Nanoscale characterization coupled to multi-parametric optimization of Hi5 cell transient gene expression

Area of Science:

• Biotechnology
• Molecular Biology
• Cell Biology

Background:

• Polyethylenimine (PEI)-based transient gene expression (TGE) is established for mammalian cells but less explored in insect cell lines.
• Hi5 insect cells offer potential for rapid recombinant protein production.

Purpose of the Study:

• To develop a fast and robust TGE methodology for suspension Hi5 cells.
• To optimize DNA:PEI polyplex formation and transfection conditions for efficient protein production.

Main Methods:

• Investigated DNA:PEI polyplex characteristics in various solutions using cryo-electron microscopy (cryo-EM).
• Employed advanced statistical designs for optimizing TGE parameters, including cell density, DNA, and PEI concentrations.
• Utilized enhanced green fluorescent protein (eGFP) as a model protein to assess transfection efficiency and productivity.

Main Results:

• Minimal complexing time (under 1 minute) in 150 mM NaCl yielded highest transfection efficiency.
• Optimal DNA:PEI polyplex size for transfection was determined to be 300-400 nm.
• Achieved 60% transfection and 0.8 μg/10^6 cells·day specific productivity under optimized conditions (1.5 × 10^6 cells/mL, 2.1 μg/mL DNA, 9.3 μg/mL PEI).

Conclusions:

• A robust and efficient TGE protocol for Hi5 cells was successfully developed.
• This baculovirus-free method provides a valuable approach for rapid recombinant protein production.
• The optimized protocol demonstrates significant potential for diverse protein expression applications.