ES2737681A1 - DEVICE AND PROCEDURE FOR PREPARING CELLS FOR ULTRA-FAST VITRIFICATION BY DEHYDRATION (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Device and procedure for preparing cells for ultra-rapid vitrification by dehydration. Vitrification is widely used in the area of reproductive medicine for the cryopreservation of eggs and embryos. Reducing the time required to complete the vitrification process avoids exposing samples to sub-optimal culture conditions, and allows for improved workflow. In the present invention, it is established that the optimal duration of exposure of human eggs and embryos to hypertonic solutions of cryoprotectants used to prepare them for vitrification, is the one in which the cells reach the maximum point of dehydration, corresponding to the minimum point. of the volume excursion. Two consecutive 60 second exposures to two standard hypertonic solutions with increasing concentration of cryoprotectants (20% v/v and 45% v/v, respectively) is sufficient to prepare these cells for vitrification with the SafeSpeed Closed Device.

Description

DESCRIPTION

Device and procedure for preparing cells for ultrafast vitrification by dehydration

SECTOR OF THE TECHNIQUE

The procedure described focuses on the biotechnology sector and the cryopreservation of biological material.

BACKGROUND OF THE INVENTION

When the liquids present in biological systems are subjected to cooling below their melting point, a favorable thermodynamic environment will be generated so that a phase change occurs to the solid state, usually by crystallization (Wowk, 2010). However, this formation of ice crystals can cause damage to biological structures and compromise their viability.

That is why for cryopreservation of cells it is intended, with different techniques, that their cellular aqueous content (cytosol) vitrify, that is, reach the glass transition, minimizing or completely preventing the nucleation and growth of ice crystals. The vitrification allows the cytosol water molecules to remain immobilized maintaining the disorderly configuration of the liquid state, allowing to preserve the intact cellular structures. Whether this intracellular glass transition occurs will depend on the characteristics of the cytosol, such as viscosity and solute concentration, and on the rates at which cooling and reheating occurs. Currently, 3 techniques are used mainly for cell cryopreservation that allow the cytosol glass transition to be achieved: slow-freezing, balance-based vitrification and ultra-rapid vitrification (MAZUR, 1963; Pegg, Wang, & Vaughan, 2006; Rall & Fahy, 1985).

The technique that concerns the present invention is ultrafast vitrification, not based on thermodynamic equilibrium. By cooling and reheating at high speeds (thousands or hundreds of thousands of degrees per minute, hence its name), it is intended to reach the glass transition temperature (Tg) without nucleation or growth of ice crystals, or within Not out of the cell. Despite that during cooling the ice formation is thermodynamically favorable, simply, due to lack of time, the water molecules fail to form the crystalline structure. These remain in a disorganized state until they reach the state of amorphous solid characteristic of vitrification and the biological reactions are stopped, the cell being cryopreserved (Baudot, Alger, & Boutron, 2000; Rall, 1987)

During an ultrafast vitrification process, whether or not undesirable ice formation occurs will depend on the cooling and reheating rates achieved, and on certain characteristics of the solution, such as solute concentration and viscosity. These characteristics will determine the colligative properties of the solution and the tendency to nucleation of ice crystals, that is, the ability of the molecules to organize and form a crystalline structure. Finally, the volume and geometry of the solution where the cells are suspended will determine the efficiency of heat transfer during cooling and reheating (Risco, Elmoazzen, Doughty, He, & Toner, 2007)

Current cryopreservation procedures of cells by ultrafast vitrification can be divided, in general, into 6 phases:

i) preparation of the cells for vitrification. It is the stage of the vitrification process that is improved in the present invention. The cells are exposed to hypertonic solutions to osmosis modify the characteristics of the cytosol so as to favor the tendency to vitrify it. The main mechanisms involved are the evacuation of intracellular water and the permeation of cryoprotectants. The objective is to reach the intracellular concentration of critical solutes: that which allows a successful vitrification at certain cooling and reheating rates.

