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Human heart valve allografts continue to represent almost perfect substitutes for heart valves. They have optimal hemodynamic characteristics and are highly resistant to infections. The first clinical use of allograft heart valves was as homovitals being transplanted after antibiotic incubation without any preservation. Since 1968, relatively standardized frozen cryopreservation (SFC) has been employed, including storage in vapor-phase liquid nitrogen. Disadvantages, particularly in pediatric patients, are limited availability due to organ scarcity, inability to grow, degeneration, immune response, and long-term failure. However, in contrast to alternative prosthetic or bioprosthetic heart valve replacements, they represent the best pediatric and juvenile replacement options for the pulmonary valve. Application of multiphoton imaging analysis for three-dimensional visualization of elastin and collagen by induction of autofluorescence without chemical fixation, embedding, and staining has revealed partial destruction of elastic and collagenous matrix in SFC valves. As the overall amount of collagen and elastin remains unchanged, the observed destruction is attributed to freezing-induced extracellular matrix damages due to ice crystal formation during SFC. The objective of this review is an assessment of current allograft preservation methods and the potential of novel preservation techniques to avoid ice formation with accompanied better long-term function.

Vitrification, a kinetic process of liquid solidification into glass, poses many potential benefits for tissue cryopreservation including indefinite storage, banking, and facilitation of tissue matching for transplantation. To date, however, successful rewarming of tissues vitrified in VS55, a cryoprotectant solution, can only be achieved by convective warming of small volumes on the order of 1 ml. Successful rewarming requires both uniform and fast rates to reduce thermal mechanical stress and cracks, and to prevent rewarming phase crystallization. We present a scalable nanowarming technology for 1- to 80-ml samples using radiofrequency-excited mesoporous silica-coated iron oxide nanoparticles in VS55. Advanced imaging including sweep imaging with Fourier transform and microcomputed tomography was used to verify loading and unloading of VS55 and nanoparticles and successful vitrification of porcine arteries. Nanowarming was then used to demonstrate uniform and rapid rewarming at >130°C/min in both physical (1 to 80 ml) and biological systems including human dermal fibroblast cells, porcine arteries and porcine aortic heart valve leaflet tissues (1 to 50 ml). Nanowarming yielded viability that matched control and/or exceeded gold standard convective warming in 1- to 50-ml systems, and improved viability compared to slow-warmed (crystallized) samples. Last, biomechanical testing displayed no significant biomechanical property changes in blood vessel length or elastic modulus after nanowarming compared to untreated fresh control porcine arteries. In aggregate, these results demonstrate new physical and biological evidence that nanowarming can improve the outcome of vitrified cryogenic storage of tissues in larger sample volumes., (Copyright © 2017, American Association for the Advancement of Science.)

In regard to evaluating tissue banking methods used to preserve or otherwise treat (process) soft allograft tissue, current tests may not be sufficiently sensitive to detect potential damage inflicted before, during, and after processing. Using controlled parameters, we aim to examine the sensitivity of specific biomechanical, electrical, and biological tests in detecting mild damage to collagen. Fresh porcine pulmonary heart valves were treated with an enzyme, collagenase, and incubated using various times. Controls received no incubation. All valves were cryopreserved and stored at -135 °C until being rewarmed for evaluation using biomechanical, permeability, and cell viability tests. Statistically significant time dependent changes in leaflet ultimate stress, (p = 0.006), permeability (p = 0.01), and viability (p ≤ 0.02, four different days of culture) were found between heart valves subjected to 0-15 min of collagenase treatment (ANOVA). However, no statistical significance was found between the tensile modulus of treated and untreated valves (p = 0.07). Furthermore, the trends of decreasing and increasing ultimate stress and viability, respectively, were somewhat inconsistent across treatment times. These results suggest that permeability tests may offer a sensitive, quantitative assay to complement traditional biomechanical and viability tests in evaluating processing methods used for soft tissue allografts, or when making changes to current validated methods. Multiple test evaluation may also offer insight into the mechanism of potential tissue damage such as, as is the case here, reduced collagen content and increased tissue porosity., Competing Interests: KGMB is an owner and employee of Tissue Testing Technologies, ZC, EDG, and LHC are employees of Tissue Testing Technologies. None of the other authors of this paper have any potential conflicts of interest that might be construed as affecting the conduct or reporting of the work presented.

Insulin-dependent diabetes mellitus is one of the leading causes of death world wide. Donor-derived pancreas and islet of Langerhans transplantation are potential cures, however, postmortem ischemia impacts islet quality. The murine βt3 cell line was used as a model to study apoptosis after hypothermic storage by comparing Unisol™ with Belzer's machine perfusion solution (BMPS) and the University of Wisconsin (UW) solution. The objective was to determine which of these solutions provided the best support for βt3 cells and which solution demonstrated the least amount of apoptotic activity. Several apoptosis markers were measured that included the translocation of phosphatidylserine, caspase activity, and the formation of DNA laddering. In addition, metabolic activity and membrane integrity were also measured. The results demonstrated that the three solutions behaved similarly during overnight cold storage at 4°C. However, Unisol was consistently better than UW solution and BMPS, demonstrating better cell viability and recovery, and lower levels of apoptotic activity when all three parameters were measured. These results demonstrated that apoptosis plays an important role in the survival of cells and tissues during cold storage. Development of solutions to help prevent or decrease the levels of apoptosis after cold storage will likely improve overall cell and tissue recovery and survival in a clinical setting.

The first Organ Banking Summit was convened from Feb. 27 - March 1, 2015 in Palo Alto, CA, with events at Stanford University, NASA Research Park, and Lawrence Berkeley National Labs. Experts at the summit outlined the potential public health impact of organ banking, discussed the major remaining scientific challenges that need to be overcome in order to bank organs, and identified key opportunities to accelerate progress toward this goal. Many areas of public health could be revolutionized by the banking of organs and other complex tissues, including transplantation, oncofertility, tissue engineering, trauma medicine and emergency preparedness, basic biomedical research and drug discovery - and even space travel. Key remaining scientific sub-challenges were discussed including ice nucleation and growth, cryoprotectant and osmotic toxicities, chilling injury, thermo-mechanical stress, the need for rapid and uniform rewarming, and ischemia/reperfusion injury. A variety of opportunities to overcome these challenge areas were discussed, i.e. preconditioning for enhanced stress tolerance, nanoparticle rewarming, cyroprotectant screening strategies, and the use of cryoprotectant cocktails including ice binding agents., (Copyright © 2015.)

