Electroporation Effect on Growth of HeLa Cells Mohamed A. Milad Zaltum , Mohamad Nazib Adon, and Muhammad Mahadi Abdul Jamil

Department of Electronic Engineering, Faculty of Electrical and Electronics Engineering, Universiti Tun Hussein Onn Malaysia

Batu Pahat, Johor, Malaysia Email: mahadi@uthm.edu.my

Abstract— Electroporation is a process of the bio-physical effect on cells exposed to an external electrical field is gaining applications in medical treatments, especially to create pores through a cell membrane and allow uptake of DNA into a cell. Therefore, in the theoretical evaluation of electroporation, transmembrane potential and characteristics of the cells growth is the target of the analysis. In this study, we used cervical cancer cells (HeLa cells) to be sample for electroporation, because HeLa cells is one of the most well-known cell lines, and easy to researched by continuous harvesting of large numbers of HeLa cells for in-vitro experimental tests. In this study we demonstrate the activity of HeLa cells induced with high voltage of 2700V/cm via a pulse length of 10μs. As a summary, it was found that the HeLa cell growth rate increased up to 50% faster when applied EP incomparison to the cell without EP treatment.

Keywords— electroporation; HeLa cell; proliferation; cell culture and electroporation

I. INTRODUCTION Electricity has been used in medicine for centuries, even

long before the effect of electric and magnetic fields on biological tissue were in anyway understood. As the knowledge of biological structures steadily increased, so has our understanding of the electric fields that our bodies generate and the effects external electric fields have on the body’s internal structures [1]. In the last decade’s modern science and technology have made the use of electromagnetic devices in medicine. Measurements of internal electric fields are taken routinely in diagnostics and electric stimulation of excitable tissues is used to sustain life, rehabilitate injuries and improve the quality of life in general [2].

Electric fields can affect not only excitable tissues, such as muscles and nerves, but also non-excitable tissues, either thermally, by generating heat inside the tissue or by inducing structural changes down to cellular membranes. Numerous studies in the 1960s and 1970s have demonstrated that appropriate electric pulses can achieve electropermeabilization of biological cells that is followed by inflow/outflow of different molecules [3]. This phenomenon was later termed electropermeabilization or electroporation, after a theory that explained the observed changes in membrane permeability in terms of formation of hydrophilic pores [4]. By controlling the electroporation parameters, it is possible to either transiently permeabilize cell membranes, which is called reversible

electroporation [5], or to kill cells, which is called irreversible electroporation [6]. Reversible electroporation allows transient molecular transport through the pores; after a few minutes cellular membranes reseal and cell functions are restored [7].Electroporation can be achieved in any cell type, which is one of the reasons why it has become a widespread technique for loading cells with substances that are otherwise difficult to load into cells [8]. Reversible electroporation is widely used in biotechnology and medicine to introduce various molecules and agents into cells and tissues and for cell fusion [9]. In this study the Electroporation experimental studies conducted to show that there is an electric field effect on the HeLa cells. The studies on EP are conducted using in-vitro techniques, by using HeLA cells as the sample and the result shows the proliferation activity of HeLa cells induced at high voltage of 2700V/cm with a pulse length of 10μs.

II. Experimental Method A. HeLa Cell lines and cell culture procedure

In this study we have used HeLa cells. HeLa cells cultured in Dulbecco's Modified Eagle Medium (Gibco) with 10% fetal bovine serum(FBS) until 90% confluence, are first harvested by incubation with trypsin (Invitrogen) for 7 min and suspended in RPMI 1640 medium (Sigma) with 10% fetal bovine serum to neutralize the trypsin. Finally, kept in the CO2 incubator for further investigation.

The steps when the tissue culture flask reaches full confluence. Remove media from the tissue culture flask. Then, add 5ml from phosphate buffered saline (PBS) in tissue culture flask for 3 minute to wash the cells. Add 2 ml of trypsin to make the cells detach from the bottom of the flask. Trypsin works best in a warm surrounding, so the flask is incubated for seven minutes at 37 °C. After that, an appropriate number of cells in suspension (0.5 ml) are then transferred to the new flask, fresh media (10ml) is added to each flask, and the new plates are incubated for the next growth phase. Adherent cells are grown in culture flasks, with FBS containing culture media at 37 °C with 5 % CO2.

