The use of high-intensity focused ultrasound for the rewarming of cryopreserved biological material

599 The Use of High-Intensity Focused Ultrasound for the Rewarming of Cryopreserved Biological Material Alberto Olmo , Pablo Barroso, Fátima Barroso, and Ramón Risco Abstract —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. Index Terms —Cryopreservation, FEM simulations, heat transfer, high-intensity focused ultrasound (HIFU). I. INTRODUCTION CRYOPRESERVATION, i.e., the preservation of biological material at low temperatures, can transform current medical practices, sorting out many of the problems found today in organ transplantation. The effective cryopreservation and recovery from cryogenic temperatures of complex tissues and organs have not been achieved yet [8]. The most important remaining challenge to this end is to avoid devitrification [12], [15] that consists of the growth of small ice nuclei (formed in the cooling process) during rewarming. This ice growth causes damage in the different tissues and prevents its correct physiological operation after transplantation [5]. Manuscript received June 3, 2020; accepted August 12, 2020. Date of publication August 17, 2020; date of current version February 24, 2021. This work was supported by the MINECO, ONCE, and Junta de Andalucía. (Alberto Olmo and Pablo Barroso contributed equally to this work.) (Corresponding author: Ramón Risco.) Alberto Olmo is with the Department of Electronic Technology, Escuela Técnica Superior de Ingeniería Informática, Universidad de Sevilla, 41012 Seville, Spain, and also with the Instituto de Microelectrónica de Sevilla, IMSE-CNM-CSIC, 41092 Seville, Spain. Pablo Barroso, Fátima Barroso, and Ramón Risco are with the Department of Applied Physics, Escuela Técnica Superior de Ingeniería, Universidad de Sevilla, 41092 Seville, Spain, and also with the Centro Nacional de Aceleradores (CNA-CSIC), 41092 Seville, Spain (e-mail: ramon. @. us.es). Digital Object Identifier 10.1109/TUFFC.2020.3016950 In practical terms, to avoid devitrification, samples must be rewarmed at a faster rate than the one used during cooling. This rate depends on the concentration of cryoprotectant used in the process (usually, the lower the concentration, the higher the warming rate). With the aim of increasing this rewarming rate and the homogeneity of the temperature field, new methods have been proposed. Electromagnetic microwaves between 300 MHz and 30 GHz have been applied to reach a rapid rewarming of the biological material [7], [18]. However, this question presents several problems for its application. The appearance of maximums and minimums of the stationary wave, dependent on the geometry of the resonant cavity, produces a heterogeneity of heating in the material [18]. Furthermore, the phenomenon of “thermal runaway” is presented, where hot spots absorb more and more electromagnetic radiation, favoring the heterogeneity of heating, damaging the biological material [6]. This rapid increase in the temperature of hot spots is very dependent on the initial boundary conditions and is difficult to control [6]. Another proposed approach is the use of magnetic nanoparticles to optimize the increase in the rate and the homogeneity of rewarming, particularly in the vascular system [13]. The use of magnetic particles coated with antifreeze proteins (magnetic Ice Binding Proteins - mIBP) has also been proposed [17]. However, the complexity of these methods and the toxicity of nanoparticles make it convenient to develop cleaner, faster, and more effective technologies. Therefore, none of the techniques and approaches developed so far have reported routinely and reproducible results to the point of being applicable. This intensifies the study and development of innovative approaches to overcome these obstacles and achieve a safer and more efficient rewarming process, essential for the creation of tissues and organ banks. High-power ultrasound (HPU) has acquired special relevance during the last years, and commercial applications on a large scale have been developed. Among HPU variants, high-intensity focused ultrasound (HIFU) has recently gained popularity in medicine. This technique consists in the application of a focalized energy ultrasound beam to produce tissue heating until temperatures of up to 100 ◦C, usually used for thermal ablation, with the particularity that the surrounding tissues are not affected [19]. 