Published: 23 oct. 2024(Updated: 23 oct. 2024)

•9 min

•9 min read

•Kayla Spivey, Rui de Paula, Mike Lesch•5•0

Optimizing HEK293 cell cultures for gene therapy applications: the role of incubator flexibility

Gene therapy is a promising approach for treating various genetic disorders and diseases. A critical component of gene therapy is the production of viral vectors, which serve as delivery vehicles for therapeutic genes. Human Embryonic Kidney 293 (HEK293) cells have become a widely used platform for viral vector production due to their efficiency in transfection and ability to support viral replication. However, optimizing HEK293 cell cultures for large-scale production of viral vectors remains a challenge in making gene therapies more accessible and cost-effective.

This article summarizes key concepts discussed in a webinar by Rui de Paula, Scientist and Instructor at the Jefferson Institute for Bioprocessing (JIB), and Mike Lesch, Regional Sales Manager at INFORS HT. The webinar focused on the role of incubator flexibility in optimizing HEK293 cell cultures for gene therapy applications, particularly in transitioning from adherent to suspension culture systems.

Viral vectors in gene therapy

One of the Gene Therapy modalities uses viral vectors to deliver therapeutic genes to target cells. This can be achieved through two main approaches: in vivo (direct injection of viral particles into the patient) or ex vivo (removal of patient cells, genetic modification in the lab, and reinfusion). Both methods require the production of viral particles that are both technically and economically viable. The webinar discussed viral vectors used in gene therapy, mentioning adenoviruses, adeno-associated viruses, and lentiviruses as examples. Adenoviruses were noted for their prevalence in clinical trials, with 575 trials reported in 2021.

HEK293 cells as a production platform

HEK293 cells are the preferred host for viral vector production due to several factors, including their well-characterized nature for transfection, infection and viral production, extensive knowledge base in literature and industry, and the fact that it is of human origin, which facilitates compatibility with patient human cell infection for gene therapy.

HEK293 cells as a production platform for viral vector gene therapy:

High transfection efficiency: HEK293 cells readily incorporate foreign DNA, making them ideal for viral vector production
Better compatibility: Human cell lines are often more suitable for producing viral vectors that target human cells
Extensive characterization: HEK293 cells are well-studied and characterized, providing a robust platform for process development

The webinar specifically mentioned using the Gibco 293-F cell line, which is adapted for suspension culture and offers benefits for large-scale production.

Adherent vs. suspension culture systems

Traditionally, HEK293 cells have been cultured in adherent systems using T-flasks or cell stacks. While these systems often yield high product-per-cell ratios (Yp/x), they present significant challenges for large-scale production. These challenges include limited scalability, increased clean room space requirements, higher labor intensity, reduced process control and automation, and specialized operator training requirements. In contrast, suspension culture systems offer several advantages for large-scale production, such as improved scalability, reduced clean room space requirements, increased automation potential, and enhanced process control. To illustrate the difference, 600 cell stack units produce a similar cell mass as a 200-liter suspension bioreactor, highlighting the potential for significant process intensification.

Transitioning to suspension culture: challenges and considerations

Adapting HEK293 cells to suspension culture introduces new variables that require optimization, including agitation rate, aeration, orbital diameter, and shear stress. These parameters either do not exist or are very limited in adherent systems, necessitating careful optimization to maintain cell growth and productivity while minimizing stress on the cells.

Efficiently optimize suspension HEK293 cultures

To efficiently optimize suspension HEK293 cultures, researchers require incubation systems that allow for the simultaneous testing of multiple culture conditions. The INFORS HT Multitron incubator shaker, used in the JIB study, offers several key features, such as multiple independent cabinets with individual parameter control, adjustable set points for temperature, humidity, agitation, and CO₂, and variable orbital diameter configurations. These features enable researchers to efficiently test different combinations of culture parameters, accelerating the optimization process for suspension HEK293 cultures.

Experimental design and methods

The JIB study utilized a comprehensive experimental setup to investigate the effects of three key parameters on HEK293 cell growth and viability: agitation rate, orbital diameter, and relative humidity. The culture medium consisted of Gibco Freestyle 293 Expression Medium supplemented with FBS, heparin (to prevent cell clumping), GlutaMAX, and antibiotics. The cell line used was Gibco 293-F, adapted for suspension culture.

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Experiments were conducted in 1-liter plain bottom vented shake flasks with a 300 mL working volume. Multiple flask positions within each cabinet of the INFORS HT Multitron incubator shaker were tested to ensure uniformity of culture conditions. Analytical methods included cell density and viability measurements using the NucleoCounter NC-202, glucose and lactate analysis with the YSI biochemical analyzer, and osmolality measurements using the OsmoPRO XT.

