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Long-term, large scale cryopreservation of insect cells at −80 °C

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Abstract

Standard tissue culture methods advise freezing cells in small aliquots (≤1 × 107 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 × 108 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.

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Acknowledgments

The baculovirus expressing green fluorescent protein was a gift from Vangelis Christodoulou. The work was funded by Cancer Research UK programme grants to David Barford, Laurence H. Pearl and Dale Wigley. We would also like to thank David Barford and Jane Sandall for assistance with the critical reading of the manuscript.

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Author notes

  1. Kirstin J. L. Wood

    Present address: 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 and Affiliations

  1. Section of Structural Biology, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK

    Lucie Vyletova, La’Verne P. Rennalls, Kirstin J. L. Wood & Valerie M. Good

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  1. Lucie Vyletova
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  2. La’Verne P. Rennalls
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  3. Kirstin J. L. Wood
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Correspondence to Valerie M. Good.

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La’Verne P. Rennalls and Kirstin J. L. Wood contributed equally to the work.

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Vyletova, L., Rennalls, L.P., Wood, K.J.L. et al. Long-term, large scale cryopreservation of insect cells at −80 °C. Cytotechnology 68, 303–311 (2016). https://doi.org/10.1007/s10616-014-9781-5

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