Investigation and circumvention of transfection inhibition by ferric ammonium citrate in serum-free media for Chinese hamster ovary cells

Biotechnol Prog. 2020 May;36(3):e2954. doi: 10.1002/btpr.2954. Epub 2019 Dec 30.


While reliable transfection methods are essential for Chinese hamster ovary (CHO) cell line engineering, reduced transfection efficiencies have been observed in several commercially prepared media. In this study, we aimed to assess common media additives that impede efficiency mediated by three chemical transfection agents: liposomal-based (Lipofectamine 2000), polymer-based (TransIT-X2), and lipopolyplex-based (TransIT-PRO). An in-house GFP-expressing vector and serum-free medium (BCR-F12: developed for the purposes of this study) were used to analyze transient transfection efficiencies of three CHO cell lines (CHO-K1, DG44, DP12). Compared to a selection of commercially available media, BCR-F12 displayed challenges associated with transfection in vendor-prepared formulations, with no detection when liposomal-based methods were used, reduced (<3%) efficiency observed when polymer-based methods were used and only limited efficiency (25%) with lipopolyplexes. Following a stepwise removal protocol, ferric ammonium citrate (FAC) was identified as the critical factor impeding transfection, with transfection enabled with the liposomal- and polymer-based methods and a 1.3- to 7-fold increased lipopolyplex efficiency observed in all cell lines in FAC-depleted media (-FAC), although lower viabilities were observed. Subsequent early addition of FAC (0.5-5 hr post-transfection) revealed 0.5 hr post-transfection as the optimal time to supplement in order to achieve transfection efficiencies similar to -FAC medium while retaining optimal cellular viabilities. In conclusion, FAC was observed to interfere with DNA transfection acting at early stages in all transfection agents and all cell lines studied, and a practical strategy to circumvent this problem is suggested.

Keywords: cationic polymer; ferric ammonium citrate (FAC); lipopolyplexes; liposomes; transfection.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CHO Cells / drug effects*
  • Cell Survival / drug effects
  • Cricetulus
  • Culture Media, Serum-Free / chemistry
  • Culture Media, Serum-Free / pharmacology*
  • Ferric Compounds / chemistry
  • Ferric Compounds / pharmacology*
  • Liposomes / chemistry
  • Liposomes / pharmacology
  • Quaternary Ammonium Compounds / chemistry
  • Quaternary Ammonium Compounds / pharmacology*
  • Transfection / methods*


  • Culture Media, Serum-Free
  • Ferric Compounds
  • Liposomes
  • Quaternary Ammonium Compounds
  • ferric ammonium citrate