Closed hollow-fiber bioreactor: a new approach to retroviral vector production.
BACKGROUND
The ability to obtain high-titer and large quantities of retroviral vector production in a 'closed' system would have profound implications in clinical and experimental gene therapy.


METHODS
We studied the cell growth and vector production of three retroviral packaging cell lines in a variety of conditions using hollow-fiber bioreactors designed as an 'artificial capillary system' (ACS) and enhanced with the application of a hermetically sealing device for sterile welding of connecting plastic tubings. Vector titer, fetal bovine serum (FBS) concentration, volume and the duration of productivity were assessed to optimize vector production.


RESULTS
In this pilot study, we observed that retroviral vector production (frozen-and-thawed) from cultures containing as low as 2.5% FBS yielded titers up to 2.2 x 10(7) cfu/ml, 14.4-fold higher than titers obtained from control dish cultures. Up to 3 liters of vector supernatant were generated during a 2-month large-scale production run. There was a potential to double this volume of higher-titer supernatant by increasing the frequency of harvest. It seemed that a lower metabolic rate (i.e. lactate production) in the packaging cell culture was associated with higher vector producing ability.


CONCLUSIONS
These data demonstrated the feasibility of producing retroviral vector with enhanced titers and clinically useful quantities in a 'closed' ACS. Thus a new approach for large-scale retroviral vector production is developed.
Pan
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Gene Therapy Program, Institute of Human Genetics and Department of Pediatrics, University of Minnesota, Minneapolis 55455, USA.
Whitley
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D000818:Animals; D019149:Bioreactors; D002417:Cattle; D018929:Cell Culture Techniques / Q000295:instrumentation*; D002470:Cell Survival; D003470:Culture Media; D017077:Culture Media, Conditioned; D004203:Disinfection; D004867:Equipment Design; D005240:Feasibility Studies; D005312:Fetal Blood; D005822:Genetic Vectors / Q000302:isolation & purification*; D010865:Pilot Projects; D012190:Retroviridae / Q000254:growth & development* / Q000302:isolation & purification; D014776:Virus Cultivation / Q000295:instrumentation / Q000379:methods*
D003470:Culture Media; D017077:Culture Media, Conditioned