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Concept: Cryobiology


It is widely considered that most organisms cannot survive prolonged exposure to temperatures below 0°C, primarily because of the damage caused by the water in cells as it freezes. However, some organisms are capable of surviving extreme variations in environmental conditions. In the case of temperature, the ability to survive subzero temperatures is referred to as cryobiosis. We show that the ozobranchid leech, Ozobranchus jantseanus, a parasite of freshwater turtles, has a surprisingly high tolerance to freezing and thawing. This finding is particularly interesting because the leach can survive these temperatures without any acclimation period or pretreatment. Specifically, the leech survived exposure to super-low temperatures by storage in liquid nitrogen (-196°C) for 24 hours, as well as long-term storage at temperatures as low as -90°C for up to 32 months. The leech was also capable of enduring repeated freeze-thaw cycles in the temperature range 20°C to -100°C and then back to 20°C. The results demonstrated that the novel cryotolerance mechanisms employed by O. jantseanus enable the leech to withstand a wider range of temperatures than those reported previously for cryobiotic organisms. We anticipate that the mechanism for the observed tolerance to freezing and thawing in O. jantseanus will prove useful for future studies of cryopreservation.

Concepts: Temperature, Thermodynamics, Solid, Liquid, Melting point, Freezing, Cryobiology, Cryopreservation


It has long been suspected that sleep is important for regulating body temperature and metabolic-rate. Hibernation, a state of acute hypothermia and reduced metabolic-rate, offers a promising system for investigating those relationships. Prior studies in hibernating ground squirrels report that, although sleep occurs during hibernation, it manifests only as non-REM sleep, and only at relatively high temperatures. In our study, we report data on sleep during hibernation in a lemuriform primate, Cheirogaleus medius. As the only primate known to experience prolonged periods of hibernation and as an inhabitant of more temperate climates than ground squirrels, this animal serves as an alternative model for exploring sleep temperature/metabolism relationships that may be uniquely relevant to understanding human physiology.

Concepts: Energy, Physiology, Climate, Temperature, Heat transfer, Cryobiology, Hibernation, Fat-tailed Dwarf Lemur


Background Ovarian aging and cytotoxic treatments are the most common causes for fertility loss in women. With increasing numbers of young female survivors following cytotoxic cancer treatments, the issue of fertility preservation has assumed greater importance. Methods We review the literature on the causes of female fertility loss as well as the recent advances in fertility preservation options and strategies that might be of interest to oncologists. Currently, several methods and techniques exist for fertility preservation of female patients with cancer including embryo freezing, ovarian protection techniques, oocyte cryopreservation, ovarian tissue cryopreservation followed by autotransplantation, and recently in vitro culture of ovarian tissue, follicles, and oocytes. Each method or technique has advantages and disadvantages related to current success rate, required delay in cancer treatment, sperm requirement, and risk of reintroducing cancer cells. Results To date, embryo freezing is the only established method successfully and widely used for fertility preservation of female patients with cancer. The other methods are promising but still considered experimental. Conclusion Patient awareness, physician knowledge, early counseling, costs management, international registry, interdisciplinary networks, and research development are necessary to improve the current care in the field of female fertility preservation.

Concepts: Cancer, Oncology, Fertility, Spermatozoon, Zygote, Cryobiology, Cryopreservation, Oocyte cryopreservation


