Development and Optimization of Novel Single Mode Electromagnetic Resonant (SMER) Techniques for Cryopreservation

Abstract

Since 1949, scientists have made significant efforts to advance cryopreservation techniques for a wide range of biological materials, including deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins, biofluids, cells, tissues, and organs. Cryopreservation typically involves five major steps: cryoprotective agent (CPA) loading, cooling, storage, warming, and CPA unloading. Despite decades of progress, the field still relies heavily on manual, dropwise CPA addition and removal methods. Current dilution protocols often employ isotonic solutions or multi-step dilution approaches, which are labor-intensive and may not be optimized for different cell types. There is a critical need for fully automated systems that can perform CPA addition and removal in a controlled, cell-type-specific manner—taking into account factors such as volumetric flow rate and hypotonic solution selection to minimize osmotic stress. Furthermore, a post-dilution concentration step that eliminates the need for centrifugation would greatly enhance the clinical utility of cryopreserved cell suspensions, particularly in cellular therapy applications.For the cooling process, vitrification offers an effective strategy to prevent ice crystal formation, which is particularly important for preserving complex biological systems such as tissues and organs containing multiple cell types. However, conventional vitrification typically requires high concentrations of small-molecule cryoprotectants like dimethyl sulfoxide (DMSO) or glycerol, which can cause significant osmotic and toxic injury to cells and tissues. To address this challenge, recent efforts have focused on reducing the use of small-molecule CPAs in vitrification protocols. In this work, we introduce a novel approach utilizing high concentrations of polyvinylpyrrolidone (PVP) with varying molecular weights to minimize DMSO content while maintaining vitrification efficacy. This strategy demonstrated successful preservation outcomes in both cell suspensions and tissue samples. Preliminary experiments on mouse liver tissue confirmed the feasibility of this CPA formulation. Using this protocol, 4 mL of cell suspension could be directly plunged into liquid nitrogen for vitrification and subsequently rewarmed in a water bath, resulting in high post-thaw recovery and proliferation rates. Conventional water bath rewarming is inadequate for large vitrified samples due to its reliance on low-efficiency convective heat transfer. In contrast, electromagnetic (EM) waves can penetrate biological materials and enable volumetric heating, offering a more effective solution for rapid and uniform rewarming. To optimize EM-based rewarming, three key parameters must be considered: operating frequency, dielectric properties of the sample, and electric field intensity (determined by input power). A single-mode electromagnetic resonant (SMER) cavity was developed to concentrate energy through mode selection, enabling efficient and localized energy deposition. A generalized design methodology was established for constructing both electric and magnetic field–dominant cavities at their respective fundamental modes. To address the frequency drift caused by temperature-dependent changes in dielectric properties, an auto-resonance tracking system was implemented to maintain resonance during the warming process. This system also monitors the voltage standing wave ratio (VSWR) to quantify reflected power. The complete system was successfully applied to rewarm both 10 mL and 20 mL frozen and vitrified samples, demonstrating superior performance compared to conventional rewarming techniques. In addition to cell suspensions, this work extends to the cryopreservation of tissues and organs. Plans for future development are also outlined in this dissertation, including the advancement of next-generation cryoprotective agents, the design of an integrated perfusion system, and the construction of an improved electromagnetic cavity. The proposed cavity will incorporate features such as real-time temperature monitoring, user-friendly sample loading, antenna coupling optimization, electric field uniformity enhancement, and an integrated shaking platform to promote thermal homogeneity. Ultimately, this research aims to advance toward the successful cryopreservation and rewarming of whole organs.