Reversible and irreversible electroporation of cell suspensions flowing through a localized DC electric field

Experiments on reversible and irreversible cell electroporation were carried out with an experimental setup based on a standard apparatus for horizontal electrophoresis, a syringe pump with regulated cell suspension flow velocity and a dcEF power supply. Cells in suspension flowing through an orifice in a barrier inserted into the electrophoresis apparatus were exposed to defined localized dcEFs in the range of 0-1000 V/cm for a selected duration in the range 10-1000 ms. This method permitted the determination of the viability of irreversibly electroperforated cells. It also showed that the uptake by reversibly electroperforated cells of fluorescent dyes (calcein, carboxyfluorescein, Alexa Fluor 488 Phalloidin), which otherwise do not penetrate cell membranes, was dependent upon the dcEF strength and duration in any given single electrical field exposure. The method yields reproducible results, makes it easy to load large volumes of cell suspensions with membrane non-penetrating substances, and permits the elimination of irreversibly electroporated cells of diameter greater than desired. The results concur with and elaborate on those in earlier reports on cell electroporation in commercially available electroporators. They proved once more that the observed cell perforation does not depend upon the thermal effects of the electric current upon cells. In addition, the method eliminates many of the limitations of commercial electroporators and disposable electroporation chambers. It permits the optimization of conditions in which reversible and irreversible electroporation are separated. Over 90% of reversibly electroporated cells remain viable after one short (less than 400 ms) exposure to the localized dcEF. Experiments were conducted with the AT-2 cancer prostate cell line, human skin fibroblasts and human red blood cells, but they could be run with suspensions of any cell type. It is postulated that the described method could be useful for many purposes in biotechnology and biomedicine and could help optimize conditions for in vivo use of both reversible and irreversible electroporation

Avoiding the side effects of electric current pulse application to electroporated cells in disposable small volume cuvettes assures good cell survival

Abstract Background The harmful side effects of electroporation to cells due to local changes in pH, the appearance of toxic electrode products, temperature increase, and the heterogeneity of the electric field acting on cells in the cuvettes used for electroporation were observed and discussed in several laboratories. If cells are subjected to weak electric fields for prolonged periods, for example in experiments on cell electrophoresis or galvanotaxis the same effects are seen. In these experiments investigators managed to reduce or eliminate the harmful side effects of electric current application. Methods For the experiments, disposable 20\ua0\u3bcl cuvettes with two walls made of dialysis membranes were constructed and placed in a locally focused electric field at a considerable distance from the electrodes. Cuvettes were mounted into an apparatus for horizontal electrophoresis and the cells were subjected to direct current electric field (dcEF) pulses from a commercial pulse generator of exponentially declining pulses and from a custom-made generator of double and single rectangular pulses. Results More than 80% of the electroporated cells survived the dcEF pulses in both systems. Side effects related to electrodes were eliminated in both the flow through the dcEF and in the disposable cuvettes placed in the focused dcEFs. With a disposable cuvette system, we also confirmed the sensitization of cells to a dcEF using procaine by observing the loading of AT2 cells with calceine and using a square pulse generator, applying 50\ua0ms single rectangular pulses. Conclusions We suggest that the same methods of avoiding the side effects of electric current pulse application as in cell electrophoresis and galvanotaxis should also be used for electroporation. This conclusion was confirmed in our electroporation experiments performed in conditions assuring survival of over 80% of the electroporated cells. If the amplitude, duration, and shape of the dcEF pulse are known, then electroporation does not depend on the type of pulse generator. This knowledge of the characteristics of the pulse assures reproducibility of electroporation experiments using different equipment

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

The recently described method of cell electroporation by flow of cell suspension through localized direct current electric fields (dcEFs) was applied to identify non-toxic substances that could sensitize cells to external electric fields. We found that local cationic anesthetics such as procaine, lidocaine and tetracaine greatly facilitated the electroporation of AT2 rat prostate carcinoma cells and human skin fibroblasts (HSF). This manifested as a 50% reduction in the strength of the electric field required to induce cell death by irreversible electroporation or to introduce fluorescent dyes such as calcein, carboxyfluorescein or Lucifer yellow into the cells. A similar decrease in the electric field thresholds for irreversible and reversible cell electroporation was observed when the cells were exposed to the electric field in the presence of the non-toxic cationic dyes 9-aminoacridine (9-AAA) or toluidine blue. Identifying non-toxic, reversibly acting cell sensitizers may facilitate cancer tissue ablation and help introduce therapeutic or diagnostic substances into the cells and tissue

Recommendations on the use of innovative medical technologies in cardiology and cardiac surgery and solutions leading to increased availability for Polish patients

There is a great need for innovative technologies that will improve the health and quality of life (QoL) of Polish patients with cardiac problems. It is important that the safety and effectiveness of the technology are confirmed by scientific evidence on which guidelines and clinical recommendations are based. Scientific evidence for medical devices is also increasingly important for decision-making in finance approval from public funds. New technologies in cardiology and cardiac surgery contribute to improved patient QoL, increased treatment effectiveness and facilitated diagnosis. Hence, it is necessary to increase accessibility to such technologies, primarily through the development of clinical recommendations, and education of medical personnel in conjunction with public funding. The aim of this publication is to present the recommendations of leading experts in the field of cardiology and cardiosurgery, supported by clinical research results, regarding the use of the cited innovative medical technologies and solutions leading to their increased availability for Polish patients.

Heart failure in Poland: Left ventricular assist device destination therapy and other challenges of interventional cardiology and cardiac surgery

Patients with severe heart failure (HF), who are not eligible for cardiac transplantation and receive optimal medical management, based mainly on the use of pharmacological treatment and devices such as resynchronization therapy (implantable cardioverter-defibrillator), achieve poor clinical outcomes and constitute a group with extremely poor prognosis. Currently, the technology used in the latest generation left ventricular assist devices (LVADs), such as the HeartMate 3, makes it possible to achieve patient survival at the level obtained by patients after heart transplantation, and they can be used not only in patients eligible for heart transplantation as a bridge to transplant, but also in those with significantly worse prognosis, who are ineligible for heart transplantation as destination therapy. The objective of this publication is to present recommendations from experts in cardiology and cardiac surgery, supported by clinical trial results, on the use of LVADs as a destination therapy in HF patients who are not eligible for cardiac transplantation. The paper also presents the issue of cardiac transplantation and extracorporeal membrane oxygenation therapy in Poland, as well as current challenges faced by interventional cardiology and cardiac surgery in Poland