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John K Critser - One of the best experts on this subject based on the ideXlab platform.

  • rationally optimized cryopreservation of multiple mouse embryonic stem cell lines i comparative fundamental Cryobiology of multiple mouse embryonic stem cell lines and the implications for embryonic stem cell cryopreservation protocols
    Cryobiology, 2014
    Co-Authors: Corinna M Kashuba, James D Benson, John K Critser
    Abstract:

    The post-thaw recovery of mouse embryonic stem cells (mESCs) is often assumed to be adequate with current methods. However as this publication will show, this recovery of viable cells actually varies significantly by genetic background. Therefore there is a need to improve the efficiency and reduce the variability of current mESC cryopreservation methods. To address this need, we employed the principles of fundamental Cryobiology to improve the cryopreservation protocol of four mESC lines from different genetic backgrounds (BALB/c, CBA, FVB, and 129R1 mESCs) through a comparative study characterizing the membrane permeability characteristics and membrane integrity osmotic tolerance limits of each cell line. In the companion paper, these values were used to predict optimal cryoprotectants, cooling rates, warming rates, and plunge temperatures, and then these predicted optimal protocols were validated against standard freezing protocols.

  • the Cryobiology of spermatozoa
    Theriogenology, 2012
    Co-Authors: James D Benson, Erik J Woods, Eric M Walters, John K Critser
    Abstract:

    Abstract The impact of successful cryopreservation of spermatozoa can be found in many fields, including agriculture, laboratory animal medicine, and human assisted reproduction, providing a cost-effective and efficient method to preserve genetic material for decades. The success of any cryobiologic protocol depends critically on understanding the fundamentals that underlie the process. In this review, we summarize the biophysical fundamentals critical to much of the research in sperm Cryobiology, provide a synopsis of the development of sperm Cryobiology as a discipline, and present the current state and directions for future research in sperm Cryobiology in the three major areas outlined above—agriculture, laboratory animal medicine, and human clinical assisted reproduction. There is much room for new research, both empiric and fundamental, in all areas, including refinement of mathematical models, optimization of cryoprotective agent addition and removal procedures for spermatozoa from many species, development of effective, efficient, and facile cryopreservation protocols and freezing containers for agricultural sperm cryopreservation, and tailoring cryopreservation protocols for individual human samples.

  • special collection of papers in honor of dr john k critser the Cryobiology of spermatozoa
    2012
    Co-Authors: James D Benson, Erik J Woods, Eric M Walters, John K Critser
    Abstract:

    The impact of successful cryopreservation of spermatozoa can be found in many fields, including agriculture, laboratory animal medicine, and human assisted reproduction, providing a cost-effective and efficient method to preserve genetic material for decades. The success of any cryobiologic protocol depends critically on understanding the fundamentals that underlie the process. In this review, we summarize the biophysical fundamentals critical to much of the research in sperm Cryobiology, provide a synopsis of the development of sperm Cryobiology as a discipline, and present the current state and directions for future research in sperm Cryobiology in the three major areas outlined above—agriculture, laboratory animal medicine, and human clinical assisted reproduction. There is much room for new research, both empiric and fundamental, in all areas, including refinement of mathematical models, optimization of cryoprotective agent addition and removal procedures for spermatozoa from many species, development of effective, efficient, and facile cryopreservation protocols and freezing containers for agricultural sperm cryopreservation, and tailoring cryopreservation protocols for individual human samples.

  • the science of Cryobiology
    Cancer treatment and research, 2007
    Co-Authors: Steven F Mullen, John K Critser
    Abstract:

    The demand for effective bio-preservation methods in the medical community continues to increase with advances in transplantation and transfusion medicine [1]. In reproductive medicine, pre-implantation embryo cryopreservation has become an integral component of overall patient care, increasing the success rate per oocyte retrieval cycle [2,3]. Oocyte cryopreservation is becoming increasingly important due to legal restrictions on the creation and transplantation of supernumerary preimplantation embryos as well as ethical considerations surrounding the cryopreservation of pre-implantation embryos [4,5]. Early investigations into the effects of sub-physiologic temperatures on living cells have been reviewed in great detail [6]. The current chapter will attempt to provide a broad overview of Cryobiology, and refer to the reproductive biology literature when appropriate. Readers interested in learning more details are directed at several excellent texts and reviews on the various subjects [7–20].