Results and discussion

The study revealed several key findings regarding the optimization of suspension HEK293 cultures. The optimal culture conditions were an agitation rate of 110 RPM, mm orbital diameter of 25 mm, and % relative humidity of 85%. These conditions, representing the highest levels tested for each variable, yielded the best cell growth performance.

Cell growth results

The optimal conditions yielded the highest specific growth rate and shortest doubling time, crucial factors for efficient viral vector production. All tested conditions maintained high cell viability (97-98%) at the end of the 6-day culture period, indicating that the cells tolerated the range of conditions well.

Specific cell growth and doubling time table JIB webinar.jpg

Regarding specific cell growth and doubling time for HEK293 cell culture, usually a better transfection/infection efficiency is achieved when cells are active and doing cell division at a high rate. That means it is also important to identify optimal and high specific growth rate values to decide the best condition to use in this application. In the executed experiments, the conditions with higher agitation and higher Relative Humidity really showed an increased number for specific growth rate and lower doubling time. Also importantly, no significant differences were observed in culture performance for flasks placed in different positions within the same cabinet, confirming the uniformity of conditions provided by the Multitron incubator.

Implications for viral vector production

The optimization of suspension HEK293 cultures has significant implications for viral vector production in gene therapy applications. Unlike stable producer cell lines used in monoclonal antibody production, viral vector production typically requires transient transfection or infection of HEK293 cells. The timing of this transfection is critical and depends on achieving both high cell density and a high specific growth rate.

The ability to culture HEK293 cells in suspension at high densities with rapid growth rates is crucial for scaling up viral vector production. The optimized conditions identified here can be a starting point for further process development and scale-up studies. Additionally, the multi-cabinet design of the Multitron incubator allows for simultaneous testing of multiple culture conditions, enabling rapid optimization and adaptation of processes for different viral vector types or HEK293 cell sublines.

Incubator design considerations for suspension HEK293 cultures

The study highlighted several key INFORS HT Multitron incubator shaker features that are particularly beneficial for suspension HEK293 culture optimization. The multi-cabinet design with independent control of each cabinet's temperature, CO₂, agitation, and relative humidity allows for simultaneously efficient testing of multiple culture conditions. The adjustable orbital diameter enables fine-tuning of mixing and aeration conditions, which can significantly impact cell growth and metabolism.

INFORS HT Offerings by Scale.jpg

Other notable features include the humidity control system, which uses a separate sterile water reservoir for humidification, reducing contamination risks and simplifying maintenance. The ergonomic design, with features like the slide-out tray with automatic lock/unlock mechanisms, facilitates easy access to cultures for sampling and media additions, improving workflow efficiency. The incubator's design also incorporates contamination mitigation measures, such as rounded interior corners and easily accessible surfaces for cleaning and disinfection.

The touchscreen display with event list functionality is an important process monitoring and validation feature. This allows researchers to track parameter changes, alarms, and door openings. This provides valuable data for troubleshooting and process validation, enhancing the overall reliability and reproducibility of the culture process.

Future directions and challenges

While the optimized suspension culture conditions identified in this study represent a significant step forward, several challenges and areas for further research remain. Translating optimized shake flask conditions to larger-scale bioreactors will require careful consideration of gas transfer, mixing, and shear stress.

Further refinement of culture conditions and process parameters may yield additional improvements in cell growth and viral vector production. Optimizing the interface between upstream cell culture and downstream purification processes will maximize overall process efficiency and yield.

Conclusion

The optimization of suspension HEK293 cell cultures represent a critical step in improving the scalability and cost-effectiveness of viral vector production for gene therapy applications. The study presented in this webinar demonstrates the importance of incubator flexibility in efficiently identifying optimal culture conditions. By leveraging advanced incubation systems like the INFORS HT Multitron, researchers can rapidly optimize key parameters such as agitation, orbital diameter, and humidity to maximize cell growth and prepare for efficient viral vector production.

As gene therapy continues to advance, the development of robust, scalable production processes for viral vectors will play a crucial role in making these innovative treatments more accessible to patients. The insights gained from studies like this one, combined with ongoing research in cell line engineering and bioprocess development, will pave the way for the next generation of gene therapy manufacturing platforms.

On-demand webinar

Perfecting HEK293 cells for gene therapy: the role of incubator flexibility

Join INFORS HT and Jefferson Institute for Bioprocessing (JIB) in this insightful webinar that is now available on-demand. Explore the critical role of incubator flexibility in optimizing HEK293 cell cultures for gene therapy applications.

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Interested in learning how the Multitron incubator shaker can help optimize your viral vector production? Reach out to us today.

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