An experimental and theoretical investigation of cell cryobiological characteristics and optimal cryopreservation technology

Thesis (Ph.D.)--University of Washington, 2021Cryopreservation at low temperatures (e.g., in liquid nitrogen) has been proved to be the most reliable and sometimes possibly the only way to preserve cells and tissues alive for months, years, decades, or even centuries. Generally, a cryopreservation process includes a few essential steps, such as the addition of cryoprotective agent (CPA) before the freezing process, the cooling/rewarming of the sample with optimal rates, and removal of CPA after the thawing process, in which cells experience a series of highly stressed conditions, causing injury to cells. Scientists rely on the theoretical interpretation of bio-heat and mass transfer and novel measurement techniques to design the optimal cryopreservation protocol. In this dissertation, several novel devices and methods developed to analyze the fundamentals of cryobiology, determine cryobiological properties of cells, and optimize cryopreservation protocols are discussed.A microfluidic device with on-chip cooling/heating mechanism, enabling precise and stable temperature control, was developed. A multiphysics simulation of three-dimensional laminar flow conjugated heat transfer, coupled with Joule heating, was conducted. The temperature control stability and response were tested and demonstrated. This platform achieves the following features: (1) hydrodynamic confinement of the single-cell; (2) switching extracellular medium during the cell trapping; (3) controlling surrounding medium temperature from 2 ℃ to 37℃. A simplified microfluidic device was employed to determine the cell membrane permeabilities of Jurkat cell at room temperature, where a microfluidic chip with a block structure was used to achieve steady trapping. The cell membrane permeabilities of Jurkat cells to water and CPA were determined through simulation and image processing analysis with cell volume excursion captured by a high-speed camera. A multifunctional cell processing system and related auto-generated CPA removal protocol are proposed to achieve both CPA removal and cell concentration functions. By applying optional hypertonic dilution and an advanced algorithm with cell properties, determined by devices discussed above, as parameters, the optimal CPA removal approach will be generated to minimize cell shrinkage and swelling during the post-cryopreservation process. Heat transfer models for water-bath and natural air rewarming processes for large samples were developed and validated by experiments. The validated models were then employed to calculate the temperature gradients spatially and temporally. Based on temperature gradient results, natural air rewarming is a better tissue and organ cryopreservation method with relatively more minor thermal stress during rewarming. Specific suggestions about the sample's location in the container and the size of the container were also discussed. An inverse method to determine the subzero Lp and Ea with cell recovery rate results and water transport model is proposed. The subzero Lp and Ea of Jurkat cells were determined and discussed. The methodology is meaningful for the fundamental research of cryobiology. The limitations of proposed devices and methods are discussed. Future work is presented, including more biological experiments of new-developed devices, advanced heat transfer models, and the relation of cryobiological properties