Numerical Simulation Of Multiple Cryoprotectant Loading Into Neurosphere

Neural stem cells (NSCs) have the ability of self-renewal and multi-directional differentiation as well as high reproductive rate. Its transplantation has wide prospect of clinical applications in brain damage repair, repair of spinal cord injury, drug screening test and treatment of a variety of nervous system diseases. Practice shows that it is difficult to keep long-term proliferation and enable reliable differentiation for NSCs, and the NSCs are prone to the aging, degradation, and even the mutation during the cell subculture, leading to shortage of NSCs. Consequently, extensive research has been conducted on the cryopreservation of NSCs, so as to establish the stem cells bank. Compared to cryopreservation of single cell, cryopreservation of the neurosphere helps maintain cell activity, and the cryoprotectant (CPA) loading process is particularly critical. At the same time, the existing experimental instrument is very difficult to enable precise measurement of the internal concentration and instantaneous volume of cells in neurosphere. Therefore, this dissertation aims to understand regularity of CPA penetration using numerical simulation method, providing guidance for optimization of multicomponent protective agent loading process and experimental study on cryopreservation of NSCs.In this dissertation, a mathematical model based on M-S equations and the2-P model is developed to describe the multiple cryoprotectant transport behavior in neurosphere. The volume change items are introduced in the model to simulate the cell volume chang during the single component and multicomponent protectant loading and to analyze the influence factors. With the finite volume method and Matlab software for programming and conducting data analysis, the concentration space-time distribution inside neurosphere during the single component and multicomponent protectant loading is obtained from the comparison of the simulation results. The systematic analysis is conducted for the influence of operating conditions on CPA mass diffusion.Simulation results show that the concentration near the neurosphere surface is significantly higher than that at the center. The change rate of CPA concentration in the extracellular space is faster than that in the intracellular element. Concentration difference increases initially and decreases afterwards, and surface concentration difference extremum is significantly higher than at other positions. The greater the external cryoprotectant concentration, the longer time for osmotic balance and the greater the concentration difference and volume change extremum. The larger the size of neurosphere, the slower the diffusion rate, the smaller the concentration difference, and the greater the volume change extremum. With the temperature increasing, the osmotic balance time reduces and concentration difference extremum decreases. CPA species have significant effects on the concentration distribution and cell volume change, and the optimization is substantially necessary. The multi-steps and continuous loading procedures can reduce the concentration difference and significantly affect the cell volume change, but will prolong osmotic balance time.The results also show the existence of the anti-concentration diffusion phenomenon in multicomponent mass diffusion, usually for the component with larger membrane permeability and the M-S diffusion coefficient. The effect of concentration ratio of the multicomponent cryoprotectant on mass diffusion is more complex. The difference of the impact of different components on concentration distribution and cell volume at certain conditions should be considered. For composite cryoprotective agent, the largger the apparent membrane permeability and diffusion coefficient, the faster the CPA spreads and the smaller the volume change extremum.