Allogeneic Heart Valve Storage Above the Glass Transition at 80 C

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1 Allogeneic Heart Valve Storage Above the Glass Transition at 80 C Kelvin G.M. Brockbank, PhD, Gregory J. Wright, BS, Hai Yao, PhD, Elizabeth D. Greene, LATg, Zhen Z. Chen, MS, and Katja Schenke-Layland, PhD Cell & Tissue Systems, Inc, North Charleston, South Carolina; Georgia Tech/Emory Center for the Engineering of Living Tissues, The Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia; Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, South Carolina; Clemson University, Clemson-MUSC Bioengineering Program, Charleston, South Carolina; Fraunhofer Institute for Interfacial Engineering and Biotechnology (IGB), Department of Cell and Tissue Engineering, Stuttgart, Germany; and Inter-University Centre for Medical Technology (IZST) and Department of Thoracic, Cardiac and Vascular Surgery, Eberhard Karls University, Tübingen, Germany 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. (Ann Thorac Surg 2011;91: ) 2011 by The Society of Thoracic Surgeons Transplantation of allograft heart valves was first introduced clinically in 1962 [1]. They have demonstrated exceptionally good initial hemodynamic characteristics, hardly any thromboembolic events without anticoagulation, and better resistance to endocarditis compared with bioprosthetic or mechanical valve substitutes [2, 3]. Allografts have especially benefited children with congenital heart disease. They are also used in young adults, women of child bearing age, and less frequently in older patients with memory problems who may not be relied upon to keep up with the medications required for mechanical valves. Initially, the allograft valves were collected and immediately transplanted [4]. Due to logistic issues, grafts were subsequently stored at 4 C in tissue culture medium with antibiotics for up to 6 weeks prior to implantation [5]. Eventually, cryopreservation with dimethyl sulfoxide (DMSO) and fetal bovine serum was introduced to enable long-term storage and improve Accepted for publication Feb 14, Address correspondence to Dr Brockbank, Cell & Tissue Systems, Inc, 2231 Technical Pkwy, Ste A, North Charleston, SC 29406; kbrockbank@celltissuesystems.com. infectious disease screening [6]. For the last 20 years freezing has been the worldwide choice for cryopreservation of human heart valves [7]. In pediatric patients allograft function is limited by early structural deterioration, necessitating more frequent reintervention procedures [8 10]. A variety of reasons for pediatric allograft heart valve failure were discussed in the past and most investigators have emphasized immunologic issues [11, 12]. We have previously proposed the hypothesis that the rapid deterioration observed in some allograft heart valve recipients is due to disruptive interstitial ice damage that occurs during cryopreservation and subsequently leads to accelerated valve degeneration and calcification upon implantation [13 15]. This hypothesis led to the development of cardiovascular tissue preservation methods that avoided ice formation by vitrification [13, 16 18] based upon a 55% weight/volume vitrification solution (VS55) originally developed for preservation of kidneys at the Holland Laboratories of the American Red Cross [19 21]. Vitrification, in contrast to traditional cryopreservation employing freezing methods, uses high concentrations of a cryoprotectant solution 2011 by The Society of Thoracic Surgeons /$36.00 Published by Elsevier Inc doi: /j.athoracsur

2 1830 BROCKBANK ET AL Ann Thorac Surg HEART VALVE STORAGE AT 80 C 2011;91: Abbreviations and Acronyms DMSO dimethylsulfoxide DSC differential scanning calorimetry ECM extracellular matrix PBS phosphate buffered saline QMR qualitative mean rating RFU relative fluorescent units SHG second harmonic generation VS55 vitrification solution 55% VS83 vitrification solution 83% to promote amorphous solidification rather than crystallization to restrict the amount of ice crystal formation. Excellent preservation was also reported using VS55 to maintain chondrocyte viability in rabbit articular cartilage [22]. However, when thicker porcine cartilage was employed it was necessary to utilize a more concentrated formulation, an 83% weight/volume vitrification solution (VS83), to maintain chondrocyte viability [23]. In this report, we present further studies using the VS83 formulation in which we investigated storage of heart valves below and above the formulation s glass transition temperature ( 119 C) at 80 C and 135 C, respectively. Material and Methods Sample Preparation Porcine hearts were obtained from a local slaughterhouse as bona fide excess tissues. The pigs were approximately 90 kg in weight, equally distributed male to female, and ranged in age from 4 to 6 months. These pigs were immature, since maturity is achieved at weights greater than 200 kg and 2 years of age. The postmortem warm ischemia time was approximately 30 minutes. All hearts were then rinsed with 4 C phosphate buffered saline (PBS; MediaTech, Herndon, VA), placed in PBS in sterile bags on ice, and immediately