Background: Undesirable processes of inflammation, calcification, or immune-mediated reactions are limiting factors in long-term survival of heart valves in patients. In this study, we target the modulatory effects of ice-free cryopreservation (IFC) of xenogeneic heart valve leaflet matrices, without decellularization, on the adaptive human immune responses in vitro., Methods: We tested porcine leaflet matrices from fresh untreated, conventionally cryopreserved (CFC), and IFC pulmonary valves by culturing them with human blood mononuclear cells for 5 d in vitro. No other tissue treatment protocols to modify possible immune responses were used. Matrices alone or in addition with a low-dose second stimulus were analyzed for induction of proliferation and cytokine release by flow cytometry-based techniques. Evaluation of the α-Gal epitope expression was performed by immunohistochemistry with fluorochrome-labeled B4 isolectin., Results: None of the tested leaflet treatment groups directly triggered the proliferation of immune cells. But when tested in combination with a second trigger by anti-CD3, IFC valves showed significantly reduced proliferation of T cells, especially effector memory T cells, in comparison with fresh or CFC tissue. Moreover, the cytokine levels for interferon-γ (IFNγ), tumor necrosis factor α, and interleukin-10 were reduced for the IFC-treated group being significantly different compared with the CFC group. However, no difference between treatment groups in the expression of the α-Gal antigen was observed., Conclusions: IFC of xenogeneic tissue might be an appropriate treatment method or processing step to prevent responses of the adaptive immune system., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Application of the original vitrification protocol used for pieces of heart valves to intact heart valves has evolved over time. Ice-free cryopreservation by Protocol 1 using VS55 is limited to small samples where relatively rapid cooling and warming rates are possible. VS55 cryopreservation typically provides extracellular matrix preservation with approximately 80 % cell viability and tissue function compared with fresh untreated tissues. In contrast, ice-free cryopreservation using VS83, Protocols 2 and 3, has several advantages over conventional cryopreservation methods and VS55 preservation, including long-term preservation capability at -80 °C; better matrix preservation than freezing with retention of material properties; very low cell viability, reducing the risks of an immune reaction in vivo; reduced risks of microbial contamination associated with use of liquid nitrogen; improved in vivo functions; no significant recipient allogeneic immune response; simplified manufacturing process; increased operator safety because liquid nitrogen is not used; and reduced manufacturing costs.

Expanding cryopreservation methods to include a wider range of cell types, such as those sensitive to freezing, is needed for maintaining the viability of cell-based regenerative medicine products. Conventional cryopreservation protocols, which include use of cryoprotectants such as dimethylsulfoxide (Me2SO), have not prevented ice-induced damage to cell and tissue matrices during freezing. A family of antifreeze proteins (AFPs) produced in the larvae of the beetle, Dendroides canadensis allow this insect to survive subzero temperatures as low as -26°C. This study is an assessment of the effect of the four hemolymph D. canadensis AFPs (DAFPs) on the supercooling (nucleating) temperature, ice structure patterns and viability of the A10 cell line derived from the thoracic aorta of embryonic rat. Cryoprotectant solution cocktails containing combinations of DAFPs in concentrations ranging from 0 to 3mg/mL in Unisol base mixed with 1M Me2SO were first evaluated by cryomicroscopy. Combining multiple DAFPs demonstrated significant supercooling point depressing activity (∼9°C) when compared to single DAFPs and/or conventional 1M Me2SO control solutions. Concentrations of DAFPs as low as 1 μg/mL were sufficient to trigger this effect. In addition, significantly improved A10 smooth muscle cell viability was observed in cryopreservation experiments with low DAFP-6 and DAFP-2 concentrations in combination with Me2SO. No significant improvement in viability was observed with either DAFP-1 or DAFP-4. Low and effective DAFP concentrations are advantageous because they minimize concerns regarding cell cytotoxicity and manufacturing cost. These findings support the potential of incorporating DAFPs in solutions used to cryopreserve cells and tissues., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Various preservation solutions have been evaluated for longer hypothermic cartilage storage for tissue transplantation; however, the results are mixed. This research was carried out to determine whether phosphate-buffered saline (PBS) or organ preservation solutions would preserve both the extracellular matrix and chondrocytes of articular cartilage better than culture medium during refrigerated storage in the time frame that cartilage is stored for clinical use. Porcine cartilage plugs were stored, without the underlying bone, in culture medium with and without fetal bovine serum (FBS), PBS, Belzer's and Unisol solutions for 1 month at 4°C. Metabolic activity was tested using a resazurin reduction method, and matrix permeability was evaluated by measuring electrical conductivity. Storage in culture medium with 10% FBS was shown to provide good cartilage metabolic function for 7 days, decreasing to about 36% after 1 month of storage. There was no significant difference between samples stored in culture medium with and without FBS after 1 month of storage (p = 0.5005). Refrigerated storage of cartilage in PBS and two different solutions (Belzer's and Unisol) designed for optimal refrigerated tissue and organ storage results in loss of chondrocyte function and retention of matrix permeability. In contrast, the opposite, namely significantly better retention of chondrocyte function and loss of matrix permeability, was observed with culture medium. Future research should be focused on combining retention of chondrocyte function and matrix permeability by storage solution formulation.

The aim of the present study was to characterize the hemocompatibility of ice-free cryopreserved heart valves in anticipation of future human trials. Porcine pulmonary heart valves were infiltrated with either an 83 % cryoprotectant solution followed by rapid cooling and storage at --80 °C or with 10 % DMSO and control rate freezing to --80 °C and storage in vapor phase nitrogen as conventional frozen controls. Cryopreserved leaflets were compared with fresh, decellularized and glutaraldehyde-fixed control valve leaflets using a battery of coagulation protein assays after exposure to human blood. Von Willebrand Factor staining indicated that most of the endothelium was lost during valve processing prior to cryopreservation. Hemocompatibility, employing thrombin/antithrombin-III-complex, polymorphonuclear neutrophil-elastase, beta-thromboglobulin and terminal complement complex SC5b-9, was preserved compared with both fresh and frozen leaflets. Hemocompatibility differences were observed for cryopreserved leaflets versus both decellularized and glutaraldehyde fixed controls. In conclusion, the hemocompatibility results support the use of ice-free cryopreservation as a simplified preservation method because no statistically significant differences in hemocompatibility were observed between the two cryopreservation methods and fresh untreated controls.

