Evolution from adherent to suspension – systems biology of HEK293 cell line development

Malm, Magdalena; Saghaleyni, Rasool; Lundqvist, Magnus; Giudici, Marco; Chotteau, Véronique; Field, Raymond; Varley, Paul; Hatton, Diane; Grassi, Luigi; Svensson, Thomas; Uhlén, Mathias; Nielsen, Jens; Rockberg, Johan

doi:10.1101/2020.01.29.924894

Cited by 12 publications

(11 citation statements)

References 78 publications

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“…The SV40 origin of replication is present on the H77 sE2 expression vector (pPPI4) we used [64]; however, it is highly improbable that an improvement in plasmid stability would affect the distal process of sE2 glycosylation. A recent omics study of the HEK 293 line and its progeny not only revealed transcriptome profile differences between adherent 293T cells and 293-F cells but also between 293H cells adapted to suspension and 293-F cells [65]. This indicates genes other than those involved in the adherent to suspension transition are also differentially expressed in 293 progeny cell lines.…”

Section: Discussionmentioning

confidence: 99%

Optimized cell systems for the investigation of hepatitis C virus E1E2 glycoproteins

Kalemera

Capella-Pujol

Chumbe

et al. 2021

Journal of General Virology

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Great strides have been made in understanding and treating hepatitis C virus (HCV) thanks to the development of various experimental systems including cell-culture-proficient HCV, the HCV pseudoparticle system and soluble envelope glycoproteins. The HCV pseudoparticle (HCVpp) system is a platform used extensively in studies of cell entry, screening of novel entry inhibitors, assessing the phenotypes of clinically observed E1 and E2 glycoproteins and, most pertinently, in characterizing neutralizing antibody breadth induced upon vaccination and natural infection in patients. Nonetheless, some patient-derived clones produce pseudoparticles that are either non-infectious or exhibit infectivity too low for meaningful phenotyping. The mechanisms governing whether any particular clone produces infectious pseudoparticles are poorly understood. Here we show that endogenous expression of CD81, an HCV receptor and a cognate-binding partner of E2, in producer HEK 293T cells is detrimental to the infectivity of recovered HCVpp for most strains. Many HCVpp clones exhibited increased infectivity or had their infectivity rescued when they were produced in 293T cells CRISPR/Cas9 engineered to ablate CD81 expression (293TCD81KO). Clones made in 293TCD81KO cells were antigenically very similar to their matched counterparts made parental cells and appear to honour the accepted HCV entry pathway. Deletion of CD81 did not appreciably increase the recovered titres of soluble E2 (sE2). However, we did, unexpectedly, find that monomeric sE2 made in 293T cells and Freestyle 293-F (293-F) cells exhibit important differences. We found that 293-F-produced sE2 harbours mostly complex-type glycans whilst 293T-produced sE2 displays a heterogeneous mixture of both complex-type glycans and high-mannose or hybrid-type glycans. Moreover, sE2 produced in 293T cells is antigenically superior; exhibiting increased binding to conformational antibodies and the large extracellular loop of CD81. In summary, this work describes an optimal cell line for the production of HCVpp and reveals that sE2 made in 293T and 293-F cells are not antigenic equals. Our findings have implications for functional studies of E1E2 and the production of candidate immunogens.

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Section: Discussionmentioning

confidence: 99%

Optimized cell systems for the investigation of hepatitis C virus E1E2 glycoproteins

Kalemera

Capella-Pujol

Chumbe

et al. 2021

Journal of General Virology

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“…The SV40 origin of replication is present on the H77 sE2 expression vector (pPPI4) we used (57); however, it is highly improbable that an improvement in plasmid stability would affect the distal process of sE2 glycosylation. A recent omics study of HEK293 and its progeny cell lines not only revealed transcriptome profile differences between adherent 293T cells and 293F cells but also between 293H cells adapted to suspension and 293F cells (58). This indicates genes other than those involved in the adherent to suspension transition are also differentially expressed 293 progeny cell lines.…”

Section: Discussionmentioning

confidence: 99%

Optimised cell systems for the investigation of hepatitis C virus E1E2 glycoproteins

Kalemera

Capella-Pujol

Chumbe

et al. 2020

Preprint

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These authors contributed equally to this work mannose or hybrid type glycans. Moreover, sE2 produced in HEK 293T cells is antigenically superior; exhibiting increased binding to conformational antibodies and the large extracellular loop of CD81. In summary, this work describes an optimal cell line for the production of HCVpp and reveals that sE2 made in 293T and 293F cells are not antigenic equals. Our findings have implications for functional studies of E1E2 and the production of candidate immunogens. human hepacivirus | viral glycoprotein | receptor | pseudoparticle Correspondence: j.grove@ucl.ac.uk, k.h.sliepen@amsterdamumc.nl Kalemera, Capella-Pujol, Chumbe et al. | bioRχiv | June 18, 2020 | 1-12

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“…In respect to cell physiology, serum removal and anchorage-independent growth is a major change. It mimics an epithelial-to-mesenchymal transition [ 29 , 30 ] which is one of the most complex and multilayer-leveled processes that cells can undergo [ 31 ]. In fact, it is not by chance that principal component analysis identified ‘serum-containing adherent vs. serum-free suspension’ cultures as the first source of variability in the dataset, before cell bank repository origin or CAV-2 infection ( Figure 6 A).…”

Section: Discussionmentioning

confidence: 99%

Cell Bank Origin of MDCK Parental Cells Shapes Adaptation to Serum-Free Suspension Culture and Canine Adenoviral Vector Production

Rodrigues

Fernandes

Laske

et al. 2020

IJMS

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Phenotypic variation in cultured mammalian cell lines is known to be induced by passaging and culture conditions. Yet, the effect these variations have on the production of viral vectors has been overlooked. In this work we evaluated the impact of using Madin–Darby canine kidney (MDCK) parental cells from American Type Culture Collection (ATCC) or European Collection of Authenticated Cell Cultures (ECACC) cell bank repositories in both adherent and suspension cultures for the production of canine adenoviral vectors type 2 (CAV-2). To further explore the differences between cells, we conducted whole-genome transcriptome analysis. ECACC’s MDCK showed to be a less heterogeneous population, more difficult to adapt to suspension and serum-free culture conditions, but more permissive to CAV-2 replication progression, enabling higher yields. Transcriptome data indicated that this increased permissiveness is due to a general down-regulation of biological networks of innate immunity in ECACC cells, including apoptosis and death receptor signaling, Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling, toll-like receptors signaling and the canonical pathway of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling. These results show the impact of MDCK source on the outcome of viral-based production processes further elucidating transcriptome signatures underlying enhanced adenoviral replication. Following functional validation, the genes and networks identified herein can be targeted in future engineering approaches aiming at improving the production of CAV-2 gene therapy vectors.

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Evolution from adherent to suspension – systems biology of HEK293 cell line development

Cited by 12 publications

References 78 publications

Optimized cell systems for the investigation of hepatitis C virus E1E2 glycoproteins

Optimized cell systems for the investigation of hepatitis C virus E1E2 glycoproteins

Optimised cell systems for the investigation of hepatitis C virus E1E2 glycoproteins

Cell Bank Origin of MDCK Parental Cells Shapes Adaptation to Serum-Free Suspension Culture and Canine Adenoviral Vector Production

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