In general, this phase is subdivided into 2 stages: i) the equilibration stage, in which the cells are gradually or directly exposed to one or more solutions, called equilibration, for a prolonged time. The duration of exposure, generally between 8 and 15 minutes, is intended to allow the osmotic balance between the cytosol and said solution to be achieved. In step ii), the cells are suspended in a vitrification solution, with a high concentration of cryoprotectants (usually greater than 45% w / w), where loading is carried out in the vitrification device. The exposure to this vitrification solution has a very long duration limited, usually 60 seconds. The most widely used permeable cryoprotectants are: ethylene glycol, dimethyl sulfoxide, 1-2 propanediol, glycerol. The most used non-permeable cryoprotectants are: sucrose, trehalose and ficoll.

ii) loading of the cells (s) to be cryopreserved, suspended in the vitrification solution, in a vitrification device. The volume of solution in which the cells are loaded into said device, and the characteristics thereof, will determine the cooling and reheating rates;

iii) cooling. It is usually done by immersion in liquid nitrogen. It will determine the cooling rate reached, and therefore, if it is fast enough to reach the glass transition temperature (Tg) without the nucleation and growth of ice crystals;

iv) storage at cryogenic temperatures. It is essential to maintain the vitreous state during it;

v) overheating. The way in which it is carried out will determine the speed of overheating, which should be as high as possible to avoid the risk of nucleation and growth of ice crystals;

vi) wash. The cells are rehydrated in a controlled manner by exposure to solutions with non-permeable osmotic agents, and permeable cryoprotectants that have penetrated the cytosol are expelled.

A successful process of vitrification of a living cell is defined as that during which live cryopreserved cells survive the process without affecting their competition. The factors that can damage living cells during vitrification are:

i) nucleation and growth of intracellular ice. It may occur during cooling, although the risk is greater during overheating (Seki & Mazur, 2009). The amount and size reached by the ice crystals will determine the damage caused to the cells.

ii) osmotic and mechanical stress. During the preparation phase, solutions with cryoprotectants cause osmotic stress that can compromise the survival of living cells to cryopreserve. Dehydration during the preparation phase for vitrification, and rehydration after reheating during the washing phase, cause volumetric excursions of the cell membrane and consequent mechanical stress. These processes of reduction and increase in volume, called shrinkage and swelling (shrinking and swelling, in English) they can compromise the viability of the cell, therefore the aqueous flow must be controlled.

iii) Cytotoxicity of cryoprotectants. It will depend on the concentration and the exposure time to them.

In the present invention a device and method for significantly reducing the preparation time of cells for ultrafast vitrification is described, in comparison with the currently available techniques. Applied in the field of assisted human reproduction, for the vitrification of gametes and embryos, it allows to reduce the duration of the preparation phase of these cells for vitrification by up to 90%.

DESCRIPTION OF THE INVENTION

Technical problem raised

Currently, the protocols for preparing human eggs and embryos for cryopreservation by the ultra-rapid vitrification technique, incorporate a balancing phase lasting between 8 to 15 minutes (Kuwayama, 2007; Medicine, 2012; Stoop et al. , 2012; Vanderzwalmen et al., 2009). During this phase, cells are exposed to one or more hypertonic solutions, usually referred to as equlibration solutions. By exposing the cell to a high extracellular solute concentration, rapid dehydration and cryoprotectant penetration occurs first, which results in a volumetric reduction of the cell. Next, there is a gradual recovery of the volume, as you move towards the osmotic equilibrium. The total duration of this phase is between 8 and 15 minutes, when it is considered that this balance has been restored.

After this first phase, a second solution with a higher concentration of cryoprotectants is used: the vitrification solution. The exposure to this solution is brief, it does not last until the osmotic equilibrium is reached. The exposure time to this second solution is generally limited to a maximum of 60 seconds, after which the cells are loaded into the vitrification device and cooling. During this phase there is a new dehydration and permeation of cryoprotectants due to the osmotic gradient. (Risco Delgado, R., Univ. Sevilla, ES2459893B2; Kuwayama, M., & Inoue, F. Kitazato Biopharma, ES20040727428T; Vanderzwalmen, P., IVF Zentren Prof. Zech Bregenz GmbH, Grant EP2337449B1; Forest, K., Vitrolife Ab, US20060234204A1; Stachecki, J., Tyho-Galileo Research Laboratories US20060046243A1).

