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Cytoplasmic Transfer Between Adhered Cells by Cell Fusion Through Microslit K.-I

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Cytoplasmic Transfer Between Adhered Cells by Cell Fusion Through Microslit K.-I CYTOPLASMIC TRANSFER BETWEEN ADHERED CELLS BY CELL FUSION THROUGH MICROSLIT K.-I. Wada1,2, E. Kondo1, K. Hosokawa1*, Y. Ito2 and M. Maeda1 1Bioengineering Laboratory and 2Nano Medical Engineering Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan ABSTRACT This paper describes a novel cell fusion method which induces cell fusion between adhered cells through a microslit for preventing nuclear mixing. For this purpose, a microfluidic device which had 105 cell paring structures (CPSs) making cell pairs through microslits with 2.1 ± 0.3 μm width was fabricated. After trapping NIH3T3 cells with hydrodynamic forces at the CPSs, the cells were fused through the microslit by the Sendai virus envelope method. With following timelapse observation, we discovered that the spread cells were much less susceptible to nuclear migration passing through the microslit compared with round cells. This finding will provide an effective method for cell fusion without nuclear mixing, and will lead to an efficient method for reprogramming and transdifferentiation of target cells toward regenerative medicine. KEYWORDS: Cell fusion, Microslit, Cytoplasmic transfer, Adhered cell INTRODUCTION The invention of induced pluripotent stem (iPS) cells[1] gave a great impact on regenerative medicine in term of cellular resources production, but some critical issues still remain such as slow and inefficient induction process, risk for tumorigenesis, and contamination of exogenous genes. Therefore, other various approaches to produce cellular resources should be explored. Cell fusion with differentiated[2] or embryonic stem (ES)[3] cells induces transdifferentiation or reprogramming, respectively. These results indicate a possibility that some factors in the cytoplasm have a capability to alter gene expression property to induce transdifferentiation or reprogramming. However, transfer of those factors by common cell fusion is accompanied by nuclear mixing resulting in abnormal karyotypes, thus being inapplicable to regenerative medicine. If cell fusion without nuclear mixing is realized, it will be a feasible method to transfer the cytoplasm, and thus provide a novel approach to induce transdifferentiation or reprogramming. One promising method to avoid abnormal karyotypes accompanied with cell fusion is to separate the nuclei of the fusing cells with a microstructure: cell fusion through a microfabricated aperture smaller than the nuclei. This kind of experiments has already been reported in some previous studies[4,5], however, in these studies cell fusion was induced between suspended cells. Because adhesive cells require adhesion to maintain their intrinsic functions[6] and cytoplasmic compositions are often different between adhered and suspended conditions7, fusion between the adhered cells may be essential to induce desired transdifferentiation or reprogramming. Then, in this study, we tried to induce cell fusion between adhered cells without nuclear mixing by using a newly developed microfabricated device. THEORY In order to transfer cytoplasm by cell fusion-based method keeping karyotype normal, it is required to avoid nuclear mixing accompanied by cell fusion. The theory for the prevention of nuclear mixing is illustrated in Fig. 1. After making cells pair in a microfabricated device, cells adhere and spread on Fig. 1: The theory for the prevention of nuclear mixing. Cell fusion is in- the culture substrate. Then, cell duced between adhered cells through the microslit (white arrow head). fusion is induced through a small aperture termed “microslit” to make a strictured cytoplasmic connection. This structure prevents nuclear migration to the fusion partner. In contrast, the cytoplasm is exchanged between fused cells by passive diffusion and/or active transport (i.e. cytoplasmic transfer). We expect that this cytoplasmic transfer from certain donor cells induces transdifferentiation or reprogramming of the recipient cells. The cell fusion system used in this study is shown in Fig. 2. A poly(dimethysiloxane) (PDMS) chip is placed on a 35 mm cell culture dish to provide a good cell adhesion substrate. The PDMS chip has 105 cell pairing structures (CPSs) with a microslit (2.1 ± 0.3 μm wide) in the microchannel with a 500 μm width and a 28 μm depth. The CPS is designed to achieve a cell pair through the microslit by hydrodynamic force. The heterogeneous cell pairs are made by chance when a mixed suspension of the two types of cells is injected. Except for the microslit, the paired cells are separated with a 136 μm long separation wall to avoid unwanted cell-cell contact bypassing the microslit. The solution is basically flowed by gravity. 