transferred to the laboratory for dissection. Aortic heart valves were excised under sterile conditions and gently rinsed free of any residual blood in sterile PBS. The valves were further dissected of adherent fat and most of the myocardium, leaving a thin ridge of subvalvular cardiac muscle tissue and a length of ascending thoracic aorta. All valves were incubated in an antibiotic solution consisting of Dulbecco s modified Eagle medium, containing 4.5 g/l glucose (MediaTech), and 1% penicillin-streptomycin (Sigma, St. Louis, MO) for approximately 24 hours at 4 C prior to use. Cryopreservation Protocol All valves were gradually infiltrated with precooled vitrification formulations of DMSO, formamide, and 1,2- propanediol in EuroCollins solution at 4 C in six steps of at least 15 minutes duration, consisting of 0, 12.5, 25, 50, 75, and 100% of each formulation to achieve a final cryoprotectant concentration of 55% or 83% weight per volume. The final concentration of each cryoprotectant was either 3.10M DMSO, 3.10M formamide, and 2.21M 1,2-propanediol (VS55), or 4.65M DMSO, 4.65M formamide, and 3.30M 1,2-propanediol (VS83). All tissue specimens were placed in polyethylene bags containing approximately 80 to 90 ml of precooled vitrification solution. A thermocouple was inserted into a separate dummy sample of the same vitrification solution and its output monitored by a digital thermometer throughout the cooling process. Samples were cooled to 100 C by placing the samples in a precooled bath containing isopentane in a 135 C mechanical storage freezer. Upon achieving 100 C, the specimens were removed from the bath and placed at 135 C in the mechanical storage freezer. When the heart valves reached 135 C they were transferred to their final storage temperatures of 80 or 135 C for 1 to 30 days. The tissues were rewarmed in two stages; first, slow warming to 100 C by placing them at the top of a mechanical storage freezer and then warming to 30 C in a 30% DMSO in water bath at room temperature. Tissues stored at 80 C were warmed using the second step only. After rewarming, the vitrification solutions were removed in seven sequential 15-minute steps at 4 C into Dulbecco s modified Eagle medium culture medium as previously described [16, 22, 24]. Biomechanical Testing Fresh control samples were obtained from the noncoronary leaflet of aortic valves; the remaining tissue including the coronary leaflets were then bisected, cryopreserved in 50 ml of solution as described above, and randomized to each experimental storage group (n 5to 6). A radial strip of tissue was removed from each fresh control leaflet and experimental leaflet sample after the rewarming and cryoprotectant removal procedure described above. Each strip was cut using uniformly spaced blades to obtain a width of 4.5 mm. The thickness of each sample was measured using a custom-designed current sensing micrometer. After samples were dissected they were immediately placed in cold PBS until mechanical testing. The biomechanical test methods were based upon published testing and calculation methods [25 27]. Tests were conducted on a soft tissue mechanical testing system (Bose ELF 3200; Bose Corporation, Eden Prairie, MN), exposing the tissue uniaxially to tension until failure. Samples were tested at room temperature and kept hydrated during testing using PBS. An initial tare load of 0.02N was applied to the tissue for preconditioning and the sample was loaded at a rate of 10 mm/minute until failure. Failure was considered to be a 20% reduction in force. Data were recorded as force versus displacement. The initial gauge length of the specimen was measured after preconditioning and used for calculating strain. The initial gauge length was approximately 9.0 mm for the samples tested. The initial cross-sectional area measured during dissection was used for stress calculations. Young s modulus (E) was calculated as the slope of the linear elastic portion of the stress-strain

3 Ann Thorac Surg BROCKBANK ET AL 2011;91: HEART VALVE STORAGE AT 80 C 1831 Fig 1. Gross examination of heart valves in 100 ml volumes at 135 C while in the storage freezer. The VS55 (A) sample shows extensive cracking throughout the cryoprotectant solution while the VS83 (B) sample remains grossly intact. In only one case the cracks observed with VS55 at 135 C damaged the muscle band of the heart valve (not shown). curve. Ultimate force (F max ), stress ( max ), and strain (ε max ) were also determined for each sample. Viability Assessment Fresh samples of the three main tissue types in each heart valve were taken for viability assessment prior to cryopreservation. Cryopreserved sample viability assessments were initiated within an hour of completion of the rewarming and cryoprotectant elution protocol. Valve tissue samples were incubated for 3 hours under cell culture conditions in media containing alamarblue as previously described [28, 29]. The alamarblue assay (Invitrogen, Carlsbad, CA) utilizes a water soluble fluorometric viability indicator based on the detection of metabolic activity, specifically an oxidation-reduction indicator which both fluoresces and changes