Radiol Oncol 2001; 35(3): 203-7. Electroporator for in vitro cell permeabilization Marko Puc, Karel Flisar, Stanislav Reberäek and Damijan Miklav.i. University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia The use of high voltage electric pulse technology, electroporation, in cell biology, biotechnology and medicine has attracted an enormous interest. Electroporation is a transient phenomenon that increases the permeability of cell plasma membrane. In the state of high permeability, the plasma membrane allows small and large molecules to be introduced into the cytoplasm, although the cell plasma membrane represents a considerable barrier for them in its normal state. The effectiveness of electroporation depends on many parameters that can be divided into the parameters of the electric field and the parameters that define the state of cells and their surrounding, i.e. temperature, osmotic pressure, etc. In this article, we present a prototype electroporator GT-1 for in vitro electropermeabilization that we have developed. Our electroporator offers a vast flexibility of parameters and can generate high and low voltage pulses, of which the latter ones are used for electrophoretic transfer of charged molecules through permeabilized cell plasma membrane. Key words: electroporation - instrumentation - methods; cell membrane permeabilization Introduction Viability of a cell depends on the integrity of its plasma membrane. The plasma membrane prevents the exchange of substances between intracellular and extracellular spaces. Exposing the cell to the electric field can increase the permeability of the cell plasma membrane. The increased permeability of the plasma membrane allows small and large molecules to be introduced into the cytoplasm. This phenomenon is transient and is Received 6 July 2001 Accepted 3 August 2001 Correspondence to: dr. Damijan Miklav.i., University of Ljubljana, Faculty of Electrical Engineering, Triaäka 25, SI-1000 Ljubljana, Slovenia. Phone: +386 1 4768456; Fax: +386 1 4264658; E-mail: damijan@ svarun.fe.uni-lj.si termed electropermeabilization and often also referred to as electroporation. The electroporation is widely used in different medical and biological applications i.e. electrochemotherapy, transdermal drug delivery and gene transfection. The effectiveness of all these applications depends on many parameters that can be divided into the parameters of the electric field and the parameters that define the state of cell and its surrounding, i.e. temperature, osmotic pressure, conductivity of cytoplasm and extracellular fluids etc. With respect to this, optimal parameters of electroporation have to be used to achieve best efficiency of the method.1 For in vitro experiments, where most commonly used electrodes are parallel plates with a 2 mm inner distance, the threshold voltages typically range from 120 V to 300 V1,2, with Puc M et al. / In vitro electroporator the pulse durations from several microseconds to several milliseconds.1,3 Beside this, it is often necessary to deliver more then one pulse to increase efficiency of permeabilization. In that case, pulses must be delivered in a certain period requiring repetition frequencies from 1 Hz to several hundred Hz. All these demands are fulfilled by special devices, often referred to as electroporators. Nowadays, there are a lot of commercially available electroporators that are designed for in vitro experiments. The problem of most of these electroporators is that the flexibility of the parameters of electric pulses is not sufficient, especially if we want to study the effects of different pulse parameters on the cell permeabilization, survival or average uptake of different molecules. In this paper, we present the design of electroporator for in vitro cell plasma membrane permeabilization. The device is operated by an internal computer that allows the user to choose the parameters of electric field. The computer drives the pulse generator that is composed of a digital pulse generator generating the signal, high voltage amplifier amplifying the signal, and current amplifier that provides the signal with sufficient energy to prevent the voltage amplitude from dropping during the pulse delivery. Beside this, the last version of the developed electroporator also includes a special unit that generates low voltage pulses usually used for the electrophoretic transfer of the charged molecules through the permeabilized cell plasma membrane. System design Figure 1 shows the basic system design of the electroporator. It consists of a computer, pulse generator, voltage amplifier, current amplifier and low voltage pulse generator. Besides this, the device comprises two high voltage power supplies and several low voltage devices that are necessary for normal operation but are not drawn on the figure. The internal computer of the device is in the first place used for selecting pulse parameters. Therefore, the computer includes a user interface composed of a display and a keyboard. The second task of the computer is to control pulse generation after activation has Figure 1. Block diagram of in vitro electroporator GT-1. Radiol Oncol 2001; 35(3): 203-7. Puc M et al. / In vitro electroporator Table 1. Output parameters of the developed in vitro been triggered. All pulse parameters, except electroporator pulse amplitude, are then transferred to the Value pulse generator. This subunit generates digi-Parameter Min. Max. Increment tal signal that is then amplified in the voltage Delay/Pulse ratio* E PULSES3 65535 1 Pulse amplitude 25V 500V linearHIGH VOLTAGNumber of pulses 1 128 1 Pulse current 30A Pulse durationES 5ms 9999ms 1ms Delay durationE PULS0ms 9999ms 1ms Pulse amplitude 0V 50V 10VLOW VOLTAGNumber of pulses 