Breakthrough Tissue and Organ Preservation and Transplantation Using Scaled-Up Nanowarming Technology

摘要： 将生物材料从玻璃化状态复温是获得成功冷冻保存的关键步骤。 抢救冷冻保存的大宗生物材料和器官的成功技术不仅将提供关键的 对供体器官运输、供应和匹配的改进，但也是潜在供应中缺失的一环 用于工程组织的链条。典型的冷冻过程会通过冰对生物材料造成重大损害 晶体形成和细胞脱水。然而，在低温保护剂(CPA)溶液的帮助下， 生物有机质可以稳定在玻璃体(即“玻璃”或“非晶态”)状态，从而允许长期使用 超低温保存。一些小组已经采用了成功的技术来冷却散装系统，以 玻璃体状态(包括整个兔肾)。重新加热这些玻璃化的生物材料是一项更大的工程 挑战，由于避免分离所必需的临界升温速率(数百oC/分钟)(即 结晶)在解冻期间。此外，温度场中的不均匀会产生热应力， 使脆性材料破裂，因此解冻的速度和均匀性都至关重要。 在这里，我们建议研究射频加热磁性纳米颗粒的能力，或 为了克服这一阻碍大宗冷冻保存进一步发展的主要限制，我们提出了“纳米武器” 接近了。尽管已经尝试了电磁复温，但波与组织的直接耦合 固有地导致加热不均匀，从而导致开裂和差异化生存能力。在较低位置 射频(RF&lt；1 MHz)交变磁场(AMF)可以均匀地穿透组织，而无需 衰减和可忽略的介质耦合。尽管这些较低频场将无法迅速 它们自己加热组织，它们能够通过与磁场耦合产生显著的热量(例如 氧化铁)纳米颗粒。我们已经演示了这种方法能够产生加热率 速度足够快，以避免分离(大于200 oC/分钟)，并应独立于样品大小进行扩展。 这项研究的目的是改进这一新的纳米温控技术，用于低温保存生物 用于移植的组织和完整的器官。为此，在目标1中，我们将扩大纳米颗粒的生产 工艺和射频加热装置的尺寸。在目标2中，我们将优化CPA和纳米颗粒的组成 以及用于细胞和组织(动脉)玻璃化和纳米武器的加载/卸载条件。在《目标3》中我们 将在越来越大和越来越复杂的心脏移植模型中测试这些优化的条件。 总而言之，这项提案的重点将是利用我们突破性的纳米武器技术 在能够玻璃化的放大系统中优化CPA组成和纳米颗粒输送 恢复细胞、动脉和完整的器官，着眼于未来应用于冷冻保存组织和 用于人体移植的器官。

ABSTRACT: Rewarming biomaterials from the vitrified state is a critical step in obtaining successful cryopreservation. Successful techniques for rescuing cryopreserved bulk biomaterials and organs would not only provide critical improvements for donor-organ transport, supply, and matching, but is also a missing link in the potential supply chain for engineered tissues. Typical freezing processes cause significant damage to biomaterials through ice crystal formation and cellular dehydration. However, with the aid of cryoprotectant (CPA) solutions, biospecimens can be stabilized in the vitreous (i.e. “glass” or “amorphous”) state, allowing for long-term cryopreservation. A number of groups have employed successful techniques for cooling bulk systems to the vitreous state (including entire rabbit kidneys). Rewarming these vitrified biomaterials is a greater engineering challenge, due to the critical warming rates (hundreds of oC/min) necessary to avoid devitirification (i.e. crystallization) during thaw. In addition, non-uniformity in temperature field produces thermal stresses that can crack the brittle material, and so both speed and uniformity of thaw are of critical importance. Here we propose to investigate the ability of radiofrequency heated magnetic nanoparticles, or “nanowarming,” to overcome this major limitation hindering further development of bulk cryopreservation approaches. Although electromagnetic rewarming has been tried, the direct coupling of the waves to tissue inherently results in non-uniformity in heating, which leads to cracking and differential viability. At lower radiofrequencies (RF < 1 MHz) alternating magnetic fields (AMFs) can uniformly penetrate tissues without attenuation and negligible dielectric coupling. Although these lower frequency fields will be unable to rapidly heat the tissue on their own, they are ability to produce significant heating through coupling with magnetic (e.g. iron-oxide) nanoparticles. We have already demonstrated that this approach is able to generate heating rates rapid enough to avoid devitirification (greater than 200 oC/min) and should scale independent of sample size. The objective of this study is to refine this novel nanowarming technology for use in cryopreserving biologic tissues and intact organs for transplant. To this end, in Aim 1 we will scale up the nanoparticle production process and the size of the RF heating device. In Aim 2 we will optimize CPA and nanoparticle composition and loading/unloading conditions for vitrification and nanowarming of cells and tissues (arteries). In Aim 3 we will test these optimized conditions in heart transplant models of increasing size and complexity. In summary, the focus of this proposal will be to leverage our breakthrough nanowarming technology by optimizing CPA composition and nanoparticle delivery in a scaled up system capable of vitrifying and recovering cells, arteries, and intact organs with an eye on future application for cryopreserving tissues and organs for use in human transplantation.