Ice-free Cryopreservation of Heart Valves

人类心脏瓣膜（同种或同种异体移植）是几乎完美的心脏瓣膜替代品。它们具有最佳的血流动力学特性和抗感染能力。特别是在儿科患者的缺点是有限的可用性，无法生长，退化和长期失败。后者的潜在罪魁祸首包括免疫反应和钙代谢增加。同种异体心脏瓣膜的首次临床应用是作为同种生命器官，在抗生素孵育后不作任何保存而移植。自1968年以来，标准冷冻冷冻保存（SFC）已被采用，其中包括在气相液氮中储存。多光子成像分析（MIA）的应用使弹性蛋白和胶原蛋白在新鲜组织中无需固定、包埋和染色的情况下通过诱导自身荧光实现三维（3D）可视化。MIA首次检测到SFC猪瓣膜的弹性和胶原基质的部分破坏。由于胶原蛋白和弹性蛋白的总量保持不变，上述破坏的病因被假设为冰晶引起的冷冻诱导的细胞外基质（ECM）损伤。在低温保存过程中发生的破坏性间隙冰损伤可以通过促进玻璃化、形成玻璃来避免，而不是在低温保存溶液的玻璃化转变温度下形成冰。由于保存和加热在没有任何组织破坏的情况下完成，玻璃化首次实现了心脏瓣膜细胞外基质的完整保存。为了简化所需的基础设施，我们进一步开发了无冰冷冻保存（IFC），它允许在-80℃的冰柜中储存高于玻璃化转变温度的溶液。我们在最近的一项绵羊研究中应用了IFC，并能够证明维持ECM可以改善瓣膜功能和减少变性。此外，IFC具有抗炎作用，可阻止CD3+ T细胞介导的反应。这种减弱的适应性免疫反应和纤维化的确切机制尚不清楚。最近对猪组织/人反应性外周血白细胞组合的研究表明，用IFC保存培养基处理瓣膜组织可降低小叶免疫原性，并诱导反应性单核细胞向内皮样细胞分化。因此，拟制定以下中长期目标：ECM的最佳保存与改善同种异体心脏瓣膜的长期血流动力学功能IFC在异种心脏瓣膜中的应用，克服器官稀缺性。3. 3 .省去了速冻、液氮设备和储藏，简化了保存过程。提高成本效益，以便在财政资源有限的第三世界和发展中国家应用。

Human heart valves (homo- or allografts) represent nearly perfect heart valve substitutes. They have optimal hemodynamic characteristics and are resistant to infections. Disadvantages particularly in pediatric patients are limited availability, an inability to grow, degeneration and long-term failure. Potential culprits for the latter include immunological responses and an increased calcium metabolism. The first clinical use of allograft heart valves was as homovitals that were transplanted after antibiotic incubation without any preservation. Since 1968 standard frozen cryopreservation (SFC) has been employed which includes storage in vapour phase liquid nitrogen. Application of multiphoton imaging analysis (MIA) enables three-dimensional (3D) visualization of elastin and collagen by induction of autofluorescence without fixation, embedding and staining in fresh tissue. MIA allowed for the first time detection of partial destruction of elastic and collagenous matrix in SFC porcine valves ex situ. As the overall amount of collagen and elastin remains unchanged the etiology of the above described destruction is postulated as freezing induced extracellular matrix (ECM) damage due to ice crystals. Disruptive interstitial ice damage that occurs during cryopreservation can be avoided by promoting vitrification, glass formation, instead of ice formation below the cryopreservation solutions glass transition temperature. As the preservation and warming is accomplished without any tissue destruction, vitrification enabled for the first time complete preservation of extracellular matrix in heart valves. In order to simplify the required infrastructure, we have further developed ice free cryopreservation (IFC), which permits storage above the solutions glass transition temperature in a -80 C freezer. We applied IFC in a recent sheep study and were able to demonstrate that maintaining ECM results in better valve function and diminished degeneration. Furthermore, IFC is anti-inflammatory preventing CD3+ T cell mediated responses. The exact mechanism of this attenuated adaptive immune response and fibrosis remains unknown. Recent studies in a porcine tissue/human responder peripheral blood leukocyte combination have shown that treatment of valve tissue with IFC preservation medium reduces leaflet immunogenicity and induces responder monocyte differentiation towards endothelial like cells. In consequence the following mid to long-term goals are intended:1. Optimal preservation of ECM with resulting improved hemodynamic long-term function of allograft heart valves 2. Application of IFC for xenogeneic heart valves to overcome organ scarcity. 3. Simplification of preservation process by omitting controlled rate freezing as well as liquid nitrogen equipment and storage.4. Improving cost effectiveness to allow application in third world and developing countries with limited financial resources.