Ann Thorac Surg 1998;65:1083-1086
© 1998 The Society of Thoracic Surgeons
Should the Aortic Valve Homograft Be Recryopreserved?
Akihiko Ohkado, MDa,
Mitsuhiro Hachida, MDa,
Hiroshi Furukawa, MDa,
Hua Lu, MDa,
Naoji Hanayama, MDa,
Hironobu Hoshi, BSa,
Hitoshi Koyanagi, MDa
a Department of Cardiovascular Surgery, Heart Institute of Japan, Tokyo Women’s Medical College, Tokyo, Japan
Accepted for publication November 26, 1997.
Address reprint requests to Dr Ohkado, Department of Cardiovascular Surgery, The Heart Institute of Japan, Tokyo Women’s Medical College, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162, Japan
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Abstract
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Background. The number of homograft donors is limited and the once-thawed homograft may be unsuitable for the recipient and obliged to be wasted. The purpose of this study was to investigate the possibility of recryopreserving and using the once-thawed homograft for another patient.
Methods. Canine aortic valve leaflets were frozen to -80°C by a programmed freezer, stored in liquid nitrogen, and thawed after 1 week. A subgroup of leaflets was left at 4°C for 15 minutes, re-cryopreserved, and thawed after 1 week. Pathologic and flow cytometric evaluations were performed.
Results. After thawing, by pathology, alignment of the fibers was acceptably maintained but the membrane and cytoplasm of the fibroblast were damaged. These findings were not significantly aggravated even after rethawing. By flow cytometry, fibroblast viability was 90.7% ± 1.7% immediately after thawing, 87.6% ± 1.0% after thawing for 15 minutes at 4°C, 63.7% ± 2.7% during refreezing at 0°C, and 39.4% ± 4.3% after rethawing.
Conclusions. From the standpoint of fibroblast viability, it is not possible to recryopreserve the once-cryopreserved and thawed aortic valve homograft.
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Introduction
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Human aortic allografts (homografts) have been used widely for the surgical treatment of heart diseases. However, as is seen in heart transplantation, the number of homograft donors is limited. The circumstance may occur in which a homograft that has been thawed is not suitable for the recipient and is obliged to be wasted. Therefore, it would be an attractive possibility to make use of the once-thawed homograft for another patient after recryopreservation. The purpose of the present study was to investigate the possibility of using a homograft that has been thawed and recryopreserved, by determining the viability of the fibroblasts and the integrity of the connective tissue.
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Material and methods
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The hearts were procured and the aortic valve leaflets were excised from 10 mongrel dogs (10 to 15 kg) under general anesthesia with pentobarbital. All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 85-23, revised 1985). The valve leaflets were irrigated with physiologic saline and placed into cold (4°C) TC-199 culture medium (Life Technologies Inc, Grand Island, NY) [1] and 10% dimethylsulfoxide for 15 minutes. They were frozen at a rate of -1°C/min to -80°C by a programmed freezer (Cryomed model; Forma Scientific Inc, Marietta, OH) and stored in liquid nitrogen (-196°C). One week after cryopreservation, the leaflets were thawed rapidly in a warm bath at 37°C and diluted stepwise in dimethylsulfoxide. A subgroup of the leaflets were left in their unopened package at 4°C for 15 minutes for the simulation experiment of recryopreservation. Then they were frozen again in the same way as before and were thawed 1 week later. No antibiotics were used because of their unknown influences on the experimental model.
Pathology
Histologic evaluation was performed by light and electron microscopy using the specimens obtained from the other 3 dogs at the time of procurement, immediately after the first thawing, and immediately after the second thawing. For light microscopic evaluation (Masson stain), samples were embedded in paraffin according to standard methods. The slices were stained with iron hematoxylin for the nucleus and Pansaw 3R and aniline blue for the fiber (Kanto Kagaku Co Ltd, Tokyo, Japan). They were examined on a standard light microscope. For electron microscopic evaluation, the specimens were fixed in glutaraldehyde and postfixed in osmium tetroxide. Then they were dehydrated and embedded in mixed resin of Polybed 812, nadic methyl anhydrase, dodecenylsuccinic anhydrase, and dimethylaminomethyl phenol (Polysciences Inc, Warrington, PA). Ultrathin sections were examined using a model JOEL JEM-1200EX transmission electron microscope (Nihon Denshi Co Ltd., Tokyo, Japan).
