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Cryostorage of Caenorhabditis elegans nematodes is important to maintain the many lines used for research. The standard method uses 15% of glycerol in M9-Buffer and a cooling rate of 1 °C/min; then worms can be stored in a −80 °C freezer or in liquid nitrogen. The recovery of C. elegans from stocks stored in liquid nitrogen is reported to be in the range of 35–45% and slightly decreases after years of storage. The storage at −80 °C is also considered safe, but the recovery is not as high as in liquid nitrogen. These observations have not been experimentally reported and therefore require verification. In this study, the standard methods were used in a set of experiments to compare the recovery of larvae and adult worms stored at −80 °C or in liquid nitrogen, after short- (a week) or long-term storage (3.5 years). No differences were observed in recovery, either for the time of storage or for the temperature of storage. Recovery of larvae was 32% at −80 °C and 36% in liquid nitrogen after 3.5 yr and that was not significantly different from the 7-d recovery rates. Adult worm recovery was below 5% for all treatments. These results suggest that both methods of storage can be used to successfully store C. elegans larvae for at least 3.5 years., This work was supported by Junta de Andalucía (P08-CTS-03965), the research group CryoBioTech: cryopreservation of tissues and organs (BIO289) and the Ministry of Economy and Competitiveness (RTC-2016-4733-1).

High-intensity focused ultrasound (HIFU) has been used in different medical applications in the last years. In this work, we present for the first time the use of HIFU in the field of cryopreservation, the preservation of biological material at low temperatures. An HIFU system has been designed with the objective of achieving a fast and uniform rewarming in organs, key to overcome the critical problem of devitrification. The finite-element simulations have been carried out using COMSOL Multiphysics software. An array of 26 ultrasonic transducers was simulated, achieving an HIFU focal area in the order of magnitude of a model organ (ovary). A parametric study of the warming rate and temperature gradients, as a function of the frequency and power of ultrasonic waves, was performed. An optimal value for these parameters was found. The results validate the appropriateness of the technique, which is of utmost importance for the future creation of cryopreserved organ banks.

ne of the main problems in the cryopreservation of biological samples is the formation of ice and the consequent mechanical damage to cells and tissues, due to the crystalline structure of ice and its associated mechanical damage. It is necessary to detect this deleterious formation of ice, especially in tissues and organs, because of their large volume and the complexity of their vascular system in the case of bulky organs. In this work, we propose the use of X-ray Computed Tomography (CT) to detect this ice formation inside tissues and organs. To achieve this aim, rabbit kidneys were loaded with cryoprotectant solutions containing Me2SO at low temperatures (below −140°C). Drops of water with a volume between 2 and 8 μL were then introduced inside the organs. Finally, the rabbit kidneys were cooled to −196°C. Volumes of ice of up to 1 μL were detected in our CT device, with a resolution of up to 50 μm, validating the proposed technology. On the contrary, we analyzed bovine ovarian tissues cryopreserved with a controlled-rate slow-cooling protocol. CT images showed the different structure on the extracellular ice formation according to the procedure, and even the intracellular ice that can be formed in the tissues. These positive results have a straightforward application in the control of the formation of ice, of significant importance for the creation of biobanks.

RESEARCH QUESTION: Could a modification in the ovarian tissue freezing protocol improve follicle survival after cryopreservation and xenotransplantation? DESIGN: Ovarian tissue was used from 13 adult patients, frozen either with our original protocol, or a modified version involving a higher concentration of dimethyl sulphoxide (DMSO), larger volume of cryopreservation solution and lower seeding temperature. After thawing, the ovarian fragments were xenotransplanted to six mice with severe combined immunodeficiency (SCID) for 3 weeks. RESULTS: The proportion of primordial follicles decreased, and the proportion of growing follicles increased significantly (all P < 0.01) after cryopreservation and xenografting compared with fresh controls for both protocols. Follicle density, development, ultrastructure and function were similar between treatments. CONCLUSIONS: This study showed that, although the higher DMSO concentration did not improve survival of preantral follicles, it did not seem to induce any major toxicity in the follicle population either.