Investigations involving rare human tissues that are difficult to acquire due to their scarcity are highly challenging. The need to verify microarray analysis data by additional methods such as immunohistochemical staining and quantitative PCR creates an even greater demand for these valuable tissues. Furthermore, since rare human tissues may come from different sources and may have been processed by variable methods, the comparability of these samples must be verified. The aim of this study was to determine and validate a processing method that allows the analysis of human fetal and adult cardiovascular tissues from different sources that were preserved using varying methods. Due to restricted access to fresh human tissues and the need to accumulate these samples over an extended period of time, we used formalin-fixed paraffin-embedded tissues for gene expression analyses. We analyzed RNA levels from four different age groups: fetal first and second trimester, adolescents, and adults. In this study, we present an improved standard processing procedure for tissue sample processing and analysis of rare human cardiovascular tissues., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Insulin-dependent diabetes mellitus is one of the leading causes of death world-wide. Donor-derived pancreas and Islet of Langerhans transplantation are potential cures; however, postmortem ischemia impacts islet quality. The murine βt3 cell line was employed as a model to study cell viability and proliferation after hypothermic storage by comparing Belzer's Machine Perfusion Solution with Unisol™ Solution. The objective was to determine which of these solutions provided the best base line support for βt3 cells and to screen potential cytoprotective additives to the solutions. Initial βt3 cell viability was similar in the two storage solutions; however, better proliferation was observed after storage in Unisol Solution. The caspase inhibitor, Q-VD-OPH, and α-tocopherol improved viability in both storage solutions, suggesting that apoptotic pathways may be responsible for cell death during hypothermic storage of βt3 cells. Analysis of apoptosis markers, caspase activity, and DNA laddering showed a reduction in apoptosis when these additives were included. The effects of Q-VD-OPH and α-tocopherol were also synergistic when employed together during either hypothermic exposure, post-hypothermic physiologic incubation, or combinations of hypothermic exposure and physiologic incubation. These results suggest that both supplements should be included in pancreas hypothermic storage solutions and in islet culture media during post-isolation culture prior to transplantation.

The purpose of this study was evaluation of an ice-free cryopreservation method for heart valves in an allogeneic juvenile pulmonary sheep implant model and comparison with traditionally frozen cryopreserved valves. Hearts of 15 crossbred Whiteface sheep were procured in Minnesota. The valves were processed in South Carolina and the pulmonary valves implanted orthotopically in 12 black faced Heidschnucke sheep in Germany. The ice-free cryopreserved valves were cryopreserved in 12.6 mol/l cryoprotectant (4.65, 4.65, and 3.31 mol/l of dimethylsulfoxide, formamide and 1,2-propanediol) and stored at -80°C. Frozen valves were cryopreserved by controlled slow rate freezing in 1.4 mol/l dimethylsulfoxide and stored in vapor-phase nitrogen. Aortic valve tissues were used to evaluate the impact of preservation without implantation. Multiphoton microscopy revealed reduced but not significantly damaged extracellular matrix before implantation in frozen valves compared with ice-free tissues. Viability assessment revealed significantly less metabolic activity in the ice-free valve leaflets and artery samples compared with frozen tissues (P < 0.05). After 3 and 6 months in vivo valve function was determined by two-dimensional echo-Doppler and at 7 months the valves were explanted. Severe valvular stenosis with right heart failure was observed in recipients of frozen valves, the echo data revealed increased velocity and pressure gradients compared to ice-free valve recipients (P = 0.0403, P = 0.0591). Histo-pathology showed significantly thickened leaflets in the frozen valves (P < 0.05) and infiltrating CD3+ T-cells (P < 0.05) compared with ice-free valve leaflets. Multiphoton microscopy at explant revealed reduced inducible autofluorescence and extracellular matrix damage in the frozen explants and well preserved structures in the ice-free explant leaflets. In conclusion, ice-free cryopreservation of heart valve transplants at -80°C avoids ice formation, tissue-glass cracking and preserves extracellular matrix integrity resulting in minimal inflammation and improved hemodynamics in allogeneic juvenile sheep.

We have previously demonstrated storage of ice-free cryopreserved heart valves at -80°C without the need for liquid nitrogen, with the aims of decreasing manufacturing costs and reducing employee safety hazards. The objectives of the present study were a further simplification of the ice-free cryopreservation method and characterization of tissue viability. Porcine pulmonary heart valves were permeated with an 83% cryoprotectant solution (VS83) followed by rapid cooling and storage at -80°C. The cryoprotectants were added and removed in either single or multiple steps. Fresh untreated frozen controls employing 10% dimethylsulfoxide and controlled rate freezing to -80°C, and storage in vapor phase nitrogen were also performed. After rewarming and washing, cryopreserved leaflets were compared with fresh controls using the resazurin reduction metabolism assay. Comparison of valve tissues in which the cryoprotectants were added and removed in either single or multiple steps demonstrated similar viability results for the muscle, conduit, and leaflet components. The ice-free cryopreserved conduit and leaflet components were significantly less viable than either fresh or frozen tissues. The muscle component, although less viable, was not significantly different. The changes in tissue viability were a function of cryoprotectant exposure, and resulting cytotoxicity, not temperature reduction during storage. TUNEL staining showed that ice-free cryopreservation did not induce significant amounts of apoptosis, suggesting that necrosis is the predominant cell death pathway in ice-free cryopreserved heart valves. There was very little difference in cell viability when the cryoprotectants were added and removed in a single step versus multiple steps. Ice-free cryopreserved valve tissues demonstrated very low viability compared with controls. These results support further simplification of the ice-free cryopreservation method.

Dimethylsulfoxide, the most commonly employed cryoprotectant for cells, has well documented cytotoxic effects in patients. Among the compounds available that may provide protection to cells and tissues during preservation with less cytotoxicity is trehalose. Some animals, such as brine shrimp and tardigrades, accumulate trehalose during periods of extreme environmental stress. In this study, experiments were performed to evaluate the effects of culturing a bovine endothelial cell line (ATCC #CCL-209) in the presence of trehalose prior to preservation by freezing. A number of factors were shown to contribute to cell retention of metabolic activity and proliferative potential including cell culture time with trehalose and the solution conditions during cryopreservation. Using an optimized protocol consisting of 24 h of cell culture with 0.2 M trehalose followed by cryopreservation with 0.2-0.4 M trehalose in sodium bicarbonate buffered Eagles minimum essential medium at pH 7.4 resulted in 87±4% post-preservation cell metabolic activity expressed as relative fluorescence based upon reduction of resazurin to resorufin. This new method provides an alternative preservation strategy to the more classical preservation methods employing dimethylsulfoxide available for cells and tissues., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cold preservation has greatly facilitated the use of cadaveric kidneys for transplantation but damage occurs during the preservation episode. It is well established that oxidant production increases during cold renal preservation and mitochondria are a key target for injury. Our laboratory has demonstrated that cold storage of renal cells and rat kidneys leads to increased mitochondrial superoxide levels and mitochondrial electron transport chain damage, and that addition of Mitoquinone (MitoQ) to the preservation solutions blunted this injury. In order to better translate animal studies, the inclusion of large animal models is necessary to develop safe preclinical protocols. Therefore, we tested the hypothesis that addition of MitoQ to cold storage solution preserves mitochondrial function by decreasing oxidative stress, leading to less renal tubular damage during cold preservation of porcine kidneys employing a standard criteria donor model. Results showed that cold storage significantly induced oxidative stress (nitrotyrosine), renal tubular damage, and cell death. Using High Resolution Respirometry and fresh porcine kidney biopsies to assess mitochondrial function we showed that MitoQ significantly improved complex II/III respiration of the electron transport chain following 24 hours of cold storage. In addition, MitoQ blunted oxidative stress, renal tubular damage, and cell death after 48 hours. These results suggested that MitoQ decreased oxidative stress, tubular damage and cell death by improving mitochondrial function during cold storage. Therefore this compound should be considered as an integral part of organ preservation solution prior to transplantation.