  • fundamental Cryobiology of reproductive cells and tissues
    Cryobiology, 2004
    Co-Authors: Erik J Woods, James D Benson, Yuksel Agca, John K Critser
    Abstract:

    Abstract During the last half of the 20th century there have been considerable advancements in mammalian reproductive technologies, including in vitro production of pre-implantation embryos and embryo sexing, and even cloning in some species. However, in most cases, management of non-cryopreserved reproductive cells (i.e., spermatozoa or oocytes) and tissues (i.e., testicular tissue or ovarian tissue) is problematic due to difficulties in donor–recipient synchronization and the potential for transmission of infectious pathogens, which cumulatively limits widespread application of these techniques. Therefore, there is an urgent need for the development of optimum cryopreservation methods for reproductive cells and tissues from many species. Today frozen–thawed spermatozoa and embryos have become an integral component of animal agriculture, laboratory animal genome banking, and human sperm banking and infertility programs. However, although widely implemented, the protocols currently used to cryopreserve bull sperm, for example, are still suboptimal, and cannot readily be extrapolated to other species’ sperm. Similarly, embryo-freezing protocols successfully used for mouse and cattle have yielded little success when applied to some other species’ embryos, or to a related cell type, oocytes. To date, with the exception of mouse oocytes, almost all mammalian species’ oocytes studied have proven very difficult to successfully cryopreserve. Currently, there is a growing interest to understand the underlying cryobiological fundamentals responsible for these low survival rates in an effort to develop better cryopreservation methods for oocytes. Additionally, there is growing interest in developing technologies for the optimal isolation and cryopreservation of the earliest stage of male (spermatogonia, spermatids) and female (primordial follicle) germ cells, with subsequent maturation to the desired stage in vitro. Female gamete maturation, fertilization, and embryo development entirely under in vitro conditions from primordial follicles has been achieved in mice, however techniques for this and other species are still very early in their development. Furthermore, with the recent advances made in intracytoplasmic sperm injection (ICSI), and gamete isolation and maturation, close attention has been given to cryopreservation of gametes in the form of gonadal tissue (i.e., testicular tissue and ovarian tissue) containing various developmental stages of male (spermatogonia, spermatids, and spermatozoa) and female (primordial, secondary) germ lines.

Erik J Woods - One of the best experts on this subject based on the ideXlab platform.

  • Organ Preservation: Cryobiology and Beyond
    Current Stem Cell Reports, 2016
    Co-Authors: Erik J Woods, Steven F Mullen
    Abstract:

    Advances in surgical techniques, immunology, and organ donor networking have allowed organ transplantation to evolve over the last several decades into a procedure that has saved hundreds of thousands of lives. While these dramatic advances have made organ transplantation more effective, they have also highlighted the growing shortage of donor organs. Unfortunately, many organs that could be of potential use for transplantation end up discarded due to short preservation times, often only 4–12 h for vital organs. Organ preservation systems have been investigated with renewed vigor in attempts to solve this problem, and new tools are becoming available that make solutions much more possible than ever before. The Cryobiology of organs must be understood for long-term banking solutions to be feasible, combined with greater understanding of the physio-chemical processes that take place during ischemia and reperfusion. New approaches based on natural models, stem cells, and various novel proteins and trophic factors all show great promise in accomplishing the goal of more donor organs matched with more patients in need.

