The ability to replace organs and tissues on demand could save or improve millions of lives each year globally and create public health benefits on par with curing cancer. Unmet needs for organ and tissue preservation place enormous logistical limitations on transplantation, regenerative medicine, drug discovery, and a variety of rapidly advancing areas spanning biomedicine. A growing coalition of researchers, clinicians, advocacy organizations, academic institutions, and other stakeholders has assembled to address the unmet need for preservation advances, outlining remaining challenges and identifying areas of underinvestment and untapped opportunities. Meanwhile, recent discoveries provide proofs of principle for breakthroughs in a family of research areas surrounding biopreservation. These developments indicate that a new paradigm, integrating multiple existing preservation approaches and new technologies that have flourished in the past 10 years, could transform preservation research. Capitalizing on these opportunities will require engagement across many research areas and stakeholder groups. A coordinated effort is needed to expedite preservation advances that can transform several areas of medicine and medical science.

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Abstract. In many clouds, the formation of ice requires the presence of particles capable of nucleating ice. Icenucleating particles (INPs) are rare in comparison to cloud condensation nuclei. However, the fact that only a small fraction of aerosol particles can nucleate ice means that detection and quantification of INPs is challenging. This is particularly true at temperatures above about − 20 • C since the population of particles capable of serving as INPs decreases dramatically with increasing temperature. In this paper, we describe an experimental technique in which droplets of microlitre volume containing ice-nucleating material are cooled down at a controlled rate and their freezing temperatures recorded. The advantage of using large droplet volumes is that the surface area per droplet is vastly larger than in experiments focused on single aerosol particles or cloud-sized droplets. This increases the probability of observing the effect of less common, but important, high-temperature INPs and therefore allows the quantification of their ice nucleation efficiency. The potential artefacts which could influence data from this experiment, and other similar experiments, are mitigated and discussed. Experimentally determined heterogeneous ice nucleation efficiencies for K-feldspar (microcline), kaolinite, chlorite, NX-illite, Snomax ® and silver iodide are presented.

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.

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Exposure of the yeast Saccharomyces cerevisiae to hypertonic solutions of non-permeating compounds resulted in cell shrinkage, without plasmolysis. The relationship between cell volume and osmolality was non-linear; between 1 and 4 osM there was a plateau in cell volume, with apparently a resistance to further shrinkage; beyond 4 osM cell volume was reduced further. The loss of viability of S. cerevisiae after hypertonic stress was directly related to the reduction in cell volume in the shrunken state. The plasma membrane is often considered to be the primary site of osmotic injury, but on resuspension from a hypertonic stress, which would have resulted in a major loss of viability, all cells were osmotically responsive. The effects of osmotic stress on mitochondrial activity and structure were investigated using the fluorescent probe rhodamine 123. The patterns of rhodamine staining were altered only after extreme stress and are assumed to be a pathological feature rather than a primary cause of injury. Changes in the ultrastructure of the cell envelope were examined by freeze-fracture and scanning electron microscopy. In shrunken cells the wall increased in thickness, the outer surface remained unaltered, whilst the cytoplasmic side buckled with irregular projections into the cytoplasm. On return to isotonic solutions these structural alterations were reversible, suggesting a considerable degree of plasticity of the wall. However, the rate of enzyme digestion of the wall may have been modified, indicating that changes in wall structure persist.

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