Extracellular nucleotide metabolism controls thrombosis and inflammation and may affect degeneration and calcification of aortic valve prostheses. We evaluated the effect of different decellularization strategies on enzyme activities involved in extracellular nucleotide metabolism. Porcine valves were tested intact or decellularized either by detergent treatment or hypotonic lysis and nuclease digestion. The rates of ATP hydrolysis, AMP hydrolysis, and adenosine deamination were estimated by incubation of aorta or valve leaflet sections with substrates followed by HPLC analysis. We demonstrated relatively high activities of ecto-enzymes on porcine valve as compared to the aortic wall. Hypotonic lysis/nuclease digestion preserved >80 % of ATP and AMP hydrolytic activity but reduced adenosine deamination to <10 %. Detergent decellularization completely removed (<5 %) all these activities. These results demonstrate high intensity of extracellular nucleotide metabolism on valve surface and indicate that various valve decellularization techniques differently affect ecto-enzyme activities that could be important in the development of improved valve prostheses.

Drs Heacox and Goldstein disclose that they have financial relationships with CryoLife, Inc. Drs Heacox and Goldstein disclose that they have financial relationships with CryoLife, Inc. The article by Brockbank and colleagues [1Brockbank K.G.M. Wright G.J. Yao H. et al.Allogenic heart storage above the glass transition at −80°C.Ann Thorac Surg. 2011; 91: 1829-1835Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar] identifies a streamlined method of storing heart valves. Because porcine valves were used as the model, we find the title misleading. Furthermore, the model may be inappropriate because differences in the stability of cryopreserved porcine and human aortic valve matrix have been noted [2Gerson C. Goldstein S. Heacox A.E. Retained structural integrity of collagen and elastin within cryopreserved human heart valve tissue by two-photon laser scanning confocal microscopy.Cryobiology. 2009; 59: 171-179Crossref PubMed Scopus (36) Google Scholar, 3Schenke-Layland K. Madershanian N. Riemann I. et al.Impact of Cryopreservation on Extracellular Matrix structures of Heart Valve Leaflets.Ann Thorac Surg. 2006; 81: 918-926Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 4Brockbank K.G.M. Heacox A.E. Schenke-Layland K. Guidance for the removal of fetal bovine serum from cryopreserved heart valve processing.Cells Tissues Organs. 2011; 193: 264-273Crossref PubMed Scopus (20) Google Scholar]. Although systems maintaining −80°C are widely available, suitability of this environment for long-term storage is questionable. Solutions with high cryoprotectant concentrations are not stable above their glass transition temperature [5Vigier G. Vassoille R. Ice nucleation and crystallization in water-glycerol mixtures.Cryobiology. 1987; 24: 345-354Crossref Scopus (29) Google Scholar] and tend to accumulate crystalline ice over time, which is potentially damaging to biologic structures. There is evidence for this phenomenon with the suggested storage medium as the authors report viable cells in valves stored at −80°C for 30 days. However, sheep valves stored in the same solution and conditions for 1 year were not viable [6Lisy M. Pennecke J. Brockbank K.G.M. et al.The performance of ice-free heart valve allografts in an orthotopic pulmonary sheep model.Biomaterials. 2010; 31: 5306-5311Crossref PubMed Scopus (32) Google Scholar]. This temporal difference could indicate cryoprotectant toxicity or matrix instability during storage. The safety and simplicity of this solution and process are also questionable. Formamide is a known teratogen, and valves stored in it might not be suitable to implant in women of childbearing age. Ultracold mechanical freezers are expensive, are subject to temperature fluctuations, recover slowly, and require liquid nitrogen backup against power outages. Tighter restrictions on airline transport of dry ice may limit distribution. Finally, the proven shelf life of such tissues is critical, as the time required to obtain clearance on donor and tissue safety factors generally exceeds 30 days. Stable long-term storage of heart valve allografts is necessary. Cryopreserved valves retain structure and cellular activity for more than 5 years [7Mirabet V. Carda C. Solves P. et al.Long-term storage in liquid nitrogen does not affect cell viability in cardiac valve allografts.Cryobiology. 2008; 57: 113-121Crossref PubMed Scopus (30) Google Scholar], and their use has been shown over the past 30 years to provide for comparable long-term functional outcomes and patient survival when compared with nonfrozen, antibiotic-stored valves. The method presented by Brockbank and colleagues needs verification. ReplyThe Annals of Thoracic SurgeryVol. 93Issue 2PreviewUpon first reading the letter by Heacox and Goldstein [1], we felt concern that a major error had occurred in our recent paper [2]. Upon review we find that human valves were not mentioned in the title and it was clearly stated in the abstract and main text that porcine valves were used. Full-Text PDF

SynerGraft® (SG) decellularized-cryopreserved cardiac valve allografts have been developed to provide a valve replacement option that has reduced antigenicity, retained structural integrity, and the ability to be stored long-term until needed for implantation. However, it is critical to ensure that both the SG processing and cryopreservation of these allografts do not detrimentally affect the extracellular matrix architecture within the tissue. This study evaluates the effects of SG decellularization and subsequent cryopreservation on the extracellular matrix integrity of allograft heart valves. Human aortic and pulmonary valves were trisected, with one-third of each either left fresh (no further processing after dissection), decellularized, or decellularized and cryopreserved. Two-photon laser scanning confocal microscopy was used to visualize collagen and elastin in leaflets and conduits. The optimized percent laser transmission (OPLT) required for full dynamic range imaging of each site was determined, and changes in OPLT were used to infer changes in collagen and elastin signal intensity. Collagen fiber crimp period and collagen and elastin fiber diameter were measured in leaflet tissue. Statistically significant differences in OPLT and the dimensional characteristics of collagen and elastin in study groups were determined through single factor ANOVA. The majority of donor-aggregated average OPLT observations showed no statistically significant differences among all groups, indicating no difference in collagen or elastin signal strength. Morphometric analysis of collagen and elastin fibers revealed no significant alterations in treated leaflet tissues relative to fresh tissues. Collagen and elastin structural integrity within allograft heart valves are maintained through SynerGraft® decellularization and subsequent cryopreservation.