Objective: Arterial allografts are routinely employed for reconstruction of infected prosthetic grafts. Usually, banked cryopreserved arteries are used; however, existing conventional freezing cryopreservation techniques applied to arteries are expensive. In contrast, a new ice-free cryopreservation technique results in processing, storage and shipping methods that are technically simpler and potentially less costly. The objective of this study was to determine whether or not ice-free cryopreservation causes tissue changes that might preclude clinical use., Methods: Conventionally frozen cryopreserved porcine arteries were compared with ice-free cryopreserved arteries and untreated fresh controls using morphological (light, scanning electron and laser scanning microscopy), viability (alamarBlue assay) and hemocompatibility methods (blood cell adhesion, thrombin/antithrombin-III-complex, polymorphonuclear neutrophil-elastase, β-thromboglobulin and terminal complement complex SC5b-9)., Results: No statistically significant structural or hemocompatibility differences between ice-free cryopreserved and frozen tissues were detectable. There were no quantitative differences observed for either autofluorescence (elastin) or second harmonic generation (collagen) measured by laser scanning microscopy. Cell viability in ice-free cryopreserved arteries was significantly reduced compared to fresh and frozen tissues (p < 0.05)., Conclusions: The formation of ice in aortic artery preservation did not make a difference in histology, structure or thrombogenicity, but significantly increased viability compared with a preservation method that precludes ice formation. Reduced cell viability should not reduce in vivo performance. Therefore, ice-free cryopreservation is a potentially safe and cost-effective technique for the cryopreservation of blood vessel allografts., (Copyright © 2012 S. Karger AG, Basel.)

BACKGROUND: The purpose of this research was to assess the extracellular matrix and chondrocytes of articular cartilage during refrigerated storage and to determine whether changes could be detected in the time frame that cartilage is stored for clinical use. MATHODS: Porcine cartilage was stored as either bisected femoral heads with bone attached or plugs without the underlying bone in culture medium with fetal bovine serum for 1 month at 4 °C. Metabolic activity was tested using a resazurin reduction method on intact tissue and viable cell recovery after enzymatic tissue digestion at each time point. Cartilage plug permeability was evaluated by measuring electrical conductivity. RESULTS: Storage in culture medium provided good cartilage viability and metabolic function for 7 days; however, significant changes were observed in femoral heads (p < 0.05). All mean chondrocyte assessment values were <30% of fresh controls at 28 days. Cartilage plugs tended to perform better after 7 days of storage than the femoral heads and retained significantly higher metabolic activity (mean = 94.5% vs. 70.5%; p < 0.05). Cartilage plugs demonstrated consistent changes in electrical conductivity after 28 days of storage (p < 0.05). CONCLUSION: Refrigerated storage of cartilage results in both loss of chondrocyte viability and matrix permeability.

Background: Cryopreserved allogeneic heart valves are usually stored and transported below -135°C; however, such methods require expensive equipment for both storage and transportation., Methods: In this study, vitrified porcine aortic valves were stored on either side of the cryoprotectant formulation's glass transition temperature (-119°C) at -80°C and -135°C, using a newly formulated vitrification solution (VS83) consisting of a combination of 4.65M dimethyl sulfoxide, 4.65M formamide, and 3.30M 1,2-propanediol. Three groups of valves were studied: (1) fresh; (2) VS83-preserved, stored at -80°C; and (3) VS83-preserved, stored at -135°C., Results: Using the VS83 cryoprotectant concentration formulation, cracking was not observed during valve storage. No ice-related events were detectable during 5°C rewarming by differential scanning calorimetry. All cryopreserved tissue samples demonstrated significantly less viability than fresh samples (p<0.01). No significant viability differences were observed between the VS83-preserved groups stored at -80°C and -135°C. Material testing did not reveal any significant differences among the three test groups. Multiphoton imaging of VS83-preserved heart valves stored at -80°C and -135°C demonstrated similar collagen and elastin structures., Conclusions: These results indicate that VS83-preserved heart valves can be stored and transported at temperatures in the vicinity of -80°C with retention of extracellular matrix integrity and material properties. The VS83 preservation of heart valves at -80°C without the need for liquid nitrogen should result in both decreased manufacturing costs and reduced employee safety hazards. Moreover, it is anticipated that low cell viability may result in less immunogenicity in vivo., (Copyright © 2011 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

The study of mechanisms by which animals tolerate environmental extremes may provide strategies for preservation of living mammalian materials. Animals employ a variety of compounds to enhance their survival, including production of disaccharides, glycerol, and antifreeze compounds. The cryoprotectant glycerol was discovered before its role in amphibian survival. In the last decade, trehalose has made an impact on freezing and drying methods for mammalian cells. Investigation of disaccharides was stimulated by the variety of organisms that tolerate dehydration stress by accumulation of disaccharides. Several methods have been developed for the loading of trehalose into mammalian cells, including inducing membrane lipid-phase transitions, genetically engineered pores, endocytosis, and prolonged cell culture with trehalose. In contrast, the many antifreeze proteins (AFPs) identified in a variety of organisms have had little impact. The first AFPs to be discovered were found in cold water fish; their AFPs have not found a medical application. Insect AFPs function by similar mechanisms, but they are more active and recombinant AFPs may offer the best opportunity for success in medical applications. For example, in contrast to fish AFPs, transgenic organisms expressing insect AFPs exhibit reduced ice nucleation. However, we must remember that nature's survival strategies may include production of AFPs, antifreeze glycolipids, ice nucleators, polyols, disaccharides, depletion of ice nucleators, and partial desiccation in synchrony with the onset of winter. We anticipate that it is only by combining several natural low temperature survival strategies that the full potential benefits for mammalian cell survival and medical applications can be achieved.

There are many compounds that can and have been used as cryoprotectants including disaccharides such as trehalose. Many organisms in nature use trehalose to help protect themselves at colder temperatures. Trehalose has also been used to a limited extent for the preservation of mammalian cells and tissues, but mainly as a supplement to other cryoprotectants like dimethyl sulfoxide. Recently, the use of trehalose as the primary cryoprotectant has gained much interest because of its low-potential cytotoxicity. Trehalose does not readily pass through mammalian cells membranes and research has shown that it is most effective when present on both sides of the cell membrane prior to preservation. Different strategies for introducing disaccharide sugars into cells have been investigated with limited success. In this study, two separate strategies are investigated for the introduction of disaccharide sugars into cells. Electroporation using an electric pulse to create temporary holes in the membrane so that molecules could pass through and a transport peptide (Chariot™) that covalently binds to the molecule of interest and then moves it across the membrane. Both strategies have the potential to load disaccharide sugars into cells at concentrations that would provide ample protection during preservation. In preparation for cryopreservation studies, smooth muscle cells that are difficult to cryopreserve using conventional preservation protocols were used to evaluate and compare the translocation potential of these two strategies using β-galactosidase. Assessment of each loading strategy was done by measuring viability and the presence of β-galactosidase inside the cells. The results indicate that both methods appear feasible as potential delivery systems and that treatment cytotoxicity can be minimized. The next step is definition of the best loading strategy to introduce trehalose into cells followed by preservation by freezing.

