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In the first half, this paper reviews the so-called “Plasma Gene Transfection” and “Plasma Molecular Introduction” which are the technology to introduce molecules such as plasmid DNA, proteins, and dye molecules into cells by discharge plasma treatment. In the latter half, the authors’ method with the micro discharge plasma is reviewed. This method induces the cell’s spontaneous uptake of external molecules by endocytosis which is triggered by the complex of weak stimuli produced by the micro discharge plasma treatment less than 10 ms and enables high introduction efficiency and high cell viability simultaneously. There is almost no damage to the cells and external introducing molecules, and there is almost no unintended integration of the introduced gene into the chromosome. The authors would like to call this feature “Random Genome Integration-Free.” This feature would enable safe and clean gene and molecular introduction for practical applications such as medicine and breeding.

The gene/molecule introduction method using surface discharge enables transfection into a wider area, compared with the micro-plasma method. In this research, as the first step in elucidating the gene transfer mechanism in the surface discharge method, it was revealed that endocytosis is the main introduction mechanism in the surface discharge method, and that 80% of the introduction is due to endocytosis. Furthermore, the relationship between the size of the target molecule and the transfection efficiency was investigated. As a result, it was made clear that when target molecules with a molecular weight of up to 10 000 bp, the transfection efficiency is proportional to the collision frequency between the target molecule and cell. On the other hand, it became clear that when the molecular weight exceeds 10 000 bp, the transfection efficiency decreases with increasing molecular weight, and that almost no introduction is made at 15 000 bp or more.

Plasma gene transfection is expected as a safe and useful method of gene transfection. However, in this method, there is difficulty in keeping both high transfection efficiency and less cell damage simultaneously. The authors have evaluated transfection efficiency and cell viability using four different plasma sources, such as arc discharge, plasma jet, dielectric barrier discharge (DBD), and microplasma. A high transfection efficiency was achieved by discharge forms in which the electric current flows via the cells. This suggested that an electric current plays an important role in plasma gene transfection. The total volume of gas flow must be small or zero and the area in which the cells are directly irradiated by plasma must be small in order to achieve a higher cell viability. The microplasma that satisfies these conditions achieved both the highest transfection efficiency and the highest cell viability simultaneously.

In order to clarify the mechanism of plasma gene transfection, the relationship between transfection efficiency and transferred molecular size was investigated. Molecules with low molecular mass (less than 50 kDa; dye or dye-labeled oligonucleotide) and high molecular mass (more than 1 MDa; plasmid DNA or fragment of plasmid DNA) were transferred to L-929 cells. It was found that the transfection efficiency decreases with increasing in transferred molecular size and also depends on the tertiary structure of transferred molecules. Moreover, it was suggested the transfection mechanism is different between the molecules with low (less than 50 kDa) and high molecular mass (higher than 1 MDa). For the amount of gene transfection after plasma irradiation, which is comparable to that during plasma irradiation, it is shown that H2O2 molecules are the main contributor. The transfection efficiency decreased to 0.40 ± 0.22 upon scavenging the H2O2 generated by plasma irradiation using the catalase. On the other hand, when the H2O2 solution is dropped into the cell suspension without plasma irradiation, the transfection efficiency is almost 0%. In these results, it is also suggested that there is a synergetic effect of H2O2 with electrical factors or other reactive species generated by plasma irradiation.