Palmerfrandsen0310

Frozen blood reserves are an important component in meeting blood needs. The idea behind a frozen blood reserve is twofold to freeze units of rare blood types for later use by patients with special transfusion needs and for managing special transfusion circumstances. The permeating additive glycerol is used as a cryoprotectant to protect red blood cells (RBCs) from freezing damage. The use of thawed RBCs has been hampered by a 24-h outdating period due to the potential bacterial contamination when a functionally open system is used for addition and removal of the glycerol. The introduction of an automated, functionally closed system for glycerolization and deglycerolization of RBCs improved the operational practice. More importantly, the closed process allowed for extended shelf life of the thawed RBCs. In the current chapter, a cryopreservation procedure for RBCs using a functionally closed processing system is described.Embryo cryopreservation is normally performed with great success in species like humans and cattle. The large size of in vivo-derived equine embryos and the presence of a capsule-impermeable to cryoprotectants-have complicated the use of embryo cryopreservation in equine reproduction. A breakthrough for this technique was obtained when large equine embryos could be successfully cryopreserved after collapsing the blastocoel cavity using a micromanipulation system. High pregnancy rates have been obtained when vitrification is used in combination with embryo collapse.Cryopreservation is one of the keystones in clinical infertility treatment. Especially vitrification has become a well-established and widely used routine procedure that allows important expansion of therapeutic strategies when IVF is used to treat infertility. Vitrification of human blastocysts allows us to maximize the potential for conception from any one in vitro fertilization cycle and prevents wastage of embryos. This goes even further toward to best utilize a patient's supernumerary oocytes after retrieval, maximizing the use of embryos from a single stimulation cycle. The technology can even be used to eliminate fresh embryo transfers for reasons of convenience, uterine receptivity, fertility preservation, preimplantation genetic diagnosis, or emergency management. In this chapter, the application of vitrification technology for cryopreserving human blastocyst will be revealed through step-by-step protocols. The results that are presented using the described protocols underscore the robustness of the vitrification technology for embryo cryopreservation.Cryopreserved ovarian cortex tissue can be used to improve or restore female fertility. It can be used for cancer patients to restore fertility after chemotherapy treatment or for social reasons for women who want to postpone their pregnancy wish. In order to preserve ovarian tissue viability in these cases, the tissue needs to be stored by cryopreservation. In this chapter we describe the entire process chain needed to prepare, transport, and cryopreserve human ovarian cortex tissues as well as to subsequently thaw and implant it.Genetic modifications in combination with highly sophisticated assisted reproductive technologies such as in vitro oocyte maturation and development, in vitro fertilization, intracytoplasmic sperm injection, and in vitro embryo culture have opened many research avenues and treatment options for both animals and humans. The number of genetically modified (GM) rodent strains increased considerably during the last several decades, and their numbers are expected to increase due to efficient gene editing technologies including the CRISPR/Cas9. Rodent ovarian tissues (OT) cryopreservation and transplantation procedures have several applications in biomedical field they provide a fertility restoration option for GM rodent strains in some circumstances. They also serve as models to investigate OT cryopreservation as potential alternatives for human infertility patients as well as other domestic and wildlife species for the development of improved cryopreservation and subsequent transplantation strategies. The modeling studies enable determining effective cryoprotective agents (CPA), CPA and water permeability kinetics, and cooling and warming rates during the development of OT cryopreservation procedures. Furthermore, rodent models are extremely useful for determining post-thaw OT graft sites as well as potential medical interventions in an effort to expedite angiogenesis and inhibit inflammatory/immune response, OT longevity, and follicular integrity. Here we describe methodologies for rodent OT cryopreservation and potential transplantation sites for frozen-thawed rat and mouse OT.Oocyte cryopreservation is a potent approach to keep female germplasm safe from epidemic diseases. In the last decade, we developed simple, cheap, and robust vitrification protocols which enable quick cryopreservation of immature porcine oocytes and zygotes in large numbers. In this chapter, we describe vitrification procedures for porcine oocytes and zygotes where they are vitrified in 1-2 μL aliquots of a defined (protein-free) vitrification medium and dropped either on a metal surface pre-cooled from the bottom with liquid nitrogen (solid surface vitrification) or directly into liquid nitrogen. Vitrified microdrops can be stored in cryo-vials in liquid nitrogen. Low concentrations of permeating cryoprotectants during equilibration and proper temperatures during equilibration and warming are crucial for achieving high survival rates. The device used for cooling does not seem to affect system efficacy as vitrification of oocytes or zygotes either on Cryotop® sheets or in microdrops were equally effective.Two basic methods for the laboratory-focused cryopreservation of mammalian oocytes are described, based on work with murine oocytes. 