Abstract：
Effective cryopreservation of large tissues, limbs, and organs has the potential to revolutionize medical post-trauma reconstruction options and organ preservation and transplantation procedures. To date, vitrification and directional freezing are the only viable methods for long-term organ or tissue preservation, but are of limited clinical relevance. This work aimed to develop a vitrification-based approach that will enable the long-term survival and functional recovery of large tissues and limbs following transplantation. The presented novel two-stage cooling process involves rapid specimen cooling to subzero temperatures, followed by gradual cooling to the vitrification solution (VS) and tissue glass transition temperature. Flap cooling and storage were only feasible at temperatures equal to or slightly lower than the VS Tg (i.e., -135°C). Vascularized rat groin flaps and below-the-knee (BTK) hind limb transplants cryopreserved using this approach exhibited long-term survival (>30 days) following transplantation to rats. BTK-limb recovery included hair regrowth, normal peripheral blood flow, and normal skin, fat, and muscle histology. Above all, BTK limbs were reinnervated, enabling rats to sense pain in the cryopreserved limb. These findings provide a strong foundation for the development of a long-term large-tissue, limb and organ preservation protocol for clinical use.
摘要：
大组织的有效冷冻保存，四肢,和器官有可能彻底改变医疗创伤后重建选择以及器官保存和移植程序。迄今为止,玻璃化和定向冷冻是长期保存器官或组织的唯一可行方法，但临床相关性有限。这项工作旨在开发一种基于玻璃化的方法，该方法将使大组织和四肢移植后的长期存活和功能恢复。提出的新颖的两阶段冷却过程涉及快速冷却样品到零度以下的温度，随后逐渐冷却至玻璃化溶液(VS)和组织玻璃化转变温度。襟翼冷却和储存仅在等于或略低于VSTg的温度下可行(即，-135°C)。使用这种方法冷冻保存的血管化的大鼠腹股沟皮瓣和膝盖下（BTK）后肢移植在移植到大鼠后表现出长期存活（>30天）。BTK肢体恢复包括头发再生，正常的外周血流量，正常的皮肤,脂肪，和肌肉组织学。最重要的是,BTK四肢神经得到了恢复,使大鼠能够感觉到冷冻保存的肢体的疼痛。这些发现为长期大组织的发展提供了坚实的基础，临床使用的肢体和器官保存方案。
