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Towards water-free biobanks: long-term dry-preservation at room temperature of desiccation-sensitive enzyme luciferase in air-dried insect cells

Shingo Kikuta, Shunsuke J. Watanabe, Ryoichi Sato, Oleg Gusev, Alexander Nesmelov, Yoichiro Sogame, Richard Cornette, Takahiro Kikawada
doi: https://doi.org/10.1101/120063
Shingo Kikuta
1Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan
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  • For correspondence: singo@cc.tuat.ac.jp kikawada@affrc.go.jp
Shunsuke J. Watanabe
1Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan
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Ryoichi Sato
1Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan
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Oleg Gusev
2Division of Genomic Technologies, RIKEN Center for Life Science Technologies, Yokohama, Kanagawa 230-0045, Japan
3RIKEN Preventive Medicine & Diagnosis Innovation Program, RIKEN, Wako, Saitama 351-0198, Japan
4Institute of fundamental Medicine and Biology, Kazan Federal University, Kazan 420008, Russia
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Alexander Nesmelov
4Institute of fundamental Medicine and Biology, Kazan Federal University, Kazan 420008, Russia
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Yoichiro Sogame
5Institute of Agrobiological Sciences, NARO, Tsukuba, Ibaraki 305-8634, Japan
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Richard Cornette
5Institute of Agrobiological Sciences, NARO, Tsukuba, Ibaraki 305-8634, Japan
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Takahiro Kikawada
5Institute of Agrobiological Sciences, NARO, Tsukuba, Ibaraki 305-8634, Japan
6Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-8562, Japan
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  • For correspondence: singo@cc.tuat.ac.jp kikawada@affrc.go.jp
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Abstract

Desiccation-tolerant cultured cells Pv11 derived from the anhydrobiotic Polypedilum vanderplanki embryo endure complete desiccation because of their ametabolic state and resume their metabolism after rehydration. These features led us to develop a novel dry preservation technology for enzymes as it was still unclear whether Pv11 cells preserved an exogenous enzyme in the dry state. This study shows that Pv11 cells protect an exogenous desiccation-sensitive enzyme, luciferase, preserving the enzymatic activity even after dry storage for 372 days at room temperature. A process including pre-incubation with trehalose, dehydration, storage, and rehydration allowed Pv11 (Pvll-Luc) cells stably expressing luciferase to survive desiccation and still emit luminescence caused by luciferase after rehydration. Luminescence produced by luciferase in Pvll-Luc cells after rehydration did not significantly decrease in presence of a translation inhibitor, showing that the activity did not derive from de novo enzyme synthesis following the resumption of cell metabolism. These findings indicate that the surviving Pv11 cells almost completely protect luciferase during desiccation. Lacking of the preincubation step resulted in the loss of luciferase activity after rehydration. We showed that preincubation with trehalose associated to induction of desiccation-tolerant related genes in Pv11 cells allowed effective in vivo preservation of enzymes in the dry state.

  • Abbreviation

    Luc
    luciferase
    GFP
    green fluorescent protein
    IPTG
    isopropyl-β-D-thiogalactopyranoside
    LEA
    late embryogenesis abundant
    HEK293T
    human embryonic kidney 293T
    BSA
    bovine serum albumin
    FBS
    fetal bovine serum
    D-MEM
    Dulbecco's modified Eagle medium
    ER
    endoplasmic reticulum
    RH
    relative humidity
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    Posted March 24, 2017.
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    Towards water-free biobanks: long-term dry-preservation at room temperature of desiccation-sensitive enzyme luciferase in air-dried insect cells
    Shingo Kikuta, Shunsuke J. Watanabe, Ryoichi Sato, Oleg Gusev, Alexander Nesmelov, Yoichiro Sogame, Richard Cornette, Takahiro Kikawada
    bioRxiv 120063; doi: https://doi.org/10.1101/120063
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    Towards water-free biobanks: long-term dry-preservation at room temperature of desiccation-sensitive enzyme luciferase in air-dried insect cells
    Shingo Kikuta, Shunsuke J. Watanabe, Ryoichi Sato, Oleg Gusev, Alexander Nesmelov, Yoichiro Sogame, Richard Cornette, Takahiro Kikawada
    bioRxiv 120063; doi: https://doi.org/10.1101/120063

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