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Coral reefs are experiencing unprecedented degradation due to human activities, and protecting specific reef habitats may not stop this decline, because the most serious threats are global (i.e., climate change), not local. However, ex situ preservation practices can provide safeguards for coral reef conservation. Specifically, modern advances in cryobiology and genome banking could secure existing species and genetic diversity until genotypes can be introduced into rehabilitated habitats. We assessed the feasibility of recovering viable sperm and embryonic cells post-thaw from two coral species, Acropora palmata and Fungia scutaria that have diffferent evolutionary histories, ecological niches and reproductive strategies. In vitro fertilization (IVF) of conspecific eggs using fresh (control) spermatozoa revealed high levels of fertilization (>90% in A. palmata; >84% in F. scutaria; P>0.05) that were unaffected by tested sperm concentrations. A solution of 10% dimethyl sulfoxide (DMSO) at cooling rates of 20 to 30°C/min most successfully cryopreserved both A. palmata and F. scutaria spermatozoa and allowed producing developing larvae in vitro. IVF success under these conditions was 65% in A. palmata and 53% in F. scutaria on particular nights; however, on subsequent nights, the same process resulted in little or no IVF success. Thus, the window for optimal freezing of high quality spermatozoa was short (∼5 h for one night each spawning cycle). Additionally, cryopreserved F. scutaria embryonic cells had∼50% post-thaw viability as measured by intact membranes. Thus, despite some differences between species, coral spermatozoa and embryonic cells are viable after low temperature (-196°C) storage, preservation and thawing. Based on these results, we have begun systematically banking coral spermatozoa and embryonic cells on a large-scale as a support approach for preserving existing bio- and genetic diversity found in reef systems. [ABSTRACT FROM AUTHOR]

Abstract: Dental pulp stem cells (DPSCs) are of interest to researchers and clinicians due to their ability to differentiate into various tissue types and potential uses in cell-mediated therapies and tissue engineering. Currently DPSCs are cryopreserved in suspension using Me2SO. However, preservation as two and three dimensional constructs, along with the elimination of toxic Me2SO, may be required. It was shown that intracellular ice formation (IIF), lethal to cells in suspensions, may be innocuous in cell monolayers due to ice propagation between cells through gap junctions that results in improved post-thaw recovery. We hypothesized that innocuous IIF protects confluent DPSC monolayers against injury during cryopreservation. The objective was to examine the effects of IIF on post-thaw viability of both confluent monolayers and suspensions of DPSCs. Confluent DPSC monolayers were assessed for the expression of gap junction protein Connexin-43. IIF was induced on the cryostage and in the methanol bath at different subzero temperatures. Membrane integrity and colony-forming ability were assessed post-thaw. Confluent DPSC monolayers expressed Connexin-43. In cell suspensions, 85.9±1.7% of cells were damaged after 100% IIF. In cell monolayers, after 100% IIF, only 25.5±5.5% and 14.8±3.3% of cells were damaged on the cryostage and in the methanol bath respectively. However, DPSC monolayers exposed to 100% IIF showed no colony-forming ability. We conclude that confluent monolayers of DPSCs express the gap junction-forming protein Connexin-43 and upon IIF retain membrane integrity, however lose the ability to proliferate. [ABSTRACT FROM AUTHOR]

