Embryo vitrification is a fundamental practice in assisted reproduction and fertility preservation. A key step of this process is replacing the internal water with cryoprotectants (CPAs) by transferring embryos from an isotonic to a hypertonic solution of CPAs. However, this applies an abrupt osmotic shock to embryos, resulting in molecular damages that have long been a source of concern. In this study, we introduce a standalone microfluidic system to automate the manual process and minimize the osmotic shock applied to embryos. This device provides the same final CPA concentrations as the manual method but with a gradual increase over time instead of sudden increases. Our system allows the introduction of the dehydrating non-permeating CPA, sucrose, from the onset of CPA-water exchange, which in turn reduced the required time of CPA loading for successful vitrification without compromising its outcomes. We compared the efficacy of our device and the conventional manual procedure by studying vitrified–warmed mouse blastocysts based on their re-expansion and hatching rates and transcription pattern of selected genes involved in endoplasmic reticulum stress, oxidative stress, heat shock, and apoptosis. While both groups of embryos showed comparable re-expansion and hatching rates, on-chip loading reduced the detrimental gene expression of cryopreservation. The device developed here allowed us to automate the CPA loading process and push the boundaries of cryopreservation by minimizing its osmotic stress, shortening the overall process, and reducing its molecular footprint.

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