Optimizing mRNA transfection on a high-definition electroporation microelectrode array results in 98% efficiency and...

Area of Science:

• Cell biology
• Biotechnology
• Bioengineering

Background:

• Spatially resolved intracellular delivery of molecules is crucial for accelerating biological discovery.
• High-Definition Electroporation (HD-EP) utilizes microelectrode arrays for targeted cell electrotransfection.
• Optimizing electroporation conditions is key to achieving high transfection efficiency.

Purpose of the Study:

• To optimize HD-EP conditions for high-efficiency mRNA transfection in primary fibroblasts.
• To demonstrate dosage-controlled mRNA delivery and protein expression.
• To showcase spatially resolved, sequential transfection capabilities for scalable screening.

Main Methods:

• Utilized a Design of Experiments framework to optimize five electrical pulse parameters for HD-EP.
• Transfected primary fibroblasts with mCherry-encoding mRNA using optimized HD-EP conditions.
• Employed single-electrode addressability for sequential transfection of multiple mRNAs.

Main Results:

• Achieved 98% transfection efficiency with no observed cell death, the highest reported for electroporation.
• Demonstrated modulation of fluorescence intensity by varying pulse number, indicating dosage control.
• Successfully performed spatially resolved, sequential transfection of three distinct mRNAs.

Conclusions:

• Optimized HD-EP provides a highly efficient and scalable method for intracellular delivery.
• This technique enables precise control over mRNA dosage and protein expression.
• The platform is adaptable for developing large-scale screening platforms for mRNA variants.