The effect of sucrose, maltodextrin and skim milk on survival of L. bulgaricus after drying was studied. Survival could be improved from 0.01% for cells that were dried in the absence of protectants to 7.8% for cells dried in a mixture of sucrose and maltodextrin. Fourier transform infrared spectroscopy (FTIR) was used to study the effect of the protectants on the overall protein secondary structure and thermophysical properties of the dried cells. Sucrose, maltodextrin and skim milk were found to have minor effects on the membrane phase behavior and the overall protein secondary structure of the dried cells. FTIR was also used to show that the air-dried cell/protectant solutions formed a glassy state at ambient temperature. 1-Palmitoyl 2-oleoyl phosphatidyl choline (POPC) was used in order to determine if sucrose and maltodextrin have the ability to interact with phospholipids during drying. In addition, the glass transition temperature and strength of hydrogen bonds in the glassy state were studied using this model system. Studies using poly-L-lysine were done in order to determine if sucrose and maltodextrin are able to stabilize protein structure during drying. As expected, sucrose depressed the membrane phase transition temperature (Tm) of POPC in the dried state and prevented conformational changes of poly-L-lysine during drying. Maltodextrin, however, did not depress the Tm of dried POPC and was less effective in preventing conformational changes of poly-L-lysine during drying. We suggest that when cells are dried in the presence of sucrose and maltodextrin, sucrose functions by directly interacting with biomolecules, whereas maltodextrin functions as an osmotically inactive bulking compound causing spacing of the cells and strengthening of the glassy matrix.

There is no generally accepted value for the lower temperature limit for life on Earth. We present empirical evidence that free-living microbial cells cooling in the presence of external ice will undergo freeze-induced desiccation and a glass transition (vitrification) at a temperature between −10°C and −26°C. In contrast to intracellular freezing, vitrification does not result in death and cells may survive very low temperatures once vitrified. The high internal viscosity following vitrification means that diffusion of oxygen and metabolites is slowed to such an extent that cellular metabolism ceases. The temperature range for intracellular vitrification makes this a process of fundamental ecological significance for free-living microbes. It is only where extracellular ice is not present that cells can continue to metabolise below these temperatures, and water droplets in clouds provide an important example of such a habitat. In multicellular organisms the cells are isolated from ice in the environment, and the major factor dictating how they respond to low temperature is the physical state of the extracellular fluid. Where this fluid freezes, then the cells will dehydrate and vitrify in a manner analogous to free-living microbes. Where the extracellular fluid undercools then cells can continue to metabolise, albeit slowly, to temperatures below the vitrification temperature of free-living microbes. Evidence suggests that these cells do also eventually vitrify, but at lower temperatures that may be below −50°C. Since cells must return to a fluid state to resume metabolism and complete their life cycle, and ice is almost universally present in environments at sub-zero temperatures, we propose that the vitrification temperature represents a general lower thermal limit to life on Earth, though its precise value differs between unicellular (typically above −20°C) and multicellular organisms (typically below −20°C). Few multicellular organisms can, however, complete their life cycle at temperatures below ∼−2°C.

For the clinical delivery of immunotherapies it is anticipated that cells will be cryopreserved and shipped to the patient where they will be thawed and administered. An established view in cellular cryopreservation is that following freezing, cells must be warmed rapidly (≤5 minutes) in order to maintain high viability. In this study we examine the interaction between the rate of cooling and rate of warming on the viability, and function of T cells formulated in a conventional DMSO based cryoprotectant and processed in conventional cryovials. The data obtained show that provided the cooling rate is −1 °C min −1 or slower, there is effectively no impact of warming rate on viable cell number within the range of warming rates examined (1.6 °C min −1 to 113 °C min −1 ). It is only following a rapid rate of cooling (−10 °C min −1 ) that a reduction in viable cell number is observed following slow rates of warming (1.6 °C min −1 and 6.2 °C min −1 ), but not rapid rates of warming (113 °C min −1 and 45 °C min −1 ). Cryomicroscopy studies revealed that this loss of viability is correlated with changes in the ice crystal structure during warming. At high cooling rates (−10 °C min −1 ) the ice structure appeared highly amorphous, and when subsequently thawed at slow rates (6.2 °C min −1 and below) ice recrystallization was observed during thaw suggesting mechanical disruption of the frozen cells. This data provides a fascinating insight into the crystal structure dependent behaviour during phase change of frozen cell therapies and its effect on live cell suspensions. Furthermore, it provides an operating envelope for the cryopreservation of T cells as an emerging industry defines formulation volumes and cryocontainers for immunotherapy products.

Vial design features can play a significant role in heat transfer between the shelf and the product and, consequently, in the final quality of the freeze-dried product. Our objective was to investigate the impact of the variability of some geometrical dimensions of a set of tubing vials commonly used for pharmaceuticals production on the distribution of the vial heat transfer coefficients (K) and its potential consequence on product temperature. Sublimation tests were carried out using pure water and 8 combinations of chamber pressure (4-50 Pa) and shelf temperature (-40°C and 0°C) in 2 freeze-dryers. K values were individually determined for 100 vials located in the center of the shelf. Vial bottom curvature depth and contact area between the vial and the shelf were carefully measured for 120 vials and these data were used to calculate K distribution due to variability in vial geometry. At low pressures commonly used for sensitive products (below 10 Pa), the vial-shelf contact area appeared crucial for explaining K heterogeneity and was found to generate, in our study, a product temperature distribution of approximately 2°C during sublimation. Our approach provides quantitative guidelines for defining vial geometry tolerance specifications and product temperature safety margins.

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The resistance to freezing and frozen storage of Streptococcus thermophilus was related to the fatty acid composition of the cell membrane. The effects of four experimental factors were investigated on the fatty acid concentrations and on the recovery of acidification activity of S. thermophilus stored at -20 degrees C by using a complete experimental design: incorporating oleic acid in the culture medium, fermentation pH, addition of glycerol as cryoprotective agent and duration of storage. The acidification activity decreased during the freezing and the frozen storage of S. thermophilus. The storage time slightly enhanced the unsaturated fatty acid concentrations. The addition of glycerol did not modify the fatty acid composition but increased the resistance to frozen storage. The addition of oleic acid and the decrease of the fermentation pH enhanced the ratio unsaturated:saturated fatty acids and improved the recovery of the acidification activity. These results indicate that the resistance to frozen storage was closely related to the membrane fatty acid composition. We interpreted this as an adaptation of S. thermophilus to the addition of oleic acid and the unfavorable growth conditions that corresponded to a low fermentation pH.

We have developed a method to quantify the resistance to freezing and frozen storage of lactic acid starters, based on measuring the time necessary to reach the maximum acidification rate in milk (tm) using the Cinac system. Depending on the operating conditions, tm increased during the freezing step and storage. The loss of acidification activity during freezing was quantified by the difference (Δtm) between the tm values of the concentrated cell suspension before and after freezing. During storage at −20 °C, linear relationships between tm and the storage time were established. Their slope, k, allowed the quantitation of the decrease in acidification activity during 9–14 weeks of frozen storage. The method was applied to determine the resistance to freezing and frozen storage of four strains of lactic acid bacteria and to quantify the cryoprotective effect of glycerol.