Establishment of the Carrot-Made LTB-Syn Antigen Cell Line in Shake Flask and Airlift Bioreactor Cultures

AbstractCarrot (Daucus carota) cells have been used to effectively manufacture recombinant biopharmaceuticals such as cytokines, vaccines, and antibodies. We generated the carrot cell line Z4, genetically modified to produce the LTB-Syn antigen, which is a fusion protein proposed for immunotherapy against synucleinopathies. In this work, the Z4 cell suspension line was cultivated to produce the LTB-Syn protein in a 250 mL shake flask and 2 L airlift bioreactor cultures grown for 45 and 30 days, respectively. Maximum biomass was obtained on day 15 in both the airlift bioreactor (35.00 ± 0.04 g/L DW) and shake flasks (17.00 ± 0.04 g/L DW). In the bioreactor, the highest LTB-Syn protein yield (1.52 ± 0.03 µg/g FW) was obtained on day 15; while the same occurred on day 18 for shake flasks (0.92 ± 0.02 µg/g FW). LTB-Syn protein levels were analyzed by GM1-ELISA and western blot. PCR analysis confirmed the presence of the transgene in the Z4 line. The obtained data demonstrate that the carrot Z4 cell suspension line grown in airlift bioreactors shows promise for a scale-up cultivation producing an oral LTB-Syn antigen.

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Scale up production of isoflavonoids in cell suspension cultures ofPueraria tuberosagrown in shake flasks and bioreactor

Engineering in Life Sciences ◽

10.1002/elsc.200800114 ◽

2009 ◽

Vol 9 (3) ◽

pp. 267-271 ◽

Cited By ~ 18

Author(s):

Varsha Sharma ◽

Shaily Goyal ◽

Kishan Gopal Ramawat

Keyword(s):

Cell Suspension ◽

Scale Up ◽

Cell Suspension Cultures ◽

Suspension Cultures ◽

Shake Flasks

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Transactivation of Latent Marek's Disease Herpesvirus Genes in QT35, a Quail Fibroblast Cell Line, by Herpesvirus of Turkeys

Journal of Virology ◽

10.1128/jvi.74.21.10176-10186.2000 ◽

2000 ◽

Vol 74 (21) ◽

pp. 10176-10186 ◽

Cited By ~ 31

Author(s):

T. Yamaguchi ◽

S. L. Kaplan ◽

P. Wakenell ◽

K. A. Schat

Keyword(s):

Cell Line ◽

Fibroblast Cell ◽

Northern Blotting ◽

Marek's Disease ◽

Pcr Analysis ◽

Marek’S Disease ◽

Reading Frame ◽

Reverse Transcription Pcr ◽

Herpesvirus Of Turkeys ◽

Serotype 1

ABSTRACT The QT35 cell line was established from a methylcholanthrene-induced tumor in Japanese quail (Coturnix coturnix japonica) (C. Moscovici, M. G. Moscovici, H. Jimenez, M. M. Lai, M. J. Hayman, and P. K. Vogt, Cell 11:95–103, 1977). Two independently maintained sublines of QT35 were found to be positive for Marek's disease virus (MDV)-like genes by Southern blotting and PCR assays. Sequence analysis of fragments of the ICP4, ICP22, ICP27, VP16, meq, pp14, pp38, open reading frame (ORF) L1, and glycoprotein B (gB) genes showed a strong homology with the corresponding fragments of MDV genes. Subsequently, a serotype 1 MDV-like herpesvirus, tentatively name QMDV, was rescued from QT35 cells in chicken kidney cell (CKC) cultures established from 6- to 9-day-old chicks inoculated at 8 days of embryonation with QT35 cells. Transmission electron microscopy failed to show herpesvirus particles in QT35 cells, but typical intranuclear herpesvirus particles were detected in CKCs. Reverse transcription-PCR analysis showed that the following QMDV transcripts were present in QT35 cells: sense and antisense meq, ORF L1, ICP4, and latency-associated transcripts, which are antisense to ICP4. A transcript of approximately 4.5 kb was detected by Northern blotting using total RNA from QT35 cells. Inoculation of QT35 cells with herpesvirus of turkeys (HVT)-infected chicken embryo fibroblasts (CEF) but not with uninfected CEF resulted in the activation of ICP22, ICP27, VP16, pp38, and gB. In addition, the level of ICP4 mRNA was increased compared to that in QT35 cells. The activation by HVT resulted in the production of pp38 protein. It was not possible to detect if the other activated genes were translated due to the lack of serotype 1-specific monoclonal antibodies.

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Enhanced production of taxadiene in Saccharomyces cerevisiae

10.1101/2020.06.08.139600 ◽

2020 ◽

Cited By ~ 1

Author(s):

