Introduction: Prostate, pancreatic and liver cancers are a major cause of death in Europe and including Bulgaria. Some methods for treatment include the use of electric current to create pores in the ...cells' membranes, and can be used in combination with other techniques, while an electric field with enough strength causes irreversible electroporation (IRE) and is a separate technique. Material and methods:Search was conducted for scientific articles about electroporation and IRE. Results: Most important parameters reported in scientific articles are electric field distribution, tissue characteristics and interaction, electric pulse settings. Articles that report patient outcomes suggest several possible advantages of – retaining urinary and sexual functions, possible increase of overall survival rate, low rate of serious adverse events and promising results when applied with chemotherapy. However, there are some studies that do not corroborate these results. The technique is also used for the treatment of renal cancer, and there are researches indicating a potential for use in ovarian, cervical and breast cancer. Conclusion: Studies suggest the IRE method is safe and feasible for the treatment of prostate cancer, pancreatic cancer and liver cancer, but improvements in the protocols is needed to prevent a decrease of quality of life.
Introduction: Re-Cellularization via Electroporation Therapy (ReCET™), a novel endoscopic procedure using electroporation to elicit apoptosis of the duodenal mucosa. In this study we aimed to ...eliminate insulin in T2D patients using a single ReCET procedure combined with GLP-1RA. Safety, feasibility, and efficacy of ReCET using the Endogenex system were assessed. Methods: First in human study with 14 T2D patients on basal insulin (BMI 24-40 kg/m2; HbA1c ≤ 8.0%; C-peptide ≥ 0.2 nmol/l). All underwent ReCET after which semaglutide was titrated up to 1mg. Primary endpoints were: feasibility (technical success rate, procedure time catheter in - out, % GLP-1RA tolerance); safety ((S)AE, hypoglycemic events); efficacy (off insulin at 6 mo; HbA1c ≤ 7.5%). Results: ReCET showed 100% technical success rate. Procedure time was 58 (IQR 49-79) min. Maximum semaglutide dosage was tolerated by 13 patients. No device related SAEs were observed. One non-severe hypoglycemic event occurred. At 6 mo, 12 patients were off insulin. A significant improvement in several glycemic and metabolic parameters was observed (Table 1); HbA1c decreased (7.2 to 6.6% p=0.003) and TIR increased (72 to 91% p=0.022). Conclusion: ReCET is safe and feasible. ReCET combined with semaglutide may effectively eliminate insulin in selected T2D patients, while improving glycemic control and metabolic health. Disclosure C.B.E.Busch: None. S.Meiring: None. A.C.G.Van baar: None. F.Holleman: None. M.Nieuwdorp: Advisory Panel; caelus health. J.J.Bergman: Consultant; Endogenex, Fractyl Health, Inc., Digma Medical, Research Support; Endogenex, Fractyl Health, Inc., Digma Medical.
High-frequency irreversible electroporation (H-FIRE) is an emerging electroporation-based therapy used to ablate cancerous tissue. Treatment consists of delivering short, bipolar pulses (1–10μs) in a ...series of 80–100 bursts (1 burst/s, 100μs on-time). Reducing pulse duration leads to reduced treatment volumes compared to traditional IRE, therefore larger voltages must be applied to generate ablations comparable in size. We show that adjuvant calcium enhances ablation area in vitro for H-FIRE treatments of several pulse durations (1, 2, 5, 10μs). Furthermore, H-FIRE treatment using 10μs pulses delivered with 1mM CaCl2 results in cell death thresholds (771±129V/cm) comparable to IRE thresholds without calcium (698±103V/cm). Quantifying the reversible electroporation threshold revealed that CaCl2 enhances the permeabilization of cells compared to a NaCl control. Gene expression analysis determined that CaCl2 upregulates expression of eIFB5 and 60S ribosomal subunit genes while downregulating NOX1/4, leading to increased signaling in pathways that may cause necroptosis. The opposite was found for control treatment without CaCl2 suggesting cells experience an increase in pro survival signaling. Our study is the first to identify key genes and signaling pathways responsible for differences in cell response to H-FIRE treatment with and without calcium.
•CaCl2 enhances ablation volume for H-FIRE treatment of varying pulse widths.•CaCl2 enhances permeabilization of cells compared to a NaCl control.•CaCl2 upregulates expression of eIFB5 and 60s ribosomal subunit genes.•CaCl2 treatment leads to increased signaling in pathways that may cause necroptosis.•Treatment without CaCl2 upregulates expression of NOX1/4 genes.•Treatment without CaCl2 leads to an increase in pro survival signaling.
