Mirus Bio LLC
Sponsored by Mirus Bio LLC Published in September 2010 2010 (p.9) DOI: 10.2144/000113524 Sponsored,vendor-submitted protocol
Transfection efficiencies are affected by many parameters including cell density at the time of transfection, DNA concentration and reagent dosing. Based on extensive testing performed during product development and the data presented herein our starting recommendations include an initial cell density of 0.5 - 1.0 x 106 cells/ml, 1 g of DNA per milliliter of culture volume and TransIT-PRO:PRO Boost Reagent:DNA ratio of 1:0.5:1. Optimization of transfection parameters will maximize transfection efficiency and reduce the impact of variables such as differences in cell lineage, handling and media formulation.
Cell culture growth medium has a considerable influence on transfection efficiency. To further examine these effects, suspension CHO cells were adapted to five representative growth media, transfected using multiple methods and analyzed for efficiency by luciferase reporter assay or IgG titer. The media formulation impacted transfection efficiencies by greater than 10-fold within a given transfection methodology. In addition, not all commercially available transfection reagents had broad spectrum media compatibility. A 10-fold increase in IgG yield was obtained when the TransIT-PRO Transfection Kit was used in conjunction with suspension CHO cells adapted to BD-SelectTM CD1000 medium. Notably, in many media formulations (BD SelectTM CHO, ProCHOTM5 and PowerCHO2 CD) the TransIT-PRO Transfection kit provided detectable levels of antibody production, significantly outperformed 25 kDa linear PEI and FreeStyleTM Max Transfection Reagent.
The TransIT-PRO Transfection Kit uses animal origin free components designed for high and reproducible protein yield in suspension CHO cells. Transfections using the TransIT-PRO Transfection Kit are linear in culture volumes ranging from 4-400 milliliters in shake or spinner vessels. Competitor benchmarking experiments were performed utilizing the FreeStyle Max expression system and multiple transfection methods. Western blot and ELISA detecting human IgG levels demonstrate that the TransIT-PRO Transfection Kit and FreeStyle Max Transfection Reagent perform similarly, outperforming 25 kDa linear PEI by 5-fold. The results demonstrate that maximum levels of transient expression are achieved when an effective delivery reagent is combined with optimized transfection parameters and growth conditions.