Microfluidic chips can be used to realize continuous cryoprotectants (CPA) loading/unloading for oocytes, reducing osmotic damage and chemical toxicity of CPA. In this study, five different Y-shape microfluidic chips were fabricated to realize the continuous CPA loading/unloading. The effects of flow rate, entrance angle, aspect ratio and turning radius of microchannels on the mixing efficiency of microfluidic chips were analyzed quantitatively. The experimental results showed that with the decrease of flow rates, the increase of aspect ratios and the decrease of turning raradius of microchannel, the mixing length decreased and the mixing velocity was promoted, while the entrance angle had little effect on the mixing efficiency. However, the operating conditions and structural parameters of the chips in practical application should be determined based on an overall consideration of CPA loading/unloading time and machining accuracy. These results would provide a reference to the application of microfluidic chip in CPA mixing.
In order to reduce osmotic damage and chemical toxicity of cryoprotectants (CPA) to oocytes during unloading process, the microfluidic chip was used to remove CPA from porcine MⅡ oocytes in this study. Firstly, the effects of unloading time, composition and concentration of diluting solutions of microfluidic method on survival rate and developmental capacity of oocytes were studied, then microfluidic method was compared with traditional one-step and two-step CPA unloading protocols. The results showed that when the total time is 8 minutes, the survival rate and morula rate of oocytes treated with microfluidic method could achieve 95.99% ± 4.64% and 74.17% ± 1.18%, respectively, which were not significantly different from fresh control group (98.53% ± 2.94%; 78.22% ± 1.34%). In addition, 1 mol/L sucrose diluting solutions were more beneficial than other solutions, and it was also showed that microfluidic method achieved better survival, cleavage rate of oocytes than traditional methods. Microfluidic CPA removal protocol can reduce the damage to oocytes during unloading process, and may further improve the cryopreservation effect of oocytes.