¹Wohl Virion Centre, Windeyer Institute of Medical Sciences, University College London, UK

²Virology Unit, GlaxoWellcome, Medicines Research Centre, Stevenage, UK

³Généthon III, Evry, France

Correspondence to: Y Takeuchi, Windeyer Institute of Medical Sciences, Wohl Virion Centre, University College London, 46 Cleveland Street, London W1T 4JF,UK

^aPresent address: Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02115, USA Received 22 November 2000; accepted 26 February 2001

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 10⁷ 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. Gene Therapy (2001) 8, 737-745.

MLV; packaging; suspension cell; serum resistance