Rapid Screening of Serum-Free Media for the Growth of Adherent Vero Cells by Using a Small-Scale and Non-invasive Tool

Abstract

The paper proposes a rapid screening method for a first step improvement of an animal component-free medium dedicated to the growth of the anchorage-dependent Vero cell line. A new, rapid, and non-invasive technique is presented to specifically monitor cultures of adherent cells in 96-well plates. The operating conditions of an image analyzer are adapted to take into account the decrease of cell size when the attached cell density increases. An experimental design is carried out to assess the influence of ten component groups in the original medium. Two groups including protein extracts, growth factor, insulin, glucose, and pyruvate show significant positive effects. The groups with vitamins and molecules related to nitrogenous bases display a less pronounced influence. The mixture of amino acids, B(1) vitamin, magnesium sulfate, and sodium phosphate as well as the couple sodium citrate and ferric chloride lead to a downward trend. The screening results are proved to be scalable in stirred cultures with cells on microcarriers. An improved serum-free medium, with some component groups being removed or added, can be rapidly formulated to reach respectively similar or 1.6 times higher cell density than in the original medium. The results from this global approach could be helpful to further focus experiments on identified medium components.

Kinetic characterization of vero cell metabolism in a serum‐free batch culture process

“…Vero cells were grown in a complex serum-free medium (reference medium) containing 4 mM glutamine and 22 mM glucose, as indicated in Petiot et al (2010). The basic medium referred to the same medium, but deleted from glucose and glutamine.…”

“…Indeed, Vero cells deriving from African green monkey kidney are used for mass productions of viruses such as reovirus (Butler et al, 2000), Japanese encephalitis virus (Toriniwa and Komiya, 2007;Wu and Huang, 2002), hepatitis A virus (Sun et al, 2004), Chikungunya virus (Tiwari et al, 2009), rabies virus (Frazatti-Gallina et al, 2004;Rourou et al, 2007Rourou et al, , 2009Trabelsi et al, 2006), and enterovirus (Liu et al, 2007;Wu et al, 2004). While Vero cells were used in virus production processes since several years, the formulation of specific media without animal origin components to satisfy control and safety requirements, is still under development (Petiot et al, 2010). Media optimization generally requires a high understanding of the cell metabolism, but very few reports focused on the metabolism of Vero cells (Huang et al, 2006(Huang et al, , 2007Mendonça et al, 2002;Mendonça and Pereira, 1998;Quesney et al, 2003).…”

“…As previously reported, Vero cells have been recently adapted to grow in a serum-free medium for vaccine production (Butler et al, 2000;Merten et al, 1997;Petiot et al, 2010;Quesney et al, 2001;Rourou et al, 2007), but Vero cell metabolism remains not well characterized particularly when cultivated in a serum-free medium (Quesney et al, 2003). Since this cell line is of importance for virus production, a metabolic characterization has still to be established.…”

“…Cell numeration was performed by harvesting 4 mL of the cell culture. Then, microcarriers were washed twice with PBS after settling as already described (Petiot et al, 2010).…”

Rational development of a serum-free medium and fed-batch process for a GS-CHO cell line expressing recombinant antibody

“…Medium development by changing one factor at a time is generally laborious, time-consuming and has the risk of neglecting the interactions among supplements (Petiot et al 2010). Design of experiments (DoE) and statistical analysis, which can test and indentify mixtures of components simultaneously, has therefore been implemented to increase efficiency and reliability of screening and optimization for cell culture media.…”