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Diagram of the major components of an electroporator with cuvette loaded.
Dr.Eberhard Neumann - Electroporation Founder

Electroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. It is usually used in molecular biology as a way of introducing some substance into a cell, such as loading it with a molecular probe, a drug that can change the cell's function, or a piece of coding DNA.[1]

Electroporation is a dynamic phenomenon that depends on the local transmembrane voltage at each cell membrane point. 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 0.5 V to 1 V). This leads to the definition of an electric field magnitude threshold for electroporation (Eth). That is, only the cells within areas where E≧Eth are electroporated. If a second threshold (Eir) is reached or surpassed, electroporation will compromise the viability of the cells, i.e., irreversible electroporation.[2]

In molecular biology, the process of electroporation is often used for the transformation of bacteria, yeast, and plant protoplasts. In addition to the lipid membranes, bacteria also have cell walls which are different from the lipid membranes and are made of peptidoglycan and its derivatives. However, the walls are naturally porous and only act as stiff shells that protect bacteria from severe environmental impacts. If bacteria and plasmids are mixed together, the plasmids can be transferred into the cell after electroporation. Several hundred volts across a distance of several millimeters are typically used in this process. Afterwards, the cells have to be handled carefully until they have had a chance to divide producing new cells that contain reproduced plasmids. This process is approximately ten times as effective as chemical transformation.[1][3]

This procedure is also highly efficient for the introduction of foreign genes in tissue culture cells, especially mammalian cells. For example, it is used in the process of producing knockout mice, as well as in tumor treatment, gene therapy, and cell-based therapy. The process of introducing foreign DNAs into eukaryotic cells is known as transfection.

Contents

Laboratory Practice

Cuvettes for electroporation. These are plastic with aluminium electrodes and a blue lid. They hold a maximum of 400 μl.

Electroporation is done with electroporators, appliances which create an electro-magnetic field in the cell solution. The cell suspension is pipetted into a glass or plastic cuvette which has two aluminum electrodes on its sides.

For bacterial electroporation, typically a suspension of around 50 microliters is used. Prior to electroporation it is mixed with the plasmid to be transformed. The mixture is pipetted into the cuvette, the voltage and capacitance is set and the cuvette inserted into the electroporator. Immediately after electroporation, one milliliter of liquid medium is added to the bacteria (in the cuvette or in an eppendorf tube), and the tube is incubated at the bacteria's optimal temperature for an hour or more to allow recovery of the cells and expression of antibiotic resistance, followed by spreading on agar plates.

The success of the elecroporation depends greatly on the purity of the plasmid solution, especially on its salt content. Solutions with high salt concentrations might cause an electrical discharge (known as arcing), which often reduces the viability of the bacteria.

For a further detailed investigation of the process more attention should be paid to the output impedance of the porator device and the input impedance of the cells suspension (e.g. salt content). As the process needs direct electrical contact between the electrodes and the suspension, and is inoperable with isolated electrodes, obviously the process involves certain electrolytic effects, due to small currents and not only fields.

The Electroporators

Electroporators come in two flavors - hand-held and bench-tops.

Benchtop electroporators are generally used as common lab equipments, residing atop a central bench or hood. They offer the advantage of electroporating multiple samples at the same time. They can also be set to different operating parameters depending on whether the cell has a cell-wall or not. Unlike them, the handheld electroporators are cordless, rechargeable and use disposable pipectrodes, which combine elements of both cuvettes and pipettes. It's operating parameters are pre-set to the optimal parameters for transforming either bacteria or mammalian cells.

Both types of electoporators have been used on a wide range of cells - including E. coli (for transformation) and mammalian cells such as neurons, astrocytes, neuroglia, lymphocytes, monocytes, fibroblasts, epithelial and endothelial cells from humans, mice, rats and monkeys (for transfection).

Medical Applications

A higher voltage of electroporation was found in pigs to irreversibly destroy target cells within a narrow range while leaving neighboring cells unnaffected, and thus represents a promising new treatment for cancer, heart disease and other disease states that require removal of tissue.[4]

Electroporation can also be used to help deliver drugs or genes into the cell by applying of short and intense electric pulses that transiently permeabilize cell membrane, thus allowing transport of molecules otherwise not transported through a cellular membrane. This proceedure is referred to as electrochemotherapy when the molecules to be transported is a chemotherapeutic agent or gene electrotransfer when the molecule to be transported is DNA.

Physical Mechanism

Electroporation allows cellular introduction of large highly charged molecules such as DNA which would never passively diffuse across the hydrophobic bilayer core.[1] This phenomenon indicates that the mechanism is the creation of nm-scale water-filled holes in the membrane. Although electroporation and dielectric breakdown both result from application of an electric field, the mechanisms involved are fundamentally different. 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.

Schematic showing the theoretical arrangement of lipids in a hydrophobic pore (top) and a hydrophilic pore (bottom).

Electroporation is a multi-step process with several distinct phases.[5] First, a short electrical pulse must be applied. Typical parameters would be 300-400 mV for < 1 ms across the membrane (note- the voltages used in cell experiments are typically much larger because they are being applied across large distances to the bulk solution so the resulting field across the actual membrane is only a small fraction of the applied bias). 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. Evidence for the existence of such pre-pores comes mostly from the “flickering” of pores, which suggests a transition between conductive and insulating states.[6] It has been suggested that these pre-pores are small (~3 Å) hydrophobic defects. 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[7] which in turn depends on the applied field, local mechanical stress and bilayer edge energy.

References

  1. ^ a b c Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH (1982). "Gene transfer into mouse lyoma cells by electroporation in high electric fields". Embo J. 1 (7): 841–5. PMID 6329708. 
  2. ^ Antoni Ivorra, Boris Rubinsky. "Gels with predetermined conductivity used in electroporation of tissue USPTO Applicaton #: 20080214986 - Class: 604 21 (USPTO)". http://www.freshpatents.com/Gels-with-predetermined-conductivity-used-in-electroporation-of-tissue-dt20080904ptan20080214986.php. 
  3. ^ Sugar IP, Neumann E (1984). "Stochastic model for electric field-induced membrane pores. Electroporation". Biophys. Chem. 19 (3): 211–25. doi:10.1016/0301-4622(84)87003-9. PMID 6722274. 
  4. ^ Sarah Yang (2007-02-12). "New medical technique punches holes in cells, could treat tumors". http://www.berkeley.edu/news/media/releases/2007/02/12_IRE.shtml. Retrieved 2007-12-13. 
  5. ^ J. C. Weaver and Y. A. Chizmadzhev."Theory of electroporation: A review " Biochemistry and Bioenergetics. 41. (1996) 135-160.
  6. ^ K. C. Melikov, V. A. Frolov, A. Shcherbakov, A. V. Samsonov, Y. A. Chizmadzhev and L. V. Chernomordik."Voltage-Induced Nonconductive Pre-Pores and Metastable Single Pores in Unmodified Planar Lipid Bilayer " Biophysical Journal. 80. (2001) 1829-1836.
  7. ^ R. P. Joshi and K. H. Schoenbach."Electroporation dynamics in biological cells subjected to ultrafast electrical pulses: A numerical simulation study." Physical Review E. 62. (2000) 1025-1033.

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