  • 8 Cryobiology past present and future
    Cryobiology, 2015
    Co-Authors: Erik J Woods
    Abstract:

    Before 1949, the majority of mammalian cells could not be kept in storage at low temperatures and recovered with high functional viability. Artificial cryopreservation was observed and published in 1948 by Polge, Smith, and Parkes through the serendipitous discovery of the cryo-protective properties of glycerol for foul sperm and, subsequently, for red cells. Lovelock (1954) expanded the concept and use of cryoprotective agents (CPAs) by proposing that the protective action of glycerol was shared with a number of other neutral solutes of low molecular weight, including methanol, acetamide and glyceryl monoacetate. This led to the introduction of dimethyl sulfoxide (DMSO) and the first report of its protective action against freezing damage to human and bovine red blood cells and to bull spermatozoa. The ready permeability of DMSO to a wide array of cell types ultimately allowed for widespread use of cryopreservation of cells for research, conservation and clinical use. Scientists interested in the natural phenomena and biomedical applications associated with freezing biological systems have continued to investigate the fundamental processes governing the relationship and the biological, chemical and physical underpinnings. However, as this has been applied over the years with the increasing reliance on cryopreserved cells for agricultural production, biobanking for research/diagnostic reasons and cryopreservation of cells for clinical use, some misconceptions surrounding the science have become problematic. In some ways, the initial successes with cryopreservation of various cell models arguably caused some complacency among practitioners and led to a disconnect between fundamental Cryobiology research and end users of the technology. This has become most apparent in the widespread use of sub-optimal “one size fits all” cryopreservation approaches and with respect to defining shelf life of cryopreserved cell products. Most scientific advances are built upon incremental refinements in methodology and are consequently iterative. As a result, for the most part, even with all of these advances cryopreservation protocols today look very similar to those first discussed over 50 years ago. The future of Cryobiology will need to go beyond applying new technologies to the same biophysical problems. The explosion of various technologies to assess and manipulate cells and tissues at a phenotypic, cytologic, biochemical and molecular level will allow the next wave of disruptive cryopreservation technologies to take root.

  • 002 Cryobiology in the future
    Cryobiology, 2013
    Co-Authors: Erik J Woods, Herman B Wells
    Abstract:

    Enhanced understanding of biology coupled with raw computing power and improved technologies in the physical and chemical sciences has empowered cryobiologists as never before. These tools have led to many advances that have been based on fundamental understanding and application of the biophysics of Cryobiology, and manipulating well described variables such as the type/concentration of cryoprotectant, addition/removal methods and using controlled rate cooling combined with induction of ice nucleation and predictions of intracellular ice formation. These advances have culminated in the widespread use of Cryobiology to facilitate agriculture and medicine. Countless lives have been improved indirectly through the results of Cryobiology, and hundreds of thousands of people today are the direct result of cryopreservation technologies applied to reproductive medicine. This has also allowed the dramatic increase in the use of “biobanks” to conserve massive quantities of biologic and biotechnological resources for conservation and medical technologies. Human diseased and non-diseased tissue repositories are now wide spread, as are repositories for other organisms including plants, corals, and other wildlife species both for reproductive and other broad ranging applications. Most scientific advances are built upon incremental refinements in methodology and are consequently iterative. As a result, for the most part, even with all of these advances cryopreservation protocols today look very similar to those first discussed in the Society for Cryobiology 50 years ago. The future of Cryobiology will need to go beyond applying new technologies to the same biophysical problems. In recent years there has been an explosion in various emerging approaches to assess and manipulate genetic integrity of cells and tissues at the phenotypic, cytologic, biochemical, and molecular level, especially with respect and relevance to stability. A better understanding of the implications of epigenetics, combined with renewed interest in cell differentiation and de-differentiation has opened new philosophies of biological mechanisms that can be exploited to the advantage of future cryobiologists as they become better understood. For instance, new understanding of induction of pluripotency in somatic cells (e.g. iPS cells) has led to a better understanding of some fundamental mechanisms of cellular repair and propagation. Cellular reprogramming may be able to modulate these pathways, thus inducing a rejuvenated state capable of restoring primary energy metabolism while avoiding free radical production post thaw. Better understanding of how to manipulate these mechanisms could potentially allow “induction of cryotolerance” (e.g. iCT cells) in difficult to freeze models to avoid quiescence or apoptosis post thaw. While these and other breakthroughs ensure that the future is indeed bright for the next generation of cryobiologists, they will need to continuously address the gap that inevitably develops between those who study and those who practice. While scientific research can be expected to considerably outpace widespread technological application, Cryobiology historically has been an area exquisitely sensitive to this phenomenon. Translation will be crucial for discoveries to find their ways into practical applications across all disciplines- some of which surely not yet contemplated. Source of funding: None declared. Conflict of interest: None declared. Erik.Woods@CookGBT.com