Bovine serum is commonly used in cryopreservation of allogeneic heart valves; however, bovine serum carries a risk of product adulteration by contamination with bovine-derived infectious agents. In this study, we compared fresh and cryopreserved porcine valves that were processed by 1 of 4 cryopreservation formulations, 3 of which were serum-free and 1 that utilized bovine serum with 1.4 M dimethylsulfoxide. In the first serum-free group, bovine serum was simply removed from the cryopreservation formulation. The second serum-free formulation had a higher cryoprotectant concentration, i.e. 2 M dimethylsulfoxide, in combination with a serum-free solution. A colloid, dextran 40, was added to the third serum-free group with 2 M dimethylsulfoxide due to theoretical concerns that removal of serum might increase the incidence of tissue cracking. Upon rewarming, the valves were inspected and subjected to a battery of tests. Gross pathology revealed conduit cracking in 1 of 98 frozen heart valves. Viability data for the cryopreserved groups versus the fresh group demonstrated a loss of viability in half of the comparisons (p < 0.05). No significant differences were observed between any of the cryopreserved groups, with or without bovine serum. Neither routine histology, autofluorescence-based multiphoton imaging nor semiquantitative second-harmonic generation microscopy of extracellular matrix components revealed any statistically significant differences. Biomechanics analyses also revealed no significant differences. Our results demonstrate that bovine serum can be safely removed from heart valve processing and that a colloid to prevent cracking was not required. This study provides guidance for the assessment of changes in cryopreservation procedures for tissues., (Copyright © 2010 S. Karger AG, Basel.)

Cryopreservation by freezing is usually employed for storage of allogeneic valves destined for clinical use; however, disruption of leaflet extracellular matrices by ice may occur. This study was performed to determine the effects of cryoprotectants employed in VS55 for ice-free, vitreous cryopreservation upon adherent porcine heart valve leaflet-derived myofibroblasts in culture. Low-passage myofibroblasts exhibiting strong actin and myosin staining were employed for these experiments. Three cryoprotectants, dimethylsulfoxide, formamide, and propanediol, were tested individually and in combination. Exposure experiments demonstrated that the individual cryoprotectants (0-5 M) were generally cytotoxic in a dose-dependent manner with little if any loss of cells as determined by measuring metabolic activity and DNA content. Exposure to formamide resulted in the greatest loss of cells and reduction in viability. Combination of the three cryoprotectants demonstrated that the cytotoxic effects of each cryoprotectant were not cumulative. Cell viability and DNA content were equivalent to dimethylsulfoxide and propanediol and higher than formamide alone over most of the 0-5 M dose-response curve. After cryopreservation by slow-rate freezing, the benefits of the combination of cryoprotectants over individual cryoprotectants were demonstrated at the 4 M concentration range for both cell viability and cell retention. In conclusion, these studies demonstrated that, with the exception of the lower concentrations of propanediol, the combination of cryoprotectants employed in these studies result in equivalent or better cell viability and attachment than individual cryoprotectants.

Tissue-engineered blood vessels may provide a solution to the lack of suitable blood vessels for coronary and peripheral vessel bypass grafting. Cyclic strain can be used to provide a more physiological environment that may result in tissue that more closely resembles native artery. In this study, cyclic strain is applied to a collagen-based, tissue-engineered vascular medium. An increased culture time was used to allow the tissue to adhere to the silastic sleeve and to eliminate longitudinal compaction. Cyclic strain improved tissue strength through increased collagen content as well as some radial tissue compaction. Mechanical stimulation promoted a more contractile phenotype and led to a greater contractile response to the vasoconstrictor endothelin-1.

The property of vasoactivity is important for both resistance vessels and larger arteries. Evaluation of smooth muscle cell phenotype is often done in place of functional testing in engineered tissues, assuming a direct correlation between cell phenotype and tissue contractile force. In this study we look at a large panel of vasoactive agents to determine the functionality of our collagen-based tissue. The engineered vascular media elicited a measurable change in force in response to seven of the nine agents used. As part of this characterization, TGF-β1 and TNF-α were used to promote a more contractile and synthetic cell phenotype respectively. Both smooth muscle α-actin and vasoconstriction were evaluated in ring sections. Due to large differences in cell-compaction and cell distribution in the tissues, no correlation was found between α-actin expression and contractile strength. This highlights the need for functional testing of engineered tissue and the importance of cell-matrix interactions in vasoactivity.

Transplantation of cryopreserved heart valves (allografts) is limited by immune responses, inflammation, subsequent structural deterioration and an expensive infrastructure. In previous studies we demonstrated that conventional frozen cryopreservation (FC) is accompanied by serious alterations of extracellular matrix (ECM) structures. As the main culprit of the observed damages ice crystal formation was identified. Objective of this study was the application principles of cryoprotection as observed in nature, occurring in animals or plants, for ice-free cryopreservation (IFC) of heart valves. Using IFC, valves were processed and stored above the glass transition temperature of the cryoprotectant formulation (-124 degrees C) at -80 degrees C to avoid any ice formation, tissue-glass cracking and preserving ECM. After implantation in the orthotopic pulmonary position in sheep, we demonstrate that IFC resulted in cell free matrices, while maintaining crucial ECM-components such as elastin and collagen, translating into superior hemodynamics. In contrast, we reveal that FC valves showed ECM damage that was not restored in vivo, and T-cell inflammation of the stroma with significant leaflet thickening. Compared to currently applied FC practice IFC also reduced infrastructural needs for preservation, storage and shipping. These results have important implications for clinical valve transplantation including the promise of better long-term function and lower costs., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The limited availability of fresh osteochondral allograft tissues necessitates the use of banking for long-term storage. A vitrification solution containing a 55% cryoprotectant formulation, VS55, previously studied using rabbit articular cartilage, was evaluated using porcine articular cartilage. Specimens ranging from 2 to 6 mm in thickness were obtained from 6mm distal femoral cartilage cores and cryopreserved by vitrification or freezing. The results of post-rewarming viability assessments employing alamarBlue demonstrated a large decrease (p<0.001) in viability in all three sizes of cartilage specimen vitrified with VS55. This is in marked contrast with prior experience with full thickness, 0.6 mm rabbit cartilage. Microscopic examination following cryosubstitution confirmed ice formation in the chondrocytes of porcine cartilage vitrified using VS55. Experiments using a more concentrated vitrification formulation (83%), VS83, showed a significant treatment benefit for larger segments of articular cartilage. Differences between the VS55 and the VS83 treatment groups were significant at p<0.001 for 2 mm and 4 mm plugs, and at p<0.01 for full thickness, 6 mm plugs. The percentage viability in fresh controls, compared to VS55 and VS83, was 24.7% and 80.7% in the 2 mm size group, 18.2% and 55.5% in the 4 mm size group, and 5.2% and 43.6% in the 6 mm group, respectively. The results of this study continue to indicate that vitrification is superior to conventional cryopreservation with low concentrations of dimethyl sulfoxide by freezing for cartilage. The vitrification technology presented here may, with further process development, enable the long-term storage and transportation of living cartilage for repair of human articular surfaces., (Copyright 2009 Elsevier Inc. All rights reserved.)

