Shen Ren
Published: 2020
Total Pages: 151
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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.