color in response to chemical reduction of the growth medium caused by cell metabolism. Samples were read on a spectrofluorometer at 590 nm. The data are expressed as relative fluorescent units /mg dry weight of tissue. Multiphoton and Second Harmonic Generation (SHG) Imaging Using unprocessed fresh and vitrified heart valves, extracellular matrix (ECM) quality of leaflet, artery, and cardiac muscle was assessed by multiphoton-excited autofluorescence and SHG microscopy as described before in detail [3]. Using these imaging modalities, collagen and elastic fibers can be nondestructively detected at two different laser excitation wavelengths [31]. Exposure to laser pulses at 760 nm reveal two-photon excited autofluorescent elastic fibers. Imaging of the same intratissue regions at 840 nm shows collagen fibers. Due to femtosecond laser-induced SHG processes, these structures emit blue light at a peak emission wavelength of 420 nm, which can then serve as a quantitative measure of structural preservation or damage of the vitrified cardiac tissues [30]. Noninvasive optical horizontal sections of four different areas of each of the specimens were taken at depths of 10 and 20 m on each side of the leaflet, the intimal surface of the aortic artery, and the associated cardiac muscle. A semiquantitative scale was employed to assess SHG intensities (detected as grey value intensities as a measure of tissue structure integrity as previously described [31]. A qualitative mean rating scale (1 being complete destruction of the majority of fiber structures and 10 being no structural change of fiber structure) was determined for the elastin and collagen fiber structure using blinded samples. The operator did not know to which group the samples belonged. Differential Scanning Calorimetry Modulated differential scanning calorimeter thermograms (TA Instruments Q1000; TA Instruments, Lukens, DE) were acquired during rewarming of precooled (5 C/ minute from 20 C to 140 C with a 5-minute hold at 140 C) 5- L aliquots of vitrification formulations that were hermetically sealed in aluminum sample pans. The samples were rewarmed from 140 C at 5 C/minute to 20 C. Statistic Analyses Statistical analyses were performed using the Mann- Whitney nonparametric t test or one-way analysis of variance using the nonparametric Kruskal-Wallis method with Dunn s post test. The p values 0.05 or less were considered statistically significant and all data are expressed as mean standard deviation. Fig 2. The graph displays differential scanning calorimetry thermograms for VS55 (upper trace) and VS83 (lower trace) during warming from 140 C to 20 C at 5 C/minute after rapid cooling (approximately 40 C/minute). The thermogram for VS55 shows glass transition at 123 C followed by a devitrification peak at 62 C and subsequent melt at 42 C. The devitrification peak at 62 C is indicative of appreciable ice crystallization in the sample at this clinically relevant warming rate. Conversely, devitrification is not observed in the VS83 sample after a glass transition at 119 C.

4 1832 BROCKBANK ET AL Ann Thorac Surg HEART VALVE STORAGE AT 80 C 2011;91: Fig 3. Representative stress-strain curve, derived from cryopreserved sample No. 5 after storage at 80 C, from which ultimate stress, ultimate strain, and Young s Modulus were calculated. Results The experiments in this report were initiated because of three observations: (1) Cracking was observed in experiments involving storage of heart valves in VS55 below the solution glass transition temperature (Fig 1A); (2) substitution of VS83 for VS55 resulted in crack-free samples during storage of heart valves at 135 C (Fig 1B); and (3) differential scanning calorimetry studies demonstrated that VS55 was subject to ice formation and melting upon rewarming at 5 C/minute. In contrast, no ice formation or melting was observed during rewarming with VS83 at 5 C/minute. Representative thermograms are shown in Figure 2. The glass transition for VS55 was approximately 123 C and VS83 was approximately 119 C. Biomechanical testing was performed on fresh untreated control leaflets and leaflets cryopreserved and stored at 80 C and 135 C. A representative stressstrain curve for the tested samples is shown in Figure 3 illustrating how Young s modulus and ultimate stress (strain) were measured. There were no significant differences observed between the 80 C and 135 C stored leaflets for any of the mechanical test comparisons, similarly neither group differed significantly when compared with the fresh untreated control leaflet samples (Table 1). We also assessed the impact of VS83 preservation upon cell viability and ECM integrity at two storage temperatures, 80 C and 135 C. The viability results of leaflet, aortic artery tissues, and cardiac muscle from fresh samples compared with the two groups of cryopreserved heart valves are presented in Figure 4. Accordingly, both groups of cryopreserved tissue samples demonstrated significantly less viability than fresh samples (p 0.01). Viability was especially low in the tissues that were stored at 80 C. Leaflet, artery, and muscle retained only 1.4%, 12.7%, and 29.9% viability