  • 046 society for Cryobiology science application and relevance of Cryobiology today
    Cryobiology, 2013
    Co-Authors: Erik J Woods
    Abstract:

    Cryobiology was established necessarily as an interdisciplinary endeavor bringing together the biological and physical sciences to solve problems seemingly impossible to many lay observers. As Peter Mazur has described, difficulties in cryopreservation stem from a “complex concatenation of conflicting variables”. No single discipline is adequately equipped on its own to resolve the problems encountered in the field. As a result, many advances have been made based on fundamental understanding and application of Cryobiology. Through manipulating variables such as the type and concentration of cryoprotectant, addition and removal methods, the use of controlled rate cooling combined with induction of ice nucleation and predictions of intracellular ice formation, these advances have culminated in the widespread use of Cryobiology to facilitate research, agriculture and medicine. Indeed, application of the basic science of Cryobiology readily lends itself to translation. Any cell or tissue of relevance benefits from the flexibility of shelf life that Cryobiology can offer. This requires cryobiologists to interact with practitioners in many technical areas. The recently emerging field of regenerative medicine demonstrates such an example. The large scale manufacturing and banking of cells has been the backbone of this evolving discipline. The current technology for laboratory scale manufacturing and cryopreserved storage of cells has been convincingly successful; yet many important technical and medical issues remain. Many cell types of interest to cellular therapy still exhibit exquisite sensitivity to the thermal stresses they experience during the cryopreservation process, and existing approaches for preserving cells are labor-intensive and operator-dependent; consequently, as allogeneic “off-the-shelf” clinical transplantation or transfusion applications continue to be developed, large-scale manufacturing methods with appropriate quality assurance and quality control has become paramount. Despite its promise, however, the development of efficient and commercially viable processes for industrial scale cell manufacturing and banking is still in its infancy. And while manufacturing or processing of patient-specific cells may not have the same scale-up requirements that large allogeneic production requires, the same quality issues with respect to traceability, documentation, and the ultimate functional viability are just as imperative. The traditional understanding of cryopreservation damage is often focused on immediate post-thaw structural preservation, whereas cryopreservation-induced stress may increase over time, often resulting in the delayed onset of cell death. Since apoptotic cell death takes at least 24 h post-thaw to manifest the “true” or “functional” viability, the development of methods to evaluate this, and subsequently mitigate it are also needed. This is of particular importance where the cells may not succumb until hours or days post-transplant, resulting in seemingly acceptable post thaw results but yielding poor clinical efficacy. Other technical considerations involve the sample container, which must be scalable and should be at least a functionally closed system to meet safety and regulatory requirements. New strategies involving injectable cryoprotectants are also highly desirable for successful application of therapeutic cells. For cell based regenerative medicine to reach its potential in mainstream clinical applications, solutions based in sound scientific understanding of the biology and physical systems will need to continue through translation to ultimately solve these technical issues. Source of funding: None declared. Conflict of interest: None declared. Erik.Woods@CookGBT.com

  • the Cryobiology of spermatozoa
    Theriogenology, 2012
    Co-Authors: James D Benson, Erik J Woods, Eric M Walters, John K Critser
    Abstract:

    Abstract The impact of successful cryopreservation of spermatozoa can be found in many fields, including agriculture, laboratory animal medicine, and human assisted reproduction, providing a cost-effective and efficient method to preserve genetic material for decades. The success of any cryobiologic protocol depends critically on understanding the fundamentals that underlie the process. In this review, we summarize the biophysical fundamentals critical to much of the research in sperm Cryobiology, provide a synopsis of the development of sperm Cryobiology as a discipline, and present the current state and directions for future research in sperm Cryobiology in the three major areas outlined above—agriculture, laboratory animal medicine, and human clinical assisted reproduction. There is much room for new research, both empiric and fundamental, in all areas, including refinement of mathematical models, optimization of cryoprotective agent addition and removal procedures for spermatozoa from many species, development of effective, efficient, and facile cryopreservation protocols and freezing containers for agricultural sperm cryopreservation, and tailoring cryopreservation protocols for individual human samples.

