PEI-Mediated Transient Gene Expression in CHO Cells.

Area of Science:

• Biotechnology
• Cell Biology
• Molecular Biology

Background:

• Transient transfection is crucial for recombinant protein production in mammalian cells.
• Optimizing transfection protocols for suspension-adapted cells like CHO-DG44 is essential for scalability.
• Existing methods may lack efficiency or scalability for diverse applications.

Purpose of the Study:

• To develop and describe a robust, scalable method for polyethyleneimine (PEI)-mediated transient transfection of suspension-adapted CHO-DG44 cells.
• To optimize transfection parameters for efficient protein expression across a wide volume range (2 mL to 2 L).
• To provide practical guidelines for implementing the method in research and bioproduction settings.

Main Methods:

• Transfection of CHO-DG44 cells at high density (5 × 10^6 cells/mL) using direct addition of plasmid DNA (pDNA) and PEI.
• Incubation at 31°C with orbital shaking for optimal gene expression.
• Utilized specific concentrations: 0.3 mg/L coding pDNA, 2.7 mg/L filler DNA, and 15 mg/L PEI.
• Production phase conducted at 31°C with 0.25% N,N-dimethylacetamide (DMA).

Main Results:

• Successfully demonstrated PEI-mediated transient transfection for protein expression in suspension CHO-DG44 cells.
• The method is validated for scales ranging from 2 mL to 2 L.
• Detailed information on culture vessels, working volumes, and shaking speeds is provided for different scales.

Conclusions:

• The described method offers a scalable and efficient approach for transient gene expression in CHO-DG44 cells.
• This protocol facilitates high-level protein production applicable to various research and biomanufacturing scales.
• The optimization of transfection conditions and inclusion of practical guidance enhance its utility.