Freeze-Dried, Viable Allogeneic Heart Valves

冻干的活同种异体心脏瓣膜

• 批准号: 7051872
• 负责人: Kelvin G.M. Brockbank
• 金额: $ 17.08万
• 依托单位: CELL AND TISSUE SYSTEMS, INC.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2007-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7051872
• 关键词: animal tissue; apoptosis; calcification; cell population study; cryopreservation; cytotoxicity; fibroblasts; heart preservation; heart valve transplantation; heart valves; homologous transplantation; laboratory rat; lyophilization; nonhuman therapy evaluation; smooth muscle; technology /technique development

DESCRIPTION (provided by applicant): Allogeneic human heart valves are used in approximately 20% of the tissue heart valve procedures performed annually. Most allogeneic human heart valves (also known as allografts or homografts) are cryopreserved and stored in the vapor phase of liquid nitrogen storage freezers. During shipping from the preservation facility to the surgical point of use, the heart valves are placed either in a liquid nitrogen dry shipper or in dry ice impregnated with liquid nitrogen. If surgery is not immediate, the heart valves must then be placed back in a vapor phase nitrogen environment, causing undesired thermal cycling and the risk of mechanical damage to the graft. Furthermore, liquid nitrogen storage freezers have been known to fail resulting in the loss of valuable inventory. Cell & Tissue Systems has been investigating alternative storage methods for tissues. Our ultimate goal in this proposal is to scale-up a freeze-drying process to human valves that will provide viable valves at either refrigeration or ambient room temperatures. In this proposal, sheep aortic valve leaflet fibroblasts and aortic conduit smooth muscle cells will be used for optimization of freeze-drying, using a prototype freeze-drying formulation, in cell culture using 96-well plates. Cell viability will be tested by employing florescent live/dead staining techniques, apoptosis markers, and a metabolic assay over periods up to 5-days, in vitro, post-rewarming and rehydration. Progression from cell studies to rat studies will be based upon demonstration of equivalent cell viability in freeze dried experimental and cryopreserved control groups. Subsequently, viable rat aortic heart valves processed by freeze-drying will be compared with two control groups, "conventionally" cryopreserved (frozen) heart valves and fresh heart valves. Freeze-dried heart valves will be compared with control valves by transplantation in rat models of structural deterioration and calcification to determine unanticipated consequences of freeze-drying. Progression from Phase I to Phase II will be based upon results indicating freeze-dried rat valves demonstrate no greater propensity for calcification and structural deterioration than traditional cryopreserved heart valves. Phase II studies will be performed to develop formulations and methods that permit storage at refrigeration or ambient temperatures and scale-up the resulting freeze-drying technology to sheep and human heart valves.

描述（由申请人提供）：每年进行的组织心脏瓣膜手术中大约有 20% 使用同种异体人类心脏瓣膜。大多数同种异体人类心脏瓣膜（也称为同种异体移植物或同种移植物）都被冷冻保存并储存在液氮冷冻柜的气相中。在从保存设施运输到手术使用点的过程中，心脏瓣膜被放置在液氮干燥运输器中或浸有液氮的干冰中。如果不立即进行手术，则必须将心脏瓣膜放回气相氮气环境中，从而导致不希望的热循环以及移植物机械损坏的风险。此外，众所周知，液氮储存冷冻机会发生故障，导致宝贵的库存损失。 Cell & Tissue Systems 一直在研究组织的替代储存方法。我们在该提案中的最终目标是将冷冻干燥工艺扩大到人体瓣膜，从而在冷藏或环境室温下提供可行的瓣膜。在该提案中，绵羊主动脉瓣叶成纤维细胞和主动脉导管平滑肌细胞将用于优化冷冻干燥，使用原型冷冻干燥制剂，在使用 96 孔板的细胞培养中。将通过采用荧光活/死染色技术、细胞凋亡标记物以及长达 5 天的体外、复温和补液后的代谢测定来测试细胞活力。从细胞研究到大鼠研究的进展将基于冻干实验组和冷冻保存对照组中等效细胞活力的证明。随后，将通过冷冻干燥处理的活大鼠主动脉心脏瓣膜与两个对照组（“传统”冷冻保存（冷冻）心脏瓣膜和新鲜心脏瓣膜）进行比较。将通过在结构恶化和钙化的大鼠模型中移植来将冻干心脏瓣膜与控制瓣膜进行比较，以确定冻干的意外后果。从第一阶段到第二阶段的进展将基于以下结果：与传统冷冻保存的心脏瓣膜相比，冻干大鼠瓣膜没有表现出更大的钙化和结构恶化倾向。将进行第二阶段研究，以开发允许在冷藏或环境温度下储存的配方和方法，并将由此产生的冷冻干燥技术扩大到绵羊和人类心脏瓣膜。