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The development of successful cryopreservation technology has been restricted to small biological materials due to the limited effective rewarming techniques. Either for conventional cryopreservation techniques which remain a certain fraction of ice crystals or vitrification which aims to exclude ice crystal in the subzero temperature range completely, the rewarming rapidity plays a significant role in the recovery of the biological materials after cryopreservation. Additionally, for larger materials such as tissues and organs, the uniformity of the temperature profile is indispensable to maintain the integrity of the cryopreserved biological matrix. The major challenges for traditional convective rewarming method, i.e., water bath, lie in that the high specific heat of cryopreserved materials likely result in a slow warming by the unsatisfactory heat source; and a large temperature gradient is induced by conductive heat transfer within the sample. The cryopreserved cells are exposed to cryoinjuries for a longer time if rewarming is insufficiently rapid in the case of conventional cryopreservation. For tissue or organ preservation, even if a vitrified state has been achieved by the addition of high concentration cryoprotective agent (CPA) solutions and/or designed cooling process, the slow rewarming can lead to devitrification and recrystallization, introducing considerable ice crystals that should be avoided. On the other hand, the non-uniform temperature profile brought by the convective heating method leads to the thermal stress induced fractures, which can tear apart the vitrified tissues. Hence, an effective rewarming should be both rapid and uniform, to guarantee the successful cryopreservation or vitrification of biological materials. Potentially it may be the most promising to achieve the goal by utilizing the electromagnetic waves, but several issues remain to inhibit the further practical applications. This dissertation reports on research in which an electromagnetic resonance rewarming technique is developed and optimized aiming for the organ and tissue preservation. An optimization procedure is established as the following steps: (1) determination of the essential physical properties of the CPA/vitrification solutions; (2) analysis and estimation of the combined electromagnetic and heat transfer phenomenon; (3) theoretical simulation and investigation of the rewarming process based on the measured properties; and (4) practical setup of an electromagnetic resonance system and enhancement by adding superparamagnetic nanoparticles. First, the effectiveness of vitrification of bulk biological material is mainly dominated by the composition and concentration of the vitrification solutions. To avoid devitrification or fractures which relies upon the absorption of electromagnetic energy and heat diffusion, the fundamental electric properties, thermal properties of vitrification solutions are indispensable for the following estimation of the electromagnetic rewarming efficacy. The dielectric properties were determined by a designed measurement system adopting the cavity perturbation method. The thermal conductivities were measured, and differential scanning calorimeter was used to determine the specific heat. The analysis of these preliminary measurement results was conducted for a selection of CPA/vitrification solutions. Due to the nonlinearity of the coupled electromagnetic and heat transfer equations, an accurate analytic prediction is hardly achieved to the electromagnetic rewarming outcomes. Hence, a numerical simulation model was established to estimate the warming procedure and select CPA solutions, optimal geometry of the cryopreserved materials and several other parameters before conducting experiments. In particular, a hybrid electromagnetic-conduction rewarming concept was raised and tested to maintain the higher warming rate and reduce the temperature non-uniformity of the cylindrical cryopreserved materials. The numerical model can simulate the field and optimize the parameters of the electromagnetic rewarming system, to achieve a higher power utilization efficiency. With the assistance of a numerical model, the electromagnetic resonance rewarming system was designed, assembled, and optimized, particularly the coupling between the system source and resonance chamber. In addition, a frequency tracking component was added to ensure the resonant state during the rewarming process, when the cryopreserved materials shift the resonance as temperature increases. Finally, to fully utilize the electromagnetic field energy provided by the dynamic controlled electromagnetic resonance rewarming system, we took advantage of magnetic nanoparticles (MNPs) to absorb magnetic field energy to further enhance the energy conversion efficiency, which overcame the low electromagnetic energy absorption ability problem that previous attempts suffer from. An ultra-high power utilization efficiency was obtained and we achieved over 200 °C min−1 rewarming rate for tens of mL cryopreserved samples. In addition, we also investigated the effect of nanoparticle size and concentration on the rewarming results and thermal properties. The closed system preventing electromagnetic radiation outwards reduced the possible concomitant side effects when increasing nanoparticles or raising the electromagnetic power. With the remarkably low dosage of nanoparticles (0.1 mg mL−1 Fe) compared to other MNPs based rewarming applications (over 1 mg mL−1 Fe), this study opens a door for new approaches to explore novel rewarming techniques for the tissue and organ preservation.
