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Ann Thorac Surg 2000;70:792-795
© 2000 The Society of Thoracic Surgeons
a Department of Cardiovascular Surgery, University of Tokushima School of Medicine, Tokushima, Japan
Address reprint requests to Dr Tominaga, Department of Cardiovascular Surgery, University of Tokushima School of Medicine, 2-50-1 Kuramoto, Tokushima, 770-8503 Japan
e-mail: ymasuda{at}clin.med.tokushima-u.ac.jp
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Abstract |
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Methods. Cryopreserved porcine aortic cusps were observed in an unfixed fresh condition with a confocal laser scanning microscope using fluorescent dye. Porcine cusps and cultured human umbilical vein endothelial cells were divided into three groups, including fresh, cold-preserved, and cryopreserved specimens, and cytosolic esterase activity and mitochondrial dehydrogenase activity were analyzed in each.
Results. Confocal laser scanning microscope findings disclosed a widely distributed fluorescence in the cusp. Cytosolic esterase activity within human umbilical vein endothelial cells (28% ± 9.0%) after cryopreservation was significantly less than that it was in the cusps (72% ± 21%). Mitochondrial dehydrogenase activity of cryopreserved human umbilical vein endothelial cells and that of cusps fell to 44% ± 6.1% and 64% ± 17% respectively; the difference between the two values was not significant.
Conclusions. Cryopreservation appeared to produce serious damage to cytosolic and mitochondrial functions of endothelial cells. The cytosolic function of cusps, mainly consisting of fibroblasts, was comparatively preserved after cryopreservation, but mitochondrial function of the cusps was more diminished.
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Introduction |
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The present investigation, undertaken to study the cellular viability of cryopreserved valve, was designed to examine the possibility of two unproved explanations regarding valve failure of cryopreserved allograft. There is a difference of viability between the fibroblast and the endothelial cell, and even more, of the cytosolic function and the mitochondrial function after cryopreservation.
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Material and methods |
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All cusps and HUVECs were further subdivided into three groups: fresh specimens, cold-preserved specimens, and cryopreserved specimens. Fresh specimens were analyzed immediately after preparation according to the techniques described below. The cold-preserved specimens were stored in cold TC-199 culture medium with 5% HEPES buffer, 10% calf serum, and antibiotic agents (240 µg/ml cefazolin, 120 µg/ml lincomycin, 50 µg/ml vancomycin, and 100 µg/ml polymyxin B) at 4°C for 24 hours. Cryopreserved specimens were transferred to TC-199 culture medium with 5% HEPES buffer, 10% calf serum, and 10% dimethyl sulfoxide (DMSO; Fisher Scientific Co, Pittsburgh, PA,).
The specimens were frozen with a programmed freezer (Cryomed model 1050; Forma Scientific, Marietta, OH) that lowered the temperature 1°C per minute down to a temperature of -80°C. The specimens were immediately immersed in the vapor phase of a liquid nitrogen freezer (-196°C). After 24 hours of storage, the specimens were thawed immediately in a 40°C shaken water bath and analyzed.
Confocal laser scanning microscopic observation of whole amounts of cusp
The excised cusps were stained with phosphate buffer solution (PBS) containing 10 µg/ml fluorescent diacetate (FDA) and 5 µg/ml propidium iodide (PI) (Sigma Co, St. Louis, MO) for 40 minutes at 37°C in a 35-mm culture dish. Propidium iodide is known as a dye selective for nuclei [9]. After staining, the cusp were washed twice with fresh PBS solution to remove any free dye. The stained cusps were then mounted on a glass slide and enclosed under a cover glass with 2% N-propyl gallate dissolved in the PBS. The stained cusps were examined with a confocal laser scanning microscope (CLSM) (Leica TCS 4D, Heidelberg, Germany) equipped with a krypton/argon laser with an excitation line of 488 nanometers (nm). A 520-nm (green) band-pass filter was used to identify FDA and a 625-nm (red) band-pass filter to identify PI. Sixteen consecutive optical sections were observed at 40- to 50-µm intervals through the cusp layer at the basal portion. Cells positive for FDA were considered to be viable cells having cytosolic esterase (CE) activity.
Quantitative analysis of cytosolic activity
Cusps and HUVECs in the 35-mm dishes were suspended with PBS containing 10 µg/ml FDA. After staining for 60 minutes at 37°C with the FDA, 2% N-propyl gallate dissolved in 1 ml PBS was added to these dish, and all specimens in the dish were homogenized and centrifuged to collect the supernatant (200 µL). The supernatant was placed into 96-well microplates, and emissions of 520 nm, which caused excitations at 490nm, were measured for quantitative analysis of CE activity using a fluorometer (model MTP-32 microplate reader, Corona Electric, Hitachinaka, Ibaragi, Japan).
Quantitative analysis of mitochondrial activity
Cusps and HUVECs in the 35-mm dishes were suspended with PBS containing 100 µL of prepared tetrazolium salt (WST-8) with 1-methoxy PMS (1-methoxy-5-methylpenazium methylsulfate) (Dojindo, Kumamoto, Japan) for 90 minutes at 37°C in 5% CO2. The tetrazolium assays based on the mitochondrial dehydrogenase (MD) activity resulted in the formation of a colored, water soluble formazan dye. To extract the formazan dye from the cusps and HUVECs, 0.62 N HCl and 0.5% trypsin/EDTA (ethylenediaminetetraacetic acid) were added to each dish. After digestion, the supernatant (200 µL) was placed into 96 well microplates. Optical density for the formazan was read at 415 nm with a reference wavelength of 630 nm.
