Nikta Ziaie - Embryo Biotechnology Laboratory (EmBio Lab), Department of Animal Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran
Sina Azizmohseni - Embryo Biotechnology Laboratory (EmBio Lab), Department of Animal Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran
Mojtaba Dashtizad - Embryo Biotechnology Laboratory (EmBio Lab), Department of Animal Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran

Embryo cryopreservation is a fundamental and widely used method in the field of assisted reproductive techniques. The way of loading/unloading of cryoprotectant agents, which require to be applied for dehydrating the embryo and consequently reduction the risk of ice crystal formation, has still remained as a matter of concern. In the conventional cryoprocedure, embryos are sequentially transferred from an isotonic solution into a highly concentrated solution of CPAs through a few discrete steps which can be a major source of osmotic shock and mechanical injuries. Thus, providing a platform to gradual increase the concentration of CPAs can effectively reduce the osmotic shock. Therefore, to achieve this, we designed and fabricated a microfluidic device to generate CPAs concentration gradient over time. Our design allows to inject the initial hypertonic CPAs solution into the inlet by using the controllable syringe pumps, and achieve four different final CPAs concentration (۶, ۹, ۱۲, ۱۵%) into the embryo chambers throughout time. Afterward, the accuracy of the concentration gradient of CPAs on the fabricated chip were examined in ۱۰ minutes by using image processing. The results showed that ۱۵%, ۱۲%, ۹% and ۶% assigned chambers reached acceptable concentrations of ۱۴.۵%, ۱۱.۹%, ۹% and ۵.۲% after ۱۰ minutes, respectively. To compare the efficacy of our device and the conventional procedure, we studied the volume changes and shrinkage rate of embryos. Our finding confirmed that these parameters significantly improve in those embryos that expose to on-chip CPAs loading. In conclusion, our developed microfluidic chip can effectively reduce osmotic shock and the concequent damages, which may provide a new path for vitrification of embryos in microfluidic systems by determining the optimum time/concentration point in the microfluidic system.

Osmotic Shock, Microfluidic Chip, Cryoprotectant agents, mouse embryo, Vitrification