Study On Transmembrane Mass Transfer And Freezing Properties Of Cryoprotective Solutions In Cryopreservation Of Cells

Cryobiology is an inter-and trans-disciplinary area which combines thermal science, engineering, biology and medicine. Cryopreservation and cryosurgery are two main application techniques of cryobiology. Specifically, cryopreservation has great implication for the long-term storage of stem cells, human blood, and rare germplasm resources, as well as the transplant of human organs. The low-temperature injury is considered as the most critical factor that limits the post-cryostorage viability. Generally, it includes the mechanical injury by intracellular ice formation and growth, the "solute injury" and so forth. The cryoprotective agents play an indispensible role in cryopreservation by reducing the freezing injury and noticeably enhancing the post-thaw viability. A typical programmed slow-freezing approach consists of five steps. They are:the loading of permeable cryoprotectants, programmed freezing, long-term storage at the temperature of liquid nitrogen, thawing and the removal of cryoprotectants. In almost every step, biological cells and tissues will perform complicated physical, chemical and biological responses which are induced by the changes in temperature, pressure, the solution composition and concentration, protein activity, metabolism rate, etc.. Therefore, to predict the change of the abovementioned physical and chemical parameters has great importance for clarifying the low-temperature injury and developing new methods for enhancing the post-thaw viability, thereby becoming one of the most challenging tasks for researchers in the field of cryogenics engineering.This dissertation studies the transmembrane mass transfer occurring in the cryopreservation of cells and the freezing properties of cryoprotective solutions (i.e., aqueous alcohols and dimethyl sulfoxide). To these aims, the theories of physical chemistry, thermodynamics and mass transfer, differential scanning calorimetry and molecular dynamics simulation technique are employed. Based on the phase diagrams of cryoprotective solutions, a non-ideal model is proposed for predicting the water loss of cells which experience the slow-freezing protocol. It is found that the conventional ideal models can underestimate the intracellular water content of cells in the slow-freezing protocol. From the Gibbs free energy, a thermodynamics model is established to analyze the transport of water and cryoprotectants across cell membranes during cooling and warming procedures. This model is established for the real solution and can be simplified for ideal and dilute solutions. It can abandon the assumption of the ideal semi-permeable membrane made by conventional models. It is used to predict the volumetric change of ICR mouse spermatozoa and human corneal keratocytes. This dissertation also measures the phase diagrams of two ternary systems, i.e., methanol/sodium chloride/water and propylene glycol/sodium chloride/water, by differential scanning calorimeter. Not only can the results extend the application scope of the models proposed in this dissertation but further prove the validity of synthesizing ternary phase diagrams by corresponding binary ones for a limited range of concentration.This dissertation measures the unfrozen water content of frozen alcohol and dimethyl sulfoxide solutions by differential scanning calorimeter. The relationship between the enthalpy of fusion of ice in cryoprotective solutions and the initial concentration is clarified. It is shown that the unfrozen water content increases as the initial molality rises. This dissertation also probe the internal molecular mechanisms for the macroscopic results given by differential scanning calorimeter by analyzing the self-diffusion coefficient of water and the hydrogen-bonding characteristics of the abovementioned solutions. It is found that the unfrozen water content is positively correlated with the percentage of the solute-water hydrogen bonds in the solution, which proves that the solute-water hydrogen bonds is directly responsible for the unfrozen water existing in frozen cryoprotective solutions.The models proposed in this dissertation can predict the biophysical responses of cells in cryopreservation more precisely and calculate the intracellular contents of cryoprotectants and water. The thermodynamics data of cryoprotective solutions are enriched here. Moreover, the results can offer both macroscopic and microscopic insights into the cryoprotective mechanisms of alcohols and dimethyl sulfoxide. The results in this dissertation will benefit the reasonable design of the cryopreservation protocols.