Microfluidic Approach For Loading And Unloading Of Cryoprotective Agents

Cryopreservation is an important technology widely used in the fields of cytotherapy,tissue engineering,regenerative medicine,organ transplantation,and assisted reproduction.Cryopreservation involves the inevitable use of cryoprotective agents(CPAs),which might induce osmotic injury,mechanical injury and toxic injury during loading and unloading processes,reducing the cell viability,and even causing the cells to die.This is especially true for vitrification which requires high CPAs concentration.Therefore,it's necessary to investigate the processes of CPA loading and unloading and find out the best strategy for successful cryopreservation.In this study,the researches on dealing with the loading and unloading of CPAs were conducted.The human umbilical vein endothelial cells(HUVECs)were used as cell model for handling loading and unloading processes of small cell suspension.A flow-focusing microfluidic device was designed and fabricated to achieve continuous concentration variation based on the laminar flow-diffusion principal in the microchannel.The concentration profile along the microchannel was verified by using the fluorescent dye solution.Comparative experiments were conducted with conventional one step or multistep method under three different concentrations.The influence of osmotic injury,toxic injury and mechanical injury were analyzed using a theoretical model.The microfluidic device optimizes the processes of CPA loading and unloading,also simplifies the manipulating process.More importantly,cell viability is improved with the micro-device method.Moreover,to deal with the CPA loading and unloading processes for single cell(such as human oocyte),another microfluidic device was designed and fabricated.Computational finite element simulation and fluorescent dye solution were both conducted for characterizing the concentration variation and verifying the effectiveness of the device.Human mature M ? oocytes that failed IVF were used as model cells.The microfluidic device includes oocyte capture part and gradient generation part for measurement of cell membrane parameters and observation of cell volume response under different CPA types and concentrations.The results provide an insight for optimization of CPA loading and unloading processes,and should be helpful for future studies on human oocyte as well.