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Only microalgae (algae with cell/filament/thallus size <2 mm) are considered in this chapter. However, the methods described are applicable to a wide variety of prokaryotic cyanobacteria and eukaryotic microalgae. Most culturable cyanobacteria and soil microalgae that have been examined can be cryopreserved with relatively high viability. Furthermore, many freshwater and marine eukaryotic algae can also be cryopreserved, but usually with lower viability. Marine diatoms can be cryopreserved, and often have high viability (Day and Brand 2005), although there has been limited success preserving freshwater diatoms (McLellan 1989). It has been observed that chlorarachniophytes, eustigmatophytes, pelagophytes, phaeothamniophytes and ulvophytes often have very high success rates, comparable to the other green algae and cyanobacteria (Day and Brand 2005). However, to date, most morphologically complex taxa including members of the dinoflagellates, cryptophytes, synurophytes and raphidophytes cannot be successfully cryopreserved. It is anticipated that further research on the basic mechanisms of freezing damage and the empirical development of improved protocols will continue to expand the number and diversity of algal taxa responsive to cryopreservation techniques. [ABSTRACT FROM AUTHOR]

The cryopreservation of mammalian embryos has expanded over the past 20 yr by encompassing a range of sophisticated methods to deal with different developmental stages and different sensitivities to low-temperature exposure. We have described a method for slow, controlled-rate freezing of early stage embryos based on exposure to 1,2-propanediol and sucrose, while the method for late-stage (blastocyst) embryos employs mixtures of glycerol and sucrose. Both methods have been used for animal and human embryos. A third rapid cooling or "vitrification" technique is described, which depends on brief but controlled exposure of multicellular embryos to mixtures of glycerol and 1,2-propanediol at high concentrations. This technique is used for successful animal embryo cryopreservation but is not yet widely applied in the clinic. [ABSTRACT FROM AUTHOR]

Two methods for the cryopreservation of mammalian oocytes are described. One method uses a relatively low concentration of the cryoprotectant propanediol plus sucrose and requires controlled-rate cooling equipment to achieve a slow cooling rate. Such a method has produced live births from cryopreserved human oocytes. The second method described employs a high concentration of the cryoprotectant dimethyl sulfoxide plus a low concentration of polyethylene glycol. This method involves cooling by plunging standard straws into liquid nitrogen vapor, hence avoiding the need for specialized equipment, but requires technical ability to manipulate the oocytes quickly in the highly concentrated cryoprotectant solutions. Murine oocytes vitrified, using this technique, have resulted in live births. [ABSTRACT FROM AUTHOR]

Mammalian spermatozoa were among the very first cells to be successfully cryopreserved and over the last five decades the use of frozen-thawed semen for artificial insemination has come to play an important role in domestic livestock production. More recently, semen freezing has increasingly been utilized in the establishment of genetic resource banks for endangered species. Semen is collected, most commonly either by use of an artificial vagina or by electroejaculation of an anaesthetized animal, and basic sperm parameters assessed. Semen is extended using a TEST-egg yolk-glycerol diluent, packaged in 0.25-mL plastic straws and slowly cooled to 5°C over a period of 1-2 h. Cooled straws are frozen by suspending within liquid nitrogen vapor above the liquid nitrogen surface before plunging into the liquid phase. Straws are thawed briefly in air before immersing in a 35°C water bath for 15 s, and often are used directly for insemination without any further processing. [ABSTRACT FROM AUTHOR]

Blood cells can be regarded as a classical field of application of low-temperature biology. Cryopreservation methods have been developed for different categories of blood cells namely red blood cells (RBCs) (erythrocytes), platelets (thrombocytes), mononuclear cells (i.e., lymphocytes, monocytes), and hematopoietic progenitor cells. This chapter outlines the four most commonly applied techniques for RBCs and two for platelets. [ABSTRACT FROM AUTHOR]

Two different approaches have been adopted for the cryopreservation of human embryonic stem cells (hESCs): vitrification and conventional slow cooling/rapid warming. The vitrification method described here is designed for hESCs that grow as discrete colonies on a feeder cell monolayer, and are subcultured by manual subdivision of the colonies into multicellular clumps. hESCs that are subcultured by enzymatic dissociation can more conveniently be cryopreserved by conventional slow cooling/rapid warming methods. Although both methods are suitable for use in a research context, neither is suitable for cryopreservation of embryonic stem cells destined for clinical diagnostic or therapeutic uses without modification. [ABSTRACT FROM AUTHOR]

Cells isolated directly from tissues (primary cultures) have many applications in research and applications in the fields of toxicology, pharmacology, and virology. Where such preparations remain the only scientific option to achieve necessary results it is still possible to refine and reduce the use of animal and human sources of the tissue by cryopreservation of the primary cells for later use, this reduces the need for fresh tissues and enables improvements in standardization through the ability to provide the same cell preparation at different times and to different laboratories. The methods described provide options for adherent cultures as monolayers, harvested cell suspensions, and also lymphocytes isolated from peripheral blood. [ABSTRACT FROM AUTHOR]

