Cryopreservation of spin-dried mammalian cells.

This study reports an alternative approach to achieve vitrification where cells are pre-desiccated prior to cooling to cryogenic temperatures for storage. Chinese Hamster Ovary (CHO) cells suspended in a trehalose solution were rapidly and uniformly desiccated to a low moisture content (<0.12 g of water per g of dry weight) using a spin-drying technique. Trehalose was also introduced into the cells using a high-capacity trehalose transporter (TRET1). Fourier Transform Infrared Spectroscopy (FTIR) was used to examine the uniformity of water concentration distribution in the spin-dried samples. 62% of the cells were shown to survive spin-drying in the presence of trehalose following immediate rehydration. The spin-dried samples were stored in liquid nitrogen (LN(2)) at a vitrified state. It was shown that following re-warming to room temperature and re-hydration with a fully complemented cell culture medium, 51% of the spin-dried and vitrified cells survived and demonstrated normal growth characteristics. Spin-drying is a novel strategy that can be used to improve cryopreservation outcome by promoting rapid vitrification.

Spatial distribution of the state of water in frozen mammalian cells.

We describe direct determination of the state of intracellular water, measurement of the intercellular concentration of a cryoprotectant agent (dimethylsulfoxide), and the distribution of organic material in frozen mammalian cells. Confocal Raman microspectroscopy was utilized at cryogenic temperatures with single live cells to conduct high spatial resolution measurements (350 × 350 × 700 nm), which yielded two, we believe, novel observations: 1), intracellular ice formation during fast cooling (50°C/min) causes more pronounced intracellular dehydration than slow cooling (1°C/min); and 2), intracellular dimethylsulfoxide concentration is lower (by as much as 50%) during fast cooling, decreasing the propensity for intracellular vitrification. These observations have a very significant impact for developing successful biopreservation protocols for cells used for therapeutic purposes and for cellular biofluids.

Hydrogen bonding and compartmentalization of water in supercooled and frozen aqueous acetone solutions.

Temperature ramp Fourier transform infrared (FTIR) microspectroscopy was utilized to examine hydrogen bonding (HB) in and crystallization of supercooled aqueous acetone solutions. We showed that temperature and concentration-dependent transitions between different water populations, representing distinct HB organization, played very significant roles in ice crystallization and formation of distinct thermodynamic phases in supercooled aqueous solutions. At cryogenic temperatures, mainly three different coexisting thermodynamic phases were identified: a hexagonal ice phase, which exhibited linear planar growth with supercooling; a low water content supercooled solution rich in water monomers and dimers, which froze at lower temperatures as a result of a significant increase in HB networking; and a high water content frozen solution. We show that spectroscopic analysis water in polar solutions at cryogenic temperatures present a unique platform to explore the freezing/melting/vitrification behavior of water.

Hydrogen bonding and kinetic/thermodynamic transitions of aqueous trehalose solutions at cryogenic temperatures.

Carbohydrates play important roles in the survival of freeze-tolerant organisms. In order to understand the role of carbohydrates on hydrogen bonding (HB) and thermodynamic/kinetic transitions, aqueous trehalose solutions at cryogenic temperatures were analyzed using FTIR spectroscopy. Distinct changes in water-water and water-carbohydrate HB organization were identified during supercooling, freezing, and vitrification. FTIR spectroscopy revealed the kosmotropic effect of trehalose and the presence of two distinct water families in supercooled carbohydrate solutions, (1) water molecules directly associated with the carbohydrate, forming its hydration layer(s) and (2) water molecules that are involved in water-water HB in small clusters. The latter showed characteristics of water in hydrophilic confinement.

Roles of membrane structure and phase transition on the hyperosmotic stress survival of Geobacter sulfurreducens.

Geobacter sulfurreducens is a delta-proteobacterium bacteria that has biotechnological applications in bioremediation and as biofuel cells. Development of these applications requires stabilization and preservation of the bacteria in thin porous coatings on electrode surfaces and in flow-through bioreactors. During the manufacturing of these coatings the bacteria are exposed to hyperosmotic stresses due to dehydration and the presence of carbohydrates in the medium. In this study we focused on quantifying the response of G. sulfurreducens to hyperosmotic shock and slow dehydration to understand the hyperosmotic damage mechanisms and to develop the methodology to maximize the survival of the bacteria. We employed FTIR spectroscopy to determine the changes in the structure and the phase transition behavior of the cell membrane. Hyperosmotic shock resulted in greatly decreased membrane lipid order in the gel phase and a less cooperative membrane phase transition. On the other hand, slow dehydration resulted in increased membrane phase transition temperature, less cooperative membrane phase transition and a small decrease in the gel phase lipid order. Both hyperosmotic shock and slow dehydration were accompanied by a decrease in viability. However, we identified that in each case the membrane damage mechanism was different. We have also shown that the post-rehydration viability could be maximized if the lyotropic phase change of the cell membrane was eliminated during dehydration. On the other hand, lyotropic phase change during re-hydration did not affect the viability of G. sulfurreducens. This study conclusively shows that the cell membrane is the primary site of injury during hyperosmotic stress, and by detailed analysis of the membrane structure as well as its thermodynamic transitions it is indeed possible to develop methods in a rational fashion to maximize the survival of the bacteria during hyperosmotic stress.