Measurement of Trehalose Loading of Mammalian Cells Porated With a Metal-Actuated Switchable Pore Jason P. Acker, 1 Xiao-ming Lu, 1 Vernon Young, 2 Stephen Cheley, 3 Hagan Bayley, 3 Alex Fowler, 1,4 Mehmet Toner 1 1 The Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Harvard Medical School, and Shriners Hospitals for Children, 51 Blossom Street, Room 266, Boston, Massachusetts 02114; telephone: 617-371-4883; fax: 617-371-4950; e-mail: mtoner@sbi.org 2 Laboratory of Human Nutrition, School of Science and Clinical Research Center, Massachusetts Institute of Technology, Cambridge, Massachusetts 3 Department of Medical Biochemistry and Genetics, Texas A & M University, College Station, Texas 4 Department of Mechanical Engineering, University of Massachusetts, Dartmouth, Massachusetts Received 10 June 2002; accepted 31 October 2002 DOI: 10.1002/bit.10599 Abstract: Efforts to improve the tolerance of mammalian cells to desiccation have focused on the role that sugars have in protecting cells from lethal injury. Among the key determinants of desiccation tolerance is the intracellu- lar trehalose concentration, and thus quantifying the amount and rate of trehalose accumulation has now be- come very critical to the success of these desiccation approaches. We introduced trehalose into 3T3 fibro- blasts, human keratinocytes, and rat hepatocytes using a genetically engineered mutant of the pore-forming -he- molysin from Staphylococcus aureus. Manipulating the extracellular Zn 2+ concentration selectively opens and closes this pore (∼2 nm) and enables controlled loading of cells with sugars. We quantified intracellular trehalose using gas chromatography–mass spectroscopy (GC-MS) to examine the trimethylsilyl derivative of intracellular trehalose. Using the GC-MS method, we demonstrate that the switchable characteristics of H5 -hemolysin permit controlled loading of the high concentrations of trehalose (up to 0.5 M) necessary for engineering desic- cation tolerance in mammalian cells. © 2003 Wiley Periodi- cals, Inc. Biotechnol Bioeng 82: 525–532, 2003. Keywords: gas chromatography (GC); -hemolysin; in- tracellular trehalose; anhydrobiosis; biopreservation INTRODUCTION The development of effective long-term preservation tech- niques has become a critical barrier to the successful clinical and commercial application of emerging cell-based tech- nologies (Karlsson and Toner, 1996). Although cryopreser- vation has been the traditional approach used to preserve cells and tissues, practical constraints placed on the manipu- lation, storage, and transportation of samples at cryogenic temperatures (Karlsson and Toner, 1996; Mazur, 1984) have led researchers to explore alternative preservation methods. Anhydrobiosis is a natural strategy used by a va- riety of organisms to preserve biological activity through times of environmental stress. By studying the complex series of internal physiological and biochemical changes and adaptations that permit anhydrobiotic organisms to sur- vive severe dehydration (Crowe et al., 1992; Potts, 1994), it has been proposed that controlled desiccation of mamma- lian cells permits long-term preservation at ambient tem- peratures. Efforts to improve the tolerance of mammalian cells to desiccation have focused on the role that intracellular sugars have in protecting cells from lethal injury. As trehalose has been shown to be important in stabilizing and preserving anhydrobiotic organisms (reviewed in Crowe et al. [1987]), it has been suggested that this nonreducing disaccharide may have an important role in the preservation of dried mammalian cells (Crowe et al., 1992). Indeed, trehalose is effective in preserving dried liposomes (Crowe et al., 1987; Sun et al., 1996), bacteria (Conrad et al., 2000; Israeli et al., 1993; Leslie et al., 1995; Potts, 1994), yeast (Leslie et al., 1994), and retroviruses (Bieganski et al., 1998). Trehalose needs to be present on both sides of the membrane to protect against the damaging effects of dehydration (Chen et al., 2001; Crowe et al., 1985; Eleutherio et al., 1993; Womers- ley et al., 1986). Among the key impediments to using trehalose during the desiccation of mammalian cells has Correspondence to: M. Toner Contract grant sponsors: Defense Advanced Research Projects Agency/ Naval Research Laboratories; National Institute of Health; Shriners Hos- pitals for Children Contract grant numbers: N00173-01-1 G011; DK46270; NS26760 © 2003 Wiley Periodicals, Inc.