James D Benson - One of the best experts on this subject based on the ideXlab platform.

  • foundations of modeling in Cryobiology iii inward solidification of a ternary solution towards a permeable spherical cell in the dilute limit
    Cryobiology, 2020
    Co-Authors: James D Benson, Daniel M Anderson, Anthony J Kearsley
    Abstract:

    Abstract In the previous two manuscripts we outlined the general theory of heat and mass transport in a cell-liquid-ice system with general boundaries and nonideal and nondilute assumptions. Here we simplify the models considerably by presenting a reduction to a spherically symmetric system—a spherical cell with an encroaching spherical ice front. We also reduce to linear approximations of the nonideal nondilute models, essentially assuming dilute and ideal conditions. We derive the resulting nondimensional combined heat and mass transport model for a ternary solution and present numerical solutions. We include an analysis of the effects of varying some nondimensional parameters on rates of ice growth with comments on the necessity of models that account for spatially varying quantities in Cryobiology.

  • foundations of modeling in Cryobiology ii heat and mass transport in bulk and at cell membrane and ice liquid interfaces
    Cryobiology, 2019
    Co-Authors: James D Benson, Daniel M Anderson, Anthony J Kearsley
    Abstract:

    Abstract Modeling coupled heat and mass transport in biological systems is critical to the understanding of Cryobiology. In Part I of this series we derived the transport equation and presented a general thermodynamic derivation of the critical components needed to use the transport equation in Cryobiology. Here we refine to more cryobiologically relevant instances of a double free-boundary problem with multiple species. In particular, we present the derivation of appropriate mass and heat transport constitutive equations for a system consisting of a cell or tissue with a free external boundary, surrounded by liquid media with an encroaching free solidification front. This model consists of two parts–namely, transport in the “bulk phases” away from boundaries, and interfacial transport. Here we derive the bulk and interfacial mass, energy, and momentum balance equations and present a simplification of transport within membranes to jump conditions across them. We establish the governing equations for this cell/liquid/solid system whose solution in the case of a ternary mixture is explored in Part III of this series.

  • Foundations of modeling in Cryobiology—II: Heat and mass transport in bulk and at cell membrane and ice-liquid interfaces
    Cryobiology, 2019
    Co-Authors: Daniel M Anderson, James D Benson, Anthony J Kearsley
    Abstract:

    Abstract Modeling coupled heat and mass transport in biological systems is critical to the understanding of Cryobiology. In Part I of this series we derived the transport equation and presented a general thermodynamic derivation of the critical components needed to use the transport equation in Cryobiology. Here we refine to more cryobiologically relevant instances of a double free-boundary problem with multiple species. In particular, we present the derivation of appropriate mass and heat transport constitutive equations for a system consisting of a cell or tissue with a free external boundary, surrounded by liquid media with an encroaching free solidification front. This model consists of two parts–namely, transport in the “bulk phases” away from boundaries, and interfacial transport. Here we derive the bulk and interfacial mass, energy, and momentum balance equations and present a simplification of transport within membranes to jump conditions across them. We establish the governing equations for this cell/liquid/solid system whose solution in the case of a ternary mixture is explored in Part III of this series.

  • foundations of modeling in Cryobiology i concentration gibbs energy and chemical potential relationships
    Cryobiology, 2014
    Co-Authors: James D Benson, Daniel M Anderson, Anthony J Kearsley
    Abstract:

    Abstract Mathematical modeling plays an enormously important role in understanding the behavior of cells, tissues, and organs undergoing cryopreservation. Uses of these models range from explanation of phenomena, exploration of potential theories of damage or success, development of equipment, and refinement of optimal cryopreservation/cryoablation strategies. Over the last half century there has been a considerable amount of work in bio-heat and mass-transport, and these models and theories have been readily and repeatedly applied to Cryobiology with much success. However, there are significant gaps between experimental and theoretical results that suggest missing links in models. One source for these potential gaps is that Cryobiology is at the intersection of several very challenging aspects of transport theory: it couples multi-component, moving boundary, multiphase solutions that interact through a semipermeable elastic membrane with multicomponent solutions in a second time-varying domain, during a two-hundred Kelvin temperature change with multi-molar concentration gradients and multi-atmosphere pressure changes. In order to better identify potential sources of error, and to point to future directions in modeling and experimental research, we present a three part series to build from first principles a theory of coupled heat and mass transport in cryobiological systems accounting for all of these effects. The hope of this series is that by presenting and justifying all steps, conclusions may be made about the importance of key assumptions, perhaps pointing to areas of future research or model development, but importantly, lending weight to standard simplification arguments that are often made in heat and mass transport. In this first part, we review concentration variable relationships, their impact on choices for Gibbs energy models, and their impact on chemical potentials.

