Intracellular trehalose improves the survival of cryopreserved mammalian cells

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.

[1]  C. Angell,et al.  Phase relations and vitrification in saccharide-water solutions and the trehalose anomaly , 1989 .

[2]  J. Morgan,et al.  Methods for the use of genetically modified keratinocytes in gene therapy. , 1997, Methods in molecular medicine.

[3]  J. Vacanti,et al.  Tissue engineering : Frontiers in biotechnology , 1993 .

[4]  H. Bayley.  Building doors into cells. , 1997, Scientific American.

[5]  H. Bayley,et al.  An intermediate in the assembly of a pore-forming protein trapped with a genetically-engineered switch. , 1995, Chemistry & biology.

[6]  J. R. Morgan,et al.  Stabilization of Active Recombinant Retroviruses in an Amorphous Dry State with Trehalose , 1998, Biotechnology progress.

[7]  M. Takai,et al.  Protective effect of disaccharides on restriction endonucleases during drying under vacuum. , 1995, Journal of biochemistry.

[8]  J. Vacanti,et al.  Tissue engineering. , 1993, Science.

[9]  P. Mazur.  Freezing of living cells: mechanisms and implications. , 1984, The American journal of physiology.

[10]  Silvia Moreno,et al.  Stabilization of the Restriction Enzyme EcoRI Dried with Trehalose and Other Selected Glass‐Forming Solutes , 1997 .

[11]  J. Crowe,et al.  Preservation of freeze-dried liposomes by trehalose. , 1985, Archives of biochemistry and biophysics.

[12]  Ana D. Lopez,et al.  Trehalose: A Cryoprotectant That Enhances Recovery and Preserves Function of Human Pancreatic Islets After Long-Term Storage , 1997, Diabetes.

[13]  J. Gouaux,et al.  Structure of Staphylococcal α-Hemolysin, a Heptameric Transmembrane Pore , 1996, Science.

[14]  B. Lighthart,et al.  Protection of freeze-dried Escherichia coli by trehalose upon exposure to environmental conditions. , 1993, Cryobiology.

[15]  J. Carpenter,et al.  Stabilization of phosphofructokinase with sugars during freeze-drying: characterization of enhanced protection in the presence of divalent cations. , 1987, Biochimica et biophysica acta.

[16]  R. Mulligan,et al.  The basic science of gene therapy. , 1993, Science.

[17]  S. Bhakdi,et al.  Staphylococcus aureus alpha-toxin. Dual mechanism of binding to target cells. , 1991, The Journal of biological chemistry.

[18]  P. Mazur,et al.  A two-factor hypothesis of freezing injury. Evidence from Chinese hamster tissue-culture cells. , 1972, Experimental cell research.

[19]  J. Crowe,et al.  Effects of carbohydrates on membrane stability at low water activities. , 1984, Biochimica et biophysica acta.

[20]  H. Bayley,et al.  A pore-forming protein with a metal-actuated switch. , 1994, Protein engineering.

[21]  N. Janel.  Methods in molecular medicine : vascular disease, molecular biology and gene therapy protocols , 2001 .

[22]  Mehmet Toner,et al.  Reversible permeabilization of plasma membranes with an engineered switchable pore , 1997, Nature Biotechnology.

[23]  J. Farrant,et al.  Survival of hamster tissue culture cells after freezing and thawing. Interactions between protective solutes and cooling and warming rates. , 1969, Cryobiology.

[24]  M. Mohamadzadeh,et al.  Staphylococcal alpha-toxin kills human keratinocytes by permeabilizing the plasma membrane for monovalent ions , 1993, Infection and immunity.