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

2021 ◽  
Vol 9 ◽  
Author(s):  
Evan Tan ◽  
Cara Sze Hui Chin ◽  
Zhi Feng Sherman Lim ◽  
Say Kong Ng
Keyword(s):  
Host Cell ◽  
Cell Lines ◽  
Recombinant Proteins ◽  
Human Cell ◽  
Viral Vectors ◽  
Animal Cell ◽  
Hek293 Cells ◽  
Human Cell Lines ◽  
Post Translational Modifications ◽  
Serum Free Suspension

Animal cell-based expression platforms enable the production of complex biomolecules such as recombinant proteins and viral vectors. Although most biotherapeutics are produced in animal cell lines, production in human cell lines is expanding. One important advantage of using human cell lines is the increased potential that the resulting biotherapeutics would carry more “human-like” post-translational modifications. Among the human cell lines, HEK293 is widely utilized due to its high transfectivity, rapid growth rate, and ability to grow in a serum-free, suspension culture. In this review, we discuss the use of HEK293 cells and its subtypes in the production of biotherapeutics. We also compare their usage against other commonly used host cell lines in each category of biotherapeutics and summarise the factors influencing the choice of host cell lines used.

scholarly journals Evaluation and Design of Genome-wide CRISPR/Cas9 Knockout Screens

2017 ◽  
Author(s):  
Traver Hart ◽  
Amy Tong ◽  
Katie Chan ◽  
Jolanda Van Leeuwen ◽  
Ashwin Seetharaman ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Viral Vectors ◽  
Human Cancer ◽  
Gene Knockout ◽  
Essential Genes ◽  
Human Cell Lines ◽  
Protein Coding ◽  
Mammalian Cell Lines ◽  
Genome Scale

AbstractThe adaptation of CRISPR/Cas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs), targeting human protein-coding genes and encoded in viral vectors, have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we re-analyze 17 genome-scale knockout screens in human cell lines from three research groups using three different genome-scale gRNA libraries, using the Bayesian Analysis of Gene Essentiality (BAGEL) algorithm to identify essential genes, to refine and expand our previously defined set of human core essential genes, from 360 to 684 genes. We use this expanded set of reference Core Essential Genes (CEG2), plus empirical data from six CRISPR knockout screens, to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines.

scholarly journals Treatment of human cell lines with 5-azacytidine may result in profound alterations in clonogenicity and growth rate.

1985 ◽  
Vol 100 (2) ◽  
pp. 508-513 ◽  
Author(s):  
L Olsson ◽  
C Due ◽  
M Diamant
Keyword(s):  
Cell Growth ◽  
Cell Lines ◽  
Human Cell ◽  
Agar Medium ◽  
Animal Cell ◽  
Resistant Cell ◽  
Population Doubling ◽  
Gene Repertoire ◽  
Human Cell Lines ◽  
Growth Properties

Liquid medium cultures of three human cell lines (B-lymphoma, myeloma, and squamous lung carcinoma) with population-doubling times (PDT) and cloning efficiencies (CE) in the range of 32-43 h and 0.01-5.6%, respectively, were exposed to 5-azacytidine (5-azaC) for 3 d. The doses used (1-3 microM) were found to be nontoxic as measured by cell growth in liquid and semisolid agar medium and to be nonmutagenic as measured by the rate of generation of ouabain- and 6-thioguanine-resistant cell variants. After 5-azaC treatment, cell samples were subsequently harvested every day and assayed for their CE in semisolid agar medium. For each cell line, 30 to 42 individual clones were harvested at the day of maximal CE and expanded in liquid culture medium. PDT and CE were determined for each subclone about every 6 wk for 12 mo. The majority of the subclones had unaltered PDT and CE compared to the original lines. However, several clones had profoundly changed proliferative activity with PDT on approximately 12-14 h and/or CE 5 to greater than 50%. Some of the clones with altered growth properties reverted to PDT and/or CE values of untreated clones. However, a few clones of each line had stable alterations with PDT on 12-14 h and CE 5 to greater than 50%; these clones were all significantly hypomethylated. It is concluded that the human gene repertoire does contain genes that appropriately activated can result in growth properties with very short PDT and high CE (and comparable to animal cell lines), and that this activation may be obtained by 5-azaC treatment. It is conceivable that the procedure here described to alter growth properties of human cell lines may be applied to experimental situations, where alterations of cell growth properties are desired.

scholarly journals Isogenic Human Cell Lines for Drug Discovery: Regulation of Target Gene Expression by Engineered Zinc-Finger Protein Transcription Factors

2005 ◽  
Vol 10 (4) ◽  
pp. 304-313 ◽  
Author(s):  
Pei-Qi Liu ◽  
Siyuan Tan ◽  
Matthew C. Mendel ◽  
Richard J. Murrills ◽  
Bheem M. Bhat ◽  
...  
Keyword(s):  
Cell Lines ◽  
Zinc Finger ◽  
Human Cell ◽  
Target Gene ◽  
Zinc Finger Protein ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Hek293 Cells ◽  
Human Cell Lines ◽  
Finger Protein

Isogenic cell lines differing only in the expression of the protein of interest provide the ideal platform for cell-based screening. However, related natural lines differentially expressing the therapeutic target of choice are rare. Here the authors report a strategy for drug screening employing isogenic human cell lines in which the expression of the target protein is regulated by a gene-specific engineered zinc-finger protein (ZFP) transcription factor (TF). To demonstrate this approach, a ZFP TF activator of the human parathyroid hormone receptor 1 (PTHR1) gene was identified and introduced into HEK293 cells (negative for PTHR1). Following induction of ZFP TF expression, this cell line produced functional PTHR1 protein, resulting in a robust and ligand-specific cyclic adenosine monophosphate (cAMP) response. Reciprocally, the natural expression of PTHR1 observed in SAOS2 cells was dramatically reduced by the introduction of the appropriate PTHR1-specific ZFP TF repressor. Moreover, this ZFP-driven PTHR1 repression selectively eliminated the functional cAMP response invoked by known ligands of PTHR1. These data establish ZFP TF–generated isogenic lines as a general approach for the identification of therapeutic agents specific for the target gene of interest.

