Mohamad Nazib Adon

Study of effect of microsecond pulsed electric fields on threshold area of HeLa cells

Microsecond pulse electric field (μsPEF) application development substantially affected the development of research process including controlling cell functions by using pulses of electrical fields to create pores through a cellular membrane causes cell lysis and apoptosis commonly known as electroporation. Here we demonstrate the influence of the μsPEF on the threshold area (TA) of human cervical cancer cells (HeLa) membrane. The electric field for μsPEF is 3 kV/cm while the pulse interval is 100 ms. The pulse length and the number of pulses were fixed at 10 μs and 5, respectively. While the cultured skin cells are placed in 9 mm-gap EP electrode chamber for allowing real time observation of membrane permeability changes and cellular physiology. In order to initiate higher cell viability rate, high transfection efficiency, lower sample contamination and smaller Joule heating effect the modification of EP chamber need to be done which can be controlled by pH scale, temperature and humidity. The experiment using high pulse electrical field with simply repetitive pulses shows the threshold area of cell membrane was decreasing gradually to 44.59 μm, and is settled within hundreds of second. We found that the threshold area of cells membrane was affected when exposed to high voltage pulse electric field. The dependence of the threshold area on the HeLa cell membrane might be associated with the electrical impedance of the plasma membrane that begins to fluctuate after the application of a certain level of μsPEF.

Study of electroporation effect on HT29 cell migration properties

Pulse electric field exposures on human cancer cells have shown an alternative towards drug free wound treatments. The earlier investigations have shown interesting findings on usage of this electrical means generated via high voltage pulse generator system. Therefore, the aim of the study is to investigate the effect of pulse electric field on the migration properties of colon cell line HT29 towards the enhancement of wound healing process. The HT29 cells were treated with an electric field of 600V/cm for 500us after a wound model was created in vitro with the aid of a sterile needle. A time-lapse live imaging of the migration properties of the HT29 cell was carried out using integrated devices for live cell imaging that was equipped with CCD camera, temperature and CO2 controllers and an inverted microscope. The result of cell migration properties showed that electroporation has an influence on migration speed of HT29 cell. The electroporated cells were found to close the wound completely in 105 minutes, at an average migration speed of 0.50 μm/min. Whereas the non-electroporated cells closed the wound in 240 minutes, at an average migration speed of 0.24 μm/min. Revealed by the live video microscopy, electroporation was also found to increase the proliferation rate of the HT29 cells. The cells in both treated and control collectively migrate in the direction of the wound closure. Thus, this can further be investigated for the use of pulse electric field in facilitating wound healing process.

Studying the influence of electroporation on HT29 cell line interaction with micro-patterned extracellular matrix protein (fibronectin)

Micro-contact printing (MCP) is a scheme that allows a substrate or surface to be functionalized freely with a protein of extracellular matrix (ECM), in a well-defined manner. MCP can be used to regulate cell adhesion geometry on a substrate and in controlling wound healing process. In this study, human colon cancer cell line, HT29 were grown on a micro-contact printed pattern of fibronectin with repeat gratings of 25µm, 50µm, and 100µm wide, for 48 hours. The cells alignments to the patterned substrates were then computed, where 0° means 100% alignment to the pattern. This was done with the purpose of finding those pattern that stimulated the best degree of cell alignment. The quantitative analysis of the study revealed that HT29 cells aligned most readily to the 50µm and 100µm pattern with a mean angle of alignment of 4.8°±1.5SD and 5.7°±1.4SD respectively. On the other hand, HT29 poorly aligned to 25um and the un-patterned fibronectin-coated substrate with a mean angle of alignment of 33.4°±8.4SD and 51.1°±9.5SD respectively. Furthermore, the 50µm stamp pattern was used to investigate the influence of electric field on the HT29 alignment to the fibronectin patterned substrate. Electroporated and non-electroporated HT29 cells were cultured on the 50µm patterned substrates for 48 hours. The result revealed that there is no significant difference in the degree of alignment between the electrically treated with a mean angle of 4.2°±1.4SD and the untreated cells with a mean angle of 4.8°±1.5SD. However electrically treated cell aligned faster (within 31 hours of seeding) as compared to the untreated cells that aligned after 44 hours of seeding. Hence, the result of the study revealed that Micro-contact printing technique together with pulse electric field could offer a potential faster method of controlling wound healing process.

Investigation a critical selection of pulse duration effect on growth rate of HeLa cells

Electroporation is a process of the biophysical 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 measured the relation between amplitude and duration, 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. Cells were submitted to single pulses, at constant field strength of 2kV/cm, at various pulse durations from 30μs to 600μs. As a summary, it was found pulse duration equal to100μs is associated the HeLa cell growth rate faster confluence in comparison to the cell with 30μs, 300μs and 600μs pulse duration.