## 7 Discussion  
In vitro experiments provide a fundamental approach to designing protocols for clinical studies. Statistical significance plays a key part in proposing any protocol for which researchers tend to adopt multiple attempts at the same setup. A way to deal with multiple attempts of the same biological experiment is to run parallel setups under the same controlled conditions. Hence, most of the research works undergo experiments in multiple culture well plates. Multiple electrically bio-interfacing samples in a parallel setup pose a challenge that is addressed in many ways, as reported in the literature. Commercially available setups, like C-Dish\({}^{\text{TM}}\), provide a standard bipolar platform. However, the issue of the configurability, scalability, and multiplexing of the setup remains unanswered. This work implemented an easy-to-design multi-electrode system for biological samples in multiple culture well dishes. The efficacy of the system has been determined by analyzing the distribution of the electric field generated due to the injection of a constant current through a biological sample. A finite element model of the cell culture well and electrode was designed in COMSOL Multiphysics. The current density and electric field distribution at various regions in the culture well were analyzed for different surface areas of electrodes to find the optimum electrode size and to maximize stimulus through the cell layer. The results show that a higher electric field is generated through the biological sample located at the bottom of the culture well if the electrodes are placed close to the bottom surface of the well with a small surface area. An increasing electrode surface area  
Figure 8: Human dermal fibroblasts: (a) 0 h; (b) 24 h; (c) 48 h.  
of results in a decrease in the electric field at the region of interest (_through the cells_). When analyzing the current density for different surface areas of electrodes, it can be seen that a higher current density is achieved with electrodes having a smaller surface area are placed at the bottom of the culture well. It can also be concluded that in order to maintain the optimal current density with an electrode of a larger surface area, the injection current should be increased accordingly. Most of this current would tend to flow through the ionic growth medium and only a portion would take part in the biomodulation process. Hence, by decreasing the size of the electrodes and by placing them near the bottom of the culture wells, it is possible to maximize the biomodulation effect in in vitro experiments. The system was designed with basic PCB fabrication technology. It has four independent electrodes per well, giving a tetrapolar arrangement. However, the connectors of each electrode can be configured to provide bipolar setups. When using the setup, the impedance measurement of a uniform medium showed a constant impedance profile over a range of frequencies, suggesting good interfacing between the electrodes and media. For biocompatibility, the electrode surfaces were coated with gold. Prolonged contact with live cells in a culture medium shows regular cell proliferation with no corrosion from the electrodes. The proposed setup was designed for a 24-well culture dish, although it can easily be replicated for any set of standard culture dishes. Moreover, as each electrode has its own connector, multiple wells can be connected together to provide a singular stimulation from one source. The setup is designed to be used in a controlled laboratory environment with the following conditions: 37 \({}^{\circ}\)C, 5% CO2, and 95% humidity, with very small deviations. The PCB structure is made from FR4 PCB board, which is made from low-moisture-absorbing polymers that perform well in humid conditions. Within the controlled environment, the setup showed data consistency in consecutive trials, asserting no effect from the external environment. Apart from in vitro stimulation cells, the setup can also be implemented in the in vitro measurement of bioimpedance.