title: Effects of ROCK inhibitor Y-27632 on cell fusion through a microslit.
abstract: We previously reported a direct cytoplasmic transfer method using a microfluidic device, in which cell fusion was induced through a microslit (slit-through-fusion) by the Sendai virus envelope (HVJ-E) to prevent nuclear mixing. However, the method was impractical due to low efficiency of slit-through-fusion formation and insufficient prevention of nuclear mixing. The purpose of this study was to establish an efficient method for inducing slit-through-fusion without nuclear mixing. We hypothesized that modulation of cytoskeletal component can decrease nuclear migration through the microslit considering its functions. Here we report that supplementation with Y-27632, a specific ROCK inhibitor, significantly enhances cell fusion induction and prevention of nuclear mixing. Supplementation with Y-27632 increased the formation of slit-through-fusion efficiency by more than twofold. Disruption of F-actin by Y-27632 prevented nuclear migration between fused cells through the microslit. These two effects of Y-27632 led to promotion of the slit-through-fusion without nuclear mixing with a 16.5-fold higher frequency compared to our previous method (i.e., cell fusion induction by HVJ-E without supplementation with Y-27632). We also confirmed that mitochondria were successfully transferred to the fusion partner under conditions of Y-27632 supplementation. These findings demonstrate the practicality of our cell fusion system in producing direct cytoplasmic transfer between live cells.
authors.affiliation: Bioengineering Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan. ki-wada@riken.jp.
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Wada
authors.affiliation: Bioengineering Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Hosokawa
authors.affiliation: Nano Medical Engineering Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Ito
authors.affiliation: Bioengineering Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
authors.forename: Mizuo
authors.initials: M
authors.lastname: Maeda
mesh_terms: D000577:Amides / Q000378:metabolism*; D000818:Animals; D002459:Cell Fusion / Q000379:methods*; D004791:Enzyme Inhibitors / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects*; D056656:Lab-On-A-Chip Devices; D051379:Mice; D041681:NIH 3T3 Cells; D011725:Pyridines / Q000378:metabolism*
chemical_list: D000577:Amides; D004791:Enzyme Inhibitors; D011725:Pyridines; C108830:Y 27632
keywords: F-actin; Y-27632; cell fusion; cytoplasmic transfer; microfluidic device; microslit