600 The HIFU technique has recently been used in different medical fields: physiotherapy [1], cardiology [9], endocrinology [11], Parkinson’s disease [21] or oncology. The field of oncology is where more applications have been developed during the last decades [10], [19], always with the aim of eliminating target cells by the elevated temperatures achieved (thermal ablation). Despite the proven effectiveness of HIFU in this type of medical applications, to date there is no evidence of the application in the field of cryopreservation. In this work, we present the use of HIFU to achieve the rewarming of tissues and organs in a sufficiently fast and homogeneous way to avoid the phenomenon of devitrification, thus ensuring the viability of the biological material. Today, with commercial transducers, a temperature increase of about 20 ◦C/s is reached, with an approximate focus of about 1 mm in diameter [19]. The warming rate reported by referenced works would be appropriate for cryopreservation applications [1], [9], [10], [19]. However, the cryopreserved sample (tissue or organ) has a volume several orders of magnitude higher. We propose to achieve this increase in the focal area by using an array of transducers of specific dimensions. In this work, we theoretically study this problem. COMSOL Multiphysics software is used. An HIFU system, with 26 ultrasonic transducers, has been simulated. The objective of these simulations is to verify whether the HIFU system tested can heat a model of organ from cryogenic temperatures to be used in cryopreservation applications. According to the state-of-the- art in cryopreservation, the heating process can be considered fast enough for practical applications if the heating rate is equal or superior to 100 ◦C/min and temperature difference between areas in the organ is between 5◦and 7◦[7], [18]. Different power intensities for ultrasonic transducers and different frequencies have been simulated to find out the optimal values. Section II describes the methodology followed to model the organ and design the HIFU system, selecting the appropriate power and frequency, together with the description of the equations solved by COMSOL. Section III describes the results of our simulations. Finally, Section IV summarizes the conclusions achieved and future research steps in the utilization of HIFU. II. MATERIALS AND METHODS A. HIFU System Design and Organ Model There are several alternatives to control the size of the focus of the ultrasound signal in HIFU: modifying the geometry of the system (radius of curvature of the transducer or the number of transducers used) or modifying the phase delay in an array of several flat-faced transducers. Our approach for the HIFU system design is based on the use of an array of transducers of specific dimensions, all in phase, to achieve a significant focus area for the rewarming of the selected biological material. An HIFU system with 26 transducers has been simulated, as shown in Fig. 1. The number of transducers was selected to enable the surrounding of the organ model, aiming to reach Fig. 1. Organ model and HIFU system. (a) Complete system. An elliptical organ of 2 cm ×1.2 cm ×1.2 cm was used. The HIFU system is composed of 26 ultrasonic transducers with the dimensions and geometry shown in Table I. The ultrasonic transducers (blue) are attached to the organ model with a cylinder, modeled as ice (gray). There is a layer of water and cryoprotectant between the transducer and the ice. (b) Transducers, layer of water and cryoprotectant, and organ model, with the ice cylinders removed. The different parts of the ovary (cortex, medulla, and ovarian follicles) can be seen. TABLE I GEOMETRIC DATA a focal area in the order of magnitude of the organ. Each ultrasonic transducer has the geometric dimensions shown in Table I. As organ model, a sheep ovary has been chosen, as there are currently important results in the restoration of the ovarian function and natural fertility following cryopreservation protocols [4]. The shape of an ellipsoid has been considered as a OLMO et al. : THE USE OF HIFU FOR THE REWARMING OF CRYOPRESERVED BIOLOGICAL MATERIAL 601 TABLE II ATTENUATION COEFFICIENTS VERSUS FREQUENCY simplification, with a size of 2 ×1.2 ×1.2 cm3. An estimation of the attenuation of ultrasonic waves in ice and in the organ is made following the Biot’s theory in porous media, in accordance with different publications [2], [14], following an initial physical study [3]. This is an approximate model for the attenuation since it considers a porous matrix whose structure is ice and a small minimum amount of water, which in our case is around 10% in volume. The different transducers were attached to the elliptical organ through different cylinders (modeled with the thermal and acoustic properties of ice), as shown in Fig. 1. This model simulates current research cryopreservation protocols [4]. A layer of water and cryoprotectant [dimethyl sulfoxide (DMSO), 10% in volume] is placed between the transducer and the ice cylinders (20% of the ice cylinder thickness). This layer is important in order to avoid unwanted reflections of the ultrasonic waves on the interface between the transducer and the solid-vitreous sample. The