compared with fresh unprocessed tissues, while tissues stored at 135 C retained 3.6%, 16.4%, and 43.7% viability, respectively. There were no significant differences in viability for cryopreserved groups stored at 80 C and 135 C (Fig 4). Multiphoton imaging of VS83-preserved heart valves stored at 80 and 135 C demonstrated branched elastic fibers and wavy bundles of collagen in almost all vitrified heart valve leaflets, aortic trunk, and cardiac muscle specimens (Fig 5). There were no significant differences in SHG signal intensities between valve tissues stored at the two temperatures (Table 2). In addition, no significant differences were observed based on the qualitative mean rating scores on tissue quality determined by the operator during imaging. The mean scores were and for 80 C and 135 C stored valves, respectively. Comment We have previously observed little or no ice formation in cardiovascular tissues, blood vessels, and heart valves preserved in VS55, using sample volumes of less than 10 ml [13, 16, 17]. However, when volumes 10 ml or greater were employed, sporadic gross cracking of the cryoprotectant solution was seen during 135 C storage. The cracking rarely involved the tissues, but gave rise to concerns about tissue cracking that might occur during shipping in nitrogen-cooled dry shippers, where much lower temperatures are achieved and the cryoprotectant solution would become more brittle. Due to this concern during vapor phase nitrogen storage and because we had discovered that VS83 reduced ice formation and increased chondrocyte viability in vitrified porcine articular cartilage [23], we decided to assess VS83 for heart valve storage. We studied rewarming events using differential scanning calorimetry at relatively slow cooling and warming rates (approximately 5 C/minute), simulating the rates achieved for a large sample volume employed for heart valve preservation. We observed devitrification, due to ice formation, and melting phenomena in VS55. Neither phenomenon occurred in VS83. These observa- Table 1. Summary of the Biomechanical Test Results Group Ultimate Force F max (N) Ultimate Stress max (MPa) Ultimate Strain ε max Young s Modulus E (MPa) Control a o C o C a Data presented as the means 1 standard deviation, n 5-6, of each individual sample mean. There were no significant differences.

5 Ann Thorac Surg BROCKBANK ET AL 2011;91: HEART VALVE STORAGE AT 80 C 1833 Fig 4. Comparison of viability in (A) leaflet and (B) aortic tissues as well as cardiac muscle of fresh (n 12) and VS83-preserved aortic heart valves stored at 80 C and 135 C (each n 6). Data are presented as mean standard error. (,, p 0.01 versus fresh control valves). tions suggested that we might be able to store tissues in VS83 at 80 C, above the glass transition temperature. Therefore, we studied the impact of storage in VS83 at 80 C and 135 C upon cell viability, material properties, and ECM component integrity of heart valve tissues. Our results demonstrated no significant differences in material properties, ECM preservation, or cell viability for valves stored in the 83% vitrification formulation at each temperature. This was in great contrast to our recent results on cartilage preservation where the best cell viability was observed using VS83 solution and storage at 135 C [23]. We hypothesize that these results are due to differences in the cryoprotectant permeability based on the very diverse anatomy and composition of the cartilage versus cardiovascular tissue ECM. This hypothesis is supported by the tissue viability results in which the thinner leaflet tissues demonstrated much lower viability than the aortic artery samples, which are intermediate in thickness, and the cardiac muscle which is thickest. The residual viability in leaflet and artery samples is believed to be due to survival of small numbers of interstitial cells. Preliminary scanning electron microscopy studies indicated that heart valve tissues exposed to VS83 were devoid of endothelial cells [32]. Low cell viability may translate into reduced immunogenicity in vivo. If the storage temperature is below the glass transition point of the cryopreservation solution (approximately 123 C for DMSO-based cryoprotectant formulations), little, if any, change occurs in biological materials [33, 34]. It has been demonstrated that traditionally frozen cryopreserved human heart valve leaflets retain their ability to synthesize proteins for at least 2 years when stored below 135 C (35). However, degradative processes may occur at and above the solution s glass transition temperature. The American Association of Tissue Banks Standards for Tissue Banking [7] states that cryopreserved cardiac allografts shall be maintained at temperatures of 100 C or colder. Heart valves are usually stored at 135 C or less in vapor phase nitrogen. Most heart valves are transported using dry shippers that maintain Fig 5. Overlays of multiphoton-induced autofluorescence images showing VS83-preserved heart valve tissue components, stored at either 80 C or 135 C, depicting collagen structures (red, 840 nm) and elastic fibers (green, 760 nm). Scale bar equals 40 m.