  • rationally optimized cryopreservation of multiple mouse embryonic stem cell lines i comparative fundamental Cryobiology of multiple mouse embryonic stem cell lines and the implications for embryonic stem cell cryopreservation protocols
    Cryobiology, 2014
    Co-Authors: Corinna M Kashuba, James D Benson, John K Critser
    Abstract:

    The post-thaw recovery of mouse embryonic stem cells (mESCs) is often assumed to be adequate with current methods. However as this publication will show, this recovery of viable cells actually varies significantly by genetic background. Therefore there is a need to improve the efficiency and reduce the variability of current mESC cryopreservation methods. To address this need, we employed the principles of fundamental Cryobiology to improve the cryopreservation protocol of four mESC lines from different genetic backgrounds (BALB/c, CBA, FVB, and 129R1 mESCs) through a comparative study characterizing the membrane permeability characteristics and membrane integrity osmotic tolerance limits of each cell line. In the companion paper, these values were used to predict optimal cryoprotectants, cooling rates, warming rates, and plunge temperatures, and then these predicted optimal protocols were validated against standard freezing protocols.

Steven F Mullen - One of the best experts on this subject based on the ideXlab platform.

  • Organ Preservation: Cryobiology and Beyond
    Current Stem Cell Reports, 2016
    Co-Authors: Erik J Woods, Steven F Mullen
    Abstract:

    Advances in surgical techniques, immunology, and organ donor networking have allowed organ transplantation to evolve over the last several decades into a procedure that has saved hundreds of thousands of lives. While these dramatic advances have made organ transplantation more effective, they have also highlighted the growing shortage of donor organs. Unfortunately, many organs that could be of potential use for transplantation end up discarded due to short preservation times, often only 4–12 h for vital organs. Organ preservation systems have been investigated with renewed vigor in attempts to solve this problem, and new tools are becoming available that make solutions much more possible than ever before. The Cryobiology of organs must be understood for long-term banking solutions to be feasible, combined with greater understanding of the physio-chemical processes that take place during ischemia and reperfusion. New approaches based on natural models, stem cells, and various novel proteins and trophic factors all show great promise in accomplishing the goal of more donor organs matched with more patients in need.

  • the science of Cryobiology
    Cancer treatment and research, 2007
    Co-Authors: Steven F Mullen, John K Critser
    Abstract:

    The demand for effective bio-preservation methods in the medical community continues to increase with advances in transplantation and transfusion medicine [1]. In reproductive medicine, pre-implantation embryo cryopreservation has become an integral component of overall patient care, increasing the success rate per oocyte retrieval cycle [2,3]. Oocyte cryopreservation is becoming increasingly important due to legal restrictions on the creation and transplantation of supernumerary preimplantation embryos as well as ethical considerations surrounding the cryopreservation of pre-implantation embryos [4,5]. Early investigations into the effects of sub-physiologic temperatures on living cells have been reviewed in great detail [6]. The current chapter will attempt to provide a broad overview of Cryobiology, and refer to the reproductive biology literature when appropriate. Readers interested in learning more details are directed at several excellent texts and reviews on the various subjects [7–20].

Mary Hagedorn - One of the best experts on this subject based on the ideXlab platform.

