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

Area of Science:

• Biotechnology and Biomedical Engineering
• Cell Biology and Diabetes Research

Background:

• Efficiently delivering large plasmids into insulin-producing beta cells is crucial for diabetes research.
• Conventional transfection methods often exhibit high cytotoxicity or low efficiency, hindering research outcomes.
• Nanostraw electroporation presents a gentler alternative, promising high transfection efficiency and cell viability.

Purpose of the Study:

• To optimize nanostraw electroporation for efficient transfection of clonal beta cells.
• To investigate the impact of voltage, cell density, and cargo solution conductivity on transfection efficiency.
• To compare nanostraw electroporation with nanopore electroporation for beta cell transfection.

Main Methods:

• Utilized GFP-encoding DNA plasmids and intercalating dyes for immediate transfection analysis.
• Conducted experiments varying voltage, cell density, and cargo buffer conductivity.
• Employed simulations to model the effect of buffer conductivity on voltage drop during electroporation.

Main Results:

• MilliQ water as the cargo buffer demonstrated the highest transfection efficiency in both experimental and simulation results.
• Optimized cell density is critical to maximize cell-nanostraw interface and prevent cell stacking.
• While nanostraws injected more plasmid DNA, nanopores resulted in higher GFP fluorescence and cell viability post-transfection.

Conclusions:

• Nanostraw electroporation can be optimized for efficient beta cell transfection by adjusting parameters like buffer conductivity and cell density.
• High plasmid injection volumes via nanostraws may negatively impact cell proliferation and viability.
• Nanopore electroporation emerges as a viable, simpler alternative for efficient and gentle clonal beta cell transfection.