Development of a suspension packaging cell line for production of high titre, serum-resistant murine leukemia virus vectors.
To date, only adherent cell lines have been used for the generation of packaging cells for the production of type C retrovirus vectors. The large-scale production of high titre retrovirus vectors could benefit from the development of packaging cells growing in suspension. Here, we describe the ability of two different lymphoid cell lines, one B- and one T-lymphoblastoid cell line (Namalwa and CEM, respectively), to produce MLV-based vectors. Upon transfection with a third generation packaging construct, the virus particle production by Namalwa cells was characterised by low RT-activity, and by CEM cells as high RT activity as previously established adherent packaging cells. An amphotropic packaging cell line (CEMFLYA) was therefore established from CEM cells. Upon introduction of a lacZ vector genome, the novel packaging cell line produced vector particles routinely in the region of 10(7) infectious units/ml. The vectors were helper-free and highly stable in fresh human serum. The potential for scaled up vector production was demonstrated by continuous culture of the new packaging cells for 14 days in a 250 ml spinner flask. These suspension packaging cells should be applicable to large bioreactor systems to bulk produce high titre, complement-resistant retrovirus vectors for gene therapy.
Pizzato
M
M
Wohl Virion Centre, Windeyer Institute of Medical Sciences, University College London, UK.
Merten
O W
OW
Blair
E D
ED
Takeuchi
Y
Y
D001402:B-Lymphocytes / Q000821:virology*; D002460:Cell Line; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D009052:Leukemia Virus, Murine / Q000235:genetics*; D013601:T-Lymphocytes / Q000821:virology*