In the area of reproductive medicine, in in vitro fertilization laboratories, the traceability of the samples to be cryopreserved is vital. This factor means that the procedures for vitrification of human eggs and embryos never overlap in the same workplace; There is a physical and resource limitation that determines the number of vitrification procedures that can be performed in a period of time. Therefore, reducing the time required to complete the vitrification process is desirable and an object of scientific interest.

This is the technical problem that is solved in the present invention. First, prolonged exposure to potentially cytotoxic cryoprotectants poses a risk to living cells to cryopreserve. On the other hand, the prolonged duration of cell preparation procedures for cryopreservation limit the scalability and efficiency of the ultrafast vitrification technique. The following describes a device and procedure that allows reaching the critical intracellular concentration of solutes necessary for successful vitrification with a significant reduction in the preparation time of the cells for the process.

Resolution of the technical problem raised

The hydraulic conductivity of the plasma membrane and its specific permeability to cryoprotectants such as ethylene glycol or dimethyl sulfoxide is high in the temperature range (between 21-37 ° C) at which the cell preparation process for vitrification is carried out (Paynter, Cooper, Gregory, Fuller, & Shaw, 1999). For this reason, the evacuation of the aqueous content and the permeation of cryoprotectants through the plasma membrane of human ovules and embryos occurs rapidly. In a short time, less than 60 seconds with the solutions described in the examples of the present invention, the minimum point of intracellular aqueous content is reached. This point of maximum dehydration presents an increase in the concentration of native cellular solutes (proteins, polysaccharides, nucleic acids, salts, etc.) and a reduction in water-free molecules, which the tendency to vitrification of the cytosol increases. In addition, in the time of exposure to the hypertonic solution at which this point of maximum dehydration is reached, the permeable cryoprotectants present in said solution, if any, will have penetrated the cytosol contributing to the total intracellular solute concentration significantly .

Therefore, the relevant osmotic action to reach the concentration of intracellular critical solutes necessary for vitrification occurs in the first seconds of exposure of the cells to said solutions, while the cell reduces its volume (Figure 1). Once this first stretch of volumetric reduction is completed, at the point we call maximum dehydration, the cells begin to recover their volume as they move towards the osmotic balance (Figure 1). During this second section of the curve, the concentration of intracellular solutes is not significantly improved. Therefore, solute concentrations at the point of minimum volume of the dehydration-rehydration curve, and at the end point of the 8-15 minute exposure, considered as the osmotic equilibrium point, can be considered equivalent. Consequently, in the present invention it is established that it is unnecessary to prolong the exposure of the cells to the hypertonic solutions of preparation to reach said osmotic balance; The most efficient way to increase the intracellular concentration of solutes in order to prepare the cells for vitrification is to perform one or more exposures to hypertonic solutions, with a duration limited to the minimum volume point of the resulting volumetric excursion.

It should be taken into account that the evacuation of intracellular aqueous content during this procedure should not produce dehydration of such magnitude that compromises cell survival. A volumetric flow of water and cryoprotectants should be caused to maintain the integrity of the plasma membrane and other relevant biological ultrastructures of the cells.

The hypertonic solutions used must be composed of permeable cryoprotectants such as ethylene glycol, dimethylsulfoxide, a combination thereof or the like and non-permeable cryoprotectants such as sucrose, trehalose, ficoll or the like. They must also have a phosphate buffered saline base (PBS) or any similar vehicle solution that allows the cryoprotectants to be diluted. Other A component of such solutions is the synthetic polymer hydroxypropyl cellulose with an average molecular mass of 80,000 Dalton or similar, or any other polymer or protein that acts as a thickener to achieve a suitable viscosity and as a surfactant to allow the manipulation of living cells in vitro. The vitrification solution must have a tendency to vitrification sufficient to prevent the formation of harmful extracellular ice during cooling and reheating.

