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Dry preservation has become an attractive approach for the long-term storage of biologics. By removing water from the matrix to solidify the sample, refrigeration needs are reduced, and thus storage costs are minimized and shipping logistics greatly simplified. This chapter describes two energy deposition technologies, namely, microwave and laser systems, that have recently been used to enhance the rate and nature of solution densification for the purpose of anhydrous preservation of feline oocytes, sperm, and egg white lysozyme in trehalose glass. Several physical screening methodologies used to determine the suitability of an amorphous matrix for biopreservation are also introduced in this chapter.

Mass transfer of protectant chemicals is a fundamental aspect of cryopreservation and freeze-drying protocols. As such, mass transfer modeling is useful for design of preservation methods. Cell membrane transport modeling has been successfully used to guide design of preservation methods for isolated cells. For tissues, though, there are several mass transfer modeling challenges that arise from phenomena associated with cells being embedded in a tissue matrix. Both cells and the tissue matrix form a barrier to the free diffusion of water and protective chemicals. Notably, the extracellular space becomes important to model. The response of cells embedded in the tissue is dependent on the state of the extracellular space which varies both spatially and temporally. Transport in the extracellular space can also lead to changes in tissue size. In this chapter, we describe various mass transfer models that can be used to describe transport phenomena occurring during loading of tissues with protective molecules for cryopreservation applications. Assumptions and simplifications that limit the applicability of each of these models are discussed.

From early dry-ice-based freezers and passive coolers, cryopreservation devices have come a long way. With increasing interest in the field of cryobiology from new scientific applications, the importance of reliable, traceable, and reproducible cold chain devices is sure to increase, ensuring more precise cryopreservation and enabling better post-thaw outcomes, both for the user and for biological samples. As with any cryopreservation process, it is important to optimize each part of the cold chain for each lab's biological samples, cryocontainers used, and logistical restraints. In this chapter we describe how freezing technology can be used for cryopreservation of cells.

Based on our long-standing experience with in vitro culture of Hypericum perforatum, a clonal multiplication system and vitrification-based cryopreservation protocols have been applied to several Hypericum species: H. humifusum L., H. annulatum Moris, H. tomentosum L., H. tetrapterum Fries, H. pulchrum L., and H. rumeliacum Boiss. The shoot tips were cryopreserved using a uniform procedure that includes pretreatment with abscisic acid (ABA), PVS3 cryoprotection, and direct immersion into the liquid nitrogen (LN). The freezing-tolerant Hypericum species were pre-exposed to the cold acclimation conditions performed by a 7-day exposure to 4 °C. The content of naphtodianthrones (hypericins) including hypericin, pseudohypericin, and their protoforms was quantified by HPLC. Ploidy of plants was determined by both flow cytometry of leaf tissue and chromosome counts of root tip meristematic cells. We have shown that the post-thaw recovery rate of the shoot tips, pretreated with 0.076 μM ABA for 7 days at room temperature, led to the post-cryogenic survival from 5 % in H. tomentosum to 21 % in H. annulatum. As compared to the untreated (control) plants, the content of hypericins in plants regenerated after cryopreservation remained unchanged or decreased in H. perforatum, H. humifusum, H. annulatum, H. tomentosum, H. tetrapterum, and H. rumeliacum. However, the pre-exposition of the freezing-tolerant H. perforatum to cold acclimation prior to excision of the shoot tips has improved the post-thaw recovery to 45 % and resulted in threefold increase of the total hypericin content.

Garlic is an important medicinal herb of culinary value by imparting its flavors and odors to the food. Allicin, a notable flavonoid in garlic, is a powerful antibiotic and antifungal compound. Due to poor bioavailability, garlic is of limited use for oral human consumption. Being sexually sterile, propagation of garlic is done by individual cloves from a bulb which increases the chances of transfer of viral diseases. In this chapter, an efficient and improved regeneration protocol for explant establishment and shoot multiplication under in vitro conditions is described. A high rate of shoot multiplication is obtained on MS medium supplemented with 0.5 mg/l BAP, 1.0 mg/l KN, and 2.0 mg/l GA3. Addition of 1.0 mg/l NAA to MS medium resulted in rooting at the shoot bases. A detailed method for encapsulation of explant in sodium alginate beads and their cryopreservation using encapsulation-dehydration is also described.