Cartilage damage was studied using non-invasive multiphoton-excited autofluorescence and quantitative second harmonic generation (SHG) microscopy. Two cryopreservation techniques based upon freezing and vitrification methods, respectively, were employed to determine whether or not the collagen fiber structure of full thickness porcine articular cartilage was affected by cryopreservation and whether the level of collagen damage could be determined quantitatively in non-processed (non-fixed, non-sliced, non-stained) tissues. Multiphoton-induced autofluorescence imaging revealed the presence of chondrocytes, as well as collagenous structures in all fresh, vitrified and frozen cryopreserved cartilage samples. SHG imaging of the frozen cryopreserved specimens showed a dramatic loss of mean gray value intensities when compared to both fresh and vitrified tissues (P<0.05), indicating structural changes of the extracellular matrix, in particular the deformation and destruction of the collagen fibers in the analyzed articular cartilage. A 0.9974 correlation coefficient was observed between the metabolic cell activity assessed by the alamarBlue technique, and retention of collagen structure between the three experimental groups. These studies suggest that multiphoton-induced autofluorescence imaging combined with quantitative SHG signal profiling may prove to be useful tools for the investigation of extracellular matrix changes in preserved cartilage, giving insights on the structural quality prior to implantation.

With the development of cell-based assays and therapies, the purity of reagents used to grow and maintain cells has become much more important. In particular, the use of fetal calf serum for culturing cells presents a direct path for potential contamination of cell cultures. In recent years, much research has focused on the development of serum-free culturing systems, not only to alleviate difficulties due to availability and cost of fetal calf serum but also to prevent the transmission of potentially fatal diseases to human patients. Additionally, methods need to be developed for long-term storage of cell stocks that also reduce the risk of exposure to harmful diseases. As most methods employ fetal calf serum in their freezing formulations, solutions that avoid the use of fetal calf serum while providing equivalent or better recovery of cells upon thawing would be ideal. In this study, two vascular cell lines have been cryopreserved as adherent cell populations in two widely used cryoprotectants, dimethyl sulfoxide and 1,2-propanediol, and two vehicle solutions, Euro-Collins and Unisol-cryoprotectant vehicle specifically formulated for the maintenance of cell homeostasis at temperatures below 37 degrees C. The addition of serum to these formulations was also evaluated to determine if its presence provided any additional benefit to the cells during cryopreservation. The results demonstrated that using vehicle solutions designed for lower temperatures produced viable cells that retained cell population viability values up to 75% of unfrozen controls. These results also demonstrated that including serum in the formulation provided no additional benefit to the cells and in some cases actually produced lower cell viability after cryopreservation. In conclusion, the development of solutions designed for low-temperature storage of cells provides a viable alternative to more conventional cryopreservation protocols and eliminates the necessity of including serum in these formulations.

Background: Cryopreservation of human tissues, particularly heart valves, is widespread in clinical practice although the effects of this process on underlying tissue structures and its potential impact on valve durability have been poorly studied. Multiphoton imaging and second-harmonic generation (SHG) microscopy permit high-resolution, noninvasive analysis of living tissues at a subcellular level. In the present study we used these novel imaging modalities to compare the effects of vitreous and frozen cryopreservation on the extracellular matrix (ECM) of cardiac tissues., Methods: Conventional histology, electron microscopy, and multiphoton imaging to obtain autofluorescence and SHG images were performed on cardiac tissues to characterize the ECM in fresh, vitrified, and frozen cryopreserved tissues., Results: Autofluorescence and particularly SHG images revealed that conventional frozen cryopreservation of cardiac valves, when compared with fresh or vitrified tissues, leads to the loss of normal ECM structures in valve leaflets. Similar results were found in all other cardiac tissues suggesting that structural deterioration of the ECM is a common consequence of frozen cryopreservation., Conclusions: Our results demonstrate that conventional cryopreservation, when compared with fresh or vitrified tissues, causes more destruction of normal ECM structure, which might contribute to eventual graft dysfunction. Whether vitrification preservation will translate into greater durability or less valve failure will need to be determined.

This study documents renal biochemistry during hypothermic machine perfusion of kidneys. It is intended to demonstrate that a comprehensive evaluation of organ viability during ex-vivo preservation is needed to increase the number of organs available for transplantation and to reduce the current renal discard rate. Porcine kidneys were hypothermically machine perfused for 72 h with either Unisol-UHK or Belzer-Machine Perfusion Solution, (Belzer-MPS). Renal perfusate samples were periodically collected and biochemically analyzed. Significant differences were measured in the renal metabolic activity between the two experimental groups while similar values for traditional parameters such as renal flow rate and vascular resistance values were recorded. The effluent of UHK perfused kidneys showed strong metabolites and NH(4)(+) dynamics (P<0.05 vs. baseline), while the Belzer-MPS kidneys metabolic activity led to little or no change of the effluent biochemistry relative to baseline.

It is well established that, in multicellular systems, conventional cryopreservation results in damaging ice formation, both in the cells and in the surrounding extracellular matrix. As an alternative to conventional cryopreservation, we performed a feasibility study using vitrification (ice-free cryopreservation) to cryopreserve tissue-engineered blood vessels. Fresh, frozen, and vitrified tissue-engineered blood vessels were compared using histological methods, cellular viability, and mechanical properties. Cryosubstitution methods were used to determine the location of ice in conventionally cryopreserved engineered vessels. Ice formation was negligible (0.0 +/- 0.0% of vessel area) in the vitrified specimens, and extensive (68.3 +/- 4.5% of vessel area) in the extracellular matrix of frozen specimens. The metabolic assay and TUNEL staining results indicated that vitrified tissue had similar viability to fresh controls. The contractility results for vitrified samples were >82.7% of fresh controls and, in marked contrast, the results for frozen samples were only 10.7% of fresh controls (p < 0.001). Passive mechanical testing revealed enhanced tissue strength after both freezing and vitrification. Vitrification is a feasible storage method for tissue-engineered blood vessel constructs, and their successful storage brings these constructs one step closer to clinical utility.