Human cell lines for the production of recombinant proteins: on the horizon

2015 ◽  
Vol 32 (6) ◽  
pp. 673-679 ◽  
Author(s):  
Lukas Fliedl ◽  
Johannes Grillari ◽  
Regina Grillari-Voglauer
Keyword(s):  
Cell Lines ◽  
Recombinant Proteins ◽  
Human Cell ◽  
Human Cell Lines

scholarly journals Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors

2011 ◽  
Vol 39 (21) ◽  
pp. e143-e143 ◽  
Author(s):  
Ashok D. Bandaranayake ◽  
Colin Correnti ◽  
Byoung Y. Ryu ◽  
Michelle Brault ◽  
Roland K. Strong ◽  
...  
Keyword(s):  
Cell Lines ◽  
Recombinant Proteins ◽  
Human Cell ◽  
Lentiviral Vectors ◽  
Human Cell Lines ◽  
Rapid Production

Morally Illicit Cells in Medical Research

2019 ◽  
Vol 44 (6) ◽  
pp. 3-4
Author(s):  
Alan B. Moy ◽  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Catholic Church ◽  
Animal Cell ◽  
Human Cell Lines ◽  
Efficacy And Safety ◽  
Safe Alternative ◽  
Scientific Rigor ◽  
Considerable Research ◽  
Or Gene

The Catholic Church missed an opportunity to be more proactive and change the course of secular biotechnology when unethical cell lines were first introduced several decades ago. No ethical alternative human cell lines to the HEK293, WI-38, and MRC-5 have been generally accepted by the scientific community. While some animal cell lines are used in creating safe alternative vaccines, no alternative human cell lines for producing vaccines, biologics, or gene therapy have met the scientific rigor of efficacy and safety of these cell lines. It is both possible and within reach to create ethical human cell lines to replace current morally objectionable lines used for producing biologics (proteins and vaccines), but it will take considerable research that requires financial support. Dignitas personae should be backed by leadership and supported by stakeholders.

scholarly journals Distinctive Evasion Mechanisms to Allow Persistence of Borrelia burgdorferi in Different Human Cell Lines

2021 ◽  
Vol 12 ◽  
Author(s):  
Kati Karvonen ◽  
Jonna Nykky ◽  
Varpu Marjomäki ◽  
Leona Gilbert
Keyword(s):  
Borrelia Burgdorferi ◽  
Host Cell ◽  
Cell Lines ◽  
Immune Evasion ◽  
Human Cell ◽  
Post Infection ◽  
Host Cells ◽  
Infected Host ◽  
Human Cell Lines ◽  
Dose Dependent

Lyme borreliosis is a multisystemic disease caused by the pleomorphic bacteria of the Borrelia burgdorferi sensu lato complex. The exact mechanisms for the infection to progress into a prolonged sequelae of the disease are currently unknown, although immune evasion and persistence of the bacteria in the host are thought to be major contributors. The current study investigated B. burgdorferi infection processes in two human cell lines, both non-immune and non-phagocytic, to further understand the mechanisms of infection of this bacterium. By utilizing light, confocal, helium ion, and transmission electron microscopy, borrelial infection of chondrosarcoma (SW1353) and dermal fibroblast (BJ) cells were examined from an early 30-min time point to a late 9-days post-infection. Host cell invasion, viability of both the host and B. burgdorferi, as well as, co-localization with lysosomes and the presence of different borrelial pleomorphic forms were analyzed. The results demonstrated differences of infection between the cell lines starting from early entry as B. burgdorferi invaded BJ cells in coiled forms with less pronounced host cell extensions, whereas in SW1353 cells, micropodial interactions with spirochetes were always seen. Moreover, infection of BJ cells increased in a dose dependent manner throughout the examined 9 days, while the percentage of infection, although dose dependent, decreased in SW1353 cells after reaching a peak at 48 h. Furthermore, blebs, round body and damaged B. burgdorferi forms, were mostly observed from the infected SW1353 cells, while spirochetes dominated in BJ cells. Both infected host cell lines grew and remained viable after 9 day post-infection. Although damaged forms were noticed in both cell lines, co-localization with lysosomes was low in both cell lines, especially in BJ cells. The invasion of non-phagocytic cells and the lack of cytopathic effects onto the host cells by B. burgdorferi indicated one mechanism of immune evasion for the bacteria. The differences in attachment, pleomorphic form expressions, and the lack of lysosomal involvement between the infected host cells likely explain the ability of a bacterium to adapt to different environments, as well as, a strategy for persistence inside a host.

Investigations of the influence of microgravity on the state of human cell lines

2004 ◽  
Vol 10 (5-6) ◽  
pp. 226-228
Author(s):  
L.M. Nosach ◽  
◽  
O.Yu. Povnitsa ◽  
V.L. Zhovnovata ◽  
◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
The State ◽  
Human Cell Lines
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