To date, more than 25,000 hematopoietic transplants have been carried out across Europe for hematological disorders, the majority being for hematological malignancies. At least 70% of these are autologous transplants, the remaining 30% being allogeneic, which are sourced from related (70% of the allogeneic) or unrelated donors. Peripheral blood mobilized with granulocyte colony stimulating factor is the major source of stem cells for transplantation, being used in approx 95% of autologous transplants and in approx 65% of allogeneic transplants. Other cell sources used for transplantation are bone marrow and umbilical cord blood. One crucial advance in the treatment of these disorders has been the development of the ability to cryopreserve hematopoietic stem cells for future transplantation. For bone marrow and mobilized peripheral blood, the majority of cryopreserved harvests come from autologous collections that are stored prior to a planned infusion following further treatment of the patient or at the time of a subsequent relapse. Other autologous harvests are stored as backup or "rainy day" harvests, the former specifically being intended to rescue patients who develop graft failure following an allogeneic transplant or who may require this transplant at a later date. Allogeneic bone marrow and mobilized peripheral blood are less often cryopreserved than autologous harvests. This is in contrast to umbilical cord blood that may be banked for directed or sibling (related) hematopoietic stem cell transplants, for allogeneic unrelated donations, and for autologous donations. Allogeneic unrelated donations are of particular use for providing a source of hematopoietic stem cells for ethnic minorities, patients with rare human leukocyte antigen types, or where the patient urgently requires a transplant and cannot wait for the weeks to months required to prepare a bone marrow donor. There are currently more than 200,000 banked umbilical cord blood units registered with the Bone Marrow Donors Worldwide registry. In this chapter, we describe several protocols that we have used to cryopreserve these different sources of hematopoietic stem/progenitor cells, keeping in mind that the protocols may vary among transplant processing centers. [ABSTRACT FROM AUTHOR]

The long-term storage of human and animal cell lines is a relatively new branch of the science of cryopreservation of living organisms. The need to maintain the increasing numbers of cell lines, as they started to emerge from research laboratories from the mid-1900s, necessitated a radical approach to the problem. The realization that they could survive cryopreservation, and that a slow rate of cooling is essential for this survival, led to the eventual discovery of cryoprotectants. Subsequent development of mechanical freezers, which can accurately control the rate of cooling, now allows cells to be cryopreserved at their maximum viability. This chapter outlines the essentials steps for the successful preparation, freezing, and storage of cell lines. [ABSTRACT FROM AUTHOR]

This chapter describes a simple system for cryopreservation of avian spermatozoa as pellets, formed by dropping volumes of sperm suspension directly into liquid nitrogen with dimethylacetamide as cryoprotectant. The method originates from the group at the Research Institute of Farm Animal Breeding and Genetics at St. Petersburg-Pushkin and is described here for chicken spermatozoa, but has also been adapted successfully for other species, such as Houbara bustards and pheasants. [ABSTRACT FROM AUTHOR]

This chapter provides detailed descriptions of the protocol used for fish sperm cryopreservation. The principles described can be applied to any species, but details are also given for individual fish species. Successful cryopreservation of fish spermatozoa depends on a range of factors including the collection of high quality sperm, equilibration conditions, choice of cryoprotectant medium, cooling/thawing regimes, and conditions for fertilization. Even though some general rules can be applied to any fish species, optimization of the protocol is needed for each individual species. Furthermore, because sperm derived from marine and freshwater fish differs markedly, the described protocol is mainly focused on the cryopreservation of sperm for freshwater fish because this poses the greater technical challenge. [ABSTRACT FROM AUTHOR]

The cryopreservation of desiccation-tolerant seeds depends on two key steps: specimen dehydration in an environment that ensures the attainment of water contents below the high-moisture freezing limit; and transfer and maintenance at a subzero temperature that may be optimized in relation to the seed-lot moisture content and species. Temperatures about 70°C below the glass transition temperature (Tg-70) or lower are recommended for seed storage. For fully desiccation-tolerant (type I) seeds, cryopreservation success tends to be independent of cooling regime and subzero (°C) temperature treatment when the seeds are dry. However, to maximize recovery of partially desiccation-tolerant (type II) seeds there can be a requirement for slowcooling, avoidance of storage at certain subzero temperatures, and controlled imbibition following cryopreservation, particularly for endospermic seeds that are high in lipid. [ABSTRACT FROM AUTHOR]

Most culturable cyanobacteria and soil microalgae can be cryopreserved with relatively high viability. Furthermore, many freshwater and marine eukaryotic algae can also be cryopreserved, but typically with lower post-thaw viability levels. However, to date, most dinoflagellates, cryptophytes, synurophytes, and raphidophytes cannot be successfully cryopreserved. Marine diatoms can be cryopreserved, and often have high viability, although freshwater diatoms have thus far proven more problematic. Large numbers of strains have been examined, most notably at the four major protistan collections: Culture Collection of Algae and Protozoa (CCAP) (UK), The Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP) (USA), Sammlung von Algenku Huren Göttingen (SAG) (Germany), and The Culture Collection of Algae at the University of Texas at Austin (UTEX) (USA), and it has been observed that chlorarachniophytes, eustigmatophytes, pelagophytes, phaeothamniophytes, and ulvophytes also have very high success rates, comparable with the other green algae and cyanobacteria. It has been noted that virtually all algae with a large cell size, as well as most filamentous strains, cannot as yet be cryopreserved. There are no known fundamental reasons why large and more complex algae cannot be successfully cryopreserved. Thus, it is anticipated that further research on the basic mechanisms of freezing damage and the empirical development of improved protocols will continue to expand the number and diversity of algal taxa that can be successfully cryopreserved. [ABSTRACT FROM AUTHOR]