Electroporation of zygotes represents a rapid alternative to the elaborate pronuclear injection procedure for CRISPR/Cas9-mediated genome editing in mice. However, current protocols for ...electroporation either require the investment in specialized electroporators or corrosive pre-treatment of zygotes which compromises embryo viability. Here, we describe an easily adaptable approach for the introduction of specific mutations in C57BL/6 mice by electroporation of intact zygotes using a common electroporator with synthetic CRISPR/Cas9 components and minimal technical requirement. Direct comparison to conventional pronuclear injection demonstrates significantly reduced physical damage and thus improved embryo development with successful genome editing in up to 100% of living offspring. Hence, our novel approach for Easy Electroporation of Zygotes (EEZy) allows highly efficient generation of CRISPR/Cas9 transgenic mice while reducing the numbers of animals required.
Pulsed-field ablation (PFA) is an emerging non-thermal ablation method based on the biophysical phenomenon of electroporation. Data on PFA cardiac selectivity nature and tissue-specific thresholds ...are lacking. We aim to compare the in vivo differential effect of high-frequency irreversible electroporation (HF-IRE) protocols on various tissues.
Twenty-three Sprague-Dawle rodents were allocated into three different protocols of 300, 600, and 900 V, respectively, while delivering twenty 100 µs bursts of a 150 kHz biphasic square wave to five tissues; cardiac muscle, skeletal muscle, liver, carotid artery and sciatic nerve. Lesions were evaluated quantitatively by histologic analysis and by morphometric evaluation. There were eight, seven and eight animals in the 300, 600, and 900 V protocols, respectively. High-frequency electroporation protocols showed a graded effect on myocardial tissue with larger lesions in the 900 V protocol compared with the other two protocols as demonstrated by width (P = 0.02), length (P = 0.01) and fibrosis ratio (P = 0.001). This effect was not observed for other tissues with attenuated degree of damage. No damage to the carotid artery was observed in all protocols. Partial damage to the sciatic nerve was observed in only two samples (25%) in the 600 V group and in one sample (14.3%) in the 900 V group.
Electroporation effect is tissue-specific such that myocardium is more prone to electroporation damage compared with neural and vascular tissues. Our results suggest no neural or vascular damage with using a low-amplitude HF-IRE protocol. Further investigation is warranted to better identify other tissue-specific thresholds.
Exposing cells to an electric field leads to electroporation of the cell membrane which has already been explored and used in a number of applications in medicine and food biotechnology (e.g. ...electrochemotherapy, gene electrotransfer, extraction of biomolecules). The extent of electroporation depends on several conditions, including pulse parameters, types of cells and tissues, surrounding media, temperature etc. Each application requires a specific level of electroporation, so it must be explored in advance by employing methods for detecting electroporation. Electroporation detection is most often done by measuring increased transport of molecules across the membrane, into or out of the cell. We review here various methods of electroporation detection, together with their advantages and disadvantages. Electroporation detection can be carried out by using dyes (fluorophores or colour stains) or functional molecules, by measuring the efflux of biomolecules, by impedance measurements and voltage clamp techniques as well as by monitoring cell swelling. This review describes methods of detecting cell membrane electroporation in order to help researchers choose the most suitable ones for their specific experiments, considering available equipment and experimental conditions.
•We review here different methods for electroporation detection.•The advantages and disadvantages of the methods are also described.•Each detection method can serve to specific experimental conditions and equipment.
Transfection of cells to alter their genotype or phenotype is crucial in a variety of life science applications. A diverse array of transfection methods are available, and selecting the best approach ...often depends on compatibility with the specific application. Electroporation is a physical transfection strategy that uses an electrical pulse to create temporary pores in cell membranes through which nucleic acids or proteins can pass into cells. It is a highly efficient and powerful tool that has been shown to have superior performance with gene editing-based payloads, such as CRISPR and TALEN. Thermo Fisher offers the research scale Neon NxT Electroporation System with either 10uL or 100uL kit and the GMP-compliant Xenon Electroporation System with either the 1mL SingleShot or the 5-25 mL MultiShot consumable. Both instruments share the same core technology. In this study, we have demonstrated that these platforms are highly flexible and show compatibility with a wide range of mammalian cell types with different payloads. Moreover, we have demonstrated the scalability of electroporation conditions between the two instruments. Gene editing conditions on the Neon NxT Electroporation System can be scaled up to the CTSTM XenonTM Electroporation System, which is a platform designed for GMP-compliant cell therapy manufacturing.
