Metabolic active-high density VERO cell cultures on microcarriers following apoptosis prevention by galactose/glutamine feeding.
The control of cell death occurring in high density cultures performed in bioreactors is an important factor in production processes. In this work, medium nutrient removal or feeding was used to determine at which extension apoptosis could be, respectively, involved or prevented in VERO cell cultures on microcarriers. Glutamine and galactose present in the VERO cell culture medium was consumed after, respectively, 6 and 12 days of culture. Kinetics studies showed that fresh medium replacement and, to some extent, galactose or glutamine depleted-fresh medium replacement provided a nutritional environment, allowing the VERO cell cultures to attain high densities. Galactose was shown to be a more critical nutrient when cultures reached a high density. In agreement with that, VERO cell cultures supplemented with galactose and/or glutamine were shown to confirm previous findings and, again at high densities, galactose was shown to be a critical nutrient for VERO cell growth. These observations also indicated that in VERO cell cultures, for feeding purposes, the glutamine could be replaced by galactose. The inverse was not true and led, at high densities, to a decrease of cell viability. In the absence of glutamine and galactose, apoptosis was observed in VERO cell cultures by cytofluorometry, Acridine orange staining or light and electron microscopy, reaching high levels when compared to cultures performed with complete medium. VERO cells apoptosis process could be prevented by the galactose and/or glutamine feeding and, at high densities, galactose was more efficient in protecting the cultures. These cultures, prevented from apoptosis, were shown to synthesize high levels of measles virus following infection. Our data show that apoptosis prevention by glutamine/galactose feeding, led to high productive and metabolic active VERO cell cultures, as indicated by the high cell density obtained and the virus multiplication leading to higher virus titers.
Mendonça
Ronaldo Z
RZ
Laboratório de Imunologia Viral, Instituto Butantan, Avenida Vital Brasil 1500, 05503-900 São Paulo, Brazil.
Arrózio
Sara J
SJ
Antoniazzi
Marta M
MM
Ferreira
Jorge M C
JM
Pereira
Carlos A
CA
D000818:Animals; D000956:Antigens, Viral / Q000096:biosynthesis; D017209:Apoptosis / Q000187:drug effects*; D019149:Bioreactors; D002455:Cell Division / Q000187:drug effects; D002522:Chlorocebus aethiops; D003470:Culture Media / Q000494:pharmacology; D005434:Flow Cytometry; D005690:Galactose / Q000494:pharmacology*; D005973:Glutamine / Q000494:pharmacology*; D008459:Measles virus / Q000254:growth & development; D009336:Necrosis; D014709:Vero Cells / Q000166:cytology / Q000378:metabolism* / Q000821:virology; D014779:Virus Replication
D000956:Antigens, Viral; D003470:Culture Media; D005973:Glutamine; D005690:Galactose