width of this layer was selected after the initial tests, to minimize the reflection of ultrasonic waves on the transducer–ice interface, without losing high percentages of power. Table II shows the attenuation coefficients used for the different parts of the model and references used for their calculation. Attenuation coefficients of the organ have been approximated using the properties of the ice, due to the lack of data found in the literature regarding the values of ultrasonic attenuation coefficients of vitrified organs perfused with cryoprotectant solutions, at different temperatures. Future experimental work will help understand the ultrasonic attenuation in vitrified organs previously perfused with cryoprotectant solutions, helping to further optimize the HIFU system used. B. Equations Solved COMSOL is a finite-element analysis and resolution software that uses different specific application modules. For this case, the acoustic and heat transfer modules have been used, so that the acoustic module allows calculating the flat wave dissipation density generated by focused ultrasound in the frequency domain and the heat transfer module, from the data obtained in the acoustic module, solves how heat is transferred to the system over time. To do this, COMSOL solves the following Helmholtz homogeneous linear equation in the acoustic module: ∇·−1 ρc (∇pt−qd)−k2 eq pt ρc =Qm(1) where ρcis the effective fluid density (a complex-valued quantity used to model damping in porous materials), ptis the total pressure exerted by acoustic waves, qdis the dipolar acoustic source, keq is the complex-valued wavenumber, and Qmthe monopolar acoustic source. The following relations are used to model the system: ρc=ρc2 c2 c (2) pt=p+pb(3) k2 eq =ω cc2 (4) keq =ω c−iln(10)α 20 (5) being cthe speed of sound in the medium, pbthe bottom pressure field, ωthe angular frequency of the ultrasonic waves, ccthe effective sound speed in the fluid (a complex-valued quantity used in the modeling of losses in the porous material), and αthe coefficient of attenuation. The absence of transverse waves and propagation effects of nonlinear acoustic waves can be supposed in the simulation to simplify. Therefore, Qm=qd=0. A value of 0 is given to the initial pressure. The normal displacement of the wave is null outside the surface of the transducers, so the equation solved is finally −n·−1 ρc (∇pt−qd)=0.(6) In the case of the surfaces of the transducers, where there is a normal displacement of the waves, COMSOL solves the equation −n·−1 ρc (∇pt−qd)=(iω)2dn(7) where nrepresents a normal vector to the surface and dn represents the normal displacement of the transducer, which is a data that are entered in the COMSOL software and is obtained from the following equations: u0=2πfd n(8) I=1 2ρcu2 0(9) I=P A(10) where u0is the speed of the particle, fis the frequency, I is the intensity over the surface of the transducer, ρis the density of the medium, Pis the power, and Ais the area of the transducer. The frequency ( f), attenuation (α), and power Authorized licensed use limited to: Universidad de Sevilla. Downloaded on January 20,2025 at 08:29:20 UTC from IEEE Xplore. Restrictions apply. 602 (P)are the parameters to modify and insert in COMSOL to obtain variations in the displacement of the transducers. In our simulations, we will vary frequency and power to study the effects on the given material. For the module corresponding to heat transfer, the program solves the equations ρcpu∇T+∇q=Q+Qted (11) q=−k∇T(12) being cpthe heat capacity at constant pressure, uthe fluid velocity vector, Tthe temperature, qthe conductive heat flow, Qted the thermoelastic cushioning, and kthe thermal conductivity. Q, the heat generated by the acoustic module, is the parameter calculated by the acoustic module, together with the acoustic pressure map. Qis inserted into the heat module to calculate the final temperature of the system, solving the following equations: ρcpu∇T+∇q=Q+Qted +Qvd (13) Q=2αIt(14) It=p2 t 2ρc(15) being Qvd the viscous dissipation and Itthe intensity of the wave. Thermal diffusivity is, therefore, being taken into account by COMSOL Multiphysics to calculate the warming rates in different points of the organ. C. Simulations Performed The heating of the elliptical organ model with the 26-transducer HIFU system, as described in Section II-B, has been simulated. The power of the HIFU system must be high enough to prevent the growth of ice crystals during rewarming. Generally, a rewarm rate around 100 ◦C per minute would be enough, although this value depends on the concentration of the cryoprotectant used, as explained above [7], [18]. However, very high powers can generate an increase in the temperature gradients inside the organ. Furthermore, ultrasonic cavitation could cause mechanical disruption in the cryopreserved