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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.

Cryoprotectants are essential to prevent ice formation during tissue cryopreservation procedures. However, the control of their concentration and spatial distribution in the tissue is necessary to avoid toxicity and other damages associated with the cryopreservation procedures, especially for bulky samples such as tissues and organs. X-ray computed tomography measures the attenuation of an X-ray beam when it passes through a substance, depending on the material properties of the samples. The high electronic density of the sulfur atom of the dimethyl sulfoxide makes it an excellent cryoprotectant to be assessed by X-ray CT, and its concentration is proportional to the X-ray attenuation either at room or cryogenic temperatures. In addition, this imaging technique also allows to detect the formation of ice and eventual fractures within tissues during the cooling and warming processes. Therefore, X-ray CT technology is an excellent tool to assess and develop new cryopreservation procedures for tissues and organs.

Cryoprotectants are essential to prevent ice formation during tissue cryopreservation procedures. However, the control of their concentration and spatial distribution in the tissue is necessary to avoid toxicity and other damages associated with the cryopreservation procedures, especially for bulky samples such as tissues and organs. X-ray computed tomography measures the attenuation of an X-ray beam when it passes through a substance, depending on the material properties of the samples. The high electronic density of the sulfur atom of the dimethyl sulfoxide makes it an excellent cryoprotectant to be assessed by X-ray CT, and its concentration is proportional to the X-ray attenuation either at room or cryogenic temperatures. In addition, this imaging technique also allows to detect the formation of ice and eventual fractures within tissues during the cooling and warming processes. Therefore, X-ray CT technology is an excellent tool to assess and develop new cryopreservation procedures for tissues and organs.

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.

The advantage of stepped vitrification (SV) is avoiding ice crystal nucleation, while decreasing the toxic effects of high cryoprotectant concentrations. We aimed to test this method for human ovarian tissue cryopreservation. Ovarian cortex was taken from 7 fertile adult women. Samples were subjected to an SV protocol performed in an automatic freezer, which allowed sample transfer to ever higher concentrations of dimethyl sulfoxide (DMSO) as the temperature was reduced. Histological evaluation of the vitrified-warmed tissue showed large numbers of degenerated follicles after 24 hours of in vitro culture. We therefore evaluated DMSO perfusion rates by X-ray computed tomography, ice crystal formation by freeze-substitution, and cell toxicity by transmission electron microscopy, seeking possible reasons why follicles degenerated. Although cryoprotectant perfusion was considered normal and no ice crystals were formed in the tissue, ultrastructural analysis detected typical signs of DMSO toxicity, such as mitochondria degeneration, alterations in chromatin condensation, cell vacuolization and extracellular matrix swelling in both stromal and follicular cells. The findings indicated that the method failed to preserve follicles due to the high concentrations of DMSO used. However, adaptations can be made to avoid toxicity to follicles caused by elevated levels of cryoprotectants., This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS) (C. A. Amorim is FRS-FNRS research associate; grant M 4/1/2/5 MCF/SD awarded to C.A. Amorim; grant 5/4/150/5 awarded to M.M. Dolmans), the Fundação de Amparo à Pesquisa do Estado e São Paulo (FAPESP) (PhD grant 2016/22947-8 awarded to E.C.R. Leonel) and donations from the Ferrero family.

Vitrification of human oocytes and embryos in different stages of development is a key element of daily clinical practice of in vitro fertilization treatments. Despite the cooling and warming of the cells is ultra-fast, the procedure as a whole is time consuming. Most of the duration is employed in a long (8–15 minutes), gradual or direct exposure to a non-vitrifying cryoprotectant solution, which is followed by a short exposure to a more concentrated vitrifying solution. A reduction in the duration of the protocols is desirable to improve the workflow in the IVF setting and reduce the time of exposure to suboptimal temperature and osmolarity, as well as potentially toxic cryoprotectants. In this work it is shown that this reduction is feasible. In silico (MatLab program using two-parameter permeability model) and in vitro observations of the oocytes’ osmotic behaviour indicate that the dehydration upon exposure to standard cryoprotectant solutions occurs very fast: the point of minimum volume of the shrink-swell curve is reached within 60 seconds. At that point, intracellular water ejection is complete, which coupled with the permeation of low molecular weight cryoprotectants results in similar intracellular and extracellular solute concentrations. This shows that prolonging the exposure to the cryoprotectant solutions does not improve the cytosolic glass forming tendency and could be avoided. To test this finding, human oocytes and zygotes that were donated for research were subjected to a shortened, dehydration-based protocol, consisting of two consecutive exposures of one-minute to two standard cryoprotectant solutions, containing ethylene glycol, dimethyl sulfoxide and sucrose. At the end of this two-minute dehydration protocol, the critical intracellular solute concentration necessary for successful vitrification was attained, confirmed by the post-warming survival and ability to resume cytokinesis of the cells. Further studies of the developmental competency of oocytes and embryos would be necessary to determine the suitability of this specific dehydration protocol for clinical practice, but based on our results, short times of exposure to increasingly hypertonic solutions could be a more time-efficient strategy to prepare human oocytes and embryos for vitrification.

