Theoretical And Experimental Study On Several Mechanical Engineering Issues In Cryopreservation Of Biomaterials

As the principal method for long-term storage of biomaterials, cryopreservation is of great significance for scientific research as well as clinic application. It has been proved that the development of cryopreservation relies on not only Biology, but also the engineering science, especially the Mechanical Engineering principles and technologies such as Heat/Mass Transfer and Mechanical Design. In this thesis, some common issues existing in cryopreservation are investigated in the view of Mechanical Engineering. Several theoretical approaches are presented accordingly and then validated by a series of experiments. The detailed research issues and results are as follows:1. Control of cooling rate. A Box-in-Box (BIB) method is investigated for reliable and low-cost control of cooling rate. The passive cooling principle used in BIB method helps to reduce energy consumption as well as structural complexity, and the configuration of flat boxes makes the heat transfer to samples be approximated to one dimensional and thus the control of cooling rate can be much simplified. In the thesis, a theoretical model is developed to evaluate the effects of several factors, and several experiments are also conducted for validation. Cell-free experiments confirm that the BIB method can provide consistent cooling processes for samples, and the predicted temperature profiles fit the recorded results quite well. In-vitro experiments about hematopoietic stem cell further demonstrate the BIB device can work as good as programmed freezer, and better than another passive cooling method.2. Addition/Removal of cryoprotectant agents (CPAs). Several membrane methods, which involve dialysis and ultrafiltration for adding or removing CPAs, are investigated. Theoretical models are established to evaluate the effects of the operation parameters to cell recovery rate and process efficiency, and then approaches are developed to determine the optimal operation protocols for given conditions, respectively. Cell-free experiments prove the models can predict actual processes accurately. The in-vitro experiments about freeze-thaw red blood cells show that the pure ultrafiltration method can achieve both efficient glycerol clearances and high cell recovery rates, and thus has significant advantage comparing to current methods. 3. Development and optimization of equipments. A BIB device and a CPA processor are developed and the procedures are described in detail. Furthermore, a model based approach is presented to optimize the assembly process and structural design for the devices as well as other equipments used in cryopreservation. The approach is validated in the optimization of the CPA processor4. Measurement of cell membrane parameters. The effects of several experimental conditions to the process of cell volume excursion are theoretically evaluated, and then validated by several experiments. Based on the results, a strategy for optimizing the experiment design for measurement of cell membrane parameters is suggested.With the study above, it is expected to achieve safer process, lower cost, more efficient and convenient operation as well as more accurate measurement in cryopreservation. The major innovation of this work can be concluded as:1. A theoretical model describing the freezing process of the ternary media cooled with BIB method is established. With the help of the model, the effects of structural parameters and application conditions can be accurately evaluated, and then the design and application of BIB can be guided to achieve the desired cooling rate.2. A novel membrane method, which involves dilution and pure ultrafiltration for processing CPAs, is presented. The method can significantly improve the efficiency while reducing cell injury. Comparing to dialysis methods, the method has further advantages in controllability as well as ability of removing large molecular substances3. An novel assembly model is presented. Based on the model, feasible assembly sequences can be generated, and subassembly stabilities, operation costs and feasible operation directions can be evaluated. Then optimal assembly sequence can be selected and structural design can also be optimized according to the quantitative criteria.4. A strategy of optimizing the experiment design for the measurement of cell membrane parameters is suggested. By properly selecting the concentrations of extra- and intracellular solutions, the cell volume change can be increased while the volume excursion can be slowed down for the best of the measurement.