An Introduction to Electroporation – A Tool for Transfection and Competent Cell Generation

Electroporation, also called electropermeabilization, is an efficient, non-viral delivery system that allows genetic material (DNA and RNA), proteins, drugs or other molecules to enter cells. It uses an accurately pulsed electrical current to create temporary pores in the cell membrane through which the molecules can then pass. This process can be used on a wide variety of cells including mammalian,¹ insect,² yeast,³ plant⁴ and bacterial cells.⁵

Once inside, drugs and other molecules may act on the cell. Genetic material may remain independent (as a plasmid) or become integrated into the host genome depending on the downstream experimental steps. While electroporation, first described in 1972⁶ but not named until 1982,⁷ has been used as an in vitro tool for many years, in 1991, the first in vivo electroporation⁸ was used to deliver genetic material and it is now being investigated for use to deliver gene and cell therapies for a range of conditions. Since then, techniques have also been developed to deliver electroporation in utero in mice⁹ and other experimental animals and in ovo in chickens and snakes.¹⁰

How does electroporation work? The electroporation machine and electroporation cuvettes

When a cell is able to take up free DNA or RNA, it is termed “competent”. Some cell types or microbial species, such as Bacillus subtilis,¹¹ are naturally competent and they can be induced to uptake DNA simply by switching nutrient concentration to induce stress. Many cells, however, are not naturally competent or may only uptake certain material, such as antimicrobial resistance cassettes, under specific conditions that are complex to reproduce in the laboratory. Chemicals can be used to induce competence in some species, such as Escherichia coli (E. coli),¹² but again, this does not work for all cell types or microbial species. Instead, the process of electroporation can make non-competent cells competent and amenable to experimental manipulation.

For in vitro electroporation, a suspension of target cells is mixed in a conductive solution with the molecule you wish to introduce into the cells and placed in a cuvette (Figure 1). Electroporation cuvettes have metal plates on either side of the sample chamber that allow an electrical current to be passed through the mixture. The cuvette is placed into a chamber of the electroporator that has corresponding electrical contacts, enabling the cuvette to form an electrical circuit. Controls on the electroporation machine allow the user to set the voltage, waveform and duration of the electrical pulse to be delivered, which should be optimized to the target cell type. An electrical pulse is then passed through the sample chamber.

Figure 1: A diagram of the main components of an electroporator with cuvette loaded. Credit: Richard Wheeler, reproduced under the GNU Free Documentation license.

The electrical pulse disturbs the phospholipid bilayer of the cell membrane,¹³ causing pores to be formed (Figure 2). This occurs asymmetrically with pores forming first on the anode side of the cell. At the same time, the electrical potential across the membrane is increased. This drives charged molecules, such as DNA, to adsorb to the membrane, first on the cathode side of the cell, and move through these pores and into the cell¹⁴ (Figure 3). The cell wall, present in plant cells and some bacteria, is a major barrier for the movement of macromolecules into cells. Consequently, protoplasts (cells from which the cell wall has been removed) are typically used for electroporation,^{4, 15} although methods for electroporation of cells with the wall intact have been developed.¹⁶

Figure 2: Schematic diagram showing disruption of the cell membrane and pore formation during electroporation. Credit: Technology Networks.

After the electrical pulse has passed, the pores begin to reseal.¹⁷ The rate¹⁸ at which this occurs varies from cell to cell but is typically in the region of milliseconds to minutes. At this stage, cells are delicate and must be treated carefully until they have had a chance to divide.

Figure 3: Schematic diagram showing the steps and associated charges during electroporation that lead to the introduction of exogenous material into the cell, in this case a plasmid. Credit: Technology Networks.