Flow cytometry
Flow cytometric evaluation was performed by the method of Niwaya and associates [2]. In short, the endothelial layer was removed from the valve leaflet using Eagle’s solution containing 0.5% type II collagenase. The fibroblast cells were extracted by collagenase treatment and centrifugation of the manually minced leaflet. They were stained first by fluorescein diacetate and then by propidium iodide, and then were analyzed by flow cytometry (Epics Elite; Coulter Co Ltd, Miami, FL). Cells that were positive for fluorescein diacetate and negative for propidium iodide were considered to be viable. The percentage of viable fibroblasts in relation to the total number of fibroblasts was recorded. Five time points were selected for evaluation: control (at procurement), immediately after thawing, 15 minutes after the thawed leaflets were left at 4°C, when the temperature in the freezer reached 0°C during the second freezing, and immediately after the second thawing. The leaflets were allotted at random so that each leaflet was analyzed at one time point. Eagle’s solution, collagenase, fluorescein diacetate, and propidium iodide were purchased from Coulter Co Ltd, Miami, FL.
Statistical methods
Results are presented as the mean plus or minus the standard error of the mean. Statistical analysis was performed with the one-way analysis of variance. Differences were considered statistically significant at a p value of less than 0.05.
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Results
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Pathology
Light microscopy
At procurement, the endothelial cells and fibroblasts were well preserved (Fig 1). The collagen or elastic fibers maintained their alignment and the fibroblasts appeared morphologically viable. After the first thawing, alignment of the endothelial cells was slightly rough, whereas that of the collagen and elastic fibers was almost maintained. After the second thawing, the morphologic features were almost the same as after the first thawing.
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Fig 1. Light micrographs of an aortic valve homograft at procurement (A), immediately after the first thawing (B), and immediately after the second thawing (C) (Masson stain; x200 before 52% reduction.)
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Electron microscopy
After the first thawing, the fibroblasts showed disruption of their plasma membrane and some degree of structural deterioration of the cytoplasm (Fig 2). However, as was seen with light microscopy, remarkable differences were not observed after the first and second thawing.
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Fig 2. Electron micrographs of an aortic valve homograft at procurement (A), immediately after the first thawing (B), and immediately after the second thawing (C) (x4,000 before 52% reduction.)
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Flow cytometry
Figure 3 illustrates the change in the proportion of viable fibroblasts in the valve leaflets. At procurement, fibroblast viability was 97.1% ± 1.0%. This is consistent with previous reports in which fibroblast viability of the fresh control was reported to be high as 100% [2, 3]. Fibroblast viability was 90.7% ± 1.7% immediately after thawing and 87.7% ± 1.0% after thawing for 15 minutes at 4°C. Therefore, cryopreservation undoubtedly decreased fibroblast viability, but it remained higher than 85%. However, fibroblast viability decreased rapidly during the second freezing, to 63.7% ± 2.7% when the temperature in the freezer reached 0°C (p < 0.001 versus the third time point) and then to only 39.4% ± 4.3% after the second thawing (p < 0.001 versus the third and fourth time points).
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Fig 3. Proportion of viable cells (percentage) by flow cytometry. Measurement time points are as follows: (A) at procurement, (B) after the first thawing, (C) 15 minutes after the thawed leaflets were left at 4°C, (D) at 0°C during recryopreservation, and (E) after the second thawing.
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Comment
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Human allografts have been used extensively for various heart diseases because they have several advantages, including lack of thrombogenicity, resistance to infections such as endocarditis, and optimum hemodynamics [4, 5]. In operations such as aortic valve replacement, adjustment of the annular size of the recipient aortic valve and the homograft valve must be precise to achieve satisfactory operative results, and careful preoperative assessment for optimum sizing as well as matching is always important. Ideally, for example, homografts of various sizes should be available and the homografts should be unpacked just after aortotomy and measurement. However, it may happen that a homograft that has been thawed and removed from its package is not suitable for use because of size mismatch. Although such cases may be clinically rare, it would be convenient and prevent wasting of homografts if a graft that had been thawed once could be recryopreserved and used for another patient. We performed this study to examine this possibility, using canine hearts and evaluating the morphology and viability of the fibroblasts in the valvular leaflets.