HSCs were meticulously isolated from mobilized leukopak through positive selection. The electroporation parameters, payload concentration, culture conditions, culture period, payload type, and knock-out locus were systematically fine-tuned in the small-scale setting to identify the optimal conditions for genome editing. A minimum of three donors were employed to ascertain the knock-out/knock-in efficiency at the B2M locus. Subsequently, we leveraged the insights gained from Neon NxT optimization to scale up the process using large-scale xenon electroporation. Remarkably, the identified conditions seamlessly translated from Neon NxT to Xenon, exhibiting up to 90% knock-out efficiency and 10-20% knock-in efficiency in Xenon, with negligible impact on cell viability. These findings highlight the successful scalability and robustness of the optimized workflow across the two electroporation systems; enabling a smooth transition from bench to manufacturing for clinical application developments.
CAR-T cell therapies have emerged as a revolutionary approach in the field of haematological malignancies, offering unprecedented clinical efficacy when patients have exhausted any other treatment ...option. Six CAR-T products are available worldwide, but many of the manufacturing challenges are yet to be addressed. A significant fraction of the cost is related to manufacturing the viral vector required to introduce the clinically relevant CAR construct. As such, non-viral gene delivery platforms such as electroporation or mechanoporation can potentially decrease the costs of manufacturing associated with CAR-T products. This study focused on establishing a scalable manufacturing method for CAR-T cell therapies using electroporation and a transposon-transposase system employed to ensure stable integration of the CAR transgene.
Several stages were undertaken to establish the scalable manufacturing process: (1) electroporation optimisation, (2) identification of suitable cell viability recovery strategy, (3) small-scale expansion studies followed by (4) scalable expansion (ongoing). The workflow used featured a screening across electroporation conditions, followed by a study focused on changing the feeding strategy to maximize cell viability recovery post-electroporation. Once these were identified, the non-viral CAR-T product was characterized using growth rate, metabolic profile, CD4:CD8 ratios, CD8 subsets and CAR expression. This work was conducted using cryopreserved CD3+ cells, RPMI supplemented with 10% FBS, 2 mM of L-glutamine and 50 IU/mL of IL-2 as expansion medium and TransAct as activation method.
Here, suitable electroporation programs that minimised cell viability loss after electroporation were identified. In addition, a DNA-to-cell ratio study demonstrated that 1 µg of each minicircle per million cells transfected is sufficient to generate CD3+ populations with viability levels above 90% with CAR expression levels reaching 30%, 48 hours past electroporation. On harvesting day, our optimized process led to a CAR-T product with a CD4:CD8 ratio of 3, with 95% of the CD8 in the central memory phenotype. CAR expression levels were shown to be above 25% on harvesting day. A total of 20-fold increase was reported during the 7-day expansion period. The next stage of this study will include the integration of the electroporation with stirred tank bioreactors to demonstrate the feasibility of modular manufacturing in the non-viral CAR-T space.
Nonthermal irreversible electroporation is a new tissue ablation technique that consists of applying pulsed electric fields across cells to induce cell death by creating permanent defects in the cell ...membrane. Nonthermal irreversible electroporation is of interest because it allows treatment near sensitive tissue structures such as blood vessels and nerves. Two recent articles report that electrolytic reaction products at electrodes can be combined with electroporation pulses to augment and optimize tissue ablation. Those articles triggered a concern that the results of earlier studies on nonthermal irreversible electroporation may have been tainted by unaccounted for electrolytic effects. The goal of this study was to reexamine previous studies on nonthermal irreversible electroporation in the context of these articles. The study shows that the results from some of the earlier studies on nonthermal irreversible electroporation were affected by unaccounted for electrolysis, in particular the research with cells in cuvettes. It also shows that tissue ablation ascribed in the past to irreversible electroporation is actually caused by at least 3 different cytotoxic effects: irreversible electroporation without electrolysis, irreversible electroporation combined with electrolysis, and reversible electroporation combined with electrolysis. These different mechanisms may affect cell and tissue ablation in different ways, and the effects may depend on various clinical parameters such as the polarity of the electrodes, the charge delivered (voltage, number, and length of pulses), and the distance of the target tissue from the electrodes. Current clinical protocols employ ever-increasing numbers of electroporation pulses to values that are now an order of magnitude larger than those used in our first fundamental nonthermal irreversible electroporation studies in tissues. The different mechanisms of cell death, and the effect of the clinical parameters on the mechanisms may explain discrepancies between results of different clinical studies and should be taken into consideration in the design of optimal electroporation ablation protocols.
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