Antifreeze proteins are a group of proteins that allow organisms to survive in subzero environments. These proteins possess thermal hysteresis and ice recrystallization inhibition activities. In the present study, we demonstrated the efficiency of a recombinant antifreeze protein from the Arctic yeast Leucosporidium sp. AY30, LeIBP, in cryopreservation of the marine diatom Phaeodactylum tricornutum, which is one of the classical model diatoms and has most widely been studied with regard to its ecology, physiology, biochemistry, and molecular biology. P. tricornutum cells were frozen by either a fast or two-step freezing method in freezing medium containing 10 % dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol, respectively, with or without LeIBP supplement. When cells were frozen using the two-step freezing method, cell survival was significantly increased and statistically the same as that of unfrozen native cells in the presence of 0.1 mg/ml LeIBP in 10 % propylene glycol or 10 % ethylene glycol at day 11 of post-thaw culture. In the presence of LeIBP, the concentration of chlorophyll a was dramatically increased to 14-, 48-, 1.6-, and 8.8-fold when cells were frozen in freezing medium containing dimethyl sulfoxide (DMSO), glycerol, propylene glycol (PG), and ethylene glycol (EG), respectively. Scanning electron microscopy observations demonstrated that the cells were also successfully preserved and epitheca or hypotheca were not deformed. These results demonstrate that LeIBP was successfully applied to improve cryopreservation of the marine diatom P. tricornutum.

Cryopreservation is the use of very low temperatures to preserve structurally intact living cells and tissues. Unprotected freezing is normally lethal and this chapter seeks to analyze some of the mechanisms involved and to show how cooling can be used to produce stable conditions that preserve life. The biological effects of cooling are dominated by the freezing of water, which results in the concentration of the solutes that are dissolved in the remaining liquid phase. Rival theories of freezing injury have envisaged either that ice crystals pierce or tease apart the cells, destroying them by direct mechanical action, or that damage is from secondary effects via changes in the composition of the liquid phase. Cryoprotectants, simply by increasing the total concentration of all solutes in the system, reduce the amount of ice formed at any given temperature; but to be biologically acceptable they must be able to penetrate into the cells and have low toxicity. Many compounds have such properties, including glycerol, dimethyl sulfoxide, ethanediol, and propanediol. In fact, both damaging mechanisms are important, their relative contributions depending on cell type, cooling rate, and warming rate. A consensus has developed that intracellular freezing is dangerous, whereas extracellular ice is harmless. If the water permeability of the cell membrane is known it is possible to predict the effect of cooling rate on cell survival and the optimum rate will be a trade-off between the risk of intracellular freezing and effects of the concentrated solutes. However, extracellular ice is not always innocuous: densely packed cells are more likely to be damaged by mechanical stresses within the channels where they are sequestered and with complex multicellular systems it is imperative not only to secure cell survival but also to avoid damage to the extracellular structure. Ice can be avoided by vitrification-the production of a glassy state that is defined by the viscosity reaching a sufficiently high value (~10(13) poises) to behave like a solid, but without any crystallization. Toxicity is the major problem in the use of vitrification methods. Whether freezing is permitted (conventional cryopreservation) or prevented (vitrification), the cryoprotectant has to gain access to all parts of the system. However, there are numerous barriers to the free diffusion of solutes (membranes), and these can result in transient, and sometimes equilibrium, changes in compartment volumes and these can be damaging. Hence, the processes of diffusion and osmosis have important effects during the introduction of cryoprotectants, the removal of cryoprotectants, the freezing process, and during thawing. These phenomena are amenable to experiment and analysis, and this has made it possible to develop effective methods for the preservation of a very wide range of cells and some tissues; these methods have found widespread applications in biology and medicine.

Modeling plays a critical role in understanding the biophysical processes behind cryopreservation. It facilitates understanding of the biophysical and some of the biochemical mechanisms of damage during all phases of cryopreservation including CPA equilibration, cooling, and warming. Modeling also provides a tool for optimization of cryopreservation protocols and has yielded a number of successes in this regard. While modern cryobiological modeling includes very detailed descriptions of the physical phenomena that occur during freezing, including ice growth kinetics and spatial gradients that define heat and mass transport models, here we reduce the complexity and approach only a small but classic subset of these problems. Namely, here we describe the process of building and using a mathematical model of a cell in suspension where spatial homogeneity is assumed for all quantities. We define the models that describe the critical cell quantities used to describe optimal and suboptimal protocols and then give an overview of classical methods of how to determine optimal protocols using these models.

Cryopreservation of red blood cell concentrates (RBCs) is an important method for maintaining an inventory of rare RBC units and managing special transfusion circumstances. The permeating additive glycerol is used as a cryoprotectant to protect RBCs against freezing damage. The use of thawed RBCs was hampered a 24-h outdating period due to potential bacterial contamination when a functionally open system was used for addition and removal of the glycerol. With the introduction of a functionally closed system for the glycerolization and deglycerolization of RBC units, extended post-thaw storage became possible. Here, we describe the cryopreservation of red blood cells according to the high-glycerol method, using a functionally closed processing system.