978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001 1329 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences 27-31 October 2013, Freiburg, Germany Fig. 2: The cell fusion system used in this study. (A) Photo, top view, and detailed view are illustrated. The PDMS microfluidic chip is confined in a 35 mm culture dish. CPS: cell paring structure. (B) Image of a CPS. Arrow head: microslit, Pi: support pillar, Po: trap pocket, Wa: separation wall. (C) Overlaid image of a achieved heterogeneous cell pair of CgNr3T3 (upper) and Nr3T3 (lower) by centrifuge-driven flow. Bars = 50 μm. EXPERIMENTAL To monitor the cell fusion and subsequent nuclear behavior, two cell lines named CgNr3T3 (cytoplasm-green and nucleus-red) and Nr3T3 (nucleus-red) were established from NIH3T3 cells by stable transfection of EGFP and H2B- mCherry expression vectors, respectively. After applying 10-40 uL of cell suspension of CgNr3T3 and Nr3T3 with 2×106/mL into the inlet, the microfluidic device was subjected to centrifuge at 60×g for 1 min to accelerate the cell trapping process. Then, inlet was rinsed twice and filled with 40 uL of the culture medium, and the microfluidic device was set on a timelapse microscopy chamber under a condition of 100% humidity, 5% CO2, and 36 °C. Then, 4 μL of Sendai virus envelope (HVJ-E) (Ishihara Sangyo, Japan) suspension was added to the culture medium in the inlet. From the addition of HVJ-E, 5 min-interval timelapse observation was performed for 5 h. RESULTS AND DISCUSSION We carried out 13 independent experiments, and selected a total of 66 heterogeneous cell pairs of CgNr3T3 and Nr3T3 for timelapse observation. The cell fusion was detected by transfer of EGFP from the CgNr3T3 cell to the Nr3T3 cell. In 14 pairs (21% of 66 pairs), the cells successfully fused through the microslit in 5 h. The other 52 cell pairs failed to fuse, mainly due to cell migration, lack of cell-cell contact, or cell death. In 7 of the 14 fused pairs, we observed suc- cessful retention of the nuclei: both of the nuclei in the fused cells did not pass through the microslit for 5 h. In these cas- es, the most cells were in spread shapes, while a few cells took round shapes in short periods. (Fig. 3A, C). In contrast, in the other 7 fused pairs, the nucleus of one of the cells migrated to its fusion partner through the microslit. With one ex- ception, the migration of the nuclei started from round cells (Fig. 3B, D). These two events—cell rounding-up and nucle- ar migration—seem to be strongly correlated, because both of the events were relatively rare in terms of time periods in which they occurred. In other words, these data suggest that spread cells are much less susceptible to nuclear migration passing through the microslit compared with round cells. CONCLUSION We succeeded in induction of cell fusion between adhered cells through the microslit by using a newly developed mi- crofluidic device, and found the importance of cell adhesion/spreading on the culture substrate for prevention of nuclear mixing. Our technique would be a unique way to study functional cytoplasmic transfer without nuclear mixing, and also would be a promising method for production of cellular resources applicable to regenerative medicine as well as basic cell biology. ACKNOWLEDGEMENTS We thank for Laboratory for Animal Resources and Genetic Engineering, Center for Developmental Biology, RIKEN Kobe for providing the pcDNA3-H2B-EGFP and the pcDNA3-H2B-mCherry vectors. 1330 Fig. 3: Cellular behavior after cell fusion through the microslit. (A) Typical timelapse images of a cell pair of CgNr3T3 (upper) and Nr3T3 (lower), in which both nuclei in the pair did not pass through the microslit. The cell fusion was judged by the transfer of EGFP (0 min). (B) Typical timelapse images of a cell pair of CgNr3T3 (upper) and Nr3T3 (lower), in which one nucleus passed through the microslit (20 min). (C) Position of nuclei in the fused cell pairs in which both nuclei did not pass through the microslit. Each nucleus was randomly selected from the two nuclei of the cell pair. (D) Position of the nuclei which passed through the microslit toward the fusion partner cell. In C and D, the nuclear position (d) was traced as a distance from the separation wall to the middle point of the nu- clei, and the blue and red lines represent the cell shapes: spread and round shapes, respectively. REFERENCES [1] K. Takahashi and S. Yamanaka, Cell, vol. 126, no. 4, pp. 663-676, Aug.2006 [2] H. M. Blau, C. P. Chiu and C. Webster, Cell, vol. 32, no. 4, pp. 1171-1180, Apr.1983 [3] M. Tada, Y. Takahama, K. Abe, N. Nakatsuji and T. Tada, Current Biology, vol. 11, no. 19, pp. 1553-1558, Oct.2001 [4] Y. Kimura, M. Gel, B. Techaumnat, H. Oana, H. Kotera and M. Washizu, Electrophoresis, vol. 32, no. 18, pp. 2496-2501, Sep.2011. [5] N. Sasaki, J. S. Gong, M. Sakuragi, K. Hosokawa, M. Maeda and Y. Ito, Japanese Journal of Applied Physics, vol. 51, no. 3, pp. 030206.1-030206.3, Mar. 2012 [6] K. Wada, K. Itoga, T. Okano, S. Yonemura and H. Sasaki, Development, vol. 138, no. 18, pp. 3907-3914, Sep.2011 CONTACT *K.
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