All editing machinery components elicited immune, stress, and apoptotic responses. Cas9 mRNA invoked the greatest amount of transcriptional change, eliciting a distinct viral response and global transcriptional downregulation, particularly of metabolic and cell cycle processes. Electroporation also induced significant transcriptional change, with notable downregulation of metabolic processes. Surprisingly, AAV6 evoked no detectable viral response. We also found Cas9/sgRNA ribonucleoprotein treatment to be well tolerated, in spite of eliciting a DNA damage signature. Overall, this data establishes a benchmark for cellular tolerance of CRISPR/Cas9-AAV6-based genome editing, ensuring that the clinical protocol is as safe and efficient as possible.

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“…The IRE intensity used in this experiment did not cause any complications, such as burns. However, necrosis is expected to occur instead of apoptosis if higher energy intensities are used [ 24 ].…”

Section: Discussionmentioning

confidence: 99%

Novel Ablation Therapy Using Endoscopic Irreversible Electroporation in the Bile Duct: A Pilot Animal Study

Lee

Choi

et al. 2021

Clin Endosc

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Background/Aims Irreversible electroporation (IRE) is a relatively new ablation method. However, the application of IRE ablation in the treatment of biliary disease has not been attempted. A minimally invasive approach using endoscopic retrograde cholangiopancreatography (ERCP) can be a novel therapeutic modality for IRE ablation. In this study, we aimed to investigate the feasibility of endoscopic IRE for the biliary tract using an animal model. Methods A new catheter-type electrode was developed for endoscopic IRE ablation of the biliary tract. We performed ERCP and endoscopic IRE ablations in the normal common bile duct of Yorkshire pigs. The experimental setting of IRE was 500 V/cm (50 pulses, 100-µs length). The animals were sacrificed after 24 hr, and the ablated bile duct was examined. Results Well-demarcated focal color changes were observed on the mucosa of the common bile duct. The depth of change after IRE was confined to the mucosal and submucosal layers. Apoptotic changes in the bile duct were observed only around the IRE ablation area. Immunohistochemistry assay showed cell death in the bile duct along the electrode. Conclusions Endoscopic IRE ablation using ERCP was successfully performed in the common bile duct. It can be a potential option for the treatment of biliary tumors.

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Real-time monitoring of cytotoxic effects of electroporation on breast and colon cancer cell lines

Cited by 20 publications

References 27 publications

Impedance analysis of adherent cells after in situ electroporation-mediated delivery of bioactive proteins, DNA and nanoparticles in µL-volumes

Impedance analysis of adherent cells after in situ electroporation-mediated delivery of bioactive proteins, DNA and nanoparticles in µL-volumes

Global Transcriptional Response to CRISPR/Cas9-AAV6-Based Genome Editing in CD34+ Hematopoietic Stem and Progenitor Cells

Novel Ablation Therapy Using Endoscopic Irreversible Electroporation in the Bile Duct: A Pilot Animal Study

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