A Simple and Sensitive Cytosensor Based Electrical Characterization of in vitro Wound Healing Assay for Keratinocytes N.Mondal, D.Mondal, C.RoyChaudhuri Department of Electronics and Telecommunication Engg. Bengal Engineering and Science University, Shibpur Shibpur, India e-mail: chirosreepram@yahoo.com A.Barui, S.Dhara, J.Chatterjee School of Medical Science and Technology, IIT Kharagpur Kharagpur, India e-mail: jds_c2000@yahoo.com Abstract—Confluent monolayers of cells in culture media are usually fragile and are susceptible to mechanical disruption. To assess the growth and migration of the cells towards recovery, the mechanical disruption is often done deliberately to perform wound healing assay. In such analysis, after a scratch in the cell monolayer, electrical characterization has been done to provide related quantitative description of the cellular behavior compared to the microscopic observation. In this direction for cellular electrical characterization, biosensors are usually designed with photolithographically patterned electrodes which are of the dimensions of the cells. This increases the cost and complexity of the analysis. Here we report the electrical characterization of in vitro wound healing for keratinocytes monolayer in DMEM-F12 medium with a low cost and sensitive macroporous silicon platform using simple electrode geometries for the first time. Impedance spectroscopy results show that there is a distinct difference between the electrical properties like the effective capacitance and the resistance of the keratinocytes (HaCaT) in the frequency range from 100Hz to 1MHz at two different time instants after wounding. The difference in the electrical properties has been qualitatively explained with the microscopic and immunocytochemical findings. This analysis may help to assess the cell behaviour during its growth and repair through a less complex and low cost electrical route. Keywords; Keratinocyte, Wound Healing Assay, Electrical Characterization, Macroporous Silicon Cytosensor I. INTRODUCTION Wound-healing assays have been performed in tissue culture for several years to monitor cell behavior, like the migration and proliferative capacities of different cells under various culture conditions. These assays generally involve growing of cells to form a confluent monolayer. The layer is then scratched (wounded) by destroying or displacing a group of cells, with a needle or micropipette tip [1]. Once a wound is achieved, the open area is then microscopically observed over time to assess the rate at which the neighboring cells are filling in the damaged area (healing). This repair can take from several hours to greater than a day, depending on the cell type, conditions and contents of culture medium and the extent of the wounded region. The results are monitored by a series of photomicrographs followed by image analysis to express it in more quantitative terms [2]. From these data, one can calculate the migration or repopulation rate. Traditional assays of this sort require extensive manipulation of the cultured cells, both in carrying out the wound and in after the subsequent repair. As might be expected, when the wounded area is not precisely controlled, the method is encumbered with problems of quantification and reproducibility. Thus alternative methods by electrical characterization are being deployed to obtain the cell migration data [3]. The method is largely based on an established process to monitor cell behavior referred to as electric cell-substrate impedance sensing (ECIS). In ECIS, cells are grown on a small gold film electrode (10 -4 cm 2 ) deposited on the bottom of a tissue culture well photolithographically; a much larger counter electrode completes the circuit by using standard tissue culture medium as an electrolyte. For such purpose, interdigitated electrode based impedance cytosensors are also employed which offer instantaneous and quantitative means to monitor cellular events, such as changes of ionic channels in cell membranes, the variations of cell membrane integrity, and cell spreading, motility, and growth [4,5] and detect analytes by converting cellular responses into a measurable electrical signal. Impedance spectroscopy has been utilized also to monitor the cytotoxicity of the mouse fibroblast cells [6]. 3D tissue culture monitoring by bipolar resistivity profiling of the cells has been recently reported [7]. Traditional impedance cytosensors often consist of photolithographically patterned electrodes whose dimensions are of the order of the cells so that averages of cell properties, such as proliferation, motility, and cell–cell separation, can be monitored over the cell population. These electrodes are often interdigitated in nature or form the configuration of a electrochemical cell [8] to increase the sensitivity of measurement. However, the fabrication of such electrodes photolithographically increase the total cost of the system and hence the analysis. In this regard there has been a recent effort in designing low cost technique for continuous monitoring of the thickness of biofilms[7]. With a view to reduce the cost of electrical analysis, we report the impedance characterization of in vitro keratinocyte cell wound healing assay using a low cost macroporous silicon platform for the first time. Macroporous silicon platform has the potential to measure the impedance of these cells cultured under different medium down to 10 3 cells/ml [9] without using inter-digitated or any 47 978-1-4577-0422-2/11/$26.00 c 2011 IEEE