On-line Treatment Of Cryoprotectant Based On Membrane Microfiltration

As the only effective long-term preservation method of cells and tissues,cryopreservation plays an important role in modern medicine,especially cryopreservation of red blood cells,stem cells and immune cells has been widely used in clinical treatment.Cryoprotective agents are necessary materials for cryopreservation,but they are toxic and have side effects at room temperature.The treatment process of cryopectant agents before clinical infusion often consumes a lot of time and leads to a large number of cell loss,which affects clinical treatment.This paper explores an "online treatment" model based on microfluidic,i.e.the real-time removal of cryopectant agents from cells during clinical infusion,which is expected to eliminate the adverse effects of existing cryopectant agents treatment mode on cell therapy.The work carried out includes the following aspects:(1)Research on on-line treatment system of cryopectant agents.In order to meet the requirements of high throughput,high removal rate and high cell recovery rate of "online processing",an online processing system based on microfiltration microfluidic was designed and manufactured in this paper.First of all,based on the theoretical analysis of the characteristics of the flow and mass transfer in the system,the structure of groove,membrane window size and window membrane material has carried on the detailed design,big flow resistance prompted system across a cell membrane in the flow resistance inside the groove,so as to realize the one-way flow of infusion fluid to cell suspension,avoid cell block system performance degradation,as a result of film hole;Furthermore,the combined structure of the online treatment system was designed based on the Fixedmolarity-step method,and through the three-dimensional simulation of the flow and mass transfer process of the system,it was confirmed that the design scheme has the ideal performance of serializing and gradient reduction of the concentration of cryopectant agents in the cell suspension side.On this basis,using the improved membrane microfluidic chip processing and assembly process,the production of online processing system is completed.(2)On-line treatment of cryopreservation stem cells.Using the designed online treatment system,the experiment of treating bone marrow mesenchymal stem cells(BMSCs)with cryopectant agents was carried out,and a single step method was used as the experimental control.The results showed that the online treatment system achieved good results under low throughput(0.2 ml/min)and high throughput(1.0 ml/min)inlet conditions: cell survival rate was higher than 81.1%,and count recovery rate was higher than 92.7%.Compared with the single-step method,the online treatment system designed in this paper realizes the gradient removal of cryopectant agents,effectively reduces the cell damage caused by osmotic pressure mutation,and the treatment process is simple and efficient.Morphological observation and CCK detection further showed that there was no significant difference between the functional properties of the cells treated online and those of the fresh stem cells and the control experiment.(3)Research on the optimization method of online processing system.The design variables of on-line processing system are complex,which supports the optimization of on-line processing system under specific conditions.A fast simulation and prediction method for on-line processing system performance is proposed in this paper.Firstly,a quasi-two-dimensional model of the mass transfer process was established based on the principle of finite volume method,and the flow/concentration distribution in the system was quickly solved based on the equivalent circuit forming method and Kirchhoff’s discrete governing equation.Furthermore,based on the obtained flow and concentration distribution,the movement process of cells in the system and the environmental concentration change conditions were predicted,and the cell volume response was calculated by Runge-Kutta algorithm.By comparing the maximum cell volumes corresponding to different system schemes,the optimal design schemes under different inlet conditions can be obtained.In this paper,the "online processing" mode based on microfluidic was studied,and the treatment experiment of cryopectant agents for BMSCs was carried out.By developing the on-line treatment system of cryopectant agents,the problems such as long time consumption,small treatment flux,low cell survival rate and incomplete removal of cryopectant agents have been solved.The results showed that the online treatment system could efficiently remove cryopectant agents from cells under the condition of high throughput,and the cell recovery rate was also better than that of the traditional methods,which provided an effective solution to the problems faced by the current treatment of cryopectant agents.