Freeze-Dried, Viable Allogeneic Heart Valves

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.