• Optimization of Transfection for Chinese Hamster Ovary (CHO) Cell Line
  • Sana Pourtabatabaei,1 Samaneh Ghanbari Mehrandooei,2 Fatemeh Davami,3,*
    1. Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran
    2. Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
    3. Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran


  • Introduction: Chinese hamster ovary (CHO) cells are the most preferred host cell system for the production of recombinant therapeutic proteins with human-like post-translational modifications. However, performing a transfection process with a high transfection efficiency and the low cytotoxic effect is a barrier against generating recombinant CHO cells. Transfection which is the process of delivering exogenous nucleic acids into eukaryotic cells is an important analytical tool for the study of the function of genes and gene products in cells. Here, we developed an efficient chemical transfection method for CHO-k1 cells by optimizing different parameters such as the presence of serum, incubation time, amount of DNA, a ratio of DNA/transfection reagent, and seeding density of cells.
  • Methods: 2.1. The expression vector and Cells Culture A DNA plasmid with a size of about 9.6 kb and harboring a protein expression cassette of GFP-2A-Puromycin was used for transfecting cells. The CHO-K1 cells were maintained in Dulbecco's Modified Eagle's Medium/Ham's Nutrient Mixture F-12 (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco) and 10% CO2 at 37°C incubator. 2.2. Transfection Process The day before transfection, approximately 1×105 cells were cultured in each well of a 24-well tissue culture plate. The following procedure was used for transfecting the cells: 1. For each transfection sample, the complexes were prepared as follows. At first, solution A was prepared by diluting 0.1-0.5 μg of DNA into 25 μl of the serum-free, antibiotic-free medium. After that, solution B was prepared by diluting DNAfectin™2100 (ABM good, Canada) in 25 μl serum-free, antibiotic-free medium with a DNA/reagent ratio between 1:3 and 1:8. Then, solutions A and B were incubated at room temperature for 5 minutes. 2. In the next step, the solutions were combined and mix gently to ensure uniform distribution and incubate for 20 minutes at room temperature. 3. After that, about 450 μl of the antibiotic-free medium was added to DNAfectin™ 2100-DNA complex and mixed. Then, the growth medium was removed from the cells and the DNAfectin™2100-DNA solution was added to the well-containing cells. 4. After 5-48 hours, remove the transfection solution and add 0.5 ml of the appropriate growth medium. 2.3. GFP expression analysis Transfection efficiency was measured based on the percentage of the population of GFP positive cells using a cell flow cytometer (Cyflow) 48-72 hours post-transfection. Sample preparation of flow cytometry analysis was done by washing 1 ×106 cells with PBS and re-suspended in 1 mL PBS containing 1% FBS.
  • Results: 3.1. Establishing an efficient transfection process In previous studies, the DNA plasmids were transiently transfected into CHO-k1 and the low number of the surviving cells displayed GFP. However, by optimizing different factors, the maximum transfection efficiency of about 85.5% was achieved in the presence of FBS, with an incubation time of 48 hours, and a DNA/reagent ratio of about 1:8. Such high transfection efficiency strongly reduces the time, cost, and amount of materials required to generate transfected cells. In the first optimization strategy, the effect of serum was examined by transfecting the cells by adding the serum to the complexes which were produced using 1:4 and 1:5 DNA/reagent ratio, and it was found that presence of serum improves the transfection efficiency from 16.85% to 19.11% and 15.5% to 27.27%. After that, the effect of incubation time was assessed by incubating DNA/complexes with the cells for 48 hours; and it was understood that the 48 hours incubation in the presence of serum boosts the transfection efficiency from 27.27% to 34.76%. In the second optimization strategy, the variable parameters were the amount of DNA and ratio of DNA/reagent. Therefore, we tried 1:6, 1:7, 1:8, and 1:9 DNA/reagent ratios. It was found that 1:8 DNA/reagent ratios with a DNA amount of 0.5 μg lead to a transfection efficiency of about 85.5%, but the 1:9 DNA/reagent ratio had a transfection efficiency of about 74%.
  • Conclusion: Improving the transfection efficiency, for different applications, such as cell line development has always been a challenge for research and industrial manufacturing. In this study, we develop an efficient method for transfection of more than 85% of cells with DNA plasmid. The results demonstrated that the presence of serum and 48 hours of incubation significantly boost the efficiency of cells.
  • Keywords: Transfection optimization, CHO cells, Transfection efficiency, Plasmid DNA