Liu, S;
Ruban, L;
Wang, Y;
Zhou, Y;
Nesbeth, DN;
(2016)
Establishing elements of a synthetic biology platform for Vaccinia virus production: BioBrickTM design, serum-free virus production and microcarrier-based cultivation of CV-1 cells.
BioRxiv
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Abstract
Vaccinia virus (VACV) is an established tool for vaccination and is beginning to prove effective as an oncolytic agent. Industrial production of VACV stands to benefit in future from advances made by synthetic biology in genome engineering and standardisation. As a step toward realising these benefits we analysed the Lister Vaccinia virus genome with respect to refactoring options and propose a VACV genome engineering BioBrickTM. We then used the CV-1 cell line to produce a conventional recombinant Lister strain VACV, VACVL-15 RFP in a serum-free process. CV-1 cells grown in 5% foetal bovine serum (FBS) Dulbecco's Modified Eagle Medium (DMEM) were adapted to growth in OptiPRO and VP-SFM brands of serum-free media. Specific growth rates of 0.047 h-1 and 0.044 h-1 were observed for cells adapted to OptiPRO and VP-SFM respectively, compared to 0.035 h-1 in 5% FBS DMEM. Cells adapted to OptiPRO and to 5% FBS DMEM achieved recovery ratios of over 96%, an indication of their robustness to cryopreservation. Cells adapted to VP-SFM showed a recovery ratio of 82%. VACV production in static culture, measured as plaque forming units (PFU) per propagator cell, was 75 PFU/cell for cells in 5% FBS DMEM. VP-SFM and OptiPRO adaptation increased VACV production to 150 PFU/cell and 350 PFU/cell respectively. Boosted PFU/cell from OptiPRO-adapted cells persisted when 5% FBS DMEM or OptiPRO medium was present during the infection step and when titre was measured using cells adapted to 5% FBS DMEM or OptiPRO medium. Finally, OptiPRO-adapted CV-1 cells were successfully cultivated using Cytodex-1 microcarriers to inform future scale up studies.
| Type: | Working / discussion paper |
|---|---|
| Title: | Establishing elements of a synthetic biology platform for Vaccinia virus production: BioBrickTM design, serum-free virus production and microcarrier-based cultivation of CV-1 cells |
| Location: | USA |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1101/042275 |
| Publisher version: | http://dx.doi.org/10.1101/042275 |
| Language: | English |
| Additional information: | Copyright © The copyright holder for this preprint is the author/funder. It is made available under a CC-BY 4.0 International license, http://creativecommons.org/licenses/by/4.0/. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1476216 |
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