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Wednesday, 24 January 2018
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Biotechnology and Bioengineering
Wiley Online Library : Biotechnology and Bioengineering

  • Glutamine synthetase gene knockout?human embryonic kidney 293E cells for stable production of monoclonal antibodies
    Previously, it was inferred that a high glutamine synthetase (GS) activity in human embryonic kidney (HEK) 293E cells results in elevated resistance to methionine sulfoximine (MSX) and consequently hampers GS?mediated gene amplification and selection by MSX. To overcome this MSX resistance in HEK293E cells, a GS?knockout HEK293E cell line was generated using the CRISPR/Cas9 system to target the endogenous human GS gene. The GS?knockout in the HEK293E cell line (RK8) was confirmed by Western blot analysis of GS and by observation of glutamine?dependent growth. Unlike the wild type HEK293E cells, the RK8 cells were successfully used as host cells to generate a recombinant HEK293E cell line (rHEK293E) producing a monoclonal antibody (mAb). When the RK8 cells were transfected with the GS expression vector containing the mAb gene, rHEK293E cells producing the mAb could be selected in the absence as well as in the presence of MSX. The gene copies and mRNA expression levels of the mAb in rHEK293E cells were also quantified using qRT?PCR. Taken together, the GS?knockout HEK293E cell line can be used as host cells to generate stable rHEK293E cells producing a mAb through GS?mediated gene selection in the absence as well as in the presence of MSX. This article is protected by copyright. All rights reserved

  • Correlation of simulation/finite element analysis to the separation of intrinsically magnetic spores and red blood cells using a microfluidic magnetic deposition system
    Magnetic separation of cells has been, and continues to be, widely used in a variety of applications, ranging from healthcare diagnostics to detection of food contamination. Typical, these technologies require cells labeled with antibody magnetic particle conjugate and a high magnetic energy gradient created in the flow containing the labeled cells (i.e. a column packed with magnetically inducible material), or dense packing of magnetic particles next to the flow cell. Such designs while creating high magnetic energy gradients, are not amenable to easy, highly detailed, mathematic characterization. Our laboratories have been characterizing and developing analysis and separation technology that can be used on intrinsically magnetic cells or spores which are typically orders of magnitude weaker than typically immunomagnetically labeled cells. One such separation system is magnetic deposition microscopy (MDM) which not only separates cells, but which deposits cells in specific locations on slides for further microscopic analysis. In this study, the MDM system has been further characterized, using finite element and computational fluid mechanics software, and separation performance predicted, using a model which combines: 1) the distribution of the intrinsic magnetophoretic mobility of the cells (spores), 2) the fluid flow within the separation device, 3) accurate maps of the values of the magnetic field (max 2.27 T), and magnetic energy gradient (max of 4.41 T2/mm) within the system. Guided by this model, experimental studies indicated that greater than 95 percent of the intrinsically magnetic bacillus spores can be separated with the MDM system. Further, this model allows analysis of cell trajectories which can assist in the design of higher throughput systems. This article is protected by copyright. All rights reserved

  • Inhibition of Apoptosis Using Exosomes in Chinese Hamster Ovary Cell Culture
    Animal cell culture technology for therapeutic protein production has shown significant improvement over the last few decades. Chinese hamster ovary (CHO) cells have been widely adapted for the production of biopharmaceutical drugs. In the biopharmaceutical industry, it is crucial to develop cell culture media and culturing conditions to achieve the highest productivity and quality. However, CHO cells are significantly affected by apoptosis in the bioreactors, resulting in a substantial decrease in product quantity and quality. Thus, to overcome the obstacle of apoptosis in CHO cell culture, it is critical to develop a novel method that does not have minimal concern of safety or cost. Herein, we showed for the first time that exosomes, which are nano?sized extracellular vesicles, derived from CHO cells inhibited apoptosis in CHO cell culture when supplemented to the culture medium. Flow cytometric and microscopic analyses revealed that substantial amounts of exosomes were delivered to CHO cells. Higher cell viability after staurosporine treatment was observed by exosome supplementation (67.3%) as compared to control (41.1%). Furthermore, exosomes prevented the mitochondrial membrane potential loss and caspase?3 activation, meaning that the exosomes enhanced cellular activities under pro?apoptotic condition. As the exosomes supplements are derived from CHO cells themselves, it is not only beneficial for the biopharmaceutical productivity of CHO cell culture to inhibit apoptosis, but also from a regulatory standpoint to diminish any safety concerns. Thus, we conclude that the method developed in this research may contribute to the biopharmaceutical industry where minimizing apoptosis in CHO cell culture is beneficial. This article is protected by copyright. All rights reserved

  • Effective Role of Medium Supplementation in Microalgal Lipid Accumulation
    The present study investigated the interaction between starch and lipid accumulation was in a green microalgae enrichment culture. The objective was to optimize the lipid content by manipulation of the medium in regular batch culture. Two medium designs were evaluated: first a high ortho?P concentration with vitamin supplement (Pi?vitamins supplemented medium), second normal growth medium (control). Both media contained a low amount of nitrogen which was consumed during batch growth in three days. The batch experiments were prolonged continued for another four days with the absence of soluble nitrogen in the medium. When the mixed microalgal culture was incubated with in the Pi?vitamin supplemented medium, the lipid and starch content of the culture increased within the first three days to 102.0 ?±?5.2?mg.l?1 (12.7?±?0.6% of DW) and 31.7?±?1.6?mg.l?1 (4.0?±?0.2% of DW), respectively. On the last day of the experiment, the lipid and starch content in Pi?vitamin medium increased to 663.1?±?32.5 (33.4?±?1.6% of DW) and 127.5?±?5.2 (6.4?±?0.3% of DW) mg.l?1. and However, the lipid and starch content in the control process, reached to 334.7?±?16.4 (20.1?±?1.0% of DW) and 94.3?±?4.6 (5.7?±?0.3% of DW), respectively. The high Pi?vitamin medium induced storing lipid formation clearly while the starch formation was not affected. The lipid contents reported here are among the high reported in the literature, note that already under full growth conditions significant lipid levels occurred in the algal enrichment culture. The high lipid productivity of the reported mixed microalgae culture provides an efficient route for efficient algal biodiesel production. This article is protected by copyright. All rights reserved

  • Ultra scale?down approaches to study the centrifugal harvest for viral vaccine production
    Large scale continuous cell?line cultures promise greater reproducibility and efficacy for the production of influenza vaccines, and adenovirus for gene therapy. This paper seeks to use an existing validated ultra scale?down tool, which is designed to mimic the commercial scale process environment using only millilitres of material, to provide some initial insight into the performance of the harvest step for these processes. The performance of industrial scale centrifugation and subsequent downstream process units is significantly affected by shear. The properties of these cells, in particular their shear sensitivity, may be changed considerably by production of a viral product, but literature on this is limited to date. In addition, the scale?down tool used here has not previously been applied to the clarification of virus production processes. The results indicate that virus infected cells do not actually show any increase in sensitivity to shear, and may indeed become less shear sensitive, in a similar manner to that previously observed in old or dead cell cultures. Clarification may be most significantly dependent on the virus release mechanism, with the budding influenza virus producing a much greater decrease in clarification than the lytic, non?enveloped adenovirus. A good match was also demonstrated to the industrial scale performance in terms of clarification, protein release and impurity profile. This article is protected by copyright. All rights reserved

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