Ann Thorac Surg 2006;82:1085-1088
© 2006 The Society of Thoracic Surgeons
New technology
A Novel Method for Preserving Human Lungs Using a Super-Cooling System
Masayoshi Abe, MD, PhDa,*,
Shiro Jimi, PhDd,
Hiroshi Hamac,
Takeshi Shiraishi, MD, PhDb,
Akinori Iwasaki, MD, PhDb,
Nobuhumi Ono, PhDc,
Takayuki Shirakusa, MD, PhDb,
Takeshi Katsuragi, PhDa
a Department of Pharmacology, Fukuoka University, Fukuoka, Japan
b Second Department of Surgery, Fukuoka University, Fukuoka, Japan
c Laboratory for Pathology and Morphology, School of Medicine, Fukuoka University, Fukuoka, Japan
d Department of Drug Information, School of Pharmaceutical Science, Fukuoka University, Fukuoka, Japan
Accepted for publication March 3, 2006.
* Address correspondence to Dr Abe, Department of Pharmacology, School of Medicine, Fukuoka University, Fukuoka, 814-0180 Japan (Email: abemasa{at}fukuoka-u.ac.jp).
 |
Abstract
|
|---|
PURPOSE: To ensure the suitable preservation of isolated lungs, a super-cooling system was used to cool water to temperatures as low as –5°C without freezing.
DESCRIPTION: After lung tissues were obtained from patients with lung cancer, they were kept at –5°C or 4°C for as many as 5 days, and then they were histologically and biochemically examined. To evaluate biochemical stability, tissues after storage were passively sensitized with immunoglobulin E and then incubated with anti-immunoglobulin-E antibody.
EVALUATION: Although tissues preserved at –5°C for 5 days had an almost normal appearance with intact cilia on bronchial epithelium and normal endothelium, tissues stored at 4°C showed degradation of these structures. Single-stranded DNA, a sign of DNA cleavage, was frequently noted in tissues stored at 4°C, but only rarely observed at –5°C. A significant amount of cysteinyl-leukotrienes was generated from tissues stored at –5°C for 3 days, but there was no response to antibody stimulation from tissues stored at 4°C.
CONCLUSIONS: Super-cooling systems may provide useful applications as a novel preserving method.
|
Introduction
|
|---|
In recent years, organ transplantation has been an almost routine treatment modality in clinical practice. However, the shortage of suitable donor organs and lack of methods for stable preservation of isolated organs are hindering the broader use of organ transplantation in the hospital setting. Moreover, there is an increasing need for human tissues to carrying out translational research and to resolve species differences in the pharmacologic responses of humans and animals, endeavors which may lead to the development of a novel drug for use in clinical practice [1]. There have been various reports on storing and preserving tissue samples for research, as well as reports on preservation of clinical usage [2]. Several methods, such as cooling, freezing, adding anti-freezing materials in subzero temperatures, fixing, drying, freeze-drying, the use of enzyme inhibitors, and cryopreservation using dimethyl sulphoxide and other compounds have been used to preserve tissue samples [3, 4]. These forms of storage usually result in poor quality tissues due to cellular lysis and further degradation. In this study we examined a novel method using super cooling to cool water to temperatures as low as –5°C below freezing to preserve human lung tissues in better condition and for longer periods than the previously reported methods [5]. Because freezing causes all cells in tissues to burst [3], a method for keeping tissues at temperatures under –5°C without the addition of exogenous compounds may have promising clinical applications.
|
Technology
|
|---|
Materials
Approval for these experiments was obtained from the ethics committees and medical product agencies of the Medical School Center of Fukuoka University Institutional Review Board on August 4, 2003. Human lungs resected from 5 patients with lung cancer were used in this study after obtaining the patients' informed consents to use these tissues for research. Super cooling was carried out with a refrigeration chamber equipped with a power voltage system ("PROKEPT" [Mebix Corp, Kanda, Tokyo]), which could maintain the temperature at –5°C [5]. The size of a super-cooling system, "PROKEPT" is 700 x 580 x 1,418 mm and the weight is approximately 98 kg. The refrigeration chamber was designed to generate unfrozen conditions below the freezing point, creating a so-called super-cooled state by briefly and stably increasing the electrical pressure to the inside materials. Although the precise mechanism is unknown at present, it is believed that the electrostatic atmosphere inside the energized chamber produces this super-cooling phenomenon by causing microvibrations at the molecular level to the inside contents. To store tissues at 4°C, we simply used a conventional refrigerator (Elegant R [Fujitsu General Corp, Kawasaki, Japan]).
A Novel Preservation of Human Lungs
Five of the patients with lung cancer received a lobectomy (cases 1 to 5). Case 1 involved an 80-year-old man who had had a left lower lobectomy. Case 2 involved a 74-year-old man who had had a right upper and middle lobectomy. Case 3 involved a 51-year-old woman who had already had a right lower lobectomy. A small portion of lung tissue that appeared macroscopically normal was obtained from the lungs of each patient immediately after the surgical resection. The tissues were suspended in Euro-Collins solution with glucose (Kobayashi Pharmaceutical Company, Tokyo, Japan) and were washed to remove blood by keeping them periodically under vacuum with a disposable syringe. Then the tissues for histological study were cut into two pieces and were kept in the super-cooling apparatus at –5°C or in the conventional refrigerator at 4°C for 5 days. Lung tissues of the remaining 2 patients were similarly stored for 3 days and were used for a biochemical study of cysteinyl-leukotriene (cysLT) production, one of the most important mediators in pulmonary inflammation [6]. Case 4 involved a 63-year-old man who had undergone a left lower lobectomy. Case 5 involved a 78-year-old woman who had already had a right upper lobectomy.
Histological Examination
After removal from the refrigerator, the lungs were fixed with 10% buffered formaldehyde solution and were embedded in paraffin. Thin paraffin sections (4 µm in thickness) were serially cut with a microtome. Some sections were subjected to hematoxylin-eosin staining, and the adjacent sections were used for immunohistochemical detection of single-stranded DNA (ssDNA) using anti-ssDNA polyclonal rabbit antibody (x100 dilution) (DakoCytomation, Kyoto, Japan) and Envision/HRP (Dako) (7). In brief, sections in 0.01 M (Mole) phosphate-buffered saline (pH 7.4) were incubated at 70°C for 20 minutes, and then immunohistochemical staining was performed according to the Envision method recommended in the manufacturer's instructions. Finally, nuclei were stained with hematoxylin-eosin.
CysLT Generation From Chopped Lung Fragments
After removal from the refrigerator, the pleura, the large blood vessels, and the bronchi were dissected and cut into small pieces (approximately 1 cm2) with scissors. The chopped lungs were centrifuged at 200 x g for 10 minutes at 4°C. This process was repeated one more time and the tissues were then cut into smaller pieces (2 to 3 mm). These small fragments were incubated overnight (15 to 16 hours) at 22°C with purified human immunoglobulin E (Chemicon International Inc, Temecula, CA) at a final concentration of 10 µg/mL. After this passive sensitization, the fragments were washed with Tyrode's buffer. Each 300 mg of lung fragment was weighed and placed in siliconized tubes on ice. Each incubation mixture containing anti-human immunoglobulin-E antibody (final concentration = 7.75 µg/mL [Chemicon Intl Inc, Temecula, CA]) in Tyrode's buffer was incubated at 37°C for 30 minutes. After addition of cold ethanol, the incubation mixture was centrifuged at 3,000 x g for 30 minutes at 4°C, and the supernatant was then stored at –80°C until analyzed.
CysLT Assay
Cysteinyl-leukotrienes (LTC4, LTD4, and LTE4) in the supernatants were measured using pre-column extraction and reversed-phase high-performance liquid chromatography with a minor modification of a previously reported method [6]. Briefly, after adding 3H-LTE4 as an internal standard and then adjusting to pH 3.0 to 3.5, the samples were loaded onto a Sep-PAK cartridge (Waters, Milford, MA). Methanol-eluted fractions passed through the mini-column and were evaporated under reduced pressure (Speed Vac Concentrator, Savant Instruments, Holbrook, NY). After re-suspension in 150 µL of HPLC solvent, 75 µL were injected onto a Novapak C18, 5 µm column (0.39 x 15 cm) (Waters). The LTC4, LTD4, and LTE4fractions were collected using a fraction collector (Model 201, Gilson, Villiers le Bel, France) and were evaporated under reduced pressure. The residues in the three fractions were collected and analyzed using a cysLT enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI). Cysteinyl-leukotriene values were normalized based on the recovery rates of 3H-LTE4 (31.6 ± 1.0 %; n = 25).