6 1834 BROCKBANK ET AL Ann Thorac Surg HEART VALVE STORAGE AT 80 C 2011;91: Table 2. Summary of Second Harmonic Generation Signal Intensities of Collagenous Structures in Heart Valve Tissues Detected By Spectral Fingerprinting After VS83 Preservation at Either 80 C or 135 C Storage Temperature Leaflet-Arterialis Leaflet-Ventricularis Artery Muscle 80 C C a a Data presented as the means 1 standard deviation, n 4, of individual sample mean maximal intensity emission values detected at peak emission wavelengths of 414 to 425 nm. There were no significant differences. low vapor phase nitrogen temperatures, in the range of 160 C. These containment devices are expensive, and the costs for two-way shipping are significant due to their size and weight. Considerable savings could be realized if mechanical freezers and dry ice shippers could be employed for storage and transport at approximately 80 C. In traditionally frozen heart valves, 10% DMSO in fetal bovine serum-containing culture medium retains significant cell viability [29]. We have previously shown that the cells in cryopreserved frozen human heart valve leaflets may be negatively affected by prolonged storage at 80 C for periods greater than 1 week [35]. A more recent paper demonstrated that transient warming from vapor phase nitrogen temperatures from 147 C to 47 C and back to vapor phase nitrogen storage resulted in a loss of collagen type I as well as cell viability [36].We have also previously demonstrated that better ECM structure preservation occurs when heart valves are vitrified compared with traditionally frozen valves [30]. Here, we compared the ECM of VS83-preserved porcine valves stored at either 80 C or 135 C. Although the detected SHG signals of the vitrified tissues were generally weaker than in previously measured valves [30], SHG signals were comparable and similarly inducible within the 80 C and 135 C stored tissues. Preliminary histochemistry results comparing traditionally frozen, VS83-preserved, and fresh untreated pig valves indicate that proteoglycans are also preserved in VS83-treated valves stored at 80 C [37]. The potential advantages of VS83 heart valve storage at 80 C includes reduced infrastructural needs for preservation, storage, and shipping in comparison with traditional freezing methods while maintaining ECM integrity and material properties. The loss of cell viability may also be a potential benefit due to reduction of immunogenicity (studies in progress). The typical methods employed for cryopreserved heart valves require a control rate freezer, storage in nitrogen-cooled tanks, and a continuous supply of liquid nitrogen. Furthermore, traditionally cryopreserved valves also require shipment in nitrogen dry shippers to the implantation site where the valves need to be kept in nitrogen-cooled freezers until implantation. Liquid nitrogen is also a safety hazard for employees and precautions must be taken during operation of nitrogen-cooled equipment. In contrast, no expensive equipment and liquid nitrogen are needed for the VS83 storage method used in this study. The only equipment required was a 80 C storage freezer and for a short amount of time a 135 C mechanical storage freezer. Shipping should be achievable using an insulated box with dry ice. The removal of liquid nitrogen from the process also reduces the training required and safety hazards for employees. In the future we anticipate removal of the 135 C step in the initial cooling procedure in order to further reduce the complexity of this method. In vivo large animal studies of VS83 preservation combined with 80 C storage employing pulmonary valves in juvenile sheep have already demonstrated better function and explant pathology than traditionally frozen cryopreserved heart valves [38]. This included minimal T-cell mediated inflammation of the valve stroma in comparison with frozen valve explants [38] suggesting that there is an immunologic benefit associated with low cell viability. Similar results have been obtained for aortic valves (unpublished data). Based upon the present pig data and the in vivo sheep study [38], it is anticipated that human valves will react in the same way. In vitro investigation of human cardiovascular tissues has been initiated in anticipation of future clinical trials. The VS83 preservation combined with 80 C storage method should be particularly beneficial in developing countries with limited financial and logistic resources that have a high incidence of aortic heart valve disease. This method may also facilitate storage and distribution of tissueengineered heart valves and other allogeneic or engineered tissues. The authors are grateful to the staff and owners of Burbage Meats, Ravenel, SC, for donating tissues. These studies were supported in part by U.S. Public Health Grant #2R44HL59731 (to Dr Brockbank) from the National Heart Lung Blood Institute of the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Heart Lung Blood Institute or the National Institutes of Health. The authors are also grateful for the financial support of the Fraunhofer-Gesellschaft Internal Programs (Grant No. Attract [to Dr Schenke-Layland]). References 1. Ross D. Homograft replacement of the aortic valve. Lancet 1962;12: O Brien MF, Stafford EG, Gardner MA, Pohlner PG, Mc- Giffin DC. 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