The described procedure establishes that the optimal duration of exposure of the cells to the hypertonic solutions used in the preparation for vitrification is that in which they reach the point of minimum volume and maximum dehydration. Thus, with a brief exposure to one or more hypertonic solutions, it is possible to reach the critical intracellular concentration of solutes necessary for vitrification, at the currently achievable cooling and reheating rates, for example, with the SafeSpeed device or the like (Risco Delgado, R., Univ. Sevilla, ES2459893B2), reducing preparation time by up to 90%.

This procedure must be carried out using a vitrification device such as SafeSpeed (Risco Delgado, R., Univ. Sevilla, ES2459893B2), which allows sufficient cooling and reheating rates to be achieved for successful vitrification with the intracellular concentration of solutes resulting from cell preparation protocol. It is with this device, previously used with conventional preparation protocols, that the present invention has been tested. Some relevant details of the SafeSpeed vitrification device are discussed below.

SafeSpeed consists of a container composed of several parts, made of thermoplastic polymer, optimized as a closed system for cryopreservation procedures, to obtain, together with the procedure described in the patent, ultrafast superheat rates. The SafeSpeed device has two features:

1) The oocyte storage takes place in a closed container that achieves the vitrification of the samples with recovery rates equal to or greater than those of any open device. The procedure is based on the use of a microcapillary of thermoplastic polymers of small dimensions and closed at both ends, called SafeSpeed.

2) The main feature of this device is that, being closed, it guarantees an overheating speed above 200,000 ° C / min (Gallardo et al., 2016).

In addition, SafeSpeed has two possible extensions: i) The existence of a possible internal barrier within the microcapillary (which can be implemented by placing another microcapillary inside it), which facilitates the positioning of the biological sample (embryo / oocyte) ; and ii) The existence of a possible funnel at the tip of the microcapillary that facilitates the loading of the biological sample within the device and that avoids any contact of the outer surface of the microcapillary with the medium in which said sample is, reducing the possibility to zero of carrying pollutants inside the storage tank, not only because the container is closed, but also because the outer surface of the device is never in contact with the medium containing the biological sample (Ramon Risco Delgado; patent, ES2459893; addition: P201201020 ).

Thus, the SafeSpeed device can be built in four versions:

- Version 1: SAFESPEED: Without barrier or funnel

- Version 2: SAFESPEED-BARRIER: With barrier, but without funnel.

- Version 3: SAFESPEED-FUNNEL: With funnel, but without barrier.

- Version 4: SAFESPEED-BARRIER-FUNNEL: With barrier and funnel.

DESCRIPTION OF THE FIGURES

Figure 1: Simulation of the relative volume and intracellular concentration of solutes of human ovules in the Metaphase-II state during exposure to hypertonic solutions. In Example A, the exposure is performed following the procedure of the present invention: the duration of exposure of the cell to hypertonic solutions is limited to 60 seconds each. In case B, one shows the current standard protocols: the duration of exposure to the first balancing solution (1) is prolonged for 10 minutes, according to the usual protocols, to allow the osmotic balance to be recovered. The exposure to the vitrification solution (2) lasts 60 seconds. The cell volume is represented by the line continues, and the intracellular concentration of solutes along the dashed line. Exposure to solution 1 (7.5% v / v Me2SO and 7.5% v / v EG) begins at t = 0, and the change to solution 2 (15% v / v Me2SO, 15% v / v EG and 0 , 5M sucrose) are indicated on the X axis. The simulation has been performed in MatLab using a two-parameter biophysical permeability model, similar to that employed by Saenz J. et al. (2009).

Figure 2: Graph of the volumetric excursion experienced in vivo by a human Metaphase-II ovule subject to PD: short preparation protocol based on the present invention, as described in Example 1, and EP: standard protocol based on balancing. The process has been recorded with a video acquisition system connected to an inverted microscope. A frame every 10 seconds has been analyzed to determine the volumetric excursion of the ovule relative to the isotonic volume. The volume has been calculated from the area observed in the frame, assuming that the ovule remains in perfect spherical shape during the process. Exposure to solution 1 (7.5% v / v Me2SO and 7.5% v / v EG) begins at t = 0, and the change to solution 2 (15% v / v Me2SO, 15% v / v EG and 0 , 5M sucrose) are indicated on the X axis.

