| Cryopreservation is widely used in biological preservation and other fields.In the process of cryopreservation,the continuous change of extracellular osmotic pressure during the loading/ unloading of cryoprotectant(CPA)can easily lead to cell damage or even death.Existing processing methods are subject to various limitations such as processing efficiency,processing throughput,and low cell recovery rate.Existing membrane microfluidic systems are also prone to cells clogging the pores of the microfiltration membrane due to pressure changes on both sides of the microfiltration membrane.In order to solve the above problems,this master thesis reduces cell damage through staged osmotic pressure changes,so that cells have time to adapt to environmental changes after each stage of osmotic pressure changes,and at the same time,unidirectional mass transfer from perfusate to cell fluid avoids microfiltration membrane contamination.Based on this idea,a sequential perfusion chip was designed,and the CPA concentration in the cell fluid was changed step by step through a specially designed structure.This chip reduces cell damage caused by osmotic pressure imbalance while efficiently completing CPA loading / unloading.The main work carried out in this master thesis are:(1)The scheme principle of sequential perfusion microfluidic chip is proposed,and the one-way perfusion into cell fluid is theoretically derived.According to the changes of the osmotic pressure of the cell fluid before and after the loading / unloading of CPA,design the staged perfusion process,design the chip structure on the basis of the theoretical model,and design the "membrane window" to realize the staged perfusion process.Processing and assembling prototype devices.(2)The mass transfer characteristics of the serialized perfusion chip were studied,and device-level simulations were performed to obtain the perfusion sequence for the transmembrane mass transfer of the perfusate to the cell fluid.The CPA concentration distribution in the flow channel is calculated through the perfusion sequence,so that the cell-level simulation is performed to calculate the dynamic response of the cell volume.Fluorescence experiments were carried out to visually explore the mass transfer characteristics of the chip.The changes in the fluorescence intensity of the fluorescein sodium salt solution under the laser reflected the changes in CPA concentration in the flow channel.The experimental results were compared with the simulation results to verify the feasibility of the simulation method.At the same time,through fluorescence experiments to help improve the chip structure and assembly process.(3)The prototype device was used for the loading/ unloading experiment of cryoprotectant(20% glycerol)of human red blood cells(RBC),and a control experiment was conducted with existing methods(single-step,multi-step,continuous method)to verify the loading / unloading of CPA performance.The experimental results were evaluated by various indicators(cell count,hemoglobin concentration).The sequential perfusion chip can obtain stable and good cell recovery rate under low-throughput(0.2ml/min)to high-throughput(0.8ml/min)inlet conditions(addition stage 100%,removal stage higher than 88%)),At the same time there is a CPA removal rate that is stable at 90%.Compared with the control group,the chip processing performance designed in this paper has been significantly improved,and can adapt to a larger inlet flow range.(4)In order to optimize the experimental conditions and improve the processing efficiency,this paper has designed an automated device that can provide a stable temperature environment for the experiment and at the same time can provide a stable inlet flow,and designed a self-holding pinch valve to control the pipeline.The sequential perfusion microfluidic chip designed in this paper has the characteristics of high safety,high throughput and high feasibility,and can be used as a highly promising solution for the safe and efficient CPA loading/unloading. |
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