A feasibility study of different commercially available serum-free mediums to enhance lentivirus and adeno-associated virus production in HEK 293 suspension cells

Çelebi, Gizem; Yetisgin, Abuzer Alp; Erdem, Cem; Cayli, Aziz; Kutlu, Özlem; Çetinel, Sibel

doi:10.1007/s10616-022-00551-1

Cited by 3 publications

(3 citation statements)

References 66 publications

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“…Because PEI alone is relatively cytotoxic to the cells [36,48], the PEI:DNA ratios must be carefully optimized to maximize transfection efficacy and minimize cytotoxicity. Once mixed, the polyplexes (complexes formed between PEI and DNA) grow in size over time [49][50][51]. Efficient polyplexes for transfection have a net positive charge and a time-sensitive size distribution.…”

Section: Production Of Rlvvs By Plasmid Transfection: Challenges and ...mentioning

confidence: 99%

“…In most optimized protocols for HEK293, PEI and DNA are mixed together, and incubated (Figure 1) for a maximum of 30 minutes (usually 10-15 minutes) in a total volume accounting for 5-10% of the cell culture volume to be transfected [46,47,53,54]. Other process-related factors, such as the medium and ion concentration used in the plasmid DNA mix, will have an impact on particle formation and transfection efficacy [49,50,[55][56][57]. Therefore, the optimal incubation time may vary between processes and products.…”

Section: Production Of Rlvvs By Plasmid Transfection: Challenges and ...mentioning

confidence: 99%

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Bioprocess Development and Bioreactor Scale-Up for the Production of Recombinant Lentiviral Viral Vectors in HEK293 Suspension Cell Culture

Robitaille,

Manceur,

Rodenbrock

et al. 2024

Technologies in Cell Culture - A Journey From Basics to Advanced Applications

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Therapeutic applications of viral vectors that initially targeted rare monogenic diseases have now grown to a broader set of indications including cell and gene therapy applications and vaccines. This has prompted the need to increase biomanufacturing capacities, which will require adjustments in the biomanufacturing space to increase yield and lower cost of goods of large-scale productions. HEK293 cells have been widely used for the production of viral vectors because they can grow rapidly in suspension and allow for different modes of production: batch, fed-batch and perfusion. Here we review methods and platforms for producing lentiviral vectors in HEK293 cells grown in serum-free media and the principles and challenges of optimizing and scaling up of bioprocesses in various bioreactors. Lentiviral vectors are particularly difficult to manufacture due to their labile nature. These challenges will be considered in view of current processes and future trends emerging to resolve bottlenecks and existing limitations.

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Section: Production Of Rlvvs By Plasmid Transfection: Challenges and ...mentioning

confidence: 99%

Section: Production Of Rlvvs By Plasmid Transfection: Challenges and ...mentioning

confidence: 99%

Bioprocess Development and Bioreactor Scale-Up for the Production of Recombinant Lentiviral Viral Vectors in HEK293 Suspension Cell Culture

Robitaille,

Manceur,

Rodenbrock

et al. 2024

Technologies in Cell Culture - A Journey From Basics to Advanced Applications

View full text Add to dashboard Cite

show abstract

“…However, the full process of transfection, including cellular uptake, intracellular tra cking, endosomal escape, and nuclear entry is still not fully understood. Multiple studies have been conducted to improve the transfection conditions with success , Srivastava, Mallela et al 2021, Torabfam, Yetisgin et al 2022, measuring marker genes or immuno uorescence labelled proteins only as nal outcome, but there is little known about the e ciency of the steps in between, especially for triple transfections in HEK-293 cells as used in gene therapy.…”

Section: Introductionmentioning

confidence: 99%

Factors affecting recombinant Adeno-Associated Virus titers during triple-plasmid transient transfection in HEK-293 cells

Pistek,

Andorfer,

Grabherr

et al. 2024

Preprint

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The efficiency of triple-plasmid transfection in recombinant Adeno-Associated Virus (rAAV) production was analyzed by examining two distinct strains of HEK-293 cells. These strains were categorized as high producer (HP) and low producer (LP) based on their differing levels of productivity under identical conditions. Analysis of RNA expression levels of viral genes revealed disparities in plasmid derived gene expression between the cell lines. Further assessment of transfection efficacy utilizing labeled plasmids revealed lower plasmid uptake and less efficient nuclear transport in LP cell line. Additionally, we observed inferior translation activity in LP, contributing to its shortcomings in overall productivity. In our attempt to optimize plasmid ratios to enhanced fully packaged rAAV particle yield, we discovered cell-strain-specific optimization potential. The findings highlight the transfection's complexity, urging tailored strategies for improved rAAV production based on each cell line's characteristics, enhancing understanding and guiding further efficiency optimization in rAAV production.

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Factors affecting rAAV titers during triple-plasmid transient transfection in HEK-293 cells

Pistek,

Andorfer,

Grabherr

et al. 2024

Biotechnol Lett

View full text Add to dashboard Cite

The efficiency of triple-plasmid transfection in recombinant Adeno-Associated Virus (rAAV) production was analyzed by examining two distinct HEK-293 cells lines. These were categorized as high producer (HP) and low producer (LP) based on their differing levels of productivity under identical conditions. Analysis of RNA expression levels of viral genes revealed disparities in plasmid derived gene expression between the cell lines. Further assessment of transfection efficiency utilizing labeled plasmids revealed lower plasmid uptake and less efficient nuclear transport in LP cell line. Additionally, we observed inferior translation activity in LP, contributing to its shortcomings in overall productivity. In our attempt to optimize plasmid ratios to enhance fully packaged rAAV particle yield, we discovered cell-line-specific optimization potential. The findings highlight the transfection's complexity, urging tailored strategies for improved rAAV production based on each cell line's characteristics, enhancing understanding and guiding further efficiency optimization in rAAV production.

show abstract

A feasibility study of different commercially available serum-free mediums to enhance lentivirus and adeno-associated virus production in HEK 293 suspension cells

Cited by 3 publications

References 66 publications

Bioprocess Development and Bioreactor Scale-Up for the Production of Recombinant Lentiviral Viral Vectors in HEK293 Suspension Cell Culture

Bioprocess Development and Bioreactor Scale-Up for the Production of Recombinant Lentiviral Viral Vectors in HEK293 Suspension Cell Culture

Factors affecting recombinant Adeno-Associated Virus titers during triple-plasmid transient transfection in HEK-293 cells

Factors affecting rAAV titers during triple-plasmid transient transfection in HEK-293 cells

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