  • Cryobiology principles species conservation and benefits for coral reefs
    Reproduction Fertility and Development, 2016
    Co-Authors: Mary Hagedorn, Virginia L Carter
    Abstract:

    Coral reefs are some of the oldest, most diverse and valuable ecosystems on Earth because they can support one-quarter of all marine life in our oceans. Despite their importance, the world’s coral reefs continue to be degraded at unprecedented rates by local and global threats that are warming and creating a more acidic ocean. This paper explores the reproductive challenges of coral for ex situ conservation, using IVF and cryopreservation, and our practical biobanking methods. Coral present challenges for cryopreservation because their reproductive period is often limited to a few nights yearly, they are mostly hermaphrodites with diverse modes of reproduction, including asexual reproduction (i.e. fragmentation and parthenogenesis) and sexual reproduction (i.e. self- and cross-fertilisation) and they express physiological toxins that can inhibit cryopreservation. We have banked spermatozoa from 12 coral species using the same field-hardy methods and have created new coral with thawed spermatozoa. In addition, we describe the cryopreservation of coral symbionts, whose physiology only permits the highest success seasonally. As part of a multidisciplinary conservation strategy, these collections may provide a major hedge against extinction for corals facing the damaging effects of climate change and loss of genetic diversity, and promise to help offset threats to our reefs worldwide.

  • C-18: Zebrafish Cryobiology: Sperm, oocytes, embryos and stem cells
    Cryobiology, 2014
    Co-Authors: Mary Hagedorn
    Abstract:

    Cryopreservation is a proven method for long-term maintenance of genetic material, nevertheless current protocols for fish are not standardized and yield inconsistent results, threatening the efficacy of large-scale genetic screening and stock centers. Previous reports have dealt with sperm cryopreservation in >200 fish species from marine and freshwater species with the most common observation from these publications being the inconsistency in the post-thaw results. Consortia are working to identify processes in zebrafish protocols to try and improve post-thaw variability. Although a great deal of work has been done examining the Cryobiology of zebrafish oocytes at various stages of maturation, the successful cryopreservation, maturation and subsequent fertilization of these oocytes has not been proven. Similarly, zebrafish embryo cryopreservation has been extensively examined for the past 25 years, and several aspects of standard cryopreservation methods have made it a challenging system to develop successful protocols for. These problems include: (1) a large overall size resulting in a low surface to volume ratio that can slow water cryoprotectant efflux and influx; (2) the presence of compartments, such as the blastoderm and yolk; with different permeability properties, (3) susceptibility to chilling injury; and (4) extreme chilling sensitivity at early relatively undifferentiated developmental stages necessitating vitrification. New laser-warming protocols may help change how we understand this complex suite of challenges in zebrafish embryo cryopreservation, especially the relative need for cryoprotectant and the potential for prevention of damaging intracellular ice crystal formation. To diversify and expand our resource collections, however, cryopreserved zebrafish spermatogonial stem cells may provide one of the most important strategies for resource centers. Researchers have successfully developed simple effective cryopreservation methods for zebrafish testes, allowing the rapid and effective preservation of mutant and transgenic lines.

  • Cryobiology of coral fragments
    Cryobiology, 2013
    Co-Authors: Mary Hagedorn, Ann Farrell, Virginia L Carter
    Abstract:

    Around the world, coral reefs are dying due to human influences, and saving habitat alone may not stop this destruction. This investigation focused on the biological processes that will provide the first steps in understanding the Cryobiology of whole coral fragments. Coral fragments are a partnership of coral tissue and endosymbiotic algae, Symbiodinium sp., commonly called zooxanthellae. These data reflected their separate sensitivities to chilling and a cryoprotectant (dimethyl sulfoxide) for the coral Pocillopora damicornis, as measured by tissue loss and Pulse Amplitude Modulated fluorometry 3 weeks post-treatment. Five cryoprotectant treatments maintained the viability of the coral tissue and zooxanthellae at control values (1 M dimethyl sulfoxide at 1.0, 1.5 and 2.0 h exposures, and 1.5 M dimethyl sulfoxide at 1.0 and 1.5 h exposures, P > 0.05, ANOVA), whereas 2 M concentrations did not (P 0.05, ANOVA), but it did not protect against the loss of zooxanthellae (P < 0.05, ANOVA). The zooxanthellae are the most sensitive element in the coral fragment complex and future cryopreservation protocols must be guided by their greater sensitivity.