DIRECT INTRODUCTION OF PLASMID INTO NUCLEUS USING ON-CHIP ELECTROPORATION

Osamu Kurosawa1,2,Yosuke Sumita1, Murat Gel2,3, Hidehiro Oana2,3, Hidetoshi Kotera2,4, Tomohisa Kato5，Junya Toguchida5 and Masao Washizu1,2,3

1Dept. Bio Engineering, The University of Tokyo, 2JST CREST, 3Dept. Mechanical Engineering, The University of Tokyo,

4Dept. Mechanical Engineering, Kyoto University, 5Institute for Frontier Medical Sciences, Graduate School of Medicine, Kyoto University,

ABSTRACT

In this paper, we visualized the motion of plasmids within a cell during pulsation using on-chip electroporation and clarified the direct introduction of plasmids into nucleus by electrophoresis. By making the orifice pitch nearly equal to nucleus diameter with guaranteeing the field constriction at a micro orifice, higher yield of transfection was experimen-tally demonstrated.

KEYWORDS: Field constriction, On-chip Electroporation, Plasmids, Cell nucleus

INTRODUCTION

Introduction of foreign genes into cells is a basic process in cellular engineering, including cell reprogramming and differentiation control through iPS cells. Presently, the retrovirus method is commonly used to create iPS cells, but it is not suitable for clinical use due to hazardous effects of viral infection. Then, more recently, the method using plasmid in-stead of retroviral vector has been tried. The problem in using plasmid is inefficiency of its yield.

We have previously reported an on-chip electroporation method based on field constriction at micro-orifices (Fig.1) [1], which essentially features high-yield and high-survivability due to the fact that the cell membrane voltage can pre-cisely be controlled regardless of cell size, shape or orientation. It has been found that plasmids are transferred into a cell by electrophoresis[2], and in some cases gene expression was observed within 2 hours after pulsing. Such a quick ex-pression is expected to occur only when the plasmids are directly driven into cell nucleus by electrophoresis. It is ex-pected that direct introduction of plasmid into cell nucleus improves the gene expression yield, because digestion by the nuclease in the cytoplasm is avoided.

In this paper, we firstly checked the motion of plasmids within a cell during pulsation and next investigated the op-timal design of orifice sheet for the on-chip electroporation chip.

EXPRIMENTAL

In order to clarify the motion of plasmids within a cell during pulsation, we made use of a microchip having an orifice on a vertical wall, whose SEM image is shown in Fig.2. Experimental set up is schematically shown in Fig.3. The device consists of PDMS chip and microelectrode on a glass coverslip. The PDMS chip was fabricated by single mask photoli-thography based on self-forming meniscus and PDMS molding [3]. Firstly, the cells were fed into one channel (right-side channel in Fig.3) and driven toward an orifice by gentle flow. Next, by applying an AC voltage of 1MHz-6Vpeak, the cell was trapped at the orifice by dielectrophoretic force. Then, plasmid labeled with fluorescent Q-dots (hereafter Qdot-DNA complex) were fed on the other side of the orifice. Fig.4 shows the bright field image of this instance. Finally, a pulse voltage (2V-100msec) was applied to the electrodes. Using EB-CCD camera, real-time motion of plasmids during pulsa-tion was observed. To examine the existence part of plasmids introduced into the cell, the depth-wise fluorescence slice view of the cell was taken.

Figure 1: Electroporation using field constriction

at a micro-orifice

Figure 2: SEM image of an orifice

978-0-9798064-3-8/µTAS 2010/$20©2010 CBMS 217 14th International Conference onMiniaturized Systems for Chemistry and Life Sciences

3 - 7 October 2010, Groningen, The Netherlands

In order to investigate the optimal design of orifice sheet for the on-chip electroporation chip, we prepared three types

of orifice sheets. The pore size and the pitch are 5-50 microns (No.1), 3-30 microns (No.2), and 2 micron of random pitch (No.3) respectively, as shown by SEM pictures in Fig.8. Adherent MSC (Mesenchymal Stem Cell) was cultured on each orifice sheet until nearly-confluent layer was formed, and then GFP plasmid was fed by 4V-200msec pulse. Gene expression of GFP plasmid was checked with fluorescence microscopy.

