S015 FREEZE-DRIED DECELLULARIZED HEART VALVES FOR HEART VALVE REPLACEMENT W. Wolkers. Leibniz University Hannover, Hannover, Germany Decellularized heart valves can be used for heart valve replacement ther- apies. If heart valves could be freeze-dried without damaging the tissue structure, this would allow long-term storage at room temperature. We have used sucrose as lyoprotectant for freeze-drying of decellularized heart valves. Diffusion studies were done using Fourier transform infrared spectroscopy (FTIR) in order to estimate incubation times needed to infiltrate heart valves with sucrose. It was estimated that 4-hour incuba- tion at 37  C is sufficient to infiltrate heart valves with sucrose. Sucrose protects decellularized heart valves against damage caused by freeze- drying. Ice crystal damage was found to be one of the main damaging events during freeze-drying resulting in pores in the tissue after rehy- dration. The extent of pore formation in rehydrated tissue decreased with increasing sucrose concentration in the freeze-drying formulation. High sucrose concentrations were needed to minimize ice crystal damage. Exposure to an 80% sucrose solution prior to freeze-drying almost completely diminished pore formation and the matrix histoarchitecture of rehydrated tissue closely resembled that of control tissue not subjected to freeze-drying. The protein denaturation profile of rehydrated tissue was found to be nearly identical compared to that of control tissue. The overall protein secondary structure, studied by FTIR, also showed that tissue proteins were little affected by the freeze-drying process. In vivo tests with freeze-dried pulmonary heart valves (ovine as well as porcine origin) in sheep demonstrated the efficacy of freeze-dried valves. Freeze-dried valves showed similar durability and repopulation with cells compared to control valves not exposed to freeze-drying. Source of funding: DFG: Cluster of Excellence REBIRTH “From regenera- tive biology to reconstructive therapy” S016 ICE-FREE CRYOPRESERVATION OF NATURAL AND ENGINEERED TISSUES K.G.M. Brockbank. Tissue Testing Technologies LLC, Charleston, South Carolina, United States Natural and tissue engineered allogeneic tissues potentially have a huge impact on orthopedic, wound care, urinary, cardiac, and vascular surgery applications. Preserved tissues using traditional freezing methods of cryopreservation are subject to ice damage. We have developed methods for preservation using ice-free vitrification. The original vitrification pro- tocol has evolved over time. Ice-free cryopreservation using VS55 with multistep addition/removal steps and storage below -135  C is limited to small samples where relatively rapid cooling and warming rates are possible. This method typically provides extracellular matrix preservation with at least 80% cell viability and tissue function compared with fresh untreated tissues. It has proven effective on natural cardiovascular tissue samples and cartilage, and a variety of engineered tissues including blood vessel and epithelial tissue constructs. In contrast, preservation of larger samples with retention of cell viability has been limited due to cryopro- tectant cytotoxicity, inability to rewarm fast enough to avoid recrystalli- zation, and stresses that occur during vitrification. Initially, for large tissues where the focus is preservation of extracellular matrix without cell viability, we employed high concentration, ±83%, cryoprotectant formu- lations. Since cell viability is not retained these methods employ simpler addition and removal protocols with long term storage at -80  C. In vitro experiments indicate that ice-free cryopreservation impacts the innate immune response reducing T-cell proliferation and release of cytokines from peripheral blood mononuclear cells. In vivo functions are also improved. The field is returning to development of methods for viable large tissues. This is due to new technologies, including analytical methods such as cryomacroscopy, micro-computed tomography imaging and mi- cro-magnetic resonance imaging, as well as innovative rewarming methods such as “nanowarming” where low radiofrequency alternating magnetic fields and Fe nanoparticles are used to warm rapidly. These technologies promise to facilitate the development of methods for reten- tion of function in large tissues and organs. Conflict of interest: Kelvin Brockbank is an employee and owner of Tissue Testing Technologies LLC. Source of funding: The National Institutes of Health grants 5R44GM106732, 1R43HL120404, and 1R43AI114486; and the US Army Medical Research and Materiel Command under Contract W81XWH-15-C- 0173. The views, opinions, and/or findings contained in this report are those of the author and should not be construed as an official National Institutes of Health or Department of the Army position, policy, or decision unless so designated by other documentation. S017 QUALITY AND IMMUNOGENICITY OF SKIN TISSUE ALLOGRAFTS FOR TRANSPLANT H. Manhani 1 , A. Halpin 1 , L. Hidalgo 1 , B. Motyka 1 , J. Pearcey 1 , L. West 1 , T. Mokoena 2 , K. Worton 2 , M. Bentley 2 , G. Dowling 2 , J. Holovati 1,2, * . 1 University of Alberta, Edmonton, Alberta, Canada; 2 Alberta Health Services, Edmonton, Alberta, Canada * Corresponding author. University of Alberta, Edmonton, Alberta, Canada. Skin allografts are currently recovered, processed, and distributed by the Comprehensive Tissue Centre (CTC) for transplant applications. The aim of this study was to evaluate the effects of refrigeration, cryopreservation, long-term storage, and glutaraldehyde treatment on structural integrity, cellular viability and alloimmunogenic antigen expression of skin allo- grafts distributed by CTC for transplant applications. Cadaveric skin tissue was recovered according to CTC standard operating procedures. After 14 days of refrigerated storage, skin allografts were cryopreserved in 10% dimethyl sulfoxide and stored in liquid nitrogen for up to 7 years. Glutaraldehyde (0.1%) treatment was either applied pre-freeze or post- thaw. Allograft structural integrity was assessed by H&E staining and brightfield microscopy. The viability of allografts was assessed by mito- chondrial cell viability assay (MTT). The immunogenicity potential of skin allografts was assessed by immunohistochemistry (IHC) staining for ABH and HLA Class I antigenic expression. Histological assessment of skin al- lografts revealed no statistically significant difference in microanatomy quality scores of refrigerated, cryopreserved, and glutaraldehyde treated skin allografts. Cellular viability of skin allografts significantly decreases during refrigerated storage (p<0.001), while the cryopreservation process maintains the pre-freeze cell viability levels. The length of liquid nitrogen storage was not a statistically significant predictor of post-thaw skin viability. IHC revealed that ABOeA or B, and HLA class I antigens are expressed in all experimental groups of skin allografts. There was no sta- tistically significant effect of glutaraldehyde treatment on skin structural quality, viability or immunogenicity. CTC skin processing procedures maintain allograft structural integrity, for both refrigerated and cry- opreserved tissue allografts. The length of hypothermic storage prior to freezing is an important parameter to consider for maintaining tissue viability properties. The length of cryopreservation storage for up to 7 years in liquid nitrogen temperatures does not negatively influence skin structural integrity or viability. Source of funding: Alberta Health Services S018 INITIATION OF INTRACELLULAR ICE FORMATION DURING FREEZING OF MICROPATTERNED ENDOTHELIAL TISSUE S.E. Harhen, J.O.M. Karlsson * . Department of Mechanical Engineering, Villanova University, Villanova, Pennsylvania, United States * Corresponding author. Previous models of intracellular ice formation (IIF) in monoculture tissue have assumed that all cells within an unfrozen tissue construct are subject to the same mechanisms of IIF initiation (such as the recently discovered IIF initiation mechanism associated with the paracellular ice penetration phenomenon), implying that the probability of IIF is the same in every cell within an unfrozen construct, and independent of tissue scale. If this assumption is valid, then it is possible to predict IIF kinetics in macroscale tissue using scaling laws to extrapolate measurements from microscale Abstracts / Cryobiology 73 (2016) 399e443 403