HEK293 Cell Line as a Platform to Produce Recombinant Proteins and Viral Vectors

Tan, E W P; Chin, Cara Sze Hui; Lim, Zhi Feng Sherman; Ng, Say Kong

doi:10.3389/fbioe.2021.796991

Cited by 102 publications

(78 citation statements)

References 84 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The human cell line HEK293 cells have been widely utilized over the last decades, not only for the basic research such as protein interaction and signal transduction studies, but also for the production of recombinant proteins and viral vectors in biopharmaceutical research due to the high transfectivity, rapid growth, and human-like post-translation attributes ( Petiot et al, 2015 ; Tripathi and Shrivastava, 2019 ; Tan et al, 2021 ). The HEK293 cell line is a naturally adherent type and is still typically cultured in serum-supplemented media.…”

Section: Introductionmentioning

confidence: 99%

The impact of serum-free culture on HEK293 cells: From the establishment of suspension and adherent serum-free adaptation cultures to the investigation of growth and metabolic profiles

Jang

Pete

Bruheim

2022

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

Serum-free cultures are preferred for application in clinical cell therapy and facilitate the purification processes of bioproducts, such as vaccines and recombinant proteins. It can replace traditional cell culture - eliminating potential issues posed by animal-derived serum supplementation, such as lot to lot variation and risks of pathogen infection from the host animal. However, adapting cells to serum-free conditions can be challenging and time-consuming, and is cell line and medium dependent. In addition, the knowledge of the impact of serum-free culture on cellular metabolism is limited. Herein, we successfully established serum-free suspension and adherent cultures through two adaptation procedures for HEK293 cells in serum-free Freestyle 293 medium. Furthermore, growth kinetics and intracellular metabolic profiles related to central carbon metabolism were investigated. The entire adaptation procedure took 1 month, and high cell viability (>90%) was maintained throughout. The serum-free adherent culture showed the best growth performance, measured as the highest cell density and growth rate. The largest differences in metabolic profiles were observed between culture modes (adherent vs. suspension), followed by culture medium condition (control growth medium vs. serum-free medium). Metabolic differences related to the adaptation procedures were only seen in suspension cultures. Interestingly, the intracellular itaconate concentration was significantly higher in suspension cells compared to adherent cells. Furthermore, when the cells back-adapted from serum-free to serum-supplemented control medium, their metabolic profiles were immediately reversed, highlighting the effect of extracellular components on metabolic phenotype. This study provides strategies for efficient serum-free cultivation and deeper insights into the cellular responses related to growth and metabolism responses to diverse culture conditions.

show abstract

Section: Introductionmentioning

confidence: 99%

The impact of serum-free culture on HEK293 cells: From the establishment of suspension and adherent serum-free adaptation cultures to the investigation of growth and metabolic profiles

Jang

Pete

Bruheim

2022

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

show abstract

“…HEK293T cells are widely used for production of recombinant proteins where a human cell source is desirable 18 . HEK293T cells were transfected with BITE plasmids using standard PEI transfection, using fluorescence microscopy to confirm successful transfection at day 1.…”

Section: Resultsmentioning

confidence: 99%

A Simplified Function-First Method for the Discovery and Optimization of Bispecific Immune Engaging Antibodies

Shepherd

Bennychen

Marcil

et al. 2022

Preprint

View full text Add to dashboard Cite

Bi-specific T-cell engager antibodies (BITEs) are synthetic soluble molecules derived from antibodies that induce active contact between T-cells and other target cells in the body. BITE therapeutics have shown great promise for the treatment of various forms of cancer; however, the current development process for BITEs is time consuming and costly. BITE development requires empirical testing and characterization of the individual antigen binding domains, followed by extensive engineering and optimization in bi-specific molecular format to generate a molecule with strong biological activity and appropriate characteristics for clinical development. Here, we sought to create a cost efficient high-throughput method for creating and evaluating BITEs using a simplified function first approach to identify bioactive molecules without purification. Using a plasmid with a modular structure to allow high efficiency exchange of either binder arm, we established a simple method to combine many novel tumour-targeting single chain variable (scFv) domains with the well-characterized OKT3 scFv CD3-targeting domain. After generating these novel plasmids, we demonstrate two systems for high throughput functional screening of BITE molecules based on Jurkat T cells (referred to as BITE-J). Using BITE-J we evaluate four EGFRvIII BITEs, identifying two constructs with superior activity. We then confirmed this activity in primary T cells, where novel EGFRvIII-BITEs induced T cell activation and antigen selective tumor killing. We also demonstrate that we can similarly exchange the CD3-interacting element of our bi-modular plasmid. By testing several novel CD3-targeting scFv elements for activity in EGFRvIII-targeted BITEs, we were able to identify highly active BITE molecules with desirable properties for downstream development. In summary, BITE-J presents a low cost, high-throughput method for the rapid assessment of novel BITE molecules without the need for purification and quantification.

show abstract

“…As various factors including cell culture methods and isolation techniques have been shown to influence downstream EV efficacy [32], we set out to dissect the effect of these factors on HEK293T EVs. HEK293T cells were chosen based on their proven ability to be integrated into scalable biotech processes as well as demonstrated ability to be engineered for therapeutic EV production [27,33]. The EVs that they secreted are also thought to be relatively therapeutically inert, especially when compared with the EVs of other cell types (e.g., mesenchymal stem cells; MSCs) [34][35][36].…”

Section: Cell Culture Condition and Ev Isolation Technique Have No Ef...mentioning

confidence: 99%

Assessment of anti-inflammatory bioactivity of extracellular vesicles is susceptible to error via media component contamination

Kronstadt

Heyningen

Aranda

et al. 2022

Preprint

View full text Add to dashboard Cite

Extracellular vesicles (EVs) are widely implicated as novel diagnostic and therapeutic modalities for a wide range of diseases. Thus, optimization of EV biomanufacturing is of high interest. In the course of developing parameters for a HEK293T EV production platform, we examined the combinatorial effects of cell culture conditions (i.e., static vs dynamic) and isolation techniques (i.e., ultracentrifugation vs tangential flow filtration vs size-exclusion chromatography) on functional characteristics of HEK293T EVs, including anti-inflammatory bioactivity using a well-established LPS-stimulated mouse macrophage model. We unexpectedly found that, depending on culture condition and isolation strategy, HEK293T EVs appeared to significantly suppress the secretion of pro-inflammatory cytokines (i.e., IL-6, RANTES) in the stimulated mouse macrophages. Further examination revealed that these results were most likely due to fetal bovine serum (FBS) EV contamination in HEK293T EV preparations. Thus, future research assessing the anti-inflammatory effects of EVs should be designed to account for this phenomenon.

show abstract

HEK293 Cell Line as a Platform to Produce Recombinant Proteins and Viral Vectors

Cited by 102 publications

References 84 publications

The impact of serum-free culture on HEK293 cells: From the establishment of suspension and adherent serum-free adaptation cultures to the investigation of growth and metabolic profiles

The impact of serum-free culture on HEK293 cells: From the establishment of suspension and adherent serum-free adaptation cultures to the investigation of growth and metabolic profiles

A Simplified Function-First Method for the Discovery and Optimization of Bispecific Immune Engaging Antibodies

Assessment of anti-inflammatory bioactivity of extracellular vesicles is susceptible to error via media component contamination

Contact Info

Product

Resources

About