organ. Therefore, a balance must be achieved between the geometry of the system, the frequency used, the power applied, and the dimensions of the organ. The initial temperature of the organ and surrounding ice cylinders was −140 ◦C. At the beginning, a frequency sweep from 100 to 400 kHz was carried out, with a power of 25 W for each transducer (a total power of 650 W), in order to find the optimal frequency for heating. When the optimal frequency is found, as shown in Section III, the frequency was fixed and the power of the ultrasonic transducers was varied to see the effect on the heating of the organ, by studying the warming rate and homogeneity of the organ temperature until it reaches the final temperature of −20 ◦C. 2-D and 3-D spatial distributions of the acoustic pressure and the temperature inside the organ were analyzed. TABLE III ORGAN,HIFUSYSTEM,AND SIMULATION PARAMETERS Table III summarizes the geometrical properties of the model used, the HIFU system, and the different parameters of the simulations performed. Finally, another set of simulations was carried out, with a sweep in the ultrasonic power in each transducer from 10 to 75 W, with the fixed optimal frequency previously calculated, to see the effect of increasing ultrasonic power on the same two relevant parameters: warming rate and homogeneity. The temporal evolution of the temperature in the different axis was obtained. D. Biological Effects of Cavitation Ultrasonic cavitation could cause mechanical disruption in the cryopreserved organ. In general, cavitation threshold increases at lower temperatures and with growing viscosity [20]. To our knowledge, cavitation thresholds of typical cryoprotectant solutions at low temperatures (below zero) have not been reported. However, the fact that the organ is in a solid-vitreous state (rewarming in our simulations starts from −140 ◦C) can avoid the presence of cavitation in this range of temperatures. An especial care to avoid ultrasonic cavitation may be required at higher temperatures (higher than −20 ◦C). Ultrasonic power should be then lowered to avoid cavitation once the organ is not solid, depending on the temperature and on the concentration of cryoprotectant used. However, at these higher temperatures, ice is not usually formed and it is, therefore, not so crucial in cryopreservation protocols [8]. III. RESULTS A. Calculation of the Optimal Frequency With a selected power of 25 W per transducer (a total power of 650 W), a frequency sweep from 100 to 400 kHz was carried out for the ultrasonic transducers. The heating rate at the middle of the organ (from −140 ◦Cto −20 ◦C) was measured for each of the frequencies, as shown in Table IV. There is an optimal value of the frequency where attenuation is low enough for ultrasonic waves to reach the focal point but high enough to produce an important focal point heating. For our organ model and the attenuation coefficients used, the highest heating rate at the center of the organ is obtained at a frequency of 300 kHz. OLMO et al. : THE USE OF HIFU FOR THE REWARMING OF CRYOPRESERVED BIOLOGICAL MATERIAL 603 Fig. 2. Total field of the acoustic pressure at 25 W per transducer and 300 kHz. (a) Acoustic pressure total field in 3-D (isosurfaces, in decibels). (b) Acoustic pressure total field (at the plane X= ¼ ). A spherical focus of approximately 1 cm in diameter is formed in the central part of the system, coinciding with the organ. The geometry of the organ is also shown. The acoustic pressure is greater in this focus, because of the convergence of ultrasonic waves of the designed HIFU system. (c) Acoustic pressure total field (at the plane Y= ¼ ). (d) Acoustic pressure total field (at the plane Z= ¼ ). TABLE IV HEATING RATE VERSUS ULTRASONIC FREQUENCY At this frequency, the warming rate (superior to 100 ◦C/min) is fast enough for many cryopreservation applications. For these reasons, we selected this frequency (300 kHz) for the next simulations. B. Study of the Velocity and Homogeneity of Organ Rewarming With these values of 25 W and 300 kHz, the spatial heating of the organ was studied, in order to evaluate the homogeneity of the organ rewarming. Fig. 2 shows the spatial distribution of the acoustic pressure, constant in time, as the result obtained by the acoustic module of COMSOL. In this figure, a spherical focus of approximately 1 cm in diameter is clearly identified, larger than that in other referenced HIFU systems and in line with the proposed cryopreservation applications sought. With the acoustic pressure results, the temperature of the system was calculated, using the Qobtained by the acoustic pressure module, which is introduced into the heat module. Fig. 3 shows the final spatial distribution of the temperature of the system, at 1 min. The homogeneity of the final temperature achieved in the organ is observed in Fig. 3. Fig. 4 shows the evolution of the temperature in the different axis, from the initial time to the final time. We can observe in this figure the homogeneity achieved in the area of the simulated organ, in different times. A difference of around 5 ◦Cin the organ area is achieved, at all times. The average heating speed is over 100 ◦C/min. Both parameters are appropriate for cryopreservation applications, validating the system design selected. 