Background and novelty
Transient transfection of HEK293 cells has traditionally been employed for protein expression for research and discovery purposes. Recently, it has gained wide interest in gene therapy applications where it serves as the powerhouse of viral vector productions. Many factors could affect the production of recombinant proteins or viral vectors, and cell culture media plays a crucial role in process run time, productivity, and product quality.
Here, we focus on the development of a prototype medium for HEK293 transient transfection that supports both high cell growth and transfection efficiency.
Experimental approach
Study workflow overview
The performance of two HyClone™ HEK293 transfection media prototypes developed previously were first evaluated based on peak viable cell density (VCD) reached and transfection efficiency. A commercially available HEK293 cell line was used for transfection.
A new transfection medium prototype, 293PT, was further developed using the mixture designs in design of experiments (DoE) based on the first two prototypes. Further analyses of 293PT were conducted to identify the component(s) at play.
Lastly, the preliminary performance of 293PT was evaluated with three HEK293 cell lines and compared with three commercial transfection media. Conclusions drawn from this study would benefit future optimization and development.
Transfection assay method
Transfection assays in this study were all performed with GFP-expressing plasmid first complexed with Polyplus PEIpro® (transfection agent), then the complex was delivered to cell cultures. Transfection efficiencies at 72 h post-transfection were measured and calculated by flow cytometer BD FACSCelesta™.
Fig 1. New transfection medium 293PT development workflow overview.
Results
New prototype exploration: mixture DoE design
The performance of two HyClone™ HEK293 transfection media prototypes was first evaluated based on cell growth and transfection efficiency (Fig 2). A mixture DoE design was employed to develop the next-generation transfection medium prototype (Fig 3). A concentration-dependent effect was observed: the higher the % of prototype 2 present in the mixture, the lower the transfection efficiency.
Fig 2. The peak viable cell density (left) and maximum transfection efficiency (right) of two HyClone™ HEK293 transfection media prototypes were evaluated. The two prototypes demonstrated opposite performance.
Fig 3. A mixture DoE design was employed to develop a new prototype. The results show a concentration-dependent effect: the higher the % of prototype 2 present in the mixture, the lower the transfection efficiency.
293PT development: key component investigation
A key component “X” in prototype 2 was further identified and tested to demonstrate its effect in transfection media (Fig 4). High concentration of “X” supported growth but had a detrimental effect on transfection efficiency. To our knowledge, “X” has not been previously reported to affect transfection efficiency at higher concentrations. The working concentration of “X” was further evaluated, and prototype 293PT was then developed (Fig 5).
Fig 4. The key component “X” was identified and its effects on transfection efficiency and growth were investigated. “X” supported growth but had a detrimental effect on transfection efficiency at higher concentration.
Fig 5. The working concentration of “X” was explored to develop 293PT. Three concentrations of “X” were studied. Con. 1 was chosen, and the new medium prototype 293PT was established for further evaluations.
293PT preliminary performance
The performance of 293PT was first assessed using three different HEK293 cell lines and compared with three commercially available transfection media from different suppliers (Fig 6).
Different media performed differently with the HEK293 cell lines. 293PT generally supported cell growth and exhibited competitive transfection efficiency when compared with other commercial media.
Commercial media used in this study:
- Comm. 1: Expi293™ Expression Media (Thermo Fisher Scientific)
- Comm. 2: OPM-293 CD03 Medium (OPM Biosciences)
- Comm. 3: BalanCD™ HEK293 (Fujifilm Irvine Scientific)
HEK293 cell lines used in this study:
- Cell line 1: Freestyle™ 293-F (Thermo Fisher Scientific)
- Cell line 2: Expi293F™ (Thermo Fisher Scientific)
- Cell line 3: HEK-293.2sus (ATCC)
Fig 6. Comprehensive comparison of 293PT with three commercially available HEK293 transfection media, using three different HEK293 cell lines*. See main text for the identities of commercial media and cell lines used.
*All studies were done at 37°C, 5% CO2, 80% humidity, and at 120 RPM (25 mm throw).
Future direction
Preliminary performance evaluations demonstrated the potential of 293PT. However, further optimization is desired to develop the next generation HEK293 transfection medium. Furthermore, the medium would need to be proven to support transient transfection and gene therapy production.
Summary
We developed a new HEK293 transfection medium prototype, 293PT, for future transient protein expression and gene therapy applications. Key discoveries:
- The study identified a key component “X” and its effect on cell growth and transfection efficiency. High concentration of “X” supported growth but had a detrimental effect on transfection efficiency. These effects haven’t been reported before, to our knowledge.
- Comprehensive comparison of 293PT with commercially available HEK293 transfection media in different cell lines illustrated the strengths and the weaknesses of the current prototype.