RESULTS AND DISCUSSION

In the experiment using the device shown in Fig.3, the cell near the orifice was moved towards the orifice by dielec-trophoretic force, by applying AC voltage. A part of the cell membrane invaded into the orifice, and formed the hernia structure (illustrated in Fig.6). The trapping was made strong enough that the cell tightly sealed the orifice and no leakage of Qdot-DNA complex occurred. During pulsation, though we could not clarify the motion of each Qdot-DNA complex, inflow of Qdot-DNA complexes into the cell was observed. Fig.5 (A to D corresponds to depth-wise position in Fig.6) shows the depth-wise fluorescence and bright field slice view of the cell. Although with a limited resolution, some Qdot-DNA complexes, which probably failed to pass through the pore, are observed to get stuck near the membrane (nucleus or cell membrane). Red arrows in the Fig.5 show the position of Qdot-DNA complex. Fig.6 is a schematic re-constructed from Fig.5, showing that the plasmids are fed into the nucleus, which cannot take place by simple diffusion in the time scale of the experiment, but most likely to be driven by electrophoresis through nuclear pores.

The result that plasmids are transferred directly into nucleus by electroporesis implies that the orifice density should be

chosen in such a way that at least one orifice exists below each nucleus. Fig.7 shows the shape and size of MSC, which was fluorescently labeled with Calcein. Its length is longer than 100μm, and width is shorter than 50μm. Probably a diameter of cell nucleus which spread on the orifice sheet is about 20μm. In Fig.8, schematic drawing of relative position between typical cell with nucleus and each orifice is shown. In each orifice sheet, at least one of orifice exists under the cell. But as for the nucleus, it is not the case. Frequency of the orifice existence un-der the nucleus increases in order of No.1, No.2, and No.3. Especially an orifice in the No.3 sheet (Fig.8-c) surely exists right under the nucleus, but it does not guarantee the field constriction. Therefore, if the yield of GFP expression depends on the plasmid influx into nucleus, No.2 sheet is ex-pected the highest among them.

GFP expressions 5 hours after pulsation are shown in Fig.8 a-c. As ex-pected, No.2 sheet with an optimized density (Fig.8-b) gives the highest yield. No.3 sheet also gives good yield (Fig.8-c), but too high orifice densi-

Figure 3: Experimental set-up

Figure 4: Bright field image of immobilized cell

Figure 5:The depth-wise fluorescence slice image of Qdot-DNA

introduced cell

Figure 6: Schematic view of observed focal plane

Figure 7: Shape and size of MSC labeled with green fluorophore

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ty reduces field constriction, which made the yield lower. The yield of GFP expression in Fig.8-b is about 50% against the total cell number on the orifice sheet. It is expected that by optimizing the size and pitch of an orifice, higher yield transfection is possible.

CONCLUSION

In conclusion, the electrophoretic effect of plasmid transfection directly into cell nuclei was clarified, and the design rule for the on-chip electroporation chip was established, through which the yield as high as 50 % was achieved for MSC cells. ACKNOWLEDGEMENTS

This research is supported by Scientific Research of Priority Areas, System Cell Engineering by Multi-scale Manipu-lation, Japanese Ministry of Education and “Development of bio/nano hybrid platform technology towards regenerative medicine” project of CREST, JST. The photomasks were prepared by the electron-beam facility at the VLSI Design and Education Centre (VDEC) of the University of Tokyo. REFERENCES [1] Osamu Kurosawa et al: "Electroporation through a micro-fabricated orifice and its application to the measurement

of cell response to external stimuli", Measur. Sci. Tech., 17, p.3127-3133, 2006 [2] Osamu Kurosawa et al: "Massively parallel on-chip Electroporation device Designed for long term post-culturing",

µ-TAS 2009, p.570-572, 2009 [3] Murat Gel et al.: "Micro-orifice based cell fusion applied for the creation of cytoplasmic hybrid cells without gene

mixing", µ-TAS 2009, p.654-656, 2009 CONTACT * M. Washizu, tel: +81-3-5841-6344; washizu@washizu.t.u-tokyo.ac.jp

Figure8: GFP expression on three types of orifice sheets 5 hours after pulsation

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