Theoretical And Experimental Study On Cryopreservation Of Human Cord Blood Stem Cells

The cryopreservation of biological materials have many applications in several fields of national economy. For a long time people are persuing how to minimize the injuries caused by freezing and rewarming and how to cryopreserve larger volumes of biological materials. With the knowledge of thermodynamics and heat and mass transfer, analysis can be made for each step of the cryopreservation process. This will facilitate the design and optimization of cryopreservation protocol. This thesis studied the cryopreservation of human cord blood stern cells(HCBSCs). Theoretical and experimental work were carried out as follows:1 A test apparatus was set up to measure the osmotic characteristics of suspended cells. Its main component was a diffusion chamber. With this apparatus, theaverage diameter, the inactive volume, the permeability to water and its activation energy, the permeability to dimethyl sulfoxide and its activation energy are measured for HCBSCs.2 Theoretical analysis was made for the addition and removal of cryoprotectants. A two-parameter model for the permeation process was introduced. The analytical solution for the extremums of cell volume and cell water volume were obtained for the case of one cryoprotectant. The approximate solution given by Katkov was analyzed for its reason of discrepancy and its applicability. The optimization of addition/removal process was discussed.3 A cryomicroscope system was built. An inverted microscope with maximum magnification of forty for the objective was employed. Cooling was supplied by cold nitrogen gas. The sample temperature was controlled by regulation of the flow rate of cold nitrogen gas and the heating rate of coated glass. The cell images during freezing process were captured by a camera. Recording and control of the sample temperature and acquisition and storage of the cell image were automatically fulfilled by a computer.4 The phenomena of intracellular ice formation for HCBSCs were studied with the cryomicroscope. Without cryoprotectant, the mechanism of intracellular ice formation for cells at isotonic state was surface catalyzed nucleation(SCN); With dimethyl sulfoxide as cryoprotectant, the mechanism of intracellular ice formation was volume catalyzed nucleation(VCN). According to the model proposed by Toner, the relevant parameters in the formula of SCN and VCN were fitted with the experimental data.5 A mathematical model was established to describe the freezing process of cells. The model includes the transportation of water and cryoprotectant across the membrane, the formation of intracellular ice and the growth of ice crystal. Simulation was done for the freezing of HCBSCs. Conclusions were drawn as follows: a) when the cell has high permeability to the cryoprotectant at low temperatures, it is not enough to consider the transportation of water only. The transportation of cryoprotectant must be considered as well, b) for the case without cryoprotectant, the probability of intracellular ice formation increases as the cooling velocity increases. The probability reaches near 100% at 5/min. The cooling velocity should be under 0.5/min so that cells will not be killed by intracellular ice. c) when 5-10% dimethyl sulfoxide is used as cryoprotectant, the probability of intracellular ice formation will vary from 0% to 100% at cooling rates from 9/min to 13 /min. The influence of the concentration of cryoprotectant is minute.6 An elevator cryocooler was made. The cryopreservation of HCBSCs was investigated with this equipment. With 5% or 10% dimethyl sulfoxide as cryoprotectant, the survival rate of HCBSCs are highest when cooling velocity is 10/min or 10.5/min. The survival rates will be seriously lowered if the cooling velocity is much greater or smaller than 10/min.7 The model of this paper well explains the impact of cooling velocity on the survival rate of HCBSCs observed. If the optimization strategy that makes cooling time minimum with intracellular ice formatio...