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Intracellular trehalose improves the survival of cryopreserved mammalian cells

Nature Biotechnology volume 18, pages 163–167 (2000)Cite this article

Abstract

We report that the introduction of low concentrations of intracellular trehalose can greatly improve the survival of mammalian cells during cryopreservation. Using a genetically engineered mutant of Staphylococcus aureus α-hemolysin to create pores in the cellular membrane, we were able to load trehalose into cells. Low concentrations (0.2 M) of trehalose permitted long-term post-thaw survival of more than 80% of 3T3 fibroblasts and 70% of human keratinocytes. These results indicate that simplified and widely applicable freezing protocols may be possible using sugars as intracellular cryoprotective additives.

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Figure 1: Micrographs of fibroblasts plunged in N2(L) and thawed at 37°C with 0.4 M trehalose.
Figure 2: 3T3 fibroblasts plunge-frozen in N2(L) and thawed at 37°C were evaluated for plating efficiency (3 h, gray bars) and population growth (18 h, black bars).
Figure 3: Effect of extracellular trehalose concentration and permeabilization technique (H5 vs. WT) on the survival and growth of cryopreserved fibroblasts.
Figure 4: Effect of extracellular trehalose and H5 permeabilization on the survival of cryopreserved human keratinocytes.

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References

  1. Langer, R. & Vacanti, J.P. Tissue engineering: the development of functional substitutes for damaged tissue. Science 260, 920–926 (1993).

    Article  CAS  Google Scholar 

  2. Mulligan, R.C. The basic science of gene therapy. Science 260, 926–932 (1993).

    Article  CAS  Google Scholar 

  3. Mazur, P. Freezing of living cells: mechanisms and implications. Am. J. Physiol. 247, C125–C142 ( 1984).

    Article  CAS  Google Scholar 

  4. Carpenter, J.F., Crowe, L.M. & Crowe, J.H. Stabilization of phosphofructokinase with sugars during freeze-drying. Biochim. Biophys. Acta 932, 109–115 (1987).

    Article  Google Scholar 

  5. Crowe, L.M., Crowe, J.H., Rudolph, A., Wormersley, C. & Appel, L. Preservation of dried liposomes by trehalose. Arch. Biochem. Biophys. 242, 240–247 (1985).

    Article  CAS  Google Scholar 

  6. Crowe, L.M., Mouradian, R., Crowe, J.H., Jackson, S.A. & Wormersly, C. Effect of carbohydrates on membrane stability at low water activities. Biochim. Biophys. Acta 769, 141–150 (1984).

    Article  CAS  Google Scholar 

  7. Green, J.L. & Angell, C.A. Phase-relations and vitrification in saccharide–water solutions and the trehalose anomaly. J. Phys. Chem. 93, 2880–2882 (1989).

    Article  CAS  Google Scholar 

  8. Bieganski, R.M., Fowler, A., Morgan & J.R. Toner, M. Stabilization of active recombinant retroviruses in an amorphos dry state with trehalose. Biotechnol. Prog. 14, 615–620 ( 1998).

    Article  CAS  Google Scholar 

  9. Israeli, E., Shaffer, B.T. & Lighthart, B. Protection of freeze-dried Escherichia coli by trehalose upon exposure to environmental conditions. Cryobiology 30 519–523 ( 1993).

    Article  CAS  Google Scholar 

  10. Rossi, S., Buera, M.P., Moreno, S. & Chirife, J. Stabilization of the restriction enzyme EcoRI dried with trehalose and other selected glass-forming solutions. Biotechnol. Prog. 13, 609–616 (1997).

    Article  CAS  Google Scholar 

  11. Uritani, M., Takai, M. & Yoshinaga, K. Protective effect of disaccharides on restriction endonucleases during drying under vacuum. J. Biochem. 117, 774–779 (1995).

    Article  CAS  Google Scholar 

  12. Miller, D.P., Pablo, J.J. & Corti, H. Thermophysical properties of trehalose and its concentrated aqueous solutions. Pharm. Res. 14, 578– 590 (1997).