Development of techniques to isolate, culture, and transplant human spermatogonial stem cells (SSCs) has the future potential to treat male infertility. To maximize the efficiency of these techniques, methods for SSC cryopreservation need to be developed to bank SSCs for extended periods of time. Although, it has been demonstrated that SSCs can reinitiate spermatogenesis after freezing, optimal cryopreservation protocols that maximize SSC proliferative capacity post-thaw have not been identified. The objective of this study was to develop an efficient cryopreservation technique for preservation of SSCs. To identify efficient cryopreservation methods for long-term preservation of SSCs, isolated testis cells enriched for SSCs were placed in medium containing dimethyl sulfoxide (DMSO) or DMSO and trehalose (50 mM, 100 mM, or 200 mM), and frozen in liquid nitrogen for 1 week, 1 month, or 3 months. Freezing in 50 mM trehalose resulted in significantly higher cell viability compared to DMSO at all thawing times and a higher proliferation rate compared to DMSO for the 1 week freezing period. Freezing in 200 mM trehalose did not result in increased cell viability; however, proliferation activity was significantly higher and percentage of apoptotic cells was significantly lower compared to DMSO after freezing for 1 and 3 months. To confirm the functionality of SSCs frozen in 200 mM trehalose, SSC transplantation was performed. Donor SSCs formed spermatogenic colonies and sperm capable of generating normal progeny. Collectively, these results indicate that freezing in DMSO with 200 mM trehalose serves as an efficient method for the cryopreservation of SSCs.

Concepts: Organ transplant, Food preservation, Dimethyl sulfoxide, Cryobiology, Cryopreservation, Cryoprotectant, Sulfoxide, Dimethyl sulfide


Vitrification of endothelial cells (MHECT-5) has not previously been compared with controlled slow freezing methods under standardized conditions. To identify the best cryopreservation technique, we evaluated vitrification and standardized controlled-rate -1°C/minute cell freezing in a -80°C freezer and tested four cryoprotective agents (CPA), namely dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), and glycerol (GLY), and two media, namely Dulbecco’s modified Eagle medium Ham’s F-12 (DMEM)and K+-modified TiProtec (K+TiP), which is a high-potassium-containing medium. Numbers of viable cells in proliferation were evaluated by the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega Corporation, Mannheim, Germany). To detect the exact frozen cell number per cryo vial, DNA content was measured by using Hoechst 33258 dye prior to analysis. Thus, results could be evaluated unconstrained by absolute cell number. Thawed cells were cultured in 25 cm2 cell culture flasks to confluence and examined daily by phase contrast imaging. With regard to cell recovery immediately after thawing, DMSO was the most suitable CPA combined with K+TiP in vitrification (99 ±0.5%) and with DMEM in slow freezing (92 ±1.6%). The most viable cells in proliferation after three days of culture were obtained in cells vitrificated by using GLY with K+TiP (308 ±34%) and PG with DMEM in slow freezing (280 ±27%).

Concepts: Ethylene glycol, Ethylene, Diol, Propylene glycol, Dimethyl sulfoxide, Glycerol, Cryobiology, Cryoprotectant


Conventional 3D bioprinting allows fabrication of 3D scaffolds for biomedical applications. In this contribution we present a cryogenic 3D printing method able to produce stable 3D structures by utilising the liquid to solid phase change of a composite hydrogel (CH) ink. This is achieved by rapidly cooling the ink solution below its freezing point using solid carbon dioxide (CO2) in an isopropanol bath. The setup was able to successfully create 3D complex geometrical structures, with an average compressive stiffness of O(1) kPa (0.49 ± 0.04 kPa stress at 30% compressive strain) and therefore mimics the mechanical properties of the softest tissues found in the human body (e.g. brain and lung). The method was further validated by showing that the 3D printed material was well matched to the cast-moulded equivalent in terms of mechanical properties and microstructure. A preliminary biological evaluation on the 3D printed material, coated with collagen type I, poly-L-lysine and gelatine, was performed by seeding human dermal fibroblasts. Cells showed good attachment and viability on the collagen-coated 3D printed CH. This greatly widens the range of applications for the cryogenically 3D printed CH structures, from soft tissue phantoms for surgical training and simulations to mechanobiology and tissue engineering.