Models for cell dehydration during a cryopreservation protocol are usually based on the hypothesis of ideal dilute solution. The strong electrolyte character of NaCl makes us revisit these models. The case of nonideal solution is analyzed by computing the dehydration curves without this additional hypothesis. The conclusion is that, in general, while the application of the ideal dilute solution hypothesis is convenient in many cases, for some specific cooling rates there exist important differences in the degree of dehydration predicted by these two models in the studied cases of mouse sperm and hepatocyte. It is shown how this finding has relevant implications for the design and optimization of cryopreservation protocols.

Human gene therapy (HGT), the treatment or prevention of disease by gene transfer is, regarded by many, as a potential revolution in medicine, because gene therapies target the causes of disease, whereas most current drugs treat the symptoms. Micro-assisted fertilization in the form of intracytoplasmatic sperm injection (ICSI) has truly revolutionized the treatment options for couples with impaired semen quality, and those with both obstructive and non-obstructive azoospermia. ICSI involves the injection of a single sperm directly into the cytoplasm of a mature egg (oocyte) using a glass needle (pipette). Fertilization with this technique occurs in 50%-80% of injected oocytes, but may damage a small percentage of them. With gene therapy, there are new and varied strategies for gene transfer and genome sequence manipulation with improved methodologies that use the technique of microinjection such as the intracytoplasmatic sperm injection-mediated transgenesis (ICSI-Tr), active transgenesis or the pronuclear microinjection technique. This review will look at these methods as well as their potential applications and limitations.

In this report we present a new instrument (a droplet nucleation analyzer) to be used in the study of the influence of ultrasonic waves on the freezing of pure water. This influence can be of great interest in the cryopreservation of biological material. Two different types of experiments have been carried out. In the first set of experiments, ultrasound waves were used during the cooling process. In the second set, ultrasound was applied prior to the cooling process, trying to quantitatively reproduce some experiments in which the ice nucleation temperature of water was successfully decreased. A theoretical discussion of the results is also presented.

From an engineering point of view, the challenge of organ cryopreservation is a problem of heat and mass transfer. However, from a technical point of view, there are four major goals: avoiding fractures, controlling ice, monitoring the cryoprotectant concentration and having a fast and uniform rewarming. Computer Tomography (CT) can help at least with the first three of these four topics; in the case of DMSO based cryoprotectant, the high number of electrons in the sulfur atom of this molecule makes it visible at energies around 70 KeV, where the photoelectric effect is dominant. Water, DMSO and air (fractures) have very different CT values, offering the possibility of drawing a 3D map of the status of the organ not only after the cryopreservation, but also during the loading and/or cooling steps. Kidneys and cryopreserved ovarian tissue have been monitored with a nanoCT. A spectacle of patterns and structures is revealed under the light of the X-rays. In this work we first analyze the theoretical basis behind the use of X-rays to detect the concentration of DMSO. The role of the three types of scattering are described: Compton, Rayleigh and Photoelectric. Four important vitrification agents, dimethyl sulfoxide, 1,2-propendiol, glycerol and ethylene glicol are studied thoroughly, with the conclusion that DMSO should be clearly visualized. Afterwards, we present the CT signals of different DMSO concentrations, making evident the capability of Computer Tomography for rendering a 3D map of the field of concentration of this cryoprotectant agent inside the tissue or organ. Next we show how it is also possible to detect ice: as ice is pure water, it gives a very different signal from that of the rest of the biological material loaded with dimethyl sulfoxide. Finally, in case of unappropriated cooling/warming rates, the presence of fractures in the samples are evident. All this three possibilities of Computer Tomography are applied not only to solutions, but also to two very different biological samples: ovarian tissue and whole kidneys. Two straightforward applications of this technology are under study: the optimization of the cryopreservation protocols of human ovarian tissue for transplantation, and the analysis and design of kidneys cryopreservation protocols. In the case of ovarian tissue, the nanoCT allows to predict the right cooling rate and (if cryopreserved by slow freezing) also the exact way of seeding and the right temperature to do it. In the case of whole kidneys, the presence of ice, fractures, and the concentration of DMSO during the cooling protocol, as well as the final state of the organ once vitrified is shown.