To meet the current clinical organ demand, efficient preservation methods and solutions are needed to increase the number of viable kidneys for transplantation. In the present study, the influence of perfusion solution buffering strength on renal pH dynamics and regulation mechanisms during kidney ex vivo preservation was determined. Porcine kidneys were hypothermically machine perfused for 72 h with either Unisol-UHK or Belzer-Machine Perfusion solution, Belzer-MP solution. Renal perfusate samples were periodically collected and biochemically analyzed. The UHK solution, a Hepes-based solution (35 mM), provided a more efficient control of renal pH that, in turn, resulted in minor changes in the perfusate pH relative to baseline, in response to tissue CO2 and HCO3- production. In the perfusate of Belzer-MP kidney group a wider range of pH values were recorded and a pronounced pH reduction was seen in response to significant rises in pCO2 and HCO3- concentrations. The Belzer-MP solution, containing phosphate (25 mM) as its main buffer, and only 10 mM Hepes, had a greater buffering requirement to attenuate larger pH changes.

Despite significant advances, some critical issues remain for the long-term storage of an engineered pancreas. In this study we employed a tissue engineered pancreatic substitute model-insulin-secreting betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate beads-to demonstrate that a prototype vitrification method can prevent ice formation and maintain cell viability/function. The results showed that the structure of the frozen samples was distorted by ice crystals throughout the matrix. In marked contrast, the vitrified samples appeared to be free of ice. Morphologic studies demonstrated extensive fractures and vacuolation in frozen specimens while there were no fractures in vitrified TEPSs. Both vitrified and frozen constructs showed some vacuolization compared to the control samples. Frozen beads showed a significantly decreased viability compared to fresh controls and the VS55 group (P < .001). There was no significant difference between the vitrified and fresh samples. Vitrification using the VS55 protocol shows similar viability and secretion properties to the control group of fresh beads. Vitrification using the PEG 400 protocol resulted in slightly lower viability and secretion properties relative to the control group; conventional freezing resulted in even significantly lower viability and secretion properties. These results combine to demonstrate feasibility of vitrification as a storage method for a tissue engineered pancreas.

Hypothermic machine perfusion (HMP) has a potential to relieve the current donor liver crisis by providing an improved and extended preservation method. This study examined the effect of HMP on hepatocellular functions, using a prototype liver transporter capable of preserving livers for 24 hours. Livers obtained from adult farm pigs (28 to 32 kg body weight) were divided into three groups: fresh control, HMP, and simple cold storage (n = 4 each). A 4-hour normothermic reperfusion of livers was conducted to assess hepato-metabolic and cellular functions. The hepatic transport function, as indicated by canalicular excretion of indocyanine green, was improved in the HMP group than in the SCS group. The overall tissue viability, as indicated by oxygen consumption levels, was notably improved in HMP and control livers as compared to the SCS group. Higher bile production in both the preserved groups as compared to the fresh control livers could be a result of biliary edema and leakage of plasma into the canaliculus. The hepato-cellular injury, measured by ALT, release was significantly greater in the SCS group as compared to the HMP and control groups. These findings suggest that HMP could be a better method to preserve hepatic function and overall tissue viability as compared to SCS. Improved hepatic functions are indirect indicators of superior microcirculation and sinusoidal endothelial cell functions. Further studies in progress will evaluate these functions to confirm the significance of these observations.

Background: Long-term storage of embryonic kidneys is crucial for the organization of transplantation and organ banking. In this study, we investigated the effects of controlled-rate freezing and ice-free vitrification on metanephroi (MN) viability., Methods: Metanephroi isolated from 15-day (E15) timed pregnant Lewis rats were either: (i) frozen, using a DMSO/FCS/RPMI solution and a controlled freezing rate of -0.3 degrees C/min, from -10 degrees to -40 degrees C; or (ii) cryopreserved in an ice-free state by rapid cooling to -100 degrees C in cryoprotectant (VS55), followed by vitrification to -120 degrees C. After cryopreservation, the metanephroi were stored at -135 degrees C for 48 hours. After storage the MN were rewarmed, resuspended in culture media, and their viability was assessed using the AlamarBlue assay and histology (light microscopy, TEM, and cryosubstitution)., Results: There was statistically no difference in embryonic kidney metabolic activity of either of the cryopreserved MN groups relative to the control untreated group. However, cryosubstitution demonstrated the presence of significant ice formation during controlled-rate freezing, yet in contrast the amount of ice was significantly reduced by vitrification. This was confirmed by TEM, where vacuolation of the cytoplasm of controlled-rate frozen metanephroi was observed, whereas vitrified metanephroi had little cytoplasmic disruption. However, vitrified metanephroi showed mitochondrial and nuclear injury at the cellular level., Conclusions: There is a need for long-term storage of organs to make MN transplantation a reality. This study demonstrates that standard freezing methods are unsuitable for this purpose. Vitrification yielded more promising results, but further development is required.

Background and Aim of the Study: The pathophysiology of allogeneic heart valve failure is not fully understood. It is hypothesized that the rapid deterioration seen in some allograft heart valve recipients is due to disruptive interstitial ice damage that occurs during cryopreservation by freezing., Methods: The hypothesis was tested by comparing a standard commercial heart valve freezing and ice-free, vitrification cryopreservation methods with fresh controls in: (i) a subcutaneous, juvenile rat implant model of calcification; and (ii) a descending thoracic aorta implant rat model for histopathology. Calcium concentrations in one- to six-week explants were determined using atomic absorption spectroscopy; calcification rates were also determined., Results: The calcification rate of frozen valves was significantly greater (p < 0.01) than that of vitrified valves in both syngeneic and allogeneic recipients, supporting prior observations that ice-free cryopreservation reduces allogeneic heart valve calcification. Cryopreservation by freezing and vitrification resulted in mild morphological changes in two- and four-week explants, a slight decrease in leaflet cellularity, and a more rapid onset of intimal hyperplasia than in fresh valve explants. The allograft explant groups exhibited similar changes, regardless of how the valves were processed., Conclusion: These findings provide only weak support for the tested hypothesis, and further studies in a large animal model are warranted.

Articular cartilage has proved refractory to satisfactory cryopreservation using conventional freezing methods. Therefore, an ice-free cryopreservation method by vitrification was tested. Osteochondral plugs from New Zealand White rabbits were preserved using either a freezing method or an ice-free vitrification method of cryopreservation. Preserved and fresh control plugs were implanted in the tibial plateau of allogeneic recipients. A modified O'Driscoll grading scale, based on gross pathology, histopathology, and histochemistry, was used to evaluate the explants.The histology of fresh and vitrified explants was essentially the same, while the frozen cryopreserved explants were devoid of chondrocytes and only fibroblastlike cells were observed. The O'Driscoll grading indicated that both fresh and vitrified plugs performed significantly better than frozen plugs (p < or =.05). The results demonstrate the feasibility of vitrification as a storage method for cartilaginous tissues.