1 1000 1 Pulse current 1A Pulse duration 5ms 5000ms1ms&50ms amplifier to the value that we set by external potentiometer. The amplified signal is then intensified by the energy from the current amplifier because we have to fulfill the ener gy requirement as defined by the load be tween electrodes.4 At this point, the genera tion of the high voltage signal that is used for permeabilization of cell plasma membrane is finished. The low voltage pulse generator, which we have constructed just recently, is used for the electrophoretic transfer of charged molecules through permeabilized cell plasma membrane. The structure of this subunit, controlled by the internal computer, is similar to that of the DC power supply. This version allows to change the amplitude in 10 V steps in a range from 0 V to 50 V. Performance and experimental results The developed in vitro electroporator, which is still a prototype, has been used in our laboratory for more than two years. During that time, we found and repaired some deficiencies in design and we also made several other improvements that reflect on greater flexibility of the parameters. The current prototype GT-1 allows the user to set high and low voltage pulses in a range that is given in Table 1. The pulse repetition frequency -o is calculated by using the following equation: 1 -o = , (1) T.(1+DR) where T is pulse duration and DR is value of the parameter Delay/Pulse ratio. Furthermore, to demonstrate the performance of the device we designed an experiment where we measured voltage and current on the output (Fig. 2). In the experiment, we ex * - parameter defines pulse repetition frequency that is calculated by the equation (1). posed 50 ml of pure SMEM, which was placed between 2mm plate electrodes, to five consecutive 100 ms pulses of 500 V at a repetition frequency of 2.5 kHz (DR = 3). Figure 2. Performance of electroporator loaded with 50 ml drop of SMEM that was placed between 2 mm plate electrodes. Electroporator was programmed to generate five consecutive 100 ms pulses of 500 V and repetition frequency of 2.5 kHz (DR = 3). Top trace (signal 1) presents voltage and bottom trace (signal 2) presents current flowing through the system. The measurements were performed using LeCroy LT9310C digital oscilloscope, a LeCroy AP015 current probe, and a Tektronix P5100 1:100 voltage probe. The voltage was 500 V, while current increased from 14 A to 19 A. Radiol Oncol 2001; 35(3): 203-7. Puc M et al. / In vitro electroporator It is evident from the Figure 2 that the instrument was able to deliver all the pulses without any distortion. Even more, it could easily increase the current between two consecutive pulses that is usually necessary due to the polarization of the sample between the electrodes. To compare the performance of the prototype GT-1 with that of Jouan GHT 1287B, we evaluated cell permeabilization, using a bleomycin method5, with both devices. The results of experiments that are shown on Figure 3 are comparable. T-test showed no statistically significant difference between both experiments. Figure 3. Comparison of cell permeabilization as a function of pulse amplitude obtained with electropul- sator Jouan GHT 1287B (l), and in vitro electroporator GT-1 (!). Besides the performance experiments that we carried out, our colleagues already published the results of the experiments using the developed electroporator.6,7 The two experiments were performed to study the influence of the parameters of electroporation (medium conductivity 6 and pulse repetition frequency 7) on the cell permeabilization, survival and average uptake. Conclusions The comparison of the developed prototype GT-1 with the commercially available device Jouan showed that GT-1 fulfils all demands of the in vitro investigations. Furthermore, it offers a vast flexibility of the parameters and has the ability to generate high and low voltage pulses, where low voltage pulses can be used for the electrophoretic transfer of charged molecules through permeabilized cell plasma membrane. Acknowledgment Our research was supported through various grants by the Ministry of Education, Science and Sports of the Republic of Slovenia and in part by IGEA s.r.l. Italy. Authors wish to thank to Maäa Kanduäar D.Sc., Tadej Kotnik D.Sc, and Gorazd Pucihar B.S. who performed many experiments with the developed prototypes and provided us with a wealth of feedback information about operation of devices. References 1. Mir LM. Therapeutic perspectives of in vivo cell electropermeabilization. Bioelectrochemistry 2000; 53: 1-10. 2. .emaiar M, Jarm T, Miklav.i. D, Ma.ek-Lebar A, Ihan A, Kopitar NA, Seräa G. Effect of electric- field intensity on electropermabilization and electrosensitivity of various tumor-cell lines in vitro. Electro Magnetobiol 1998; 17: 261-70. 3. Tsong TY. Electroporation of cell membranes. Biophys J 1991; 60: 297-306. 4. Puc M, Reberäek S, Miklav.i. D. Requirements for a clinical electrochemotherapy device - electroporator. Radiol Oncol 1997; 31: 368-73. 5. Kotnik T, Ma.ek-Lebar A, Miklav.i. D, Mir LM. Evaluation of cell membrane electropermeabilization by means of a nonpermeant cytotoxic agent. Biotechniques 2000; 28: 921-6. 6. Pucihar G, Mir LM, Miklav.i. D. The effect of pulse repetition frequency on Lucifer Yellow up- Radiol Oncol 2001; 35(3): 203-7. Puc M et al. / In vitro electroporator take by means of cell electropermeabilization in 7. Pucihar G, Kotnik T, Kanduäer M, Miklav.i. D. vitro. In Magjarevi. R, Tonkovi. S, Bilas V, The influence of medium conductivity on elecLackovi . I, editors. IFMBE Proceedings. Pula: tropermeabilization and survival of cells in vitro. Birotisak; 2001. p. 834-6. Bioelectrochemistry 2001; (accepted - in print). Radiol Oncol 2001; 35(3): 203-7.