Figure 3: Frames of the volumetric excursion of a human ovule in Metaphase-II during exposure to the following dehydration-rehydration regimen: (A) Base isotonic solution (buffered saline phosphate and hydroxypropyl cellulose); (B) ovule after 60 seconds of exposure to hypertonic dehydration solution 1 (7.5% v / v Me2SO and 7.5% v / v EG): a reduction in volume is observed by the evacuation of the aqueous content; (C) Ovule after 60 seconds of exposure to hypertonic dehydration solution 2 (15% v / v Me2SO, 15% v / v EG and 0.5M sucrose): by increasing the osmotic gradient, the volume is reduced to a new minimum point, reaching a critical solute concentration appropriate for successful vitrification at the cooling and reheating rates achieved with the SafeSpeed device or the like. (D) Ovule after 60 seconds of exposure to rehydration solution 1 (base solution with 1M sucrose), beginning rehydration; (E) Ovule after 180 seconds of exposure to rehydration solution 2 (base solution, with 0.5M sucrose), where rehydration continues in a controlled manner to avoid excessive swelling. (F) Ovule after 5 minutes in rehydration solution 3 (base solution, without osmotic agents), where the rehydration Note: The Ovule is exposed sequentially to the solutions described. The total dehydration time is 120 seconds, and the rehydration time is 540 seconds.

Figure 4: Representation of the SafeSpeed device. It contains the ideas of the basic device without extensions. The figure shows its different parts: the microcapillary, the main straw, the outer straw, the protector, the cleft, the writing area and the hairpiece. The microcapillary has a conical finish at its end. The protector also has a conical finish, in this case to prevent its displacement when its extreme positions are reached.

Detailed description of the invention

The concentration of solutes in the cytosol must reach a critical point that allows successful vitrification at certain cooling and reheating conditions. The tendency to vitrification of the cytosol will be determined mainly by the free aqueous content of the cytosol and by the presence and concentration of solutes, both of the cytosol and ice formation inhibitor molecules (cryoprotectants) that have penetrated the cell membrane during the Preparation of living cells for cryopreservation by exposure to hypertonic solutions.

When cells are exposed to hypertonic solutions, rapid dehydration occurs due to the high hydraulic conductivity of the plasma membrane: as a result, in a short period of time, the cell reaches a minimum volume in which its aqueous content is minimal. Only by this dehydration process, due to the presence of cellular proteins and polysaccharides, the cytosolic concentration of solutes increases considerably. If the hypertonic solution in question is composed of permeable cryoprotectants, there will also be a penetration of cryoprotectants towards the cytosol, which will contribute to the concentration of intracellular solutes. When the cell reaches the minimum volume in a hypertonic solution, the concentration of intracellular solutes will be, due to these two phenomena, very similar to that of extracellular solute concentration. From this low point, the osmotic gradient between the inside and outside of the cell is low, so there is a reduced flow to the cell inside of cryoprotectants and water. This is observed as a gradual volumetric recovery, until the initial isotonic volume of the living cell is apparently reached at cryopreserve, coinciding with the restoration of osmotic balance. This is known as the 'Shrink-Swell curve' or volumetric reduction and increase curve (Fahy, GM, 2007).

The present invention exploits the fact that the concentration of intracellular solutes at the point of minimum volume does not significantly improve by increasing the exposure time to the solution, that is, by allowing osmotic equilibration. Therefore, the minimum volume point of the volumetric excursion curve should be used as a reference for the optimal duration of exposure of cells to hypertonic solutions. For this reason, the most efficient and fastest way to prepare a cell for vitrification is by exposure to one or more hypertonic solutions, the duration of the most efficient exposure being that in which the cell to be cryopreserved reaches its minimum volume. The use of this paradigm makes it possible to reduce the duration of cell preparation procedures for vitrification ostensibly, as exemplified below.