Evaluation methods are required for non-heart-beating donor (NHBD) kidneys to ensure the success of transplantation. In this study, the microdialysis technique was employed for the ex-vivo assessment of hypothermically preserved NHBD kidney function. Microdialysis probes were placed in the renal cortex of 2 h warm ischaemic porcine kidneys to monitor interstitial pyruvate dynamics during hypothermic machine perfusion with perfusate containing 29.4 mM fructose-1,6-diphosphate (FDP). The presence of exogenous FDP in the perfusate induced no changes in the renal flow rate and vascular resistance, renal artery effluent biochemistry, or pyruvate concentration relative to untreated control kidneys. Significant increases in pyruvate production (P < 0.05), however, were observed after 12 h of perfusion in the interstitial fluid of FDP-treated kidneys relative to control kidneys. After 24 h of perfusion, interstitial fluid concentrations of pyruvate were 149.1 +/- 58.4 vs. 55.6 +/- 17.9 micro M (P < 0.05) in the FDP and control group, respectively. The microdialysis probe collected the interstitial fluid directly from the cellular sites of metabolic and synthetic activity, where perfusate dilution was minimal. Consequently, the biochemical changes induced by the organ metabolic activity were detected only at the interstitial level, in the microdialysates. Interstitial fluid pyruvate may be a good indicator of kidney function. The addition of FDP to the perfusion solution during ischaemic kidney preservation resulted in enhanced pyruvate production in the extracellular space, indirectly reflecting an increase in anaerobic ATP production. The pyruvate will be transformed during organ reperfusion into acetyl Co-A enzyme allowing an immediate start of aerobic metabolism. This in turn can increase the amount of ATP available to the cells and may help prevent reperfusion injury upon transplantation.

Background: Cryopreserved human heart valves are used in approximately 20% of the tissue heart valve procedures performed annually. The pathophysiology of allograft failure is not fully understood. The authors proposed the hypothesis that the rapid deterioration observed in some allograft heart valve recipients is caused by disruptive interstitial ice damage that occurs during cryopreservation and subsequently leads to accelerated valve degeneration on implantation., Methods: This hypothesis was tested by comparison of the standard commercial heart valve freezing method of cryopreservation and an ice-free, vitrification method of cryopreservation with fresh controls in a subcutaneous, juvenile rat implant model of calcification. Calcium concentration in explants was determined by atomic absorption spectroscopy., Results: Statistically significant calcification (P<0.05) was observed in both syngeneic and allogeneic cryopreserved valves relative to fresh valves. The ice-free cryopreservation method demonstrated significant reduction of allogeneic heart valve calcification (P<0.01). Comparison of fresh syngeneic and allogeneic grafts at the 3-week time point demonstrated significantly higher calcium content in allograft valve explants (P<0.005)., Conclusions: These findings demonstrate that allogeneic valve calcification is influenced by two factors, the cryopreservation method used and immunogenicity. Alternative cryopreservation methods that avoid ice formation may improve the in vivo performance of cryopreserved allogeneic heart valves.

Autogenous veins are the materials of choice for arterial reconstruction. In the absence of autogenous material, prosthetic materials are used. However, vascular prostheses of less than 0.4 cm in diameter have low long-term patency. This study was designed to determine if cells would infiltrate an engineered xenogeneic biomaterial used as a small diameter arterial graft in dogs and, if so, to determine the phenotype of the infiltrating cells. Nine acellular xenogeneic grafts (0.4 cm in diameter, 5 cm long), composed of porcine collagen derived from the submucosa of the small intestine and type I bovine collagen, were implanted as end to-end interposition grafts in femoral arteries of five male mongrel dogs (total of nine grafts). All dogs received daily aspirin (325 mg). Patency of implanted grafts was monitored weekly by Duplex ultrasonography. After 9 weeks, or earlier in case of blood flow reduction by at least 75%, grafts were explanted and prepared for light or electron microscopy to evaluate cellularization. Eight of nine grafts remained patent up to 9 weeks. At explant, diameters were 0.31 +/- 0.02 cm at the midgraft, and 0.14 +/- 0.01 and 0.19 +/- 0.01 cm at the proximal and distal anastomoses. At explant, cells of mesenchymal origin (endothelial cells, smooth muscle cells, myofibroblasts) were embedded in the extracellular matrix of the graft scaffold. Minimal evidence of cellular inflammatory reaction and no aneurysmal dilatation or thrombus formation was detected. Variable degrees of hyperplasia were present at proximal and distal anastomoses. This preliminary study demonstrates that a collagen-based xenogeneic biomaterial provides a scaffold for cellularization when used for arterial reconstruction in dogs.

Conventionally cryopreserved vascular grafts have performed poorly as arterial grafts. One possible mechanism that causes the poor function is the extracellular ice damage in tissue. We used a novel new ice-free cryopreservation (namely, vitrification) method for prevention of ice formation in cryopreserved venous grafts. This study was designed to evaluate the in vivo effects of the vitrification process on autologous vascular grafts using a short-term transplantation model and to examine the morphology and patency of vitrified grafts in correlation with control grafts. New Zealand White rabbits underwent a right common carotid interposition bypass graft. Fresh and vitrified reversed ipsilateral external jugular veins were used as autologous grafts. Animals were sacrificed at either 2 or 4 weeks after implantation, and fresh and vitrified vein grafts were harvested for histology studies. The results, comparing the patency of fresh and vitrified grafts, demonstrated similar short-term patency rates (approximately 90%). There were no signs of media disruption, aneurysm, or graft stenosis in vitrified vein grafts. Vitrification had not altered the pathophysiological cascade of events that occur when a vein graft is inserted into the arterial system. The vitrification process had no adverse effects locally or systemically in vivo. In addition, vitrification has preserved endothelial cell and smooth muscle cell integrity posttransplantation. In conclusion, this study, using an autologous animal model, clearly demonstrated a significant benefit of vitrification for preservation of graft function, and vitrification may be an acceptable approach for preservation of blood vessels or engineered tissue constructs.

Avoidance of ice formation during cooling can be achieved by vitrification, which is defined as solidification in an amorphous glassy state that obviates ice nucleation and growth. We show that a vitrification approach to storing vascular tissue results in markedly improved tissue function compared with a standard method involving freezing. The maximum contractions achieved in vitrified vessels were >80% of fresh matched controls with similar drug sensitivities, whereas frozen vessels exhibited maximal contractions below 30% of controls and concomitant decreases in drug sensitivity. In vivo studies of vitrified vessel segments in an autologous transplant model showed no adverse effects of vitreous cryopreservation compared with fresh tissue grafts.