From previous studies it has been demonstrated that the fusion of tumor cells with antigen-presenting cells generates hybrids that are known to induce anti-tumor immunity. With the advancement of scientific research and medicine, the need to produce cell-cell hybrids for cancer immunotherapy and for various other applications is substantial. Among the many methods used to generate these hybrid cells, electrofusion is a technique that is more widely used and recognized as a method to efficiently produce hybrids. Electrofusion requires two steps. In the first step, cells are brought into close adjacent contact either by a mechanical method like centrifugation or by dieletrophoresis using alternating current (AC). The second step includes the reversible breakdown and fusion of cell membranes induced by high voltage direct current (DC) pulses. The goal of this investigation was to study the use of electrical charge to bring cells into close contact with one another in the cell contact stage prior to delivering high voltage fusion pulses. The possibility of achieving considerable cell-cell contact was tested in two separate electrical systems. In the first system B16 murine melanoma cancer cells were subjected to a range of direct current (DC) voltages between 4 V/cm and 40 V/cm. With the use of DC from a small power source the response of the cells was tested in multiple fusion chambers consisting of two or four electrodes. The configurations of the chambers were varied by changing the distance between the electrodes, the thickness, material and type of coating on the electrodes. In the second system the movement of cells in the presence of corona charge was studied. B16 cells in a culture dish were confined by a circular grounded electrode and subjected to corona discharge for known periods of time. Application of corona charge (positive or negative) facilitated the contact of cells in the annular region between the two circular electrodes. After series of tests, final designs for fusion chambers to be used with DC and with corona were developed. Cell contact achieved with the DC fusion chamber was not substantial enough to produce a significant amount of fusion yield. The fusion chamber designed to be used with corona on the other hand produced exceptional cell contact results consequentially generating fusion yields as high as 40%.

Cell fusion is an important technique that is used in the field of medicine and biomedical research. For instance, fusion can be used to create hybridomas and novel types of secretory hybrid cells. It may also be used to engineer cultured insulin-secreting pancreatic B-cell lines for the treatment of diabetes. Historically, the applications listed above have been accomplished by a number of methodologies including dielectrophoresis, centrifugation, polyethylene glycol (PEG) and viral fusion proteins. However, these approaches often fail to produce the desired results due to poor cell viability, lack of 1:1 fusion, and use of non-physiological environments. It is proposed that the application of an electrical field generated by corona charge (corona fields) and subsequent treatment with direct current (DC) pulse technology will overcome these deficiencies. Isolated and pre-labeled neuronally committed human teratocarcinoma (NT2) cells in monoculture or co-culture, were seeded in chambers, constructed in the laboratory, and allowed to adhere to the chamber bottom prior to corona treatment. A corona generator, also constructed in the laboratory, was used to expose cells to positive and negative electrical charges to induce cell-cell contact. The cells were then pulsed with DC voltage to induce fusion. During the experiments, cells were photographed sequentially to record cell movement/contact and fusion. The project was designed to identify optimal corona-based electrofusion parameters for viable, 1:1 cell fusion. Optimal results for cell-cell contact were obtained using a cell density of 2.35 times ten to the fourth power cells per microliter Dulbecco's Modified Eagle Medium (DMEM) in a grounded circular plate corona chamber following at least 3 minutes of settling time. Corona charges from (+) 6.1 kilivolt and (-) 5.5 kilivolt potentials were determined as being most favorable for cell movement and viability. Fusion was best achieved by first exposing either a circular or square ungrounded corona chamber configuration to 3 minutes (+) corona charge followed by 3 minutes (--) corona charge; disturbing the cells in the chamber with mechanical force; and then exposing them to 8-15 sequences of a 2,500 Volts per centimeter DC pulse at 100 microseconds.