Vyletova Lucie, Rennalls La'Verne P, Wood Kirstin J L, Good Valerie M
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.
Cytotechnology. 2016 Mar;68(2):303-11. doi: 10.1007/s10616-014-9781-5. Epub 2014 Aug 23.
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.
标准的组织培养方法建议将细胞以小份冷冻（1毫升中≤1×10⁷个细胞），并储存在液氮中。对于利用重组杆状病毒表达来培养大量昆虫细胞的实验室而言，这并不方便，因为从小份细胞开始产生大规模培养物所需的时间较长。液氮储存需要使用专门的冻存管、个人防护装备和氧气监测系统。本文描述了在-80°C下使用标准的50毫升锥形管容纳40毫升细胞悬液对8×10⁸个昆虫细胞进行长期、大规模冷冻保存的方法。在这些条件下储存一年后，Sf9、Sf21和High 5细胞的复苏活力>90%，与在液氮中储存相同时间的细胞活力相比具有优势。添加编码绿色荧光蛋白的杆状病毒表明，细胞在解冻后立即“具备表达能力”。我们的方法能够从在-80°C下冷冻保存的储备中快速复苏大规模培养物，从而避免了与液氮相关的不便、风险和费用。
