Rabies virus production in high vero cell density cultures on macroporous microcarriers

Abstract

The purpose of the study was to investigate the rabies virus multiplication in Vero cell cultures performed on porous microcarriers, MCs (cellulose-Cytopore and gelatin-Cultispher G), which provide higher available surface area compared with solid (nonporous) MCs (DEAE-Cytodex 1). In a set of experiments performed at the same MC concentration (MCs per milliliter), cell densities regularly obtained in porous MC cultures were comparable, but almost twice as high as those in solid MC cultures. In addition, 41.1 +/- 3.9-, 35.2 +/- 2-, and 19.6 +/- 5.8-fold increases in cell concentration, relative to the initial cell number, along with maximum rabies virus titers of 6.3 +/- 0.3 x 10(4), 5 +/- 0.1 x 10(4), and 4.3 +/- 0.2 x 10(4) FFD(50)/mL were observed in Cytopore, Cultispher G, and Cytodex 1 MC cultures, respectively. When higher concentrations of MCs were employed, lower performances of virus production and MC-cell occupation (cells per MC or cells per square millimeter) were observed. Cell attachment to MCs was shown to be faster for Cytopore MCs and Cytodex 1 MCs than for Cultispher G MCs. Concerning the kinetics of cell multiplication on MCs, exponential cell growth, at similar specific cell growth rates, took place on Cytopore, Cultispher G, and Cytodex 1 MCs. In addition, cell densities as high as 2.1 +/- 0.2 x 10(6) cells/mL on Cytopore MCs, 1.8 +/- 0.1 x 10(6) cells/mL on Cultispher G MCs, and 1 +/- 0.3 x 10(6) cells/mL on Cytodex 1 MCs were regularly obtained in batch cultures. Optical as well as scanning and transmission electron microscopy studies carried out to analyze MC structure, MC cell occupation, and cell permissivity to virus infection demonstrated that there was uniform cell distribution in the external and internal areas of the MCs, suggesting an efficiency of virus synthesis. Our results indicate the usefulness of these supports for rabies virus antigen production, as well as possibilities for further optimization.

Materials in particulate form for tissue engineering. 1. Basic concepts

“…Chromatography (Attebery, 1975;Rocca and Rouchouse, 1976;Fahlvik et al, 1990;Zhang and El Rassi, 1999;Spegel et al, 2001) Imaging (Cuthbertson et al, 2003;Cavalieri et al, 2005;Huang et al, 2006;Klibanov, 2006) Filling of defects (Schepers et al, 1991;Guicheux et al, 1997;Santos et al, 1998;Schepers et al, 1998;Falaize et al, 1999;Huygh et al, 2002;Day et al, 2004;Domingues et al, 2004;Gosain, 2004) Adjuvants in vaccines (Ohagan et al, 1993;Moore et al, 1995;Nakaoka et al, 1995;Ertl et al, 1996;Heritage et al, 1996;Ohagan et al, 1997;Stertman et al, 2006) Cell culture (Malda et al, 2003b;Xu et al, 2003;Zhang et al, 2003;Liu and Wu, 2004;Yokomizo et al, 2004;Hong et al, 2005;Melero-Martin et al, 2006) Drug delivery (Herrmann and Bodmeier, 1995;Guicheux et al, 1997;Berthold et al, 1998;Herrmann and Bodmeier, 1998;Jeong et al, 1998;Cruaud et al, 1999;Ganza-Gonzalez et al, 1999;Lam et al, 2000;Lim et al, 2000;…”

Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system

“…Stirred culture systems, such as the rotary RCCS™ bioreactor used by Chen et al (8) and the spinner flasks employed by Fok and Zandstra (9), represent a significant improvement in culture techniques compared to static systems, as they offer several advantages in the scale-up process, includ-A.M. Fernandes et al www.bjournal.com.br ing: 1) a controlled environment leading to a homogeneous culture, 2) simplicity of handling, and 3) less susceptibility to contamination (10). Indeed, bioreactors have been used in a variety of bioprocesses, such as cell expansion (11)(12)(13)(14) and protein and virus production (15)(16)(17).…”

A serum-free Vero production platform for a chimeric virus vaccine candidate

“…Although Vero cells can grow in suspension as cell aggregates (Litwin 1992), they are typically propagated in static cultures or on microcarriers (Yokomizo et al 2004;Wu et al 2004;Berry et al 1999) because they are usually considered to be anchorage-dependent. Since adherent cultures of Vero cells are well-characterized, have demonstrated an absence of tumorigenicity (VincentFalquet et al 1989) and also have an excellent safety record (Montagnon and Vincent-Falquet 1998), no efforts were made in this work to adapt the anchorage-dependent Vero cells to grow in suspension culture.…”