目的:玻璃化可以长期保存卵母细胞和胚胎。这是一种技术上具有挑战性的程序,需要在潜在的细胞毒性冷冻保护剂溶液之间直接处理和移动细胞。坚持时间的变化,以及在手术过程中追踪细胞的能力,影响变暖后的生存。我们假设最大限度地减少直接处理将简化程序并提高可追溯性。为了解决这个问题,我们提出了一种新型的光聚合装置,在玻璃化过程中容纳样品。
方法:制造的设备由两个组件组成:Pod和Garage。单个小鼠卵母细胞或胚胎被安置在一个Pod中,多个豆荚停靠在一个车库。通过重复玻璃化和加温循环来评估该装置用于低温应用的适用性。使用标准做法或在Pods和Garage内将卵母细胞或胚泡早期胚胎玻璃化,并与非玻璃化对照组进行比较。变暖后,我们评估了生存率,卵母细胞发育潜力(受精和随后的发育)和代谢(自体荧光)。
结果:装置内的玻璃化发生在~3nL的冷冻保护剂内:该体积比标准玻璃化低~1000倍。与标准做法相比,我们的装置内的玻璃化和变暖显示出生存能力没有差异,发展能力,或卵母细胞和胚胎的新陈代谢。该设备在处理过程中容纳了样品,这提高了可追溯性和最小化处理。有趣的是,玻璃化加热本身,改变卵母细胞和胚胎代谢。
结论:Pod和Garage系统在玻璃化时将冷冻保护剂的体积最小化-提高了〜1000倍-可追溯性并减少了样品的直接处理。这是简化程序的重要步骤。
OBJECTIVE: Vitrification permits long-term banking of oocytes and embryos. It is a technically challenging procedure requiring direct handling and movement of cells between potentially cytotoxic cryoprotectant solutions. Variation in adherence to timing, and ability to trace cells during the procedure, affects survival post-warming. We hypothesized that minimizing direct handling will simplify the procedure and improve traceability. To address this, we present a novel photopolymerized device that houses the sample during vitrification.
METHODS: The fabricated device consisted of two components: the Pod and Garage. Single mouse oocytes or embryos were housed in a Pod, with multiple Pods docked into a Garage. The suitability of the device for cryogenic application was assessed by repeated vitrification and warming cycles. Oocytes or early blastocyst-stage embryos were vitrified either using standard practice or within Pods and a Garage and compared to non-vitrified control groups. Post-warming, we assessed survival rate, oocyte developmental potential (fertilization and subsequent development) and metabolism (autofluorescence).
RESULTS: Vitrification within the device occurred within ~ 3 nL of cryoprotectant: this volume being ~ 1000-fold lower than standard vitrification. Compared to standard practice, vitrification and warming within our device showed no differences in viability, developmental competency, or metabolism for oocytes and embryos. The device housed the sample during processing, which improved traceability and minimized handling. Interestingly, vitrification-warming itself, altered oocyte and embryo metabolism.
CONCLUSIONS: The Pod and Garage system minimized the volume of cryoprotectant at vitrification-by ~ 1000-fold-improved traceability and reduced direct handling of the sample. This is a major step in simplifying the procedure.