Optimize Transient Transfection Solution for Instantaneous Enhancement of Transfection Efficiency!

Transient transfection is a specialized technique for introducing exogenous recombinant DNA into eukaryotic cells to achieve high-level expression of target genes. With the ongoing advancements in molecular biology and cell biology research, transient transfection has evolved into a fundamental method for assessing the efficacy, mechanism of action, toxicological responses, pharmacokinetic characteristics, and drug interactions of early-stage investigational drugs in innovative drug development.

I. Transient Transfection Technique

Transient transfection refers to the method of introducing pre-constructed plasmids into mammalian cells through chemical, biological, or physical means. The exogenous genes introduced do not integrate into the host cell genome but only transiently exist in the nucleus and cytoplasm of the host cell. After transfection, the high-copy exogenous genes undergo transcription and translation, resulting in the desired target proteins. Compared to stable transfection, transient transfection offers advantages of rapidity, flexibility, and high efficiency, meeting the demands for short production cycles and large expression levels of recombinant proteins. CHO (Chinese hamster ovary) cells, a commonly used cell line for expressing recombinant proteins, are often utilized as carriers for transient transfection.

II. Important Parameters for Transfection

01 Cellular status

① Cell density: Typically, CHO-S cells are transfected during the mid-logarithmic growth phase, which is more conducive to recombinant protein expression of plasmid DNA in the cell nucleus.

② Cell viability: Cell viability, the most direct measure of cell health, should be ensured to be above 90-95% before conducting cell transfection experiments.

③ Cell passage number: The passage number of cells during transfection can affect transfection efficiency and gene expression levels. Generally, higher-passage cells may exhibit decreased transfection efficiency and expression levels compared to lower-passage cells. This is because cells undergo a series of division and replication during passage, leading to increased genomic instability, cell cycle dysregulation, chromosomal abnormalities, etc. Cell aging status can typically be assessed by evaluating indicators such as the nucleotide ratio within cells, thereby selecting cells at an appropriate passage for transfection experiments.

In summary, cell density, cell viability, and passage number are all important parameters affecting transient transfection. It is necessary to comprehensively consider their effects and strengthen research on their molecular mechanisms to achieve high yield and reproducibility in transfection experiments.

02 Plasmid DNA

Chemical transfection methods are currently the most commonly used transfection techniques. They involve the formation of transfection complexes through electrostatic interactions between positively charged cationic substances (such as PEI) and negatively charged nucleic acids (plasmid DNA), which are then taken up by cells through endocytosis.

For transient transfection, plasmid DNA is one of the most critical factors. The primary quality parameters of plasmids include the A260/A280 ratio (1.8-2.0) and the percentage of supercoiled plasmids (70-90%). The A260/A280 ratio is a commonly used indicator to measure DNA purity. A reasonable A260/A280 ratio indicates no significant protein contamination in the DNA sample. The percentage of supercoiled plasmids indicates the degree of supercoiling of the plasmid DNA. A higher proportion of supercoiled plasmids contributes to improved plasmid stability and transfection efficiency.

If the purity of the plasmid DNA is low and impurities such as salt ions, proteins, metabolites are present, the formation of transfection complexes will be significantly affected, thereby affecting transfection efficiency. In such cases, purification and concentration of the plasmid DNA should be performed before use.

03 Transfection reagents

Currently, the most commonly used chemical transfection reagents in the market are cationic liposomes and cationic polymers such as PEI.

Liposomes, known for their ability to carry large DNA fragments, are versatile for various types of DNA and RNA and are highly efficient in the in vitro gene transfection. However, liposomes themselves can interfere with cellular physiological activities, leading to dysregulated gene expression, causing cellular toxicity, and resulting in significant interference with test results.

PEI is a cationic polymer that can condense DNA molecules through electrostatic interactions, forming nano-complexes (multimers) with a net positive charge. Common types of PEI include linear PEI (LPEI) and branched PEI (BPEI), with molecular weights ranging from 1 to 1600 kDa. The transfection efficiency and cytotoxicity of PEI are mainly influenced by its molecular weight, degree of branching, and cationic charge density. As the molecular weight increases, the transfection efficiency gradually improves, but at the same time, cytotoxicity also significantly increases. Compared to branched PEI molecules, linear PEI molecules have lower cytotoxicity and stronger DNA condensation ability, leading to an increase in the average particle size of the complexes, thereby increasing the chances of cellular contact with the complexes and ultimately improving transfection efficiency.

PEI is the most widely used transfection reagent in recombinant protein or virus production, especially in large-scale applications. It is cost-effective, exhibits high transfection efficiency and expression levels, and has become the gold standard in the field of transient transfection.

04 Transient transfection process parameters

The parameters commonly used in transient transfection generally include DNA dosage, PEI/DNA ratio, incubation time after mixing DNA and PEI, and transfection duration (the time of contact between the complex and the cells).

① Different DNA concentrations ultimately affect the number of exogenous plasmid copies obtained by individual cells after transfection, thereby influencing protein expression.

② The DNA/PEI ratio greatly affects the characteristics of the complexes, such as their particle size, structure, and surface charge. An appropriate DNA/PEI ratio can facilitate complex formation and cellular uptake, thereby enhancing transfection efficiency. Typically, the recommended mass ratio of DNA to PEI ranges from 1:1 to 1:6, but the optimal ratio still needs to be optimized according to specific experiments.

③ The incubation time affects the binding and interaction between DNA and PEI molecules. Proper incubation time promotes the formation and maturation of complexes, typically ranging from 5 min to 20 min. During incubation, gentle mixing and shaking can promote the interaction between DNA and PEI.