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B. Micro second Pulse Electric Field (μsPEF) Exposure System

The commercial electroporation ECM 830 as shown in Fig. 1 made by BTX Harvard Apparatus provide two modes of operation. First, a high voltage mode ranging from 30V to 3kV and pulse length of 10μs to 600μs (lμs resolution). The second mode is a low voltage which gives an output voltage of 5V to 500V and pulse length of 10ms to 999ms (l ms resolution).

Figure 1. Picture of ECM 830 Square Wave Electroporator

C. Integrated devices of real time imaging system Fig. 2 shows the main components of the experimental

setup. A Nikon inverted research microscope (Ti series) offers improved system speed, increased flexibility and efficient multi-mode microscopy as part of a fully integrated microscope system that is ideal for high end research and live cell imaging. Thus, this system will be fit for the EP experiment purpose since we are interested with the live imaging mode as available on this system.

Figure 2. Picture of Nikon Inverted Microscope (Ti series)

D. Electroporation of HeLa cell The EF stimulation chamber system was designed and

built thus allow the direct observation of the HeLa cells changes following EP exposure applications. These chambers consist of two platinum electrodes with a distance of 9mm as shown in Fig. 3. In order to achieve the high electric field intensity of 2700kV /cm with a pulse length of 10μs, the cells were needed to be placed between the electrodes, after EP of

HeLa cell, adherent cells are grown in culture flasks, with FBS containing culture media at 37 °C with 5 % CO2.

Figure 3. Picture of 9mm - gap EP electrode chamber for μsPEF

excitation

III. RESULT AND DISCUSSION

Figure 4. Harvested by incubation with Trypsin (scale bar =50μm)

Figure 5. HeLa cell after 6 hours incubated (scale bar =100μm)

The HeLa cell detach from the bottom of the flask after

added trypsin and incubated at 37 °C as shown in fig. 4. The HeLa cell after taken 0.5ml suspension cell and transferred at new flask, added 10ml fresh media and incubated at 37 °C with 5 % CO2 it will start to reattach on the bottom of the flask as shown in fig. 5. At the beginning of the experimental work (0 to 10 hours) it shows as if there is no effect on the HeLa cell. However, experimental data shows that there is an electric field effect on the growth of HeLa cells when the cells were left longer (above 12 hours, refer Fig. 6).

Figure 6. Show the HeLa cell growth rate in percentage per time (hour) in culture flask without electroporation process (A, B, C, D, E)

and with electroporation process (F, G, H) (scale bar =100μm)

Time (hour) HeLa cell without Ep (confluence%)

HeLa cell with EP (confluence%)

12 10 30 24 20 50 48 50 90

96 70

108 90

Table 1. Quantitative result of HeLa cell growth rate

Figure 7. Analysis of HeLa cell growth rate

Further investigation revealed that the HeLa cell proliferation rate increased dramatically at the 24 hours this could be seen cleary on Fig. 6 (A, B, C, D, E) HeLa cell growing without EP incomparison to the one with EP treatment figure 6 (F, G, H). In another words, HeLa without the EP exposure will reach 90% confluence at 108 hours, rather than HeLa with EP (2700V/cm with a pulse length of 10μs) will reach 90% confluence at 48 hours as shown in figure 6 (F, G, H). Next, to further validate this findings we then performed quantitative analysis which also demonstrated that the HeLa with EP exposure does strongly has effect on the proliferation in comparison to the one without (Refer Table 1 and Fig. 7). The investigation for the HeLa with EP exposure were stopped at 48 hours since it has reached the confluence level which were no the same for the HeLa without EP exposure, monitoring were continued up to 108 hours to reach confluence. This preliminary findings clearly shows that the EP process does have effect on the proliferation rate of HeLa cell.

IV. CONCLUSION As a summary, from this work it could be concluded that

the growth rate of HeLa cell increased up to 50% than the normal proliferation rate. These were due to the EP exposure on the HeLa cell. In this study, we demonstrated preliminary results of HeLa cells proliferation rate effected by the EP exposure however, this following study requires further

investigation to identify the critical process of growth rate which might lead us on to an interesting application. Finally, the current findings do give us a dimension to explore further down to cellular level which may contribute towards wound healing applications.

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