Example electroporation protocol

When establishing an electroporation protocol, it is important to choose parameters¹⁹ that enable permeabilization while at the same time minimizing disturbance to the cell membrane to maximize cell viability, experimental reproducibility and efficiency. Factors to consider include the:

• waveform
• pulse time
• field strength

Pulses are generally either classed as square wave or exponential decay wave. Square wave pulses rise rapidly to their set voltage, maintain the level and then swiftly cut off at the pulse end. With exponential decay waves on the other hand, the voltage rises rapidly to the target value and is then allowed to decline over time. Square waves are typically used for mammalian cells, while exponential decay waves are more often used for bacterial, yeast, insect and plant cells as well as some types of mammalian cell. While transfection is possible with both waveforms in the differing cell types, transformation efficiencies tend to be low using square waves in bacterial, yeast and insect cells and exponential decay waves tend to reduce the viability of many mammalian cells.¹⁹

The duration of the pulse (normally micro- to milliseconds) can be inputted directly for square waves. However, with exponential decay waves, this is not a predetermined value but is impacted by other experimental conditions due to the nature of the wave. Instead, the pulse length is referred to in terms of the “time constant” (TC) and is the length of time it takes for the electrical pulse to decay to a third of its original voltage (Figure 4). Efficiency is typically optimal around a TC of 5.0–5.1 for cells like E.coli, with values of 4.0–5.0 considered fairly normal. However, efficiency reduces the further from this value it goes. Lower time constants can be caused by issues like poor quality DNA, insufficient washing or having too dense a sample of cells. Higher time constants can indicate that the pores have been open for too long and will likely result in higher cell mortality.

Figure 4: Graph showing the reduction in voltage of an exponential decay wave with the time constant (T) indicated where the voltage reaches a third of its initial value. Credit: Technology Networks.

If the pulse length is increased, the voltage is typically decreased and vice versa to help prevent irreparable cell damage.

The field strength is the voltage delivered across the electrode gap and is measured in kV/cm. Cuvettes are available with differing gap sizes, so it is important to take this into consideration when optimizing your voltage. If the necessary field strength is known, it is possible to calculate the voltage required for any given cuvette size using the following equation:

Other factors that can impact the field strength required for success include cell size (with smaller cells typically requiring higher voltages for a given electrode gap) and temperature (with higher voltages generally required at cooler temperatures).

In contrast to bacterial electroporation, which generally works best with short, high voltage pulses (hundreds to thousands of volts for micro- to milliseconds), electroporating cells or tissue from higher organisms often requires milder conditions.²⁰ Many electroporators offer a selection of preset programs for specific cell types that have already been optimized.

While for many cell types a single pulse is sufficient, some cell types require two or more pulses, typically delivered automatically by a multi-electroporator at preset intervals.

Let’s consider an example protocol for bacterial electroporation.

1. Grow a healthy culture of cells. For bacterial strains, mid-exponential phase cultures that are actively dividing are a good rule of thumb. For some cells, such as bacterial strains that produce large amounts of hyaluronic acid capsule, additional enzymes may need to be added to the growth media to remove the capsule to improve the process efficiency.
    
2. Cells need to be free of salts as otherwise the current will pass around them in the solution when the electrical pulse is delivered. Therefore, wash the cells to remove salts; ice cold 0.5 M sucrose can be a good option. When pelleting the cells to remove the growth media and washes, treat them gently and use a chilled centrifuge to minimize cell damage. Once washed, keep them on ice and gently resuspend in the same sucrose solution.
    
3. As well as chilling the wash and cells, place the cuvette and substance to be introduced on ice to keep things as cold as possible.
    
4. Pipette the washed cells into the cuvette and gently add the substance to be introduced, pipetting gently to mix.
    
5. Before placing the cuvette in the electroporator, ensure it is thoroughly dried. Even a few drops of water on the outside can lead to an electrical short circuit that will spoil your experiment (and produce a loud bang!).
    
6. Deliver the electrical pulse and check the time constant is within the expected parameters.
    
7. Add ice cold media to the cells and allow them to warm back up to their normal growth temperature in the incubator. Try to prevent sudden shocks as this can increase cell death and reduce the efficiency of your experiment.
    
8. Once they are warm and actively dividing again (which will depend on the cell type or strain being cultured), cells can be cultured as normal. At this point, selective agents to promote growth of cells that have taken up the target substance over those that have not can be introduced if they are being used.