Background. The extracellular matrix plays an important role in heart valve function. To improve the processing of porcine pulmonary valves for clinical use, we have studied the influence of cryopreservation, decellularization, and irradiation on extracellular matrix components.Methods. Decellularization was carried out followed by DNAseI/RNAseA digestion and isotonic washout. Valves were cryopreserved in 10% DMSO/10% fetal bovine serum, and then subjected to 25-40 kGy gamma-radiation. Extracellular matrix constituents were evaluated by histologic staining, immunohistochemistry, transmission electron microscopy, and liquid chromatography/mass spectrometry.Results. Histologic, immunohistochemical, ultrastructural, and biochemical analyses demonstrated a marked reduction in the expression of extracellular matrix components particularly in the valves that had been gamma-irradiated following decellularization and cryopreservation. In this group, histology and immunohistochemistry showed an obvious reduction in staining for chondroitin sulphates, versican, hyaluronan, and collagens. Transmission electron microscopy revealed the smallest fibril diameter of collagen, shortest D-period, and loss of compactness of collagen fiber packaging and fragmentation of elastic fibers. Biochemical analysis showed loss of collagen and elastin crosslinks. Decellularization followed by cryopreservation showed some reduction in staining for collagens and versican, smaller diameter, shorter D-period in collagen fibers, and ridges in elastic fibers. Cryopreservation alone showed minimal changes in ECM staining intensity, collagen, and elastin ultrastructure and biochemistry.Conclusion. gamma-Irradiated valves that have been decellularized and cryopreserved produces significant changes in the expression of ECM components, thus providing useful information for improving valve preparation for clinical use and also some indication as to why irradiated human heart valves were not clinically successful. (C) 2012 Elsevier Inc. All rights reserved.

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.

Biopreservation and BiobankingVol. 8, No. 3 The Experts SpeakWhat Are Three Actionable Strategies to Improve Quality in Biomedical ResearchJim Vaught, John G. Baust, Albert E. Heacox, Peter Riegman, Fay Betsou, Peter Mazur, John M. Baust, Glyn Stacey, and Michael BarnesJim VaughtSearch for more papers by this author, John G. BaustSearch for more papers by this author, Albert E. HeacoxSearch for more papers by this author, Peter RiegmanSearch for more papers by this author, Fay BetsouSearch for more papers by this author, Peter MazurSearch for more papers by this author, John M. BaustSearch for more papers by this author, Glyn StaceySearch for more papers by this author, and Michael BarnesSearch for more papers by this authorPublished Online:29 Sep 2010https://doi.org/10.1089/bio.2010.8315AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookXLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited byMaintaining a Focus on Biobanking Science and Innovation Zisis Kozlakidis and Olivier Vandenberg13 June 2022 | Biopreservation and Biobanking, Vol. 20, No. 3Biochemically Tracked Variability of Blood Plasma Thawed-State Exposure Times in a Multisite Collection Study Yueming Hu, Claire Mulot, Camille Bourreau, Diane Martin, Pierre Laurent-Puig, Loredana Radoï, Pascal Guénel, and Chad R. Borges28 September 2020 | Biopreservation and Biobanking, Vol. 18, No. 5Biobanking: the foundation of personalized medicineCurrent Opinion in Oncology, Vol. 23, No. 1 Volume 8Issue 3Sep 2010 InformationCopyright 2010, Mary Ann Liebert, Inc.To cite this article:Jim Vaught, John G. Baust, Albert E. Heacox, Peter Riegman, Fay Betsou, Peter Mazur, John M. Baust, Glyn Stacey, and Michael Barnes.What Are Three Actionable Strategies to Improve Quality in Biomedical Research.Biopreservation and Biobanking.Sep 2010.121-125.http://doi.org/10.1089/bio.2010.8315Published in Volume: 8 Issue 3: September 29, 2010PDF download

Cryopreservation is commonly used for the long-term storage of heart valve allografts. Despite the excellent hemodynamic performance and durability of cryopreserved allografts, reports have questioned whether cryopreservation affects the valvular structural proteins, collagen and elastin. This study uses two-photon laser scanning confocal microscopy (LSCM) to evaluate the effect of cryopreservation on collagen and elastin integrity within the leaflet and conduit of aortic and pulmonary human heart valves. To permit pairwise comparisons of fresh and cryopreserved tissue, test valves were bisected longitudinally with one segment imaged fresh and the other imaged after cryopreservation and brief storage in liquid nitrogen. Collagen was detected by second harmonic generation (SHG) stimulation and elastin by autofluorescence excitation. Qualitative analysis of all resultant images indicated the maintenance of collagen and elastin structure within leaflet and conduit post-cryopreservation. Analysis of the optimized percent laser transmission (OPLT) required for full dynamic range imaging of collagen and elastin showed that OPLT observations were highly variable among both fresh and cryopreserved samples. Changes in donor-specific average OPLT in response to cryopreservation exhibited no consistent directional trend. The donor-aggregated results predominantly showed no statistically significant change in collagen and elastin average OPLT due to cryopreservation. Since OPLT has an inverse relationship with structural signal intensity, these results indicate that there was largely no statistical difference in collagen and elastin signal strength between fresh and cryopreserved tissue. Overall, this study indicates that the conventional cryopreservation of human heart valve allografts does not detrimentally affect their collagen and elastin structural integrity.