title: Long-term, large scale cryopreservation of insect cells at -80 °C.
abstract: Standard tissue culture methods advise freezing cells in small aliquots (≤1 × 10(7) cells in 1 mL), and storing in liquid nitrogen. This is inconvenient for laboratories culturing large quantities of insect cells for recombinant baculovirus expression, owing to the length of time taken to produce large scale cultures from small aliquots of cells. Liquid nitrogen storage requires use of specialized cryovials, personal protective equipment and oxygen monitoring systems. This paper describes the long-term, large scale cryopreservation of 8 × 10(8) insect cells at -80 °C, using standard 50 mL conical tubes to contain a 40 mL cell suspension. Sf9, Sf21 and High 5 cells were recovered with a viability > 90 % after storage for one year under these conditions, which compared favorably with the viability of cells stored in liquid nitrogen for the same length of time. Addition of green fluorescent protein encoding baculovirus demonstrated that cells were "expression ready" immediately post thaw. Our method enables large scale cultures to be recovered rapidly from stocks cryopreserved at -80 °C, thus avoiding the inconvenience, hazards and expense associated with liquid nitrogen.
authors.affiliation: Section of Structural Biology, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK.
authors.forename: Lucie
authors.initials: L
authors.lastname: Vyletova
authors.affiliation: Section of Structural Biology, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK.
authors.forename: La'Verne P
authors.initials: LP
authors.lastname: Rennalls
authors.affiliation: Section of Structural Biology, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK.|Laboratory of Protein Crystallography, Centre for Amyloidosis and Acute Phase, Proteins, UCL Division of Medicine (Royal Free Campus), Rowland Hill Street, London, NW3 2PF, UK.
authors.forename: Kirstin J L
authors.initials: KJ
authors.lastname: Wood
authors.affiliation: Section of Structural Biology, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK. vgood@icr.ac.uk.
authors.forename: Valerie M
authors.initials: VM
authors.lastname: Good
keywords: Baculovirus expression; Cryopreservation; Green fluorescent protein; Insect cell culture