Facile and highly efficient loading and freezing of cryoprotectants for oocyte vitrification based on planar microfluidics

Vitrification is widely used in assisted reproduction technology via cell cryopreservation, which requires precise washing sequences and timings using several cryoprotectants (CPAs) to alleviate their osmotic shock. Compare with embryos, oocytes are more susceptible to osmotic stress and chemical toxicity and hence require more precise and complicated manual operations. In this paper, an automatic prototype system is established based on a novel planar microfluidic chip featuring superhydrophobic surfaces and poly-D-Lysine coating, which enables a single oocyte to be easily settled inside a ball-like droplet to facilitate the CPA exchange. Using the automatic equipment, the droplet concentration can be smoothly changed while maintaining a stable spherical shape. After the loading and removal of CPAs, the planar chip with an oocyte can be directly placed into liquid nitrogen for cryopreservation with similar oocyte survival rate as that obtained by manual operation. Compared with manual operation or other channel-based microfluidic technologies, this planar microfluidic method can avoid cell loss and the inconvenience of cell transfer and cryopreservation, which indicates its immense potential to serve as a facile and universal automatic solution for the vitrification of various precious cells.