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Process Development for Newcastle Disease Virus-Vectored Vaccines in Serum-Free Vero Cell Suspension Cultures

Tue Oct 18 14:51:54 CST 2022

2. Materials and Methods

2.1. Cell Lines and Culture Media

The Vero cell line adapted to suspension was provided by the National Research Council of Canada (NRC), and the adaptation was described in a previous work [24]. For routine passaging, cells were centrifuged at 800× g for 5 min and resuspended in fresh media with a seeding density of 3–6 × 105 cells/mL. Cell cultures were maintained at 37 °C, 135 rpm and 5% CO2 in humified Multitron orbital shakers (Infors HT, Bottmingen, Switzerland). Cells were cultured in MDXK medium (Xell AG, Bielefeld, Germany), supplemented with 4 mM GlutaMAX (Thermo Fisher Scientific, Waltham, MA, USA), at a working volume of 20 mL, 25 mL, 50 mL or 100–200 mL in polycarbonate shake flasks of volume 125 mL, 250 mL, 500 mL or 1 L (TriForest Enterprises, Irvine, CA, USA), respectively.

                FuDau 125ml Erlenmeyer Shake Flasks

HEK293 suspension cells are originated from HEK293SF (clone 293SF-3F6) cells, which derive from a GMP-grade master cell bank [25]. The cells were cultured in HEK GM medium (Xell AG, Bielefeld, Germany), supplemented with 4 mM GlutaMAX. Routine passaging and incubation in shakers was the same as for suspension Vero cells.

   FuDau 1000ml Erlenmeyer Shake Flasks

Adherent Vero cells (ATCC CCL-81.5) were routinely passaged by washing with PBS without calcium and magnesium (WISENT Inc., Saint-Jean-Baptiste, QC, Canada), detaching with TrypLE™ Express Enzyme (Gibco, Gaithersburg, MD, USA) and adding VP Serum-Free Medium (VP-SFM) (Gibco, Gaithersburg, MD, USA) with 4 mM GlutaMAX and 1% Penicillin-Streptomycin solution (WISENT Inc., Saint-Jean-Baptiste, QC, Canada) to collect. Once collected, cells are pelleted at 300× g for 5 min and resuspended in VP-SFM to remove TrypLE. Cells are plated onto T-175 flasks or 150 mm plates, at 5–10 × 106 cells and are passaged every 2–3 days.

Source: https://www.mdpi.com/2076-393X/9/11/1335/htm

The FAI climbed 5.9 percent year-on-year in the first 11 months of 2018, quickening from the 5.7-percent growth in Jan-Oct, the National Bureau of Statistics (NBS) said Friday in an online statement.

The key indicator of investment, dubbed a major growth driver, hit the bottom in August and has since started to rebound steadily.

In the face of emerging economic challenges home and abroad, China has stepped up efforts to stabilize investment, in particular rolling out measures to motivate private investors and channel funds into infrastructure.

Friday's data showed private investment, accounting for more than 60 percent of the total FAI, expanded by a brisk 8.7 percent.

NBS spokesperson Mao Shengyong said funds into weak economic links registered rapid increases as investment in environmental protection and agriculture jumped 42 percent and 12.5 percent respectively, much faster than the average.

In breakdown, investment in high-tech and equipment manufacturing remained vigorous with 16.1-percent and 11.6-percent increases respectively in the first 11 months. Infrastructure investment gained 3.7 percent, staying flat. Investment in property development rose 9.7 percent, also unchanged.