Achieving efficient clonal beta cells transfection using nanostraw/nanopore-assisted electroporation

Abstract

The prospect of being able to efficiently inject large plasmids in insulin-producing beta cells is very attractive for diabetes research. However, conventional transfection methods suffer from high cytotoxicity or low transfection efficiency, which negatively affect their outcome. In contrast, nanostraw electroporation is a gentle method that can provide a high transfection efficiency while maintaining high cell viability. While nanostraw electroporation has gone through some method optimization in the past, such as tuning the pulse frequency, amplitude, and duration, the effect of other parameters has not been thoroughly investigated. Here, we demonstrate efficient transfection of clonal beta cells and investigate the effect of voltage at a fixed inter-electrode distance, cell density, and cargo solution conductivity on transfection efficiency. We used GFP-encoding DNA plasmids stained with an intercalating dye to enable immediate analysis and assessment of the electrophoretic transport of cargo. Moreover, we ran simulations to assess how cargo buffer conductivity impacts the transfection efficiency by affecting the voltage drop on the nanostraws and cell membrane during electroporation. Both experiments and simulations show that MilliQ water as the cargo buffer yields the best transfection efficiency. We also show that the cell density should be adjusted to maximize the number of cells interfacing the nanostraws and avoid cell stacking. Finally, we compared the transfection efficiency when using nanostraws and nanopores. Whereas the amount of GFP plasmids injected using nanostraws is larger than for nanopores, the outcome in terms of GFP fluorescence 48 h after transfection was worse than for nanopores. Moreover, when using nanostraws, fewer cells were found on the substrate 48 h after transfection compared to when using nanopores. This suggests that injecting substantial amounts of plasmids in cells can affect their proliferation and/or viability, and that nanopore electroporation, as a simpler method, is an interesting alternative to nanostraws in achieving efficient and gentle clonal beta cell transfection.