2012AA02A303) (http://www.863.gov.cn/), the National Technology and Technology Major Project (No.2013ZX10004003-003-003) (http://www.nmp.gov.cn/), and the Fundamental Research Funds for the Central Universities (WF1214035) (http://jkw.mof.gov.cn/). Data Availability All relevant data are within the paper and its Supporting Information file.. the serum-free medium supported the stable subculture and growth of both adherent and suspension cells. In batch tradition, for both cell lines, the growth kinetics in the serum-free medium was similar with those in the serum-containing medium and a commercialized serum-free medium. In the serum-free medium, peak viable cell denseness (VCD), haemagglutinin (HA) and median cells culture infective dose (TCID50) titers of the two cell lines reached 4.51106 cells/mL, 2.94Log10(HAU/50 L) and 8.49Log10(virions/mL), and 5.97106 cells/mL, 3.88Log10(HAU/50 L), and 10.34Log10(virions/mL), respectively. While disease yield of adherent cells in the serum-free medium was similar to that in the serum-containing medium, suspension tradition in the serum-free medium showed a higher virus yield than adherent cells in the serum-containing medium and suspension cells in the commercialized serum-free medium. However, the percentage of infectious viruses was lower for suspension tradition in the serum-free medium. These results demonstrate the great potential of this suspension MDCK cell collection in Benzydamine HCl serum-free medium for influenza vaccine production and further improvements Benzydamine HCl are warranted. Intro In Rabbit polyclonal to Ly-6G recent years, animal cell tradition technology has gradually replaced the traditional chick embryo production process for influenza vaccine production. Currently, most of cells applied for influenza vaccine production are adherent and cultivated as monolayers. As a result, large-scale culture processes mainly rely on cultivating adherent cells on microcarriers in serum-containing medium [1C3]. For vaccine production, supplementation of serum brings about many problems, such as high cost, batch variance and risk of contamination with viruses, mycoplasmas and prions [4, 5]. In addition, the presence of serum can cause problems for downstream purification . To address these issues, serum-free medium has been exploited in vaccine production processes. Several studies reported successful development of microcarrier-based cell tradition processes using serum-free medium for influenza vaccine production [7, 8]. Although a high virus production yield can be obtained through the microcarrier-based approach, it is often demanding for scale-up due to the labor-intensive process and high cost of microcarriers [9, 10]. The use of suspension cells is anticipated to facilitate the scale-up of the production process by Benzydamine HCl eliminating trypsinization and reattachment of cells, which are normally required in the microcarrier system [11, 12]. Thus far, several suspension cell lines, including MDCK, PER. C6, AGE. CR, EB14/EB66 and CAP, have been founded and applied in influenza vaccine production [10, 12C20]. In particular, for influenza production, serum-free suspension tradition of MDCK cells has been reported [9, 18, 19]. However, low influenza disease productivity is generally acquired in simple batch tradition, albeit the productivity can be promoted by applying complex fed-batch or perfusion culture [21C23]. Therefore, there is an urgent need to develop more effective batch cell culture process for suspension culture of MDCK cells in influenza vaccine production. Previously, a few studies characterized cell growth and influenza computer virus production in different culture modes, for example, adherent and suspension cultures in either serum-containing or serum-free medium, in comparison with methods using chick embryo [12, 24, 25]. However, a direct comparison among different cell culture modes regarding cell growth and influenza computer virus production is Benzydamine HCl still missing, which should be vital to the development of suspension cell-based influenza vaccine production process. Previously, we had successfully established a suspension MDCK cell collection for influenza computer virus production process . The objective of the present study was to investigate the growth of MDCK cells during subculture and batch culture in different culture modes, including serum-containing adherent culture, serum-free adherent culture and serum-free suspension culture. The influenza computer virus production in these batch cultures was also compared. Materials and Methods Cell lines and culture conditions The adherent MDCK cells (CCL-34, ATCC) were cultivated on CytodexTM 3 microcarriers (3 g/L, GE Healthcare) in DMEM (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco) or a proprietary serum-free medium developed by the authors (MDCK-SFM1) . The adherent MDCK cells were adapted to suspension culture by the serum reduction and serial passaging approach in another proprietary serum-free medium developed by the authors (MDCK-SFM2) [26, 27], and the producing suspension MDCK cells were cultured in MDCK-SFM2 Benzydamine HCl or a commercialized serum-free medium Ex-cell MDCK (Sigma-Aldrich). The formulations of MDCK-SFM1 and MDCK-SFM2 were outlined in Furniture.