| Freeze-drying preservation is one of the effective methods for long-term storage of erythrocytes(red blood cells)at room temperature.However,in this method,the cell membrane is seriously damaged during the freezing process,and the structure integrity of the cells is destroyed during the drying process,resulting in cell death.Therefore,the addition of protective agents before freeze-drying is a critical and necessary step.Studies have shown that trehalose loaded in erythrocytes can protect erythrocytes from freezing and drying damage.However,trehalose is impermeable to the cell membrane.That is,it cannot spontaneously penetrate the erythrocyte membrane.Recently,although some methods have been developed to promote the penetration of trehalose into red blood cells,these methods are usually manually operated,have many complex processes and have a long processing time;moreover,the loading strategies are various and lack uniform standards.Thus,in this thesis,a hypotonic shock-based trehalose-loading microdevice(TLM)was developed to rapidly,continuously,and automatically produce erythrocytes with loaded trehalose.The main contents of this thesis are as follows:(1)The TLM was designed and fabricated.The TLM made of organic glass can be divided into three zones.In the loading zone,the hypotonic solution causes the membrane pore to open and trehalose is loaded through the erythrocyte membrane pores;in the encapsulation zone,the hypertonic solution caused the membrane pores to close and trehalose is encapsulated;in the membrane exchange zone,the saline is used to wash the trehalose-loaded erythrocytes for removing hemoglobin molecules and cell debris and to restore the extracellular solution to the isotonic state.Here,the loading and encapsulation time and the trehalose concentration were determined based on pre-experiments,the solutions were mixed rapidly using S-shaped mixers,and the dimension parameters were designed according to the Kirchhoff's current and voltage laws.(2)The performances of the mixers and the membrane exchange zone were analyzed theoretically and experimentally.The fluid flow was simulated using the Navier-Stokes equation and the mass transfer was simulated using the convective-diffusion equation.Then,the functions of the mixers and the membrane exchange zone were confirmed theoretically.In experiments,the performances of the mixers and the membrane exchange zone were confirmed by fluorescence microscopy imaging based on the fluorescein distribution.The substance exchange capacity of the exchange zone was further analyzed based on the change in the concentration of trehalose at outlets.The results showed that under the designated operating conditions,the mixer designed here could quickly mix the solutions and the membrane exchange efficiency reached over 50%.(3)The performance of TLM was evaluated,the factors affecting the performance of TLM were studied,and the experiments on freeze-drying and freeze-thawing erythrocytes with loaded trehalose were preliminarily performed.The results showed that under the designated operating conditions,the loading efficiency for human erythrocytes reached?21 mM in?2 min with a value of the hemolysis rate of erythrocytes(?17%)and a value of the recovery rate of hemoglobin in erythrocytes(?74%)and the loading efficiency for swine erythrocytes reached?30 mM in?2 min with a value of the hemolysis rate of erythrocytes(?15%)and a value of the recovery rate of hemoglobin in erythrocytes(?76%).The TLM had a higher loading efficiency when the hypotonic trehalose concentration was lower,the solution flow rate was smaller,and the hematocrit was lower.The freeze-drying and freeze-thawing preservations of human and swine erythrocytes with loaded trehalose were preli'minarily realized,but the recovery rate of erythrocytes needs to be improved.The TLM can replace labor to automatically load trehalose into erythrocytes.This study not only provides a method of loading trehalose into erythrocytes,but also promotes the automation process of biochips. |
PDF Full Text Request