Behnaz Nowrouzi ◽

Rachel Li ◽

Laura E. Walls ◽

Leopold d’Espaux ◽

Koray Malci ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Shake Flask ◽

Scale Up ◽

Cost Effective ◽

Cancer Drug ◽

List Type ◽

Taxadiene Synthase ◽

Enhanced Production ◽

Anti Cancer ◽

Positive Effect

AbstractCost-effective production of the highly effective anti-cancer drug, paclitaxel (Taxol®), remains limited despite growing global demands. Low yields of the critical taxadiene precursor remains a key bottleneck in microbial production. In this study, the key challenge of poor taxadiene synthase (TASY) solubility in S. cerevisiae was revealed, and the strains were strategically engineered to relieve this bottleneck. Multi-copy chromosomal integration of TASY harbouring a selection of fusion solubility tags improved taxadiene titres 22-fold, up to 57 ± 3 mg/L at 30 °C at shake flask scale. The scalability of the process was highlighted through achieving similar titres during scale up to 25 mL and 250 mL in shake flask and bioreactor cultivations, respectively. Maximum taxadiene titres of 129 ± 15 mg/L and 119 mg/L were achieved through shake flask and bioreactor cultivation, respectively, of the optimal strain at a reduced temperature of 20 °C. The results highlight the positive effect of coupling molecular biology tools with bioprocess variable optimisation on synthetic pathway development.HighlightsMaximum taxadiene titre of 129 ± 15 mg/L in Saccharomyces cerevisiae at 20 °CIntegrating fusion protein tagged-taxadiene synthase improved taxadiene titre.Consistent taxadiene titres were achieved at the micro-and mini-bioreactor scales.

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The feasibility of storing ovarian tumor cells on databasing paper: establishing a library of ovarian cancer DNA

International Journal of Gynecological Cancer ◽

10.1111/j.1525-1438.2006.00755.x ◽

2007 ◽

Vol 17 (1) ◽

pp. 94-100 ◽

Cited By ~ 8

Author(s):

K. Galaal ◽

M. Meirovitz ◽

R. Hussain ◽

L. Allcroft ◽

N. Sullivan ◽

...

Keyword(s):

Ovarian Cancer ◽

Cell Suspension ◽

Direct Sequencing ◽

Genetic Material ◽

Cell Number ◽

Pcr Analysis ◽

Tissue Banks ◽

Cancer Tissue ◽

Ovarian Cancer Tissue ◽

The Stability

The purpose of this study was to assess the feasibility of establishing a library of ovarian cancer nucleic acids using paper matrix by: 1) confirming the stability of DNA stored on paper matrix over a prolonged period of time, 2) determining the amount of genetic material required for storage, and 3) establishing the stability of RNA. Tumor tissue from 66 patients with ovarian cancer was collected intraoperatively, frozen, and dissociated with collagenase and trypsin. A cell suspension was then prepared and spotted onto the paper. The numbers of cells that were stored on the paper were counted using a hemocytometer. The cell suspension was serially diluted and spotted on the paper matrix until the minimum cell number that can be stored and produce a PCR product was determined. PCR, STR genotyping and direct sequencing were performed on tissue stored on the paper matrix. FTA® paper was used as RNA template, and RT PCR converted the RNA to cDNA. Ten to 50 mg of ovarian cancer tissue was stored on FTA® paper. We stored 7 × 104 cells on ISOcode® paper and 18 × 104 cells on FTA® and obtained extractable DNA. PCR analysis on cards with DNA stored 18 months ago enabled us to establish the stability of DNA after storage. RNA was stable for 6 months when stored on FTA® cards. Since genetic material is extractable from the paper matrices after passage of time, it could be a suitable medium for the storage of genetic material in cancer tissue banks.

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731. To Develop a Lentiviral Packaging Cell Line for Scale-Up Production of Lentiviral Vectors

Molecular Therapy ◽

10.1016/s1525-0016(16)35744-6 ◽

2014 ◽

Vol 22 ◽

pp. S282-S283

Keyword(s):

Cell Line ◽

Scale Up ◽

Lentiviral Vectors ◽

Packaging Cell Line ◽

Packaging Cell

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Influence of Light Intensity and Temperature on Cultivation of Microalgae Desmodesmus Communis in Flasks and Laboratory-Scale Stirred Tank Photobioreactor

Latvian Journal of Physics and Technical Sciences ◽

10.1515/lpts-2015-0012 ◽

2015 ◽

Vol 52 (2) ◽

pp. 59-70 ◽

Cited By ~ 1

Author(s):

J. Vanags ◽

L. Kunga ◽

K. Dubencovs ◽

V. Galvanauskas ◽

O. Grīgs

Keyword(s):

Light Intensity ◽

Shake Flask ◽

Scale Up ◽

Biomass Concentration ◽

Biomass Productivity ◽

Pilot Scale ◽

Laboratory Scale ◽

Stirred Tank ◽

Light Limitation ◽

Maximum Biomass

Abstract Optimization of the microalgae cultivation process and of the bioprocess in general traditionally starts with cultivation experiments in flasks. Then the scale-up follows, when the process from flasks is transferred into a laboratory-scale bioreactor, in which further experiments are performed before developing the process in a pilot-scale reactor. This research was done in order to scale-up the process from a 0.4 1 shake flask to a 4.0 1 laboratory-scale stirred-tank photobioreactor for the cultivation of Desmodesmus (D.) communis microalgae. First, the effect of variation in temperature (21-29 ºC) and in light intensity (200-600 μmol m-2s-1) was studied in the shake-flask experiments. It was shown that the best results (the maximum biomass concentration of 2.72 g 1-1 with a specific growth rate of 0.65 g g-1d-1) can be achieved at the cultivation temperature and light intensity being 25 °C and 300 μmol m2s-1, respectively. At the same time, D. communis cultivation under the same conditions in stirred-tank photobioreactor resulted in average volumetric productivities of biomass due to the light limitation even when the light intensity was increased during the experiment (the maximum biomass productivity 0.25 g 1-1d-1; the maximum biomass concentration 1.78 g 1-1).

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