Production of canine adenovirus type 2 in serum-free suspension cultures of MDCK cells

R. Castro, P. Fernandes, T. Laske, M. F Q Sousa, Y. Genzel, K. Scharfenberg, P. M. Alves, A. S. Coroadinha

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

The potential of adherent Madin Darby Canine Kidney (MDCK) cells for the production of influenza viruses and canine adenovirus type 2 (CAV-2) for vaccines or gene therapy approaches has been shown. Recently, a new MDCK cell line (MDCK.SUS2) that was able to grow in suspension in a fully defined system was established. In this work, we investigated whether the new MDCK.SUS2 suspension cell line is suitable for the amplification of CAV-2 under serum-free culture conditions. Cell growth performance and CAV-2 production were evaluated in three serum-free media: AEM, SMIF8, and EXCELL MDCK. CAV-2 production in shake flasks was maximal when AEM medium was used, resulting in an amplification ratio of infectious particles (IP) of 142 IP out/IP in and volumetric and cell-specific productivities of 2.1 × 108 IP/mL and 482 IP/cell, respectively. CAV-2 production was further improved when cells were cultivated in a 0.5-L stirred tank bioreactor. To monitor infection and virus production, cells were analyzed by flow cytometry. A correlation between the side scatter measurement and CAV-2 productivity was found, which represents a key feature to determine the best harvesting time during process development of gene therapy vectors that do not express reporter genes. This work demonstrates that MDCK.SUS2 is a suitable cell substrate for CAV-2 production, constituting a step forward in developing a production process transferable to industrial scales. This could allow for the production of high CAV-2 titers either for vaccination or for gene therapy purposes.

Original languageEnglish
Pages (from-to)7059-7068
Number of pages10
JournalApplied Microbiology and Biotechnology
Volume99
Issue number17
DOIs
Publication statusPublished - 18 Sep 2015

Keywords

  • Canine adenovirus
  • MDCK
  • Process monitoring
  • Serum-free media
  • Suspension cultures

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