EXAMPLE OF EMBODIMENT OF THE INVENTION

Example 1

In the present example, one of the ways in which living cells, in particular Metavase-II stage human ovules, can be prepared for ultra-rapid vitrification cryopreservation, by means of the procedure described, using the SafeSpeed device is illustrated.

The procedure and the solutions used are carried out at a temperature of 23-27 ° C. First, the ovule (or ovules) to be prepared for vitrification of the culture medium is transferred to a volume of 200 pL of a buffered saline solution supplemented with 0.06-0.125 mg / mL of hydroxypropyl cellulose (80 kDa), containing a combination 7.5% v / v of ethylene glycol permeable cryoprotectant and 7.5% v / v dimethylsulfoxide.

After one minute of exposure to this solution (see figure 1), the ovule is recovered with a pipette of suitable diameter and transferred to a second volume of 200 pL of a buffered saline solution supplemented with 0.06-0.125 mg / mL of hydroxypropyl cellulose (80 kDa), containing a combination of 15% v / v permeable cryoprotectant ethylene glycol and 15% v / v dimethylsulfoxide.

During the last 20 seconds of exposure to the solution described above, the ovule is loaded into the vitrification support: for example, in the case of safespeed vitrification support (Gallardo, M., et al., 2016), the ovule is sucked into a capillary, hermetically sealed at both ends and submerged in liquid nitrogen for cooling. For reheating, the exposed capillary is immersed in sterile water at 37 ° C, obtaining a reheating rate of approximately 120,000 ° C / min. Then, the end of the capillary is cut and the ovule is expelled in a volume of 200 ul of hypotonic solution with a 1 Molar concentration of sucrose. After one minute of exposure, it is transferred to a volume of 200 ul of hypotonic solution with a concentration of 0.5 molar of sucrose, and finally it is transferred to a volume of 200 ul of the buffered saline without osmotic agents for 5 minutes, where the egg finishes rehydrating. It can then be transferred to the appropriate culture medium.

Example 2 :

Vitrification of 34 murine zygotes FVBxCD1 on day 1 of development (24 hours post fertilization) following a preparation protocol based on the present invention as described in example 1. In summary, they are exposed for 60 seconds to a hypertonic dehydration solution 1 (7.5% v / v Me2SO and 7.5% v / v EG), followed by 60 seconds of exposure to hypertonic dehydration solution 2 (15% v / v Me2SO, 15% v / v EG and 0.5M sucrose). During the last 30 seconds of exposure to the previous solution, 4 embryos are loaded into a SafeSpeed vitrification support, following the manufacturer's recommendations, and immersed in liquid nitrogen. The vitrification support is reheated in a sterile water bath and the embryos are expelled in 1 mL of rehydration solution 1 (1 M sucrose), where they remain for 60 seconds, then being transferred immediately to rehydration solution 2 (0.5 M sucrose) for 180 seconds and finally to a solution without cryoprotectants, where 300 seconds remain.

After the experimental intervention of dehydration, cooling, reheating and rehydration, the embryos remain in culture, following the standard operational practices of the laboratory. The results are compared with a control group of 43 zygotes that have not undergone the intervention. At 2 hours post-fertilization, survival is assessed: 33/34 (97%) embryos survive the vitrification process. At 120 hours the development is evaluated by the blastocyst arrival rate: a decrease in the probability of blastocyst arrival by vitrification is not observed with the described preparation protocol (OR 0.89; 0.34 2.31) .

Example 3:

Exposure of 20 murine blastocysts FVBxCD1 to the preparation protocol for ultrafast vitrification described in Example 1. At 2 hours the morphological survival of 18/20 (90%) of the blastocysts is checked.

References:

Wowk, B. (2010). Thermodynamic aspects of vitrification. Cryobiology, 60 (1), 11-22. Rall, W. F. Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology 24, 387-402 (1987).

Baudot, A., Alger, L., & Boutron, P. (2000). Glass-forming tendency in the system water-dimethyl sulfoxide. Cryobiology, 40 (2), 151-158.

Risco, R., Elmoazzen, H., Doughty, M., He, X., & Toner, M. (2007). Thermal performance of quartz capillaries for vitrification. Cryobiology, 55 (3), 222-229.