604 Fig. 3. Final temperature of the system at 25 W per transducer and 300 kHz (◦C). (a) 3-D temperature of the system at the end of the process (1 min). (b) Temperature of the system at the plane X =0, at the end of the process (1 min). We can see that the temperature achieved is around −15 ◦C in the organ region. The ovary model is also shown, with its different parts. (c) Temperature of the system at the plane Y =0, at the end of the process (1 min). (d) Temperature of the system at the plane Z =0, at the end of the process (1 min). We can observe that most of the power is dissipated in the transducers (especially at high frequencies). C. Simulation of Increasing Ultrasonic Power Levels These final simulations were carried out with increasing values of ultrasonic power (10, 25, 50, and 75 W for each ultrasonic transducer), at 300 kHz, to see the effect of increasing ultrasonic power on the rewarming speed of the organ and on the homogeneity achieved. Table V shows the effect of an increasing ultrasonic power on the rewarming velocity of the center of the organ. We must take into account that these are the values for the ultrasonic power in each transducer and that COMSOL calculates the warming rate including the thermal diffusion of heat in the organ. We can see an increase in the heating velocity with the increasing power, as could be expected. However, a reduction in the homogeneity achieved was also found with higher power levels, as shown in Fig. 5.Adifference in temperature of up to 17 ◦C in the different regions of the organ for 50 W and 33 ◦C for 75 W is found, making these TABLE V HEATING RATE VERSUS ULTRASONIC POWER AT 300 kHz power levels unfeasible for many cryopreservation applications. Furthermore, ultrasonic cavitation ultrasonic cavitation at these power levels could cause mechanical disruption of the organ, especially at higher temperatures where the organ is no longer in its solid-vitreous state. OLMO et al. : THE USE OF HIFU FOR THE REWARMING OF CRYOPRESERVED BIOLOGICAL MATERIAL 605 Fig. 4. Evolution of the temperature of the system in the different axis, for 25 W in each transducer and 300 kHz. (a) Evolution of the temperature of the system (◦C) at the X -axis, from the initial time ( t =0, bottom line) to the final time ( t =1 min, upper line). (b) Evolution of the temperature of the system (◦C) at the Y -axis, from the initial time ( t =0) to the final time ( t =1min).(c) Evolution of the temperature of the system (◦C) at the Z -axis, from the initial time ( t =0) to the final time ( t =1 min). The homogeneity of the achieved temperature is also shown, with a difference of around 5 ◦C in the organ area (around the final temperature, −25 ◦C). The average heating speed is over 100 ◦C/min. An optimal value of 25 W per transducer is found for our HIFU system, at 300 kHz (Fig. 5), providing both a compatible heating velocity and a homogeneous rewarming for cryopreservation applications. 606 IEEE TRANSACTIONS ON ULTRASONICS, FERROELECTRICS, AND FREQUENCY CONTROL, VOL. 68, NO. 3, MARCH 2021 Fig. 5. Warming at 300 kHz with different power intensities for each transducer. (a) Evolution of the temperature of the system in the X -axis, for 10 W, from t =0, bottom line, to t =1 min, upper line. The final temperature of the center of the organ reaches around −38 ◦C, with this power level. (b) Evolution of the temperature of the system in the X -axis, for 25 W, from t =0, bottom line, to t =1 min, upper line. (c) Evolution of the temperature of the system in the X -axis, for 50 W, from t =0, bottom line, to t =1 min, upper line. A decrease in the homogeneity achieved was found, with a difference in temperature of up to 17 ◦C in the different regions of the organ. Similar results were obtained for the other two axes. (d) Evolution of the temperature of the system in the X -axis, for 75 W, from t =0, bottom line, to t =1 min, upper line. A decrease in the homogeneity achieved was found, and this time with a difference in temperature of up to 33 ◦C in the different regions of the organ. Similar results were obtained for the other two axes. IV. CONCLUSION A new HIFU system, based on the use of an array of ultrasonic transducers, is proposed for the application of rewarming of cryopreserved tissues and organs. The initial simulations are carried out in this work with the use of COMSOL Multiphysics software, with 