In the last few decades, applications of biopreservation, particularly cryopreservation, have played significant roles in scientific and clinical settings. The foundation of these approaches is the successful cryopreservation of biological samples, including deoxyribonucleic acid (DNA) / Ribonucleic acid (RNA), proteins, bio-fluids, cells, tissues, and organs. So far, we can only cryopreserve bio-samples of small volumes in the order of 1 mL, mostly due to the lack of an effective rewarming technique. For small volume systems, the convective rewarming method remains the gold standard. However, as the sample volume increased, the conventional way cannot avoid physical damage to the bio-specimen due to the cellular injuries induced by the remaining ice crystals. To prevent crystallization in the thawing process, rapid rewarming is the key. Additionally, for larger biomaterials such as the bulk volume of cell suspension, tissues, or organs, a uniform temperature profile throughout the biomaterial is required to avert the larger thermal gradient that often causes fracture or cracks within the material. Thus, an ideal rewarming method should be both rapid and uniform to achieve the successful cryopreservation of large scale biomaterials. The conversion of electromagnetic energy into heat provides a possible solution to this problem. In this dissertation, an automatic electromagnetic resonance rewarming system is developed to (1) determine the critical thermal-electrical properties of the samples, (2) simulate and investigate the coupled electromagnetic and heat transfer problem, (3) adjust the frequency and power input in real-time to ensure the resonant state, (4) automatically load cryopreserved samples from liquid nitrogen to the rewarming chamber, and (5) achieve the rapid-uniform warming of large-scale biomaterials. The cryoprotectant (CPA) is introduced to diminish the cryo-injuries during cryopreservation. The effectiveness of CPA, or vitrification solution, which is aiming to prevent ice crystals for larger samples in the lower temperature range completely, mainly depends on the composition and concentration of certain chemical agents. It is critical to understand and determine the electrical and thermal properties of CPA solutions to better utilize electromagnetic energy. The dielectric constant and dielectric loss were determined through a dynamic feedback system adopting the cavity perturbation theory. The thermal conductivities were determined with a designed micro thermal sensor, and specific heats were measured through differential scanning calorimetry (DSC). The selection of the optimal CPA/vitrification solution is based on the permeability of the cell membrane to the CPA, the toxicity of the CPA to the cells, absorptivity of the CPA to the electromagnetic power, and heat diffusion within the CPA solution. The higher concentration of CPA will reduce the ice formation in the sample during the cooling process, but meanwhile, it also introduces higher osmotic pressure and toxicity that results in the damage or death of the cells. On the other side, the previously measured electrical and thermal properties of CPA solutions were analyzed to predict the rewarming rate and temperature profile. By balancing these fundamental parameters, the optimal CPA/vitrification solution for a specific type of biological materials was decided. An exact analytic solution is hard to derive for the electromagnetic warming problem due to the complexity and nonlinearity of the electromagnetic and heat transfer theories. Therefore, a numerical simulation model built with COMSOL was used to predict the warming process, finalize the geometry of the sample holder, and optimize the power utilization efficiency. Another challenge with electromagnetic heating is maintaining the resonant state during the entire rewarming process since an off-resonant state will dramatically reduce the sample's absorption of the electromagnetic energy. A real-time resonant frequency monitoring and controlling system was developed. As the temperature of the cryopreserved sample increased, the resonant frequency shifted and was captured by the control system. The system adjusted the signal source automatically based on the feedback from the tracking components to keep the power absorption at a high level. In the meantime, an aluminum-based supporting frame was designed, manufactured, and assembled to achieve the automated sample loading process. The results showed that this sample loading system could transfer the cryopreserved specimen from the liquid nitrogen environment to the heating chamber in a short time and protect the operator from the potential frostbite. Several experimental tests of the automatic electromagnetic resonance rewarming system with cells and tissues were also performed in this dissertation. To further utilize the energy generated by the electromagnetic field, the magnetic nanoparticles (MNPs) were used to absorb magnetic field energy and extend the improvements of rapid-uniform rewarming. In the cell test, Jurkat cells were chosen because of their importance in Leukemia and HIV research and poor preservation with current protocols for large volume. With the combined electromagnetic and MNPs warming, the recovery rate of 25 mL Jurkat cell suspension was obtained at 93.7©2℗ł5.5%, almost doubled compared to the performance of the convective water bath method. The post-thaw assessment with alamarBlue assay proved the biological functionalities of Jurkat cell were well preserved with the electromagnetic resonance rewarming system. In the tissue test, rabbit jugular veins were chosen because of their similar vessel size to humans and the ease of handling. Vitrification solution was adopted to eliminate the ice crystals, post-thaw assessments including histology analysis, agonist and antagonist induced contractile and relaxation measurements, alamarBlue assay, and mitochondria membrane potential measurements were performed to qualitatively and quantitatively evaluate the performance of different rewarming methods. Plans for future improvements are also presented in this dissertation, including the design of the next generation resonance cavity, further improvements of the resonant frequency monitoring and controlling system, and optimization of the organ rewarming test.
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