Ice-free Cryopreservation of Heart Valves

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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.