④ Different transfection durations affect the contact time between cells and exogenous DNA/PEI complexes, which also influences the number of exogenous genes engulfed by the cells.

To achieve high transfection efficiency and protein expression levels, it is necessary to carefully consider and optimize these transfection conditions. Through a series of experiments and parameter optimizations, the optimal transfection parameters can be determined to meet the requirements of the specific experiment.

05 Culture medium formulations for transient transfection

The culture medium is the most fundamental element for transient transfection. Medium that supports optimal cell growth often contains compounds such as serum, growth factors, and peptides. However, the abundance of negatively charged proteins in serum can interfere with the adsorption of nucleic acids by cationic liposomes, greatly affecting transfection efficiency.

On the other hand, medium suitable for transient transfection typically includes components that promote cell aggregation and the formation of multicellular aggregates. This aggregated state is advantageous for transient transfection because cell aggregation enhances the contact and uptake of complexes by cells, thereby increasing transfection efficiency. However, compared to medium that supports growth in a single-cell form, medium in an aggregated state often does not support the maintenance of high cell density. This is because cell aggregation can lead to competitive growth among cells and nutrient limitations, restricting the increase in cell numbers. Therefore, transient transfection may need to be performed at lower cell densities to ensure sufficient contact between cells and complexes.

Therefore, the composition of the medium is closely related to the growth status of cells and transient transfection efficiency. To achieve high transfection efficiency and maintain cell density, adjustments and optimizations may be needed in the medium formulation to balance between cell growth and transfection efficiency.

III. Eminence-CHO Transient Transfection Platform

Leveraging extensive experience in CHO cell culture medium formulation, Eminence has successfully developed a highly efficient transient transfection solution tailored for ExpiCHO-S cells. This solution comprises both medium and process parameters, ensuring outstanding transfection efficiency while achieving breakthroughs in maintaining cell viability and high cell density. By precisely controlling key factors such as DNA dosage, complex formation time, and transfection duration, our process enables efficient introduction of exogenous genes and stable protein expression. The Eminence-CHO transient transfection platform offers well-established experimental conditions and proprietary medium formulations, enabling high transfection efficiency and expression levels. Here are the main advantages of this platform:

• Eminence-CHO medium series can be directly utilized for transient transfection experiments without the need for cell adaption, particularly suitable for transient expression of ExpiCHO-S cells;

• The doubling time is approximately 17 h, with uniform cell size and no clumping.

• Products: EmCD CHO-S 203 Medium and Feed

IV. Transient Transfection Steps (example for EmCD CHO-S 203 medium)

Tips: Cells should be fully adapted to EmCD CHO-S 203 medium before transfection and passaged at regular density.

Step 1: Preparation

1. Cell preparation

a) One day before transfection, adjust cell density to 2.2-2.5×10⁶ cells/mL using EmCD CHO-S 203 medium + 6 mM glutamine.

Step 2: Transfection

1. Cell density confirmation

a) On the day of transfection, adjust cell density to 5.5-6.0×10⁶ cells/mL using fresh EmCD CHO-S 203 medium + 6 mM glutamine. Prepare 28 mL of cell suspension in a 125-mL shake flask.

2. Preparation of transfection reagents

a) Allow plasmid DNA to stand at room temperature for at least 5 min.

b) Prepare transfection complexes according to Table 1 (example for a 30-mL transfection system)

Transfection complex
Complex A (1 mL)
EmCD CHO-S 203 Medium	1 mL
DNA	30 μg
Complex B (1 mL)
EmCD CHO-S 203 Medium	1 mL
Polyethylenimine (PEI, Polysciences, Cat#: 24765)	90 μg
Note: Complexes A/B should be allowed to stand at room temperature for 5 min before final mixing.

Table 1 Recommended Preparation Method for a 30-mL Transfection System

3.  Add complex B to complex A (with a total volume of 2 mL), and mix gently by vortexing or gentle pipetting 2-3 times.  

4.  Incubate the mixture at room temperature for 5 min.

5.  Slowly add the mixture to the prepared cells (Step 2, 28 mL), and gently shake the cells during addition to ensure uniform mixing.

6.  Place the cells on a shaker: 37°C, 5% CO₂, 100 rpm ± 5 (axis distance: 50 mm).

Step 3: Addition of supplements and feeds

1. Directly add the supplements listed in Table 2 after transfection, and mix by gently shaking the shaker flask during addition.

Name	Amount Added (Initial Volume %)	Example for a 30-mL Transfection System
EmCD HEK293 Plus Supplement 1	0.5%	0.15 mL

Table 2 Recommended Method for Adding Supplements

2. On Days 1-10 after transfection, supplement with the feed listed in Table 3.

Name	Amount Added (Initial Volume %)	Example for a 30-mL Transfection System	Feeding Timepoint
EmACF CHO 203 Feed	8%	2.4 mL	D1&D5

Table 3 Recommended Method for Adding Feeds

3.  Lower the temperature to 32°C on the first day after transfection.

4.  Maintain glucose concentration above 2 g/L during the culture process.

Step 4: Protein harvest

1.  Most protein expression levels reach their peak 7-10 days after transfection. The end point of cultivation can be adjusted according to the customer's original process.

2.  Optimization of the culture process helps to increase protein yield.

We look forward to you to experience the advanced CHO cell transient transfection platform from Eminence, along with optimized basal and feed media! You can contact us through the following channels:

1. Contact the regional sales representative;

2. Send your requirements via email to vip@eminencebio.com.