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J Thorac Cardiovasc Surg 1994;108:583-586
© 1994 Mosby, Inc.
SURGERY FOR ACQUIRED HEART DISEASE |
Kyoto, Japan
From the Department of Cardiovascular Surgery, Faculty of Medicine, Kyoto University, Kyoto, Japan.
Received for publication Nov. 9, 1993. Accepted for publication Feb. 4, 1994. Address for reprints: Shogo Nakayama, MD, Department of Cardiovascular Surgery, Osaka Red Cross Hospital, 5-53, Fudegasaki-cho, Tennoji-ku, Osaka, 543, Japan.
Abstract
The purpose of this study was to estimate the protective effects of fetal bovine serum on the cryopreservation of aortic valve tissues. Ten porcine aortic valves were divided into two groups according to the nutrient content of the medium used for sterilization and cryopreservation. Group A valves were preserved in Dulbecco's modified Eagle medium containing 10% fetal bovine serum, whereas group B valves were preserved in Dulbecco's modified Eagle medium alone. The fibroblast viability of the cryopreserved tissues was assessed autoradiographically with the use of tritiated proline at the following points: fresh control, after sterilization, and after storage for 1 week or 3 months. The proportion of viable fibroblasts decreased to a similar extent in both groups of tissues after 1 week of storage, 81.1%±2.5% in group A and 80.4%±3.3% in group B, and after 3 months of storage it decreased slightly to around 73% for both groups of tissues. No significant difference was observed between the two groups at any time. These results suggest that fibroblasts are adequately preserved even after 3 months of cryopreservation, and that the addition of fetal bovine serum to the medium does not further improve their viability. Thus, from a point of view of fibroblast viability, it is not necessary to use fetal bovine serum for the cryopreservation of aortic valve tissues. (J THORACCARDIOVASCSURG1994;108:583-6)
Since their introduction by Ross 
1 and Barratt-Boyes 2 in 1962, aortic valve allografts have been extensively used for aortic valve replacement, aortic root replacement, 3,4 and relief of right ventricular outflow obstruction. 5 Recently, to maintain tissue viability and better preserve the aortic valves, cryopreservation techniques have been widely adopted, although the methods for optimal tissue preservation have not been clearly defined. Though approximately 10% to 20% fetal bovine serum (FBS) is added to the freezing medium in many institutes, the necessity for FBS remains a topic of controversy because of its heterologous antigenicity. 6 In this study, we investigated the viability of aortic valve fibroblasts after sterilization and cryopreservation in nutrient media with FBS either added or omitted. To assess the viability of cryopreserved valves, we measured the incorporation of tritiated proline into fibroblasts with the use of autoradiography.
MATERIAL AND METHODS
Sterilization and cryopreservation procedures
Ten porcine aortic valves were used in this study. The aortic valves were divided into two groups such that half of them were preserved in Dulbecco's modified Eagle medium (DMEM; Nissui Seiyaku, Tokyo, Japan) containing 10% FBS (group A; n = 5) and the remainder were preserved in DMEM alone (group B; n = 5). The porcine hearts were procured at a slaughterhouse and the aortic valves were excised immediately on site. The excised aortic valves were cut into several parts. One part was taken as a fresh control without any treatment. The remaining tissues were sterilized by incubation at 37° C for 6 hours in the nutrient medium as mentioned previously, which contained low concentrations of antibiotics (240 µg/ml cefoxitin, 100 µg/ml imipenem-cilastatin, 50 unit/ml penicillin G, and 50 µg/ml streptomycin). The aortic valves were then frozen (model CMS-450A; Cryo Med, Mount Clemens, Mich.) to -80° C at a controlled rate of -1° C per minute in the nutrient medium supplemented with 10% dimethyl sulfoxide as a cryoprotective agent to which FBS was added or from which it was omitted. The tissues were stored in the vapor phase of liquid nitrogen. After storage for 1 week or 3 months, the valves were rapidly thawed in 37° C water and the dimethyl sulfoxide was removed by a 4 times stepwise dilution. The viability of the cryopreserved valves was evaluated by the fibroblast viability test and histologic examination.
Test for fibroblast viability
Fibroblast viability was evaluated autoradiographically by the method of van der Kamp and associates. 7 In brief, the aortic valves were incubated in DMEM supplemented with 10% FBS and 15 µCi/ml tritiated proline at 37° C for 5 hours. After incubation, the samples were fixed with 10% formalin and then processed for autoradiography. After samples were stained by hematoxylin and eosin stain, labeled fibroblasts were determined microscopically (x400) in five random fields of view for each valve piece. Fibroblast viability was calculated by dividing the number of labeled fibroblasts by the total number of fibroblasts. These measurements were made immediately after sterilization and after storage for 1 week or 3 months. The proportion of viable fibroblasts at each test period was expressed as a percentage of the fibroblast viability of fresh control tissue.