26 ultrasonic transducers and an elliptical organ model. After the initial tests, a frequency of 300 kHz was selected to optimize the organ rewarming. The spatial distribution of the organ was analyzed, finding a spherical focus area of approximately 1 cm of diameter, larger than that in other HIFU applications and in line with the application proposed. The velocity of the organ rewarming achieved, superior to 100 ◦C/min, is fast enough for many cryopreservation applications, avoiding the growth of ice crystals during rewarming. On another hand, the homogeneity of the rewarming (around 5◦C between the different parts of the organ) is also suitable for the practical implementation of the technology. This homogeneity decreased in our simulations with other increasing power values of the ultrasonic transducers. Ultrasonic power, which could cause mechanical disruption in the organ, should be lowered to avoid cavitation once the organ is not solid, depending on the temperature and on the concentration of cryoprotectant used. However, important benefits are found in comparison with other proposed techniques. Very high rewarming rates can be achieved (119 ◦C/min is achieved in our simulations) greater than that with traditional convective heating (reaching a maximum heating rate of 50 ◦C/min for vials with a radius of ≥0.5 cm) [13]. This achieved heating rate can also be reached with an appropriated power in microwave rewarming, but HIFU avoids the inhomogeneities caused by thermal runaway or the maximums and minimums of the stationary wave [7], [18]. Also, HIFU avoids the complexity and potential toxicity of other proposed techniques, such as the use of magnetic fields and magnetic nanoparticles perfused in the vascular system [13], [17]. Based on these initial findings, further work should be oriented to analyze and study the practical aspects of the implementation of the HIFU technology in a real case study. Different focal areas could be achieved, based on the specific biological material to be rewarmed from cryogenic temperatures and a more precise knowledge of attenuation coefficients, based on experimental findings. Practical aspects of the implementation of a real HIFU system, with its specific ultrasonic transducers, and its utilization in cryopreservation protocols should also be studied. Further experimental work is needed OLMO et al. : THE USE OF HIFU FOR THE REWARMING OF CRYOPRESERVED BIOLOGICAL MATERIAL 607 to optimize the DMSO and water layer in order to minimize the ultrasonic reflections in the transducer–ice interface. We think that the new technology proposed in this work and the excellent results simulated show a very promising field of research for the following years, in order to finally achieve the practical cryopreservation of organs. ACKNOWLEDGMENT The authors would like to thank the anonymous reviewers for their helpful suggestions in the different versions of the article and their appreciated interest in our work. REFERENCES [1] E. Aydin, E. Tastaban, I. K. Omurlu, Y. Turan, and Ö. F. ¸Sendur, “Effects of deep heating provided by therapeutic ultrasound on demyelinating nerves,” J. Phys. Therapy Sci., vol. 28, no. 4, pp. 1278–1283, 2016. [2] J. M. Carcione, O. H. Campanella, and J. E. 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Martínez-Fernández et al., “Focused ultrasound subthalamotomy in patients with asymmetric Parkinson’s disease: A pilot study,” Lancet Neurol., vol. 17, no. 1, pp. 54–63, Jan. 2018. Alberto Olmo received the Ph.D. degree in engineering from the University of Seville, Seville, Spain, in 2010. He is currently a Professor with the School of Computer Engineering, University of Seville, and a Researcher with National Center for Micro-electronics, IMSE-CNM-CSIC, where he works in the development of biomedical circuits. The application of electronic technology to cryopreservation is one of his most active research lines. Pablo Barroso received the M.Sci. degree in environmental engineering from the University of Seville, Seville, Spain, in 2014. In 2015, he joined Physics Research Group CryoBioTech, University of Seville, focusing on the FEM simulations for high-intensity focused ultrasound applications in cryopreservation. Fátima Barroso received the M.Sc. degree in pharmacy from the University of Seville, Seville, Spain, in 2004. In 2019, she joined the M.Sc. Physics research group CryoBioTech, University of Seville, focusing on pharmacokinetics in cryopreservation and biological effects of ultrasound. Ramón Risco is currently a Professor with the School of Engineering, a Researcher with the CNA-CSIC, and the Director of the research group CryoBioTech, University of Seville, focused on the study and optimization of cooling and rewarming rates for the cryopreservation of cells, tissues, organs, and complete organisms.