Kuwayama, M. (2007). Highly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method. Theriogenology, 67 (1), 73-80.

Vanderzwalmen, P., et al., (2009). Aseptic vitrification of blastocysts from infertile patients, egg donors and after IVM. Reproductive biomedicine online, 19 (5), 700-707; Stoop, D., De Munck, N., Jansen, E., Platteau, P., Van den Abbeel, E., Verheyen, G., & Devroey, P. (2012). Clinical validation of a closed vitrification system in an oocytedonation program. Reproductive biomedicine online, 24 (2), 180-185.

Medicine, A. S. I. R. (2012). The Alpha consensus meeting on cryopreservation key performance indicators and benchmarks: proceedings of an expert meeting. Reproductive biomedicine online, 25 (2), 146-167.

Seki, S., & Mazur, P. (2009). The dominance of warming rate over cooling rate in the survival of mouse oocytes subjected to a vitrification procedure. Cryobiology, 59 (1), 75 82.

Fahy, G. M. (2007). Theoretical considerations for oocyte cryopreservation by freezing. Reproductive biomedicine online, 14 (6), 709-714.

Gallardo, M., Hebles, M., Migueles, B., Dorado, M., Aguilera, L., González, M., & Risco, R. (2016). Thermal and clinical performance of a closed device designed for human oocyte vitrification based on the optimization of the warming rate. Cryobiology, 73 (1), 40-46.

Saenz, J., Toner, M., & Risco, R. (2009). Comparison between ideal and nonideal solution models for single-cell cryopreservation protocols. The Journal of Physical Chemistry B, 113 (14), 4853-4864.

Claims (13)

1. Device and procedure for preparing cells for cryopreservation by ultrafast vitrification in a time significantly less than current procedures. 2. Device and method according to claim 1 wherein the cells are ovules or embryos on day 1 to 7 of development. 3. Device and method according to claim 2 wherein the ovules or embryos on day 1 to 7 of development are human. 4. Device and procedure characterized by achieving a critical solute concentration in the cytosol of the cells to be cryopreserved by exposing them to a solution or series of hypertonic solutions. 5. Device and method according to claim 4, characterized in that the concentration of critical solutes of the cytosol is that which gives it some properties that allow a successful vitrification to occur when subjected to cooling and reheating rates such as those obtained with the device of SafeSpeed or similar vitrification, preventing the formation of extra and intracellular damaging ice during the process. 6. Device and method according to claim 4, wherein the optimum duration of exposure of the cells to be cryopreserved to a solution or series of hypertonic solutions is considered to be that which allows it to reach the minimum volumetric excursion point caused by the gradient osmotic. 7. Device and method according to claim 6 wherein it is considered that the duration of exposure of the cells to be cryopreserved to hypertonic solutions can be shortened or prolonged to accommodate the handling and loading needs of the cells in the vitrification devices used . 8. Device and method according to claim 4, characterized by not requiring the permeation of the cryoprotectants through the cell membrane to reach the Critical concentration of solutes in the cytosol, although not specifically avoided if they were able to penetrate the cells under the conditions applied. 9. Device and method according to claim 4, characterized by the use of hypertonic solutions whose molarity does not cause an osmotic or mechanical stress that compromises the survival and biological competence of the living cells to be cryopreserved. 10. Device and method according to claim 4, characterized by employing a direct or gradual exposure of living cells to hypertonic solutions to reach the critical solute concentration in the cytosol. 11. Device and method according to claim 4, characterized by the use of non-permeable cryoprotectants (such as sucrose, or the like) and / or / or permeable cryoprotectants (such as ethylene glycol, dimethylsulfoxide or the like) as osmotic agents of the solutions hypertonic 12. Device and method according to claim 4, characterized by the use of saline buffered phosphate (PBS) or the like as the basis for the hypertonic solutions used for the preparation of living cells for vitrification. 13. Device and method according to claim 5, characterized by the use of the synthetic polymer hydroxypropyl cellulose with an average molecular mass of 80,000 Da, or the like, as a surfactant and thickener of the hypertonic solutions used for the preparation of living cells for vitrification .