Histologic examination
After fresh control and stored valves were fixed in 10% formalin, paraffin sections were prepared and stained with hematoxylin and eosin and elastic van Gieson's stains. The histologic changes to the tissues were evaluated by light microscopy.
Statistical analysis
All values were expressed as the mean plus or minus the standard error. The statistical significance was determined with the Student's t test. A p value less than 0.05 was considered significant.
RESULTS
Fibroblast viability
The fibroblast viability of the fresh control tissue was 97.9% ± 0.2% in group A and 97.9% ± 0.4% in group B. The cells that did not take up tritiated proline were considered to be in the mitosis phase.
Fig. 1 illustrates the change in the proportion of viable fibroblasts in aortic valves stored in the presence (group A) or absence (group B) of FBS after sterilization and the two periods of cryopreservation. After sterilization, the proportion of viable fibroblasts decreased to 96.0% ± 1.1% in group A (p < 0.01) and 95.8% ± 1.2% in group B (p < 0.01). After 1 week of storage, fibroblast viability decreased further to 81.1% ± 2.5% in group A (p < 0.01) and 80.4% ± 3.3% in group B (p < 0.01). After 3 months of storage, the fibroblast viability declined slightly to 73.3% ± 2.4% in group A and 72.9% ± 2.5% in group B. No significant difference was observed between the two groups at any time.
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Cryopreservation has been a currently accepted technique for the preservation of aortic valve tissues. O'Brien and associates 8,9 reported that viable cryopreserved allografts showed excellent durability whereas nonviable grafts tended to degenerate in the long run. Therefore cellular viability of aortic valve allografts at implantation is considered to be one of the important factors that ensure long-term graft function. 10,11 Our results show that the fibroblasts in aortic valves remain viable even after 3 months of storage. However, there is no direct scientific evidence that cellular viability directly contributes to leaflet durability, and conflicts still exist regarding the importance of cellular viability for long-term allograft durability. Thus, at this stage, the significance of cellular viability could be viewed as a quantitative marker for successful cryopreservation, which may or may not correlate with graft survival.
Because FBS contains numerous proteins, hormonal substances, and growth factors, its addition to the nutrient medium is considered essential for the maintenance of cell growth in cell culture. FBS is also thought to contribute to the cryopreservation of aortic valve allografts, because it probably helps to balance the oncotic pressure and minimize the dilution shock that compromises cell viability. 12 However, FBS could be detrimental to the grafting process because it introduces heterologous antigenicity, and thus some institutions use high-molecular-weight colloid substitutes (for example, albumin, pasteurized plasma protein fraction, human serum) instead of FBS for cryopreservation of aortic valve allografts. 13 We have also shown that the addition of human serum to the medium does not increase fibroblast viability above that provided by 10% FBS (unpublished data).
Although our results showed no beneficial effect of FBS on fibroblast viability, at the present time it is difficult to apply these results to the clinical situation or to long-term allograft preservation. In a similar manner, some other factors such as warm ischemic time and the method of sterilization are known to decrease fibroblast viability. 14,15 Further investigation into the cryopreservation method, especially in vivo, is required.
Our results have demonstrated that the fibroblast viability of cryopreserved aortic valves is maintained to a similar extent in both serum-free medium and medium supplemented with 10% FBS. Consequently, we suggest that it is not necessary to add FBS to the cryopreservation medium from a point of view of fibroblast viability and that the use of FBS should be reviewed regarding preservation of aortic valve tissues designated for allografting.
Acknowledgments
We thank Mr. M. Fujioka for performing the autoradiographic procedures and Dr. K. Nishimura for revising the manuscript.
References
This article has been cited by other articles:
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  A. Ohkado, M. Hachida, H. Furukawa, H. Lu, N. Hanayama, H. Hoshi, and H. Koyanagi Should the Aortic Valve Homograft Be Recryopreserved? Ann. Thorac. Surg., April 1, 1998; 65(4): 1083 - 1086. [Abstract] [Full Text] [PDF]
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 Y. C. Song, L. Y. Yao, J. M. Kneebone, and F. M. Lupinetti EFFECT OF CRYOPRESERVATION AND HISTOCOMPATIBILITY ON TYPE I PROCOLLAGEN GENE EXPRESSION IN AORTIC VALVE GRAFTS J. Thorac. Cardiovasc. Surg., September 1, 1997; 114(3): 421 - 427. [Abstract] [Full Text]
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