ON-CHIP ELECTROPORATION DEVICE FOR DIRECT INTRODUCTION OF PLASMIDS INTO CELL NUCLEUS AND OBSERVATION OF CELL REPROGRAMMING PROCESS K. O. Okeyo 1* , Y. Hayashi 1 , O. Kurosawa 1 , H. Oana 1 , H. Kotera 2 , M. Washizu 1 1 University of Tokyo, JAPAN 2 Kyoto University, JAPAN ABSTRACT We have developed a novel on-chip electroporation system for introducing plasmids directly into the cell nucleus that employs electric field constriction created at micro-orifices embodied on a polymer sheet. Using the device, HeLa cells expressing Fucci, a cell cycle indicator, were electroporated with plasmids containing GFP and reprogramming factors. GFP expression was observed within 2 hours after electroporation, and correlated well with the expression Oct3/4 as determined by immunostaining. Compared with control, cells electroporated with reprogramming genes showed a higher likelihood of cell cycle arrest between S-M phases, suggesting that reprogramming factors have an immediate influence on cell cycle. KEYWORDS: On-chip Electroporation, Plasmid Delivery, Cell Reprogramming, Yamanaka Factors, Fucci, Cell Cycle INTRODUCTION Pluripotent stem cells, for instance iPSCs (induced pluripotent stem cells) are attracting great attention due to their potential application in regenerative medicine and therapy [1]. The generation of these cells involves introducing a set of four reprogramming genes, or the so-called Yamanaka factors (YF: oct3/4, Sox2, Klf 4 and cMyc), into adult somatic cells to induce cell reprogramming. However, reprogramming efficiency still low (less than 1%) and the process leading to pluripotency is not yet fully understood. Considering that cellular events such as DNA synthesis and gene expression are closely controlled and synchronized with the cell cycle, it is predictable that reprogramming efficiency should depend on the timing of the delivery of the reprogramming factors, as determined by the cell cycle. In other words, it is plausible to reason that timing gene delivery with cell cycle, for instance introducing reprogramming factors during the DNA synthesis phase (S-phase), can improve chances of reprogramming or even speed up the process. This calls for a gene delivery system that allows for timing of gene introduction to a specific phase of the cell cycle, in addition to making it possible to monitor the effect of the introduced genes right from the point of delivery. Cell cycle monitoring has been made possible by the development of Fucci (fluorescent cell cycle indicator) [2]. Electroporation remains a powerful tool for gene delivery because, compared with viral methods, it is less costly, does not depend on cell type and there are no concerns of contamination with foreign genetic materials as is the case with viral carriers. More importantly, with electroporation, it is possible to time the delivery of materials into the cell. However, conventional electroporation techniques are less desirable for plasmid delivery because of cell damage from high voltage necessary to achieve successful gene delivery. Moreover, since cells are electroporated in bulk, it is difficult to determine the state of the cell at the time of gene delivery using conventional electroporation methods. To address these issues, we are developing a low-voltage on-chip electroporation technique for direct delivery of reprogramming factors into the nucleus of adherent somatic cells. The technique employs the use of an orifice sheet sandwiched between two electrodes and on which cells to be electroporated are seeded. Electric field concentration created at the orifices enable delivery of plasmids and other materials at low voltage, and real-time monitoring of cell response is possible during and after pulsation [3]. EXPERIMENTAL A schematic of the on-chip electroporation device developed in this study is shown in Figure 1. The device consists of Figure 1: Schematic of on-chip electroporation device. Figure 2: SEM image of Orifice sheet. 978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001 113 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences 27-31 October 2013, Freiburg, Germany