    Article  CAS  Google Scholar 

  13. Russo, M.J., Bayley, H. & Toner, M. Reversible permeabilization of plasma membranes with an engineered switchable pore. Nat. Biotechnol. 15, 278–282 (1997).

    Article  CAS  Google Scholar 

  14. Walker, B., Kasianowicz, J., Krishnasastry, M. & Bayley, H. A pore-forming protein with a metal-actuated switch. Protein Eng. 7, 655–662 ( 1994).

    Article  CAS  Google Scholar 

  15. Song, L. et al. Structure of staphylococcal α-hemolysin, a heptameric transmembrane pore. Science 274, 1859– 1866 (1996).

    Article  CAS  Google Scholar 

  16. Bayley, H. Building doors into cells. Sci. Am. 277, 62–67 (1997).

    Article  CAS  Google Scholar 

  17. Beattie, G.M. et al. Trehalose: a cryoprotectant that enhances recovery and preserves function of human pancreatic islets after long-term storage. Diabetes 46, 519–523 ( 1997).

    Article  CAS  Google Scholar 

  18. Mazur, P., Farrant, J., Leibo, S.P. & Chu, E.H.Y. Survival of hamster tissue culture cells after freezing and thawing. Cryobiology 6, 1–9 (1969).

    Article  CAS  Google Scholar 

  19. Mazur, P., Leibo, S.P. & Chu, E.H.Y. A two-factor hypothesis of freezing injury. Exp. Cell Res. 71, 345–355 (1972).

    Article  CAS  Google Scholar 

  20. Walev, I. et al. Staphylococcal alpha-toxin kills human keratinocytes by permeabilizing the plasma membrane for monovalent ions. Infect. Immun. 61. 4972–4979 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Eming, S.A. & Morgan, J.R. in Methods in molecular medicine, gene therapy protocols. (ed. Robbins, P.) 265– 279 (Humana, Totowa, NJ; 1997).

    Google Scholar 

  22. Hildebrand, A., Pohl, M. & Bhakdi, S. Staphylococcus aureus α-toxin: dual mechanism of binding to target cells. J. Biol. Chem. 266, 17195–17200 (1991).

    CAS  PubMed  Google Scholar 

  23. Walker, B., Braha, O., Cheley. S. & Bayley H. An intermediate in the assembly of a pore-forming protein trapped with a genetically engineered switch. Chem. Biol. 2, 99– 105 (1995).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was partially supported by the National Institutes of Health grants DK46270 and NS26760, Shriners Hospitals for Children, ONR and DOE. The authors thank Xiaofeng Lu for technical assistance. The authors also acknowledge Dr. Jeffrey Morgan's assistance in keratinocyte culture and evaluation. Michael J. Russo was a Howard Hughes Medical Institute Pre-Doctoral Fellow.

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Author notes

  1. Ali Eroglu and Michael J. Russo: The first two authors contributed equally to this work and are listed alphabetically by their last names.

Authors and Affiliations

  1. The Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Harvard Medical School,

    Ali Eroglu, Michael J. Russo, Robert Bieganski, Alex Fowler & Mehmet Toner

  2. Shriners Hospital for Children, Boston, 02114, MA

    Ali Eroglu, Michael J. Russo, Robert Bieganski, Alex Fowler & Mehmet Toner

  3. Department of Mechanical Engineering, University of Massachusetts, Dartmouth, 02747, MA

    Alex Fowler

  4. Department of Medical Biochemistry and Genetics, Texas A&M University, College Station, 77843, TX

    Stephen Cheley & Hagan Bayley

Authors
  1. Ali Eroglu
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Correspondence to Mehmet Toner.

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Eroglu, A., Russo, M., Bieganski, R. et al. Intracellular trehalose improves the survival of cryopreserved mammalian cells. Nat Biotechnol 18, 163–167 (2000). https://doi.org/10.1038/72608

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