title: Production of canine adenovirus type 2 in serum-free suspension cultures of MDCK cells.
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 × 10(8) 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.
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901, Oeiras, Portugal.
authors.forename: R
authors.initials: R
authors.lastname: Castro
authors.forename: P
authors.initials: P
authors.lastname: Fernandes
authors.forename: T
authors.initials: T
authors.lastname: Laske
authors.forename: M F Q
authors.initials: MF
authors.lastname: Sousa
authors.forename: Y
authors.initials: Y
authors.lastname: Genzel
authors.forename: K
authors.initials: K
authors.lastname: Scharfenberg
authors.forename: P M
authors.initials: PM
authors.lastname: Alves
authors.forename: A S
authors.initials: AS
authors.lastname: Coroadinha
mesh_terms: D006516:Adenoviruses, Canine / Q000254:growth & development*; D000818:Animals; D019149:Bioreactors; D049109:Cell Proliferation; D016895:Culture Media, Serum-Free; D004285:Dogs; D061985:Madin Darby Canine Kidney Cells; D014776:Virus Cultivation / Q000379:methods*
chemical_list: D016895:Culture Media, Serum-Free