Purpose: Hydroxypropyl cellulose (HPC), a polysaccharide that forms a viscous gel under low temperatures, is a promising substitute of the blood-derived macromolecules traditionally used in cryopreservation solutions. The performance of a protein-free, fully synthetic set of vitrification and warming solutions was assessed in a matched pair analysis with donor oocytes. Methods: A prospective study including 219 donor MII oocytes was carried out, comparing the laboratory outcomes of oocytes vitrified with HPC-based solutions and their fresh counterparts. The primary performance endpoint was the fertilization rate. Secondary parameters assessed were embryo quality on days 2 and 3. Results: 70/73 (95.9%) vitrified MII oocytes exhibited morphologic survival 2 h post-warming, with 49 (70.0%) presented normal fertilization, compared to 105 of 146 (71.9%) MII fresh oocytes. Similar embryo quality was observed in both groups. A total of 18 embryos implanted, out of 38 embryos transferred (47.3%), resulting in 13 newborns.

Trabajo presentado en CRYO 2018, 55th Annual Meeting of the Society for Cryobiology, celebrado en Madrid (España), del 10 al 13 de julio de 2018

Cryopreservation and subsequent transplantation of ovarian tissue is the only option to preserve fertility in certain patients facing gonadotoxic treatment. So far, cryopreservation of ovarian tissue has been carried out mostly by a controlled rate slow cooling process, typically known as slow freezing. Even though there are still some concerns about the iatrogenic damage on the follicle population, this technique has been used in the more than 100 live births reported to date. It is well known that the control of the cryoprotectant loading in the tissue is crucial to in a cryopreservation procedure. We have used the technology of X-ray computed tomography to assess the concentration and distribution of dimethyl sulfoxide (one of the cryoprotectants most used in fertility preservation) inside pieces of bovine ovarian tissue after its cryopreservation. The low voltage used in our device (75 kV) and the high electronic density of this cryoprotectant makes the X-ray attenuation proportional to its concentration. By assessing and comparing the permeation and homogeneity of the cryoprotectant inside ovarian tissue fragments subjected to a controlled rate slow cooling process, we have characterized the effect of variations in the main parameters involved in the process, with the goal of achieving an optimized protocol with higher permeation of the cryoprotectant in the tissue. The most promissory results were obtained by increasing the initial concentration of dimethyl sulfoxide in the vehicle solution from 10 to 20., This work has been partially supported by the Junta de Andalucía, Proyectos de Investigación de Excelencia (P08-CTS-03965), Siemens Healthcare S.L.U. (2729/0708) and Retos-Colaboración RTC-2016-4733-1 (MINECO, Spain).