Pathologic analysis showed that morphologic changes in the cells and fibers were remarkably few after the first and second thawing, and there was not a significant difference in pathologic findings. However, fibroblast viability measured by flow cytometry showed different results. Although fibroblast viability does not necessarily parallel cellular function accurately, we believe that the degree of its maintenance coincides roughly with the degree of tissue damage. Fibroblast viability, which was changed little after the first thawing or even after sitting for 15 minutes at 4°C, decreased rapidly after the second freezing to only 35%. This finding suggests that, from the standpoint of fibroblast viability, it is not appropriate to refreeze the once-thawed homograft. In vessels such as the aorta, fibroblast viability may not be as important; flexibility of the aorta is not mandatory because the aorta can function as a simple conduit. However, the valvular tissue must have flexibility to function normally. Several reports have emphasized the importance of fibroblast viability of the allograft valve to its long-term durability [6–8]. Our results shows that refreezing of the valve homograft damages fibroblasts. Although there is no conclusive evidence that retained viable fibroblasts in an implanted valve homograft can regenerate, repair, and reproduce fibers, it is clear that the valvular intracellular matrix is damaged extensively by recryopreservation.
The discrepancy between the results of the pathologic and flow cytometric evaluations may be related to the fact that cellular function and morphologic changes do not necessarily coincide. Molecular biological investigation may be necessary to elucidate the mechanism of the effect of refreezing on the fibroblast. It is possible that the structure of the cellular membrane is damaged, leading to the disorder of intracellular metabolism by the repeated stress of thawing and freezing. It also is possible that dimethylsulfoxide loses its effect after the second thawing, allowing the formation of ice crystals in the cell. Finally, the level of high-energy phosphate compounds in the homograft may fall below the level critical for maintaining cellular structure. In addition, we did not use any antibiotics in our model because of their unknown influences. However, in the clinical setting, it is possible that they may cause additional damage during the process of recryopreservation.
In conclusion, at least from the standpoint of fibroblast viability, it is not possible to recryopreserve an aortic valve homograft that has been cryopreserved and thawed.
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Acknowledgments
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We sincerely appreciate and acknowledge Dr Pedro J del Nido, Boston Children’s Hospital, for his assistance in the preparation of the manuscript and Dr Yasunari Sakomura, Department of Cardiology, Heart Institute of Japan, for his assistance in the pathologic assessments.
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References
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- Morgan J.F., Morton H.J., Parker R.C. Nutrition of animal cells in tissue culture. I. Initial studies on a synthetic medium. Proc Soc Exp Biol Med 1950;73:1-9.
- Niwaya K., Sakaguchi H., Kawachi K., et al. Effect of warm ischemia and cryopreservation on cell viability of human allograft valves. Ann Thorac Surg 1995;60:S114-S117.
- Nakayama S., Ban T., Okamoto Y. Fetal bovine serum is not necessary for the cryopreservation of aortic valve tissues. J Thorac Cardiovasc Surg 1994;108:583-586.[Abstract/Free Full Text]
- O’Brien M.F., McGiffin D.C., Stafford E.G., et al. Allograft aortic replacement. Long-term comparative clinical analysis of the viable cryopreserved and antibiotic 4°C stored valve. J Cardiothorac Surg 1991;6(Suppl):534-543.
- Angell W.W., Oury J.H., Lamberti J.J., et al. Durability of the viable aortic allograft. J Thorac Cardiovasc Surg 1989;98:48-56.[Abstract]
- Gonzalez-Lavin L., Spotnitz A.J., Mackenzie J.W., et al. Homograft valve durability: host or donor influence?. Heart Vessels 1990;5:102-106.[Medline]
- O’Brien M.F., Johnston N., Stafford G., et al. A study of cells in the expanded viable cryopreserved allograft valve. J Cardiothorac Surg 1988;3(Suppl):279-287.
- O’Brien M.F., Stafford G., Gardner M., et al. The viable cryopreserved allograft aortic valve. J Cardiac Surg 1987;2(Suppl):153-167.[Medline]
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Abstract
Introduction
