| Cryopreserved cells have been widely applied in the fields of cell therapy,tissue regeneration,reproductive medicine,and so on.In the cryopreservation of cells,although cryoprotective agents(CPAs)can prolong shelf life of cells and prevent cells from cryoinjury,it is necessary to remove CPAs from cells before use because of the toxicity of CPAs to cells and the side effects of CPAs to body.Currently,approaches for unloading CPAs from cryopreserved cells mainly include centrifugation-based,dialysis-based,and filtration-based methods.The centrifugation-based method is simple to operate,but the centrifugal force may easily induce the damage to cells and the clotting of cells and then cause the loss of cells;the dialysis-based method can effectively reduce the osmotic damage to cells,but the clearance efficiency of CPAs is relatively low;the filtration-based method can remove CPAs quickly,but the recovery rate of cells is relatively low.These methods are designed usually for large volume cell samples(30?500 mL)and unfit for small volume cell samples.Recently,with the rapid progress of micro fluidics,scientists have already started to develop CPA-unloading chips to process a small volume of frozen-thawed cells.However,there are still some deficiencies in the developed chips(for example,the wash efficiency of CPAs is low).Here,therefore we developed a dilution-filtration-based microsystem to unload CPAs from cells(the microsystem is also called multistep dilution-filtration microdevice,MDFD).The core idea in the MDFD design is:based on microfluidics,the cell suspension with CPAs is diluted step by step using dilution solutions with various NaCl concentrations,the CPAs inside of cells are transported across the cell membrane to the extracellular solution,and the extracellular solution with CPAs is removed step by step by filtration.The main contents of this thesis are as follows:(1)The MDFD was designed and fabricated.Here,the MDFD was designed to unload glycerin from glycerin-loaded porcine erythrocytes.In design,the ladder network was adopted to produce dilution solutions with various NaCl concentrations,the Tesla mixers were adopted to mix solutions and the Kirchhoff's current and voltage laws were adopted to calculate the dimension parameters of the MDFD.The MDFD was fabricated with organic glass.(2)The performance of the MDFD was analyzed theoretically.First,the volume change of cells and the concentration change of glycerin in the process of unloading glycerin were simulated using the transport equations of CPAs across the cell membrane.Then,the mixing performances of the mixers in the dilution-producing region and the dilution-filtration executing region were investigated using the transport equations of diluted species.(3)The performance of the MDFD was studied experimentally.In the experiments with cells,the glycerin clearance rate(CG),the cell survival rate(Sc)and the cell recovery rate(Rc)under the setting conditions were determined and then the effects of glycerin concentration,cell density and membrane pore size on the performance of the MDFD were investigated.In the cell-free fluorescence experiments,the functions of the dilution-producing region and the dilution-filtration executing region were confirmed.The results show that in the MDFD,the volume of cells is always in the safe range of the upper and lower volume tolerance limits,the glycerin inside cells can be removed effectively,and the mixers can mix two solutions fully and quickly.Under the setting conditions(0.2 mL/min of the cell suspension flow rate,20%w/v of the glycerin concentration and 24%v/v of the cell density),CG reached?80%and SC reached?90%,but RC was relatively low(?40%).When the concentration of glycerin to be removed is lower,CG,SC,and Rc are all higher.For either a higher cell density or a larger pore membrane,CG is higher,but both Sc and Rc are lower.The work presented here can contribute to developing the biochips for the in-line unloading of CPAs from cryopreserved cells. |
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