Rabies virus production in high vero cell density cultures on macroporous microcarriers.
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.
Yokomizo
A Y
AY
Laboratório de Imunologia Viral, Instituto Butantan, 05503-900 São Paulo, Brasil. grugel@butantan.gov.br
Antoniazzi
M M
MM
Galdino
P L
PL
Azambuja
N
N
Jorge
S A C
SA
Pereira
C A
CA
D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods*; D002455:Cell Division / Q000502:physiology; D002522:Chlorocebus aethiops; D019544:Equipment Failure Analysis; D016062:Porosity; D011820:Rabies virus / Q000254:growth & development* / Q000648:ultrastructure*; D014709:Vero Cells; D014776:Virus Cultivation / Q000295:instrumentation* / Q000379:methods*; D014779:Virus Replication / Q000502:physiology*