Large-scale production of recombinant viruses by use of a large culture vessel with active gassing.
Adenovirus and adeno-associated virus (AAV) vectors are increasingly used for gene transduction experiments. However, to produce a sufficient amount of these vectors for in vivo experiments requires large-capacity tissue culture facilities, which may not be practical in limited laboratory space. We describe here a large-scale method to produce adenovirus and AAV vectors with an active gassing system that uses large culture vessels to process labor- and cost-effective infection or transfection in a closed system. Development of this system was based on the infection or transfection of 293 cells on a large scale, using a large culture vessel with a surface area of 6320 cm2. A minipump was connected to the gas inlet of the large vessel, which was placed inside the incubator, so that the incubator atmosphere was circulated through the vessel. When active gassing was employed, the productivity of the adenovirus and AAV vectors significantly increased. This vector production system was achieved by improved CO2 and air exchange and maintenance of pH in the culture medium. Viral production with active gassing is particularly promising, as it can be used with existing incubators and the large culture vessel can readily be converted for use with the active gassing system.
Okada
Takashi
T
Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Minami-Kawachi, Tochigi 329-0498, Japan. tokada@jichi.ac.jp
Nomoto
Tatsuya
T
Yoshioka
Toru
T
Nonaka-Sarukawa
Mutsuko
M
Ito
Takayuki
T
Ogura
Tsuyoshi
T
Iwata-Okada
Mayumi
M
Uchibori
Ryosuke
R
Shimazaki
Kuniko
K
Mizukami
Hiroaki
H
Kume
Akihiro
A
Ozawa
Keiya
K
D000256:Adenoviridae / Q000235:genetics / Q000254:growth & development; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D000229:Dependovirus / Q000235:genetics / Q000254:growth & development; D005260:Female; D006801:Humans; D051381:Rats; D017207:Rats, Sprague-Dawley; D014161:Transduction, Genetic / Q000379:methods