Abstract: Dental pulp is a promising source of mesenchymal stem cells with the potential for cell-mediated therapies and tissue engineering applications. We recently reported that isolation of dental pulp-derived stem cells (DPSC) is feasible for at least 120h after tooth extraction, and that cryopreservation of early passage cultured DPSC leads to high-efficiency recovery post-thaw. This study investigated additional processing and cryobiological characteristics of DPSC, ending with development of procedures for banking. First, we aimed to optimize cryopreservation of established DPSC cultures, with regards to optimizing the cryoprotective agent (CPA), the CPA concentration, the concentration of cells frozen, and storage temperatures. Secondly, we focused on determining cryopreservation characteristics of enzymatically digested tissue as a cell suspension. Lastly, we evaluated the growth, surface markers and differentiation properties of DPSC obtained from intact teeth and undigested, whole dental tissue frozen and thawed using the optimized procedures. In these experiments it was determined that Me2SO at a concentration between 1 and 1.5M was the ideal cryopreservative of the three studied. It was also determined that DPSC viability after cryopreservation is not limited by the concentration of cells frozen, at least up to 2×106 cells/mL. It was further established that DPSC can be stored at −85°C or −196°C for at least six months without loss of functionality. The optimal results with the least manipulation were achieved by isolating and cryopreserving the tooth pulp tissues, with digestion and culture performed post-thaw. A recovery of cells from >85% of the tissues frozen was achieved and cells isolated post-thaw from tissue processed and frozen with a serum free, defined cryopreservation medium maintained morphological and developmental competence and demonstrated MSC-hallmark trilineage differentiation under the appropriate culture conditions. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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. These approaches can now be exploited with respect to cryo-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 developed 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. A deeper understanding of the epigenetic influences on cell phenotype also shows great promise to help yield improved cryopreservation outcomes of cells, tissues and organs. Cellular reprogramming through molecular or environmental cues may be able to modulate cell pathways, 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 and induction of cryotolerance in difficult to freeze models to avoid quiescence or apoptosis post thaw. By combining these tools with novel chemistry and physics systems can be developed which will allow for more stable preservation through “classic” cryopreservation (e.g. slow cooling approaches), vitrification and potentially lyophilization. [ABSTRACT FROM AUTHOR]

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 24h 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 [Copyright &y& Elsevier]

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 50years 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 [Copyright &y& Elsevier]

Abstract: Partial phase diagrams are of considerable utility in the development of optimized cryobiological procedures. Recent theoretical predictions of the melting points of ternary solutions of interest to cryobiology have caused us to re-examine measurements that our group made for the ethylene-glycol–sodium chloride–water phase diagram. Here we revisit our previous experiments by measuring melting points at five ethylene-glycol to sodium chloride ratios (R values; R=5, 10, 15, 30, and 45) and five levels of concentration for each ratio. Melting points were averaged from three measurements and plotted as a function of total solute concentration for each R value studied. The new measurements differed from our original experimental values and agreed with predicted values from both theoretical models. Additionally, the data were fit to the polynomial described in our previous report and the resulting equation was obtained:where w is the total solute mass fraction. This new equation provided good fits to the experimental data as well as published values and relates the determined polynomial constants to the R value of the corresponding isopleths of the three dimensional phase diagram, allowing the liquidus curve for any R value to be obtained. [ABSTRACT FROM AUTHOR]

The vast and growing shortage of organs leads to premature death for millions and costs to society are immense. There is further suffering, death and cost due to needed immune suppression and non-ideal organ matching. Cryopreservation of organs can help solve each of these problems. Cryopreservation of complex tissues would have many other benefits. It would enable doctors to protect the ovaries of young women with curable cancer and enable more hand and limb transplants for maimed soldiers, firefighters and civilians. Preservation of human tissues for drug screening will also, increase efficiency, reduce cost and decrease the need of animals in research. Moreover, it is a valuable complement to tissue engineering, which is already increasing the demand for cryopreservation. Stem cells, sperm, and embryos have been cryopreserved for decades, and progress in cryobiology has made it possible to bank arteries, heart valves, corneas, organ slices and more. We have also seen progress with rat hearts, pig livers, sheep ovaries, pig uteri, rodent limbs, and the cryopreservation and successful transplantation of a rabbit kidney. Building on this to make organ banking routine medicine will be a challenge. But it can be broken into six sub-challenges, each of which appears translatable into an “engineering” problem: 1. Control excessive ice formation, 2. Hold cryoprotectant toxicity within acceptable levels, 3. Limit disproportionate mechanical/thermodynamic stress, 4. Control excessive chilling injury, 5. Avoid unacceptable levels of ischemic injury, and 6. Ensure acceptable repair and revival protocols. Due to the body’s own regenerative abilities, each sub-problem need not be perfectly solved. As never before, valuable understanding and tools in other - often radically accelerating - domains are empowering cryobiologists and can, via interdisciplinary collaborations, help with each of the six sub-challenges. New Organ and the Organ Preservation Alliance, incubated at SU LABS at NASA Research Park in Silicon Valley, are working to accelerate these needed breakthroughs via 1. increased research funds , 2. Grand Challenge Prizes, and 3. the first global, interdisciplinary Organ Banking Summit . The Society for Cryobiology is investigating ways to support the alliance. [ABSTRACT FROM AUTHOR]