Ovarian tissue cryopreservation is, in most cases, the only fertility preservation option available for female patients soon to undergo gonadotoxic treatment. To date, cryopreservation of ovarian tissue has been carried out by both traditional slow freezing method and vitrification, but even with the best techniques, there is still a considerable loss of follicle viability. In this report, we investigated a stepped cryopreservation procedure which combines features of slow cooling and vitrification (hereafter called stepped vitrification). Bovine ovarian tissue was used as a tissue model. Stepwise increments of the Me2SO concentration coupled with stepwise drops-in temperature in a device specifically designed for this purpose and X-ray computed tomography were combined to investigate loading times at each step, by monitoring the attenuation of the radiation proportional to Me2SO permeation. Viability analysis was performed in warmed tissues by immunohistochemistry. Although further viability tests should be conducted after transplantation, preliminary results are very promising. Four protocols were explored. Two of them showed a poor permeation of the vitrification solution (P1 and P2). The other two (P3 and P4), with higher permeation, were studied in deeper detail. Out of these two protocols, P4, with a longer permeation time at −40 °C, showed the same histological integrity after warming as fresh controls., This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS) to C. A. Amorim as a research associate at FRS-FNRS and M.M. Dolmans (grant 5/4/150/5), Fonds Spéciaux de Recherche, Fondation St Luc, Foundation Against Cancer, Coordenação de Aperfeiçoamento de Pessoal de Nível 66 Superior (CAPES –Brazil) (grant #013/14 CAPES/WBI awarded to C.M. Lucci, with F. Paulini receiving a postdoctoral fellowship), and Wallonie-Bruxelles International, and donations from the Ferrero family. Also, it has been partially supported by Siemens Healthcare S.L.U. and Retos-Colaboración RTC-2016-4733-1 (MINECO, Spain).

Trabajo presentado en CRYO 2018, 55th Annual Meeting of the Society for Cryobiology, celebrado en Madrid (Espana), del 10 al 13 de julio de 2018

Although it was qualitatively pointed out by Fahy et al. (1984), the key role of the warming rates in non-equillibrium vitrification has only recently been quantitatively established for murine oocytes by Mazur and Seki (2011). In this work we study the performance of a closed vitrification device designed under the new paradigm, for the vitrification of human oocytes. The vitrification carrier consists of a main straw in which a specifically designed capillary is mounted and where the oocytes are loaded by aspiration. It can be hermetically sealed before immersion in liquid nitrogen for vitrification, and it is warmed in a sterile water bath at 37 °C. Measured warming rates achieved with this design were of 600.000 ºC/min for a standard DMEM solution and 200.000 ºC/min with the vitrification solution for human oocytes. A cohort of 143 donor MII sibling human oocytes was split into two groups: control (fresh) and vitrified with SafeSpeed device. Similar results were found in both groups: survival (97.1%), fertilization after ICSI (74.7% in control vs. 77.3% in vitrified) and good quality embryos at day three (54.3% in control vs. 58.1% in vitrified) were settled as performance indicators. The pregnancy rate was 3/6 (50%) for the control, 2/3 (66%) for vitrified and 4/5 (80%) for mixed transfers., The study received financial support from Fundación Ginemed, Junta de Andalucia (Excellence Project, 2008) and I.N.I.A. (RTA-2012).

Trabajo presentado en CRYO 2018, 55th Annual Meeting of the Society for Cryobiology, celebrado en Madrid (Espana), del 10 al 13 de julio de 2018

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.

Cryoprotection of bulky organs is crucial for their storage and for subsequent transplantation. In this work we demonstrate the capability of the X-ray computed tomography (CT) as a non-invasive method to measure the cryoprotectant (cpa) concentration inside a tissue or an organ, specifically for the case of dymethil sulfoxide (Me2SO). It is remarkable that the use of Me2SO has been leader in techniques of cells and tissues cryopreservation. Although CT technologies are mainly based in density differences, and many cpas are alcohols with densities similar to water, the use of very low energies as acceleration voltage (~70 kV) and the sulfur atom in the molecule of Me2SO makes possible the visualization of this cpa inside tissues. As result we obtain a CT signal proportional to the Me2SO concentration with a spatial resolution up to 50 μm in the case of our device., This study has been funded by the Junta de Andalucía (Programa de Excelencia, 2008: P08-CTS-03965), supported by the Centro Nacional de Aceleradores, the University of Seville and the INIA project RTA2012-006.

This work describes the selective targeting of pigmented retinal pigment epithelial (RPE) cells by a single pulsed laser irradiation. We observed: (1) single pulsed laser irradiation caused cellular damages on pigmented, and not on non-pigmented RPE cells at laser radiant exposure up to 2550 mJ/cm(2); (2) in the mixture of pigmented and non-pigmented RPE cells, single pulsed laser-induced damage was confined to pigmented RPE cells. This study demonstrates that the pigmented RPE cells can be selectively damaged, using a single pulsed laser irradiation, without thermal coagulation to adjacent non-pigmented RPE cells.

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.

Trabajo presentado al 47TH Annual Meeting on the Society for Cryobiology (CRYO-2010) celebrado en Bristol (UK).