Use of fine capillaries for cryopreservation of Caenorhabditis elegans by vitrification.

Caenorhabditis elegans is an exceptional model organism. More than twenty thousand different strains have been developed, increasing knowledge on countless topics. However, the traditional method to cryopreserve this nematode, based on slow freezing, usually reaches recovery rates of around 35% for the L1 and L2 larval stages. Here, we propose two alternative methods to cryopreserve this nematode based on vitrification that are applicable in common laboratories and allow the selective individual cryopreservation of this organism. These new methods require ultra-high warming rates, which are achieved by employing very thin capillaries as the nematode container, and a very low final concentration of cryoprotectants, which, as compared to slow freezing, reduce toxicity damage. The recovery rate was 98.5% for larvae (L1 - L4) and 84.3% for adults. Given these results, our procedures offer an alternative to cryopreserve this nematode (larvae and adults) with higher recovery rates, avoiding expensive requirements. Indeed, it only needed a container with liquid nitrogen and a warming bath for water at 37 °C. The high performance of this approach has been revealed by preserving the long-term memory and, probably, the connectome of this nematode.

Sound waves for solving the problem of recrystallization in cryopreservation.

Organ biobanking is the pending subject of cryopreservation. Although the problem is multifaceted, advances in recent decades have largely related it to achieving rapid and uniform rewarming of cryopreserved samples. This is a physical challenge largely investigated in past in addition to cryoprotectant toxicity studies, which have also shown a great amount of advancement. This paper presents a proof-of-principle, based on the nematode Caenorhabditis elegans, of a technology capable of performing such a function: high intensity focused ultrasound. Thus, avoiding the problem of recrystallization, this worm, in its adult state, preserved at - [Formula: see text], has been systematically brought back to life after being heated with High Intensity Focused Ultrasound (HIFU) waves. The great advantage of this technology is that it is scalable; in addition, rewarming can be monitored in real time by MRI thermography and can be controlled by acoustic interferometry. We anticipate that our findings are the starting point for a possible approach to rewarming that can be used for cryopreservation of millimeter scale systems: either alone or in combination with other promising ways of heating, like nanowarming or dielectric heating, the present technology provides new ways of solving the physical aspects of the problem of recrystallization in cryopreservation, opening the door for the long-term storage of larger samples.

Long-term cryostorage does not negatively affect the recovery of Caenorhabditis elegans.

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.

Use of X-Ray Computed Tomography for Monitoring Tissue Permeation Processes.

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.

The Use of High-Intensity Focused Ultrasound for the Rewarming of Cryopreserved Biological Material.

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.

Stepped vitrification technique for human ovarian tissue cryopreservation.

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.

Human oocytes and zygotes are ready for ultra-fast vitrification after 2 minutes of exposure to standard CPA solutions.

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.

Use of X-Ray Computed Tomography for Ice Detection Applied to Organ Cryopreservation.

One 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.

Evaluation of a new freezing protocol containing 20% dimethyl sulphoxide concentration to cryopreserve human ovarian tissue.

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.

An optimized controlled rate slow cooling protocol for bovine ovarian tissue cryopreservation by means of X-ray computed tomography.

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%v/v.

Ovarian tissue cryopreservation by stepped vitrification and monitored by X-ray computed tomography.

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.

Hydroxypropyl cellulose supplementation in vitrification solutions: a prospective study with donor oocytes.

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.

Thermal and clinical performance of a closed device designed for human oocyte vitrification based on the optimization of the warming rate.

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.

Assessment of the cryoprotectant concentration inside a bulky organ for cryopreservation using X-ray computed tomography.

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.

Bioimpedance monitoring for cryopreservation process control.

This paper analyses the use of Electrical Impedance Spectroscopy (EIS) to efficiently monitor cryoprotectant concentrations in cryopreservation protocols. The proposed technique can improve methods such as Liquidus Tracking (LT), allowing vitrification without exposing tissues to damaging concentrations of cryoprotectant at relatively high temperatures, and avoiding rapid temperature changes. This work is focused to continuous monitoring of cryoprotectant concentrations by detecting changes in electrical impedance. These variations, derived from cryoprotectant perfusion inside cells and tissues, can be efficiently measure by using of EIS. Finite element simulation performed with COMSOL Multiphysics software was used to analyse the frequency response of a two-electrode system to several concentrations of Me2SO, perfused into 3T3 fibroblasts and monolayers of Mesenchymal Stem Cells (MSCs), fundamental in tissue-based therapeutics.

Comparison between ideal and nonideal solution models for single-cell cryopreservation protocols.

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.

Thermal performance of quartz capillaries for vitrification.

In this paper we report the thermal behavior of a new approach for vitrification. Thermal performance of traditional open pulled straws is compared with a new technique based on the combined use of quartz capillaries with slush nitrogen. This new method of vitrification achieved ultrafast cooling rates of 250,000 degrees C/min. As a result, a much lower concentration of cryoprotectant was needed to reach vitrification. In fact, a cryoprotectant solution typically used in oocyte slow freezing protocols was shown to remain transparent after cooling to liquid nitrogen temperatures indicating apparent "vitrification". This approach offers a new and very promising technique for vitrification of cells using low levels of cryoprotectants.