Guide Electroporation and Electrofusion in Cell Biology

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Inevitably, the basic questions and the practical applications will not keep in step. The questions are intrinsically tough. It is hard enough to analyze the action of the relatively weak fields that rotate or align cells, but it is nearly impossible to predict responses to the cell-shredding bursts of electricity that cause them to fuse or to open up to very large molecular assemblies. Even so, theoretical studies and systematic examination of model systems have produced some creditable results, ideas which should ultimately provide hints of what to try next.

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Cell electrofusion using nanosecond electric pulses

Bloggat om Electroporation and Electrofusion in Cell Proteins, the extracellular matrix, and critical structures such as blood vessels and nerves are all unaffected and left healthy by this treatment. This allows for a quicker recovery, and facilitates a more rapid replacement of dead tumor cells with healthy cells.

Before doing the procedure, scientists must carefully calculate exactly what needs to be done, and treat each patient on an individual case-by-case basis. From this information, they can approximate the volume of the tumor and decide on the best course of action including the insertion site of electrodes, the angle they are inserted in, the voltage needed, and more, using software technology.

Often, a CT machine will be used to help with the placement of electrodes during the procedure, particularly when the electrodes are being used to treat tumors in the brain. The entire procedure is very quick, typically taking about five minutes. The success rate of these procedures is high and is very promising for future treatment in humans. One disadvantage to using N-TIRE is that the electricity delivered from the electrodes can stimulate muscle cells to contract, which could have lethal consequences depending on the situation.

Therefore, a paralytic agent must be used when performing the procedure. The paralytic agents that have been used in such research are successful; however, there is always some risk, albeit slight, when using anesthetics. A more recent technique has been developed called high-frequency irreversible electroporation H-FIRE. This technique uses electrodes to apply bipolar bursts of electricity at a high frequency, as opposed to unipolar bursts of electricity at a low frequency. However, it has one distinct advantage, H-FIRE does not cause muscle contraction in the patient and therefore there is no need for a paralytic agent.

Electroporation can also be used to help deliver drugs or genes into the cell by applying short and intense electric pulses that transiently permeabilize cell membrane, thus allowing transport of molecules otherwise not transported through a cellular membrane.

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This procedure is referred to as electrochemotherapy when the molecules to be transported are chemotherapeutic agents or gene electrotransfer when the molecule to be transported is DNA. Scientists from Karolinska Institutet and the University of Oxford use electroporation of exosomes to deliver siRNAs, antisense oligonucleotides, chemotherapeutic agents and proteins specifically to neurons after inject them systemically in blood.

Because these exosomes are able to cross the blood brain barrier , this protocol could solve the problem of poor delivery of medications to the central nervous system, and potentially treat Alzheimer's , Parkinson's Disease and brain cancer , among other conditions. Bacterial transformation is generally the easiest way to make large amounts of a particular protein needed for biotechnology purposes or in medicine.

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Since gene electrotransfer is very simple, rapid and highly effective technique it first became very convenient replacement for other transformation procedures. Electroporation allows cellular introduction of large highly charged molecules such as DNA which would never passively diffuse across the hydrophobic bilayer core. In dielectric breakdown the barrier material is ionized, creating a conductive pathway.

The material alteration is thus chemical in nature. In contrast, during electroporation the lipid molecules are not chemically altered but simply shift position, opening up a pore which acts as the conductive pathway through the bilayer as it is filled with water.

Electroporation is a dynamic phenomenon that depends on the local transmembrane voltage at each point on the cell membrane. It is generally accepted that for a given pulse duration and shape, a specific transmembrane voltage threshold exists for the manifestation of the electroporation phenomenon from 0. This leads to the definition of an electric field magnitude threshold for electroporation E th.

- Electroporation and Electrofusion in Cell Biology by C.A. JORDAN

If a second threshold E ir is reached or surpassed, electroporation will compromise the viability of the cells, i. Electroporation is a multi-step process with several distinct phases.

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Upon application of this potential the membrane charges like a capacitor through the migration of ions from the surrounding solution. Once the critical field is achieved there is a rapid localized rearrangement in lipid morphology. The resulting structure is believed to be a "pre-pore" since it is not electrically conductive but leads rapidly to the creation of a conductive pore. If this theory is correct, then the transition to a conductive state could be explained by a rearrangement at the pore edge, in which the lipid heads fold over to create a hydrophilic interface.

Finally, these conductive pores can either heal, resealing the bilayer or expand, eventually rupturing it. The resultant fate depends on whether the critical defect size was exceeded [42] which in turn depends on the applied field, local mechanical stress and bilayer edge energy.

Application of electric pulses of sufficient strength to the cell causes an increase in the trans-membrane potential difference, which provokes the membrane destabilization. Cell membrane permeability is increased and otherwise nonpermeant molecules enter the cell. It was in the s that there were publications reporting that by applying an external electric field, a large membrane potential at the two pole of a cell can be created. Then in s, several groups, including Crowley, Zimmermann, Neumann reported that when this membrane potential reached a critical level, the membrane will breakdown.

By the end of s and the beginning of s, people found that when applying the electric pulse short enough, the "pores" created on the cell membrane can be resealed, and the cell can recover and survive. From Wikipedia, the free encyclopedia. Main article: Irreversible electroporation. Further information: Lipid bilayer mechanics. KGaA, doi : Biophysical Chemistry. February Nature Protocols.

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    Issue 41 doi: Trontelj, K. Cell Electrofusion Visualized with Fluorescence Microscopy. Figure 1. The ability of cell membranes to fuse non-specifically, e. Such nonspecific fusion enables production of highly valuable hybrid cells and their products, such as monoclonal antibodies, and provides information about fundamental mechanisms of fusion [2]. Electrofusion is a potentially very effective method since it can be properly adjusted to different types of cells. Electrofusion is achieved when cells in close physical contact are brought into their fusogenic state prone to fusion by means of high-voltage electric pulses.

    The efficiency of electrofusion depends on various parameters that affect two parts of the electrofusion process. First part of the electrofusion process is achievement of the close physical contact between cells, which can be obtained with different methods []. Adherence method growing cells to confluence can be used efficiently due to spontaneously established cell contacts in large zones between cells; however, it produces very large fused cells with many nuclei.

    Cell Electrofusion Visualized with Fluorescence Microscopy

    We are using the modified adherence method, where smaller cells with 2 to 5 nuclei , which are more likely to survive and proliferate, are obtained Figure 1. Contact between cells also benefit from osmotic swelling of cells, due to osmotic treatment used in the experiment [9]. Second part of the electrofusion process is the achievement of the fusogenic state of the cell membranes. Fusogenic state correlates well with electropermeabilized state of the membrane cells are non-specifically permeabilized to molecules that normally cannot pass through intact membrane and is governed by the same parameters of the electric pulses amplitude, length, number and frequency [10].

    The values of electrical parameters needed for optimal electroporation [1] and electrofusion differ between different cells and depend on cells size and their biological properties. Electrical parameters thus need to be optimized for different cell lines, which are used as fusion partners, to obtain fusion. This video represents supplementary material for the "Electroporation-based Technologies and Treatments" scientific workshop and postgraduate course, organized by the Faculty of Electrical Engineering at the University of Ljubljana, Slovenia.

    You must be signed in to post a comment. Please sign in or create an account. Hi, my name is Zaza Begiashvili, from Georgia, Tbilisi. I have good idea about using hybridomas for lysis of cancer's cells, but we haven't in my country technology and apparatus for electrofusion. This content is Open Access. A verification has been sent to.