Concepts: Oxygen, Carbon dioxide, Collagen, Extracellular matrix, Carbon, Phase transition, Inkjet printer, Cryobiology


Vitrification, a kinetic process of liquid solidification into glass, poses many potential benefits for tissue cryopreservation including indefinite storage, banking, and facilitation of tissue matching for transplantation. To date, however, successful rewarming of tissues vitrified in VS55, a cryoprotectant solution, can only be achieved by convective warming of small volumes on the order of 1 ml. Successful rewarming requires both uniform and fast rates to reduce thermal mechanical stress and cracks, and to prevent rewarming phase crystallization. We present a scalable nanowarming technology for 1- to 80-ml samples using radiofrequency-excited mesoporous silica-coated iron oxide nanoparticles in VS55. Advanced imaging including sweep imaging with Fourier transform and microcomputed tomography was used to verify loading and unloading of VS55 and nanoparticles and successful vitrification of porcine arteries. Nanowarming was then used to demonstrate uniform and rapid rewarming at >130°C/min in both physical (1 to 80 ml) and biological systems including human dermal fibroblast cells, porcine arteries and porcine aortic heart valve leaflet tissues (1 to 50 ml). Nanowarming yielded viability that matched control and/or exceeded gold standard convective warming in 1- to 50-ml systems, and improved viability compared to slow-warmed (crystallized) samples. Last, biomechanical testing displayed no significant biomechanical property changes in blood vessel length or elastic modulus after nanowarming compared to untreated fresh control porcine arteries. In aggregate, these results demonstrate new physical and biological evidence that nanowarming can improve the outcome of vitrified cryogenic storage of tissues in larger sample volumes.

Concepts: Blood, Extracellular matrix, Blood vessel, Fourier transform, Glass transition, Cryobiology, Cryopreservation, Cryoprotectant


Primordial follicles dictate a female’s reproductive lifespan and therefore are central to fertility preservation for both endangered species as well as for individuals with fertility threatening conditions. Ovarian tissue containing primordial follicles can be cryopreserved and later thawed and transplanted back into individuals to restore both endocrine function and fertility. Importantly, increasing numbers of human live births have been reported following ovarian tissue cryopreservation and transplantation. A current limitation of this technology is patient access to sites that are approved or equipped to process and cryopreserve ovarian tissue - especially in larger countries or low resource settings. Here we review empirical evidence from both animal models and human studies that suggest that ovarian tissue can be transported at cold temperatures for several hours while still maintaining the integrity and reproductive potential of the primordial follicles within the tissue. In fact, several human live births have been reported in European countries using tissue that was transported at cold temperatures for up to 20 hours prior to cryopreservation and transplantation. Ovarian tissue transport, if implemented widely in clinical practice, could therefore expand both patient and provider access to emerging fertility preservation options.

Concepts: Human, Temperature, Reproductive system, Endocrine system, Cold, Cryobiology, Cryopreservation, Ex-situ conservation


The conservation of endangered fish is of critical importance. Cryobanking could provide an effective backup measure for use in conjunction with the conservation of natural populations; however, methodology for cryopreservation of fish eggs and embryos has not yet been developed. The present study established a methodology capable of deriving functional eggs and sperm from frozen type A spermatogonia (ASGs). Whole testes taken from rainbow trout were slowly frozen in a cryomedium, and the viability of ASGs within these testes did not decrease over a 728-d freezing period. Frozen-thawed ASGs that were intraperitoneally transplanted into sterile triploid hatchlings migrated toward, and were incorporated into, recipient genital ridges. Transplantability of ASGs did not decrease after as much as 939 d of cryopreservation. Nearly half of triploid recipients produced functional eggs or sperm derived from the frozen ASGs and displayed high fecundity. Fertilization of resultant gametes resulted in the successful production of normal, frozen ASG-derived offspring. Feasibility and simplicity of this methodology will call for an immediate application for real conservation of endangered wild salmonids.

Concepts: Salmon, Salmonidae, Rainbow trout, Freezing, Cryobiology, Cryopreservation, Cryoprotectant, Ex-situ conservation


To determine the accuracy of axillary temperature relative to core rectal temperature during whole-body therapeutic hypothermia for moderate-to-severe hypoxic-ischaemic encephalopathy.

Concepts: Hypothermia, Rectum, Cryobiology