Statistical analysis
The absorbency and fluorescence values of the prepared specimens were expressed throughout this study as the percentage of the values of the fresh specimens (mean ± SD). The Student t test was used to compare values, and statistical significance was considered present with a p value less than 0.05.
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Results |
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Comment |
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In this study, we used different tissue species and types: porcine aortic valve and HUVECs. To determine the viability of endothelial cells after cryopreservation process, we attempted to isolate fresh endothelial cells of the porcine aortic valve using either Hautchen preparations or ultrasonication. However, we could not exclude the fibroblasts and obtain pure homogeneous endothelial cells, as the isolating process itself would significantly influences cytosolic function, organellar function, or both. In addition, it is known that umbilical vein tissue possesses more growth potential than mature aortic or pulmonary endothelial cells. Therefore, we used commercially available HUVECs and certificated endothelial cells. Because of the difference in cell lines and cross-species in the study design, great caution should be taken in comparing the results of the HUVECs to those of porcine aortic valve.
Conventionally established methods for the evaluation of valvular viability consist of many processes that may cause injuries to the valve not associated with cryopreservation methods themselves [1–3]. Therefore, we tried to evaluate valvular viability in an unfixed fresh tissues. Confocal laser scanning microscopy permits visualization of cells of considerable thickness and provides morphologic information not obtained by conventional fluorescence microscopy. Fluorescent diacetate, a nonpolar ester, passes through living cell membranes and is hydrolyzed by CE. Hydrolyzed FDA exhibits green fluorescence when excited. Viable cells accumulate fluorescence in cytoplasm only [10, 11]. From the observation with CLSM, the activity of the CE of cusps was maintained after cryopreservation. We could not, however, differentiate clearly between the viability of fibroblasts and that of endothelial cells in these tissues.
Propidium iodide has been used to identify nonviable cells. However, a recent article reported that the nonapoptotic cell is also stained by PI, although it differs from the stained pattern of the apoptotic cells [18]. We also observed fresh cusps positive for PI using CLMS. Therefore, in this study, we did not use PI as a marker of the cellular viability. We could not clearly distinguish the endothelial cells and the fibroblasts in every layer of the cusp treated with endothelial cell-specific marker (Ulex europaeus I) by CLSM observation. Fewer endothelial cells than fibroblasts were observed, however. Therefore, we presumed that the green fluorescence observed on CLSM is mainly produced by the fibroblasts of the cusps.
The difference between the CE activity of HUVECs and that of cusps during the storing process suggested that cold-preservation seemed to be slightly harmful only to HUVECs, whereas cryopreservation and the thawing process caused lethal injury to HUVECs, unlike the slight damage those two processes caused to cusps (Fig 2).
Mitochondria are the center of the intracellular energy source. The more mitochondrial function is aggravated by the storing process, the more the cell membrane deteriorates because of energy depletion. We think that mitochondrial damage leads to irreversible cell damage. Therefore, it is important to know the change of the MD activity associated with cryopreservation to be able to assess cryopreserved allograft valve viability.
We used WST-8 tetrazolium salt, a specific indicator of the activity of nicotinamide adenine dinucleotide (phosphate) dehydrogenases in mitochondria. It has now become a standard assay widely used in the determination of cellular viability [12, 13]. In our experiment, cryopreservation reduced the MD activities of both cusps and HUVECs.
Several researchers have studied the viability and function of donor endothelial cells of cryopreserved allograft valves [1–3]. Lupinetti and associates, for example, demonstrated that viable endothelial cells were only observed on 21 of 131 (16%) cryopreserved allografts specimens [3].
Although HUVECs are not endothelial cells of aortic valves, our results with HUVECs appear comparable to the previous reports using endothelial cells [1, 3, 4, 13, 14]. Many articles including ours have reported that loss of endothelial cells may be inevitable even by "better" cryopreservation methods. It has not yet been determined whether the loss of endothelial cells has a positive or negative effect on the long-term structure and function of cryopreserved allograft valves. In some articles, short-course immunosuppression has been recommended to prevent early failure of allograft valves. In another article, it is reported that the usual degeneration was not derived from immunologic responses [5, 6, 15].
On the other hand, Niwaya and associates reported that fibroblast viability of the cryopreserved human allograft valve was well preserved (70%) as measured by flow cytometry when it was exposed to a warm ischemic time of less than 520 minutes [9]. Lupinetti and associates demonstrated that allografts retain a persistent capacity for procollagen synthesis of fibroblast similar to that of the native aortic valve [3]. However, the histologic finding of explanted cryopreserved allografts showed acellularity, rare cellularity, or patchy cellularity of leaflets [2, 16, 17]. Because of conflicting reports, we employed two other methods to assess the viability of the cusp, demonstrating reduced MD activity in comparison with CE activity in cryopreserved cusps. Lu and associates also reported that the MD activity in porcine valves was significantly diminished after cryopreservation processing with the XTT-tetrazolium salt method [13].
In conclusion, this study demonstrates that the cytosolic function of cusps, mainly consisting of fibroblasts, was comparatively preserved after cryopreservation; mitochondrial function, however, was relatively diminished during the process. These results imply that cryopreservation leads to latent injury even in fibroblasts that may cause valve failure after implantation.
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Acknowledgments |
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