Tirgar Pouria, Sarmadi Fatemeh, Najafi Mojgan, Kazemi Parinaz, AzizMohseni Sina, Fayazi Samaneh, Zandi Ghazaleh, Ziaie Nikta, Shoushtari Zadeh Naseri Aida, Ehrlicher Allen, Dashtizad Mojtaba
Embryo Biotechnology Laboratory (EmBio Lab), Department of Animal Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran 1497716316, Iran.
Department of Bioengineering, McGill University, Montreal, Quebec H3A0B9, Canada.
Biomicrofluidics. 2021 May 18;15(3):034104. doi: 10.1063/5.0047185. eCollection 2021 May.
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.
胚胎玻璃化是辅助生殖和生育力保存中的一项基本操作。该过程的一个关键步骤是通过将胚胎从等渗溶液转移到高渗的冷冻保护剂（CPA）溶液中，用冷冻保护剂替代胚胎内部的水分。然而，这会给胚胎带来突然的渗透压冲击，导致分子损伤，长期以来一直令人担忧。在本研究中，我们引入了一个独立的微流控系统，以自动化手动操作过程，并将施加于胚胎的渗透压冲击降至最低。该装置提供与手动方法相同的最终CPA浓度，但随着时间的推移逐渐增加，而不是突然增加。我们的系统允许从CPA-水交换开始时引入脱水非渗透性CPA（蔗糖），这反过来减少了成功玻璃化所需的CPA加载时间，同时不影响其效果。我们通过研究玻璃化-解冻后的小鼠囊胚的再扩张率、孵化率以及参与内质网应激、氧化应激、热休克和细胞凋亡的选定基因的转录模式，比较了我们的装置与传统手动操作程序的效果。虽然两组胚胎的再扩张率和孵化率相当，但芯片上加载减少了冷冻保存的有害基因表达。这里开发的装置使我们能够自动化CPA加载过程，并通过最小化渗透压应激、缩短整个过程以及减少其分子影响来突破冷冻保存的界限。
