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Ann Thorac Surg 1996;61:542-545
© 1996 The Society of Thoracic Surgeons

Original Article: General Thoracic

Trehalose-Containing Solutions Enhance Preservation of Cultured Endothelial Cells

Noritaka Isowa, MD, Shigeki Hitomi, MD, Hiromi Wada, MD

Department of Thoracic Surgery, Chest Disease Research Institute, Kyoto University, Kyoto, Japan

Accepted for publication October 25, 1995.


   Abstract
Top
 Footnotes
 Abstract
Introduction
 Materials and Methods
 Results
 Comment
 References
 
Background and Methods. We have developed two types of preservation solutions containing trehalose. ET-Kyoto solution (ET-K) is an extracellular type and IT-Kyoto solution (IT-K) is an intracellular type. In the present study we examined with in vitro assays the ability of ET-K, IT-K, Euro-Collins (EC), and University of Wisconsin (UW) solutions to preserve a murine endothelial cell line. The viability of cells stored in the solutions at 4°C was determined by trypan blue exclusion and MTT assay.

Results. Trypan blue exclusion showed the viability after 48 hours of cold storage to be 49.5 ± 4.7% (mean ± standard error) in ET-K, 59.5 ± 0.7% in IT-K, 29.2 ± 2.5% in EC, and 55.3 ± 7.6% in UW (ET-K or UW versus EC, p < 0.05; IT-K versus EC, p < 0.01). MTT assay absorbance values for cells after 48 hours of cold storage were 0.366 ± 0.0066 (mean ± standard error) in ET-K, 0.358 ± 0.0044 in IT-K, 0.336 ± 0.011 in EC, and 0.362 ± 0.0019 in UW (ET-K or UW versus EC, p < 0.05). After 120 hours, absorbance values for cells were 0.303 ± 0.0038 in ET-K, 0.269 ± 0.0034 in IT-K, 0.186 ± 0.011 in EC, and 0.265 ± 0.0066 in UW (ET-K versus UW, p < 0.05; ET-K versus IT-K, p < 0.01; ET-K, IT-K or UW versus EC, p < 0.01).

Conclusions. As far as the ability to preserve a murine endothelial cell line at a low temperature was concerned, the ET-K solution was superior to the UW solution, the IT-K solution and UW solution were equal, and the ET-K and IT-K solutions were superior to the EC solution.


   Introduction
Top
 Footnotes
 Abstract
 Introduction
Materials and Methods
 Results
 Comment
 References
 
The lack of supply of donor organs has been a serious problem in the progress of organ transplantation. To perform successful transplantation with an organ that has traveled a long distance, it is crucial to extend the preservation time of donor organs. Therefore much research has been conducted on the improvement of preservation solutions. Trehalose is a nonreducing disaccharide of glucose that can be found in high concentrations in anhydrobiotic organisms [1]. Previously we showed that the Euro-Collins (EC) solution containing trehalose was more effective in preserving canine lungs for 12 hours than the standard EC solution containing glucose [2]. Organ preservation solutions that contain trehalose, gluconate, and hydroxyethyl starch were developed as a result of the above studies, and two solutions have been found to be useful: extracellular fluid-type ET-Kyoto solution (ET-K; Na 100 mmol/L, K 44 mmol/L), and intracellular fluid-type, IT-Kyoto solution (IT-K; Na 20 mmol/L, K 130 mmol/L). These two solutions and the EC solution were used to preserve canine lungs for 20 hours, and the test results showed that the ET-K solution was more effective in preserving the lungs than the other two solutions [3].

The University of Wisconsin (UW) solution is known to be effective in preserving solid organs [4], and the solution is beginning to be used in lung transplantation as well [5]. Therefore, to test the effectiveness of our solutions, we decided to compare the effectiveness of the UW and the ET-K and IT-K solutions.

MTT (C, N-diphenyl-N`-4,5-dimethyl-thiazol-2-yl-tetrazolium bromide) colorimetric assay measures the viability of cells by the amount of tetrazolium salt reduced to formazan crystals by succinate dehydrogenase, a mitochondrial enzyme [6].

F-2 is a murine endothelial cell line established from an ultraviolet light-induced tumor [7]. In the present study, EC solution, UW solution, and the trehalose-containing solutions ET-K and IT-K were examined in vitro for murine endothelial cell preservation by the MTT assay and the trypan blue dye exclusion method.


   Materials and Methods
Top
 Footnotes
 Abstract
 Introduction
 Materials and Methods
Results
 Comment
 References
 
Cells
F-2 is a murine endothelial cell line established from a tumor induced by ultraviolet light on the dorsal skin of a BALB/c x C57BL/6 F1-nu/nu nude mouse [7]. F-2 cells were maintained in 10% fetal calf serum (GIBCO Laboratories Life Technologies Inc, Grand Island, NY) containing Dulbecco's modified Eagle medium (Nissui Pharmaceutical Co Ltd, Tokyo, Japan) in a humidified atmosphere of 5% carbon dioxide at 37°C.

Preservation solutions
Four solutions were evaluated: ET-K, IT-K, EC, and UW (ViaSpan: DuPont Pharmaceuticals, Wilmington, DE) solutions. Their compositions are listed in Table 1Go.


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  		Table 1. . Composition of Preservation Solutions
 
Trypan Blue Exclusion
F-2 cells, 5 x 105/mL of 10% fetal calf serum-supplemented Dulbecco's modified Eagle medium, were plated in six-well plates (Costar, Cambridge, MA) and incubated until confluence in a humidified atmosphere of 5% carbon dioxide at 37°C. Then the culture medium was discarded. Each well was washed twice with each test solution and 3 mL of the solution was added