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. This method has also produced live births from cryopreserved human oocytes. The second method, which is described here, employs a high concentration of the cryoprotectant dimethyl sulfoxide plus a low concentration of polyethylene glycol. This is a vitrification method, which involves ultra-rapid 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. selleck chemicals llc Murine oocytes that have been vitrified using this technique have resulted in live births. Vitrification using other cryoprotectant mixtures is now a popular clinically accepted method for cryobanking of human oocytes.Spermatozoa cryopreservation is used for the management of infertility and some other medical conditions. Routinely applied cryopreservation techniques depend on permeating cryoprotectants and relatively slow freezing rates. Cryoprotectant-free vitrification is an alternative and cost-effective method that is based on rapid cooling of spermatozoa by direct plunging into a cooling agent to prevent lethal intracellular ice crystallization and the detrimental effects of high salt concentrations. One of the problems with this technique is that full sterilization of commercially produced liquid nitrogen, which could be contaminated with different pathogens, is not possible. Here we use a benchtop device for the production of sterile liquid air with the same temperature as liquid nitrogen (-195.7 °C). This has been used to develop aseptic technology for cryoprotectant-free vitrification of human spermatozoa.Marine invertebrates represent the vast majority of marine biodiversity; they are extremely diverse playing a key role in marine ecosystems, thus playing an important role at the socioeconomic level. Some invertebrates such as sea urchins, ascidians, and horse-shoe crabs are very well-known model organisms for research and biocompound discovery. In this chapter we revisit the importance of cryopreservation for the conservation and rational use in research, fisheries management, or aquaculture and provide comprehensive protocols for the cryopreservation of sperm, embryos, and larvae.Germplasm cryobanking of transgenic rodent models is a valuable tool for protecting important genotypes from genetic drift, genetic contamination, and loss of breeding colonies due to disease or catastrophic disasters to the housing facilities as well as avoiding stress associated with domestic and international live animal shipment. Furthermore, cryopreservation of germplasm enhances management efficiencies by saving animal room space, reducing workload for staff, reducing cost of maintaining live animals, reducing the number of animals used to maintain a breeding colony, and facilitating transportation of genetics by allowing distribution of frozen germplasm rather than live animals which also reduces the risk of transfer of pathogens between facilities. Thus, effective long-term preservation methods of mouse spermatozoa are critical for future reconstitution of scientifically important mouse strains used for biomedical research.Cryopreservation protocols for semen exist for bird species used in animal production, fancy and hobby species, and wild bird species. Freezing of bird oocytes or embryos is not possible. Cryopreservation of avian semen is used for preserving (genetic diversity of) endangered species or breeds. Freezing semen can also be used in the breeding industry for maintaining breeding lines, as a cost-effective alternative to holding live birds. Success and efficiency of cryopreservation of bird semen differs among species and breeds or selection lines. This chapter describes important variables of methods for collecting, diluting, cold storage, and freezing and thawing of bird semen, notably the medium composition, cryoprotectant used and its concentration, cooling rate, freezing method, and warming method. Media and methods are described for freezing semen using either glycerol or DMA as cryoprotectant, which both are known in chicken and a number of other bird species to render adequate post-thaw fertility rates.In modern livestock breeding, cryopreserved semen is routinely used for artificial insemination. Sperm cryopreservation allows for long-term storage of insemination doses and secures reproduction at a desired time point. In order to cryopreserve semen, it needs to be carefully processed to preserve its vital functions after thawing. In this chapter, we describe the processes involved in cryopreservation of bull, stallion, and boar sperm. These include preparation of diluents, dilution of sperm in primary and freezing extender, slow cooling from room temperature to 5 °C, packaging of insemination doses in straws, freezing at a defined cooling rate in liquid nitrogen vapor, cryogenic storage, and thawing. Two-step dilution approaches, with commonly used diluents, are presented, namely, TRIS-egg yolk (TEY) extender for bull sperm, skim milk (INRA-82) extender for stallion sperm, and lactose-egg yolk (LEY) extender for boar sperm. Furthermore, simple methods are presented for cooling and freezing of sperm at defined cooling rates.