|
Clinical Experience
|
|---|
Results
Three of the patients with lung cancer received a lobectomy (ie, cases 1 to 3). As shown in Figure 1, the lung tissues stored at 4°C for 5 days showed degenerative changes in the alveoli, the bronchi, and the blood vessels (Figs 1A–1C). The bronchial epithelium (arrows) was severely affected (inserted square). In contrast, the tissues stored at –5°C including the alveoli, the bronchi, and the blood vessels were well preserved (Figs 1D–1F). The bronchial epithelium retained normal morphology and apparently intact cilia (inserted square), and the endothelial cells in the pulmonary blood vessels also retained normal morphology. Single-stranded DNA was frequently found in the tissues stored at 4°C (Figs 1A–1C), but was largely absent in the tissues stored at –5°C (Figs 1D–1F). The tissues from the other 2 patients showed similar findings.
View larger version (87K):
[in this window]
[in a new window]
|
Fig 1. Histologic study to compare a novel and conventional preservation of isolated lungs. The lung tissue from each patient with lung cancer (ie, cases 1 to 3) was suspended in Euro-Collins solution and was kept at 4°C or at –5°C for 5 days. Thereafter the tissue was fixed and stained. Representative results of three cases is shown. (A–C) The tissue was stored at 4°C. (D–F) The tissue stored at –5°C were stained with hematoxylin-eosin (HE; left panel) and with anti-single-stranded DNA (ssDNA) antibody (right panel, brown-colored). The lung tissue stored at 4°C showed degenerative changes in (A) the alveoli, (B) the bronchi, and (C) the blood vessels. The bronchial epithelium (arrows) was severely affected (inserted square). In contrast, the tissues stored at –5°C, including (D) the alveoli, (E) the bronchi, and (F) the blood vessels were well preserved (inserted square). (A–C) Single-stranded DNA was frequently found in tissues stored at 4°C, but (D–F) was almost absent in tissues stored at –5°C. Original magnification x200 except for insets (x400).
|
|
Two of the patients with lung cancer received a lobectomy (ie, cases 4 and 5). The lung tissue was suspended in Euro-Collins solution and kept at –5°C or 4°C for 3 days. Thereafter, biochemical stability was evaluated. As shown in Figure 2, the control incubated without anti-immunoglobulin-E antibody showed only a trace amount of cysLT, but with the addition of the antibody cysLT production increased. The addition of the patient's serum at 5% further increased the amount of cysLTs. In contrast, lung tissues stored in the conventional refrigerator showed an abundance of cysLTs, either with or without the addition of anti-immunoglobulin-E antibody. These results suggest that the lung tissue stored in Euro-Collins solution at –5°C for 3 days responded by producing cysLTs after the anaphylactic reaction.
View larger version (13K):
[in this window]
[in a new window]
|
Fig 2. Cysteinyl-leukotriene synthesis in chopped lung fragments caused by anaphylactic reaction. Lung tissue removed from two patients (cases 4 and 5) was suspended in Euro-Collins solution and kept at –5°C (hatched column) or 4°C (black column) for 3 days. Thereafter the lung fragments were cut into small pieces with scissors and then incubated with human immunoglobulin E at 22°C for 15 hours for passive sensitization. After washing with Tyrode's buffer, the passively sensitized lung fragments were stimulated with or without anti-human immunoglobulin-E antibody (7.75 µg/nL) at 37°C for 30 minutes in the presence or absence of the patients' serum (n = 4). After the termination of the reaction, cysLTs in the supernatants were assayed by purification with column chromatography and enzyme immunoassay. Data are reported as the mean ± standard error of the mean. The statistical analysis was performed using analysis of variance, and the Tukey-Kramer test was used for multiple comparisons (*p < 0.05). ( IgE ab = anti-immunoglobulin E-antibody.)
|
|
|
Comment
|
|---|
A super-cooling system can keep tissues suspended in solution at –5°C without the addition of any anti-freezing materials. In comparison with storage in the conventional refrigerator (at 4°C), the morphological changes in the lung tissues were dramatically reduced by storage with the super-cooling system. The lung tissues stored in the conventional refrigerator for 5 days showed numerous cells positive for ssDNA in the whole lung, including alveoli, bronchi, and interstitium. In contrast, these DNA cleavage products were scarcely found in the tissues stored with the super-cooling system for the same periods. These results suggest that the enzymes responsible for the formation of ssDNA may play a role in the postmortem changes of lung tissues. It has been suggested that in apoptosis, single-strand modification precedes the appearance of double-strand breaks and the resultant cell loss may be responsible for severe organ dysfunction [7, 8]. The near absence of ssDNA suggests less frequent apoptosis after organ implantation compared with that observed in the presence of ssDNA [8]. Furthermore, lung tissues that were preserved with this system generated lipid mediators, cysLTs, after specific stimulation. Because the cysLTs are produced by a three-step process through the sequential involvement of phospholipase A2, 5-lipoxygenase, and leukotriene C4 synthase, this suggests that the entire enzymatic reaction remains intact [6]. However, although the lung tissues stored conventionally at 4°C contained a considerable amount of cysLTs, they did not respond to the anaphylactic stimulation. In conclusion, we examined the effect of a super-cooling system on human lung tissue preservation for the first time. Because this system strongly prevented not only morphological and biochemical changes for 3 to 5 days, but also DNA cleavage, results warrant further experiments to confirm the usefulness of this novel super-cooling system in preserving transplantable organs for considerable periods.
|
Disclosures and Freedom of Investigation
|
|---|
This study was supported by a Grant-in Aid for Scientific Research (to MA) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. We borrowed a super-cooling machine, "PROKEPT", from Mebix, Corp, but there was no financial conflict of interest. The home-page website for this manufacturer (ie, Mebix, Corp) is www.mebix.co.jp. The authors have performed a free and independent study of this new technology.
|
Disclaimer
|
|---|
The Society of Thoracic Surgeons, the Southern Thoracic Surgical Association, and The Annals of Thoracic Surgery neither endorse nor discourage use of the new technology described in this article.
|
Acknowledgments
|
|---|
This work was supported in part by a grant from the Ministry of Health, Labor, and Welfare of Japan.
|
References
|
|---|
- Coundouris JA, Grant MH, Engeset J, Petrie JC, Hawksworth GM. Cryopreservation of human adult hepatocytes for use in drug metabolism and toxicity studies Xenobiotica 1993;23:1399-1409.[Medline]
- McLaren AJ, Friend PJ. Trends in organ preservation Transpl Int 2003;16:701-708.[Medline]
- Costanzo JP, Lee RE, DcVries AL, Wang T, Layne JR. Survival mechanisms of vertebrate ectotherms at subfreezing temperaturesapplications in cryomedicine. FASEB J 1995;9:351-358.[Abstract/Free Full Text]
- Bottino R, Balamurugan AN, Bertera S, Pietropaolo M, Trucco M, Piganelli JD. Preservation of human islet cell functional mass by anti-oxidative action of a novel SOD mimic compound Diabetes 2002;51:2561-2567.[Abstract/Free Full Text]
- Monzen K, Hosoda T, Hayashi D, et al. The use of a supercooling refrigerator improves the preservation of organ grafts Biochem Biophys Res Commun 2005;337:534-539.[Medline]
- Murai A, Abe M, Hayashi Y, Sakata N, Katsuragi T, Tanaka K. Comparison study between the mechanisms of allergic asthma amelioration by a cysteinyl-leukotriene type 1 receptor antagonist montelukast and methylprednisolone J Pharmacol Exp Ther 2005;312:432-440.[Abstract/Free Full Text]
- Maeda M, Sugiyama T, Akai F, Jikihara I, Hayashi Y, Takagi H. Single-stranded DNA as an immunocytochemical marker for apoptotic change of ischemia in gerbil hippocampus Neurosci Lett 1998;240:69-72.[Medline]
- Fischer S, Cassivi SD, Xavier AM, et al. Cell death in human lung transplantationapoptosis induction in human lungs during ischemia and after transplantation. Ann Surg 2000;231:424-431.[Medline]
Related Article
-
Invited commentary
- Charles Hoopes
Ann. Thorac. Surg. 2006 82: 1088-1089.
[Extract]
[Full Text]
[PDF]
This article has been cited by other articles:
|
|
|
|
 
C. Hoopes
Invited commentary.
Ann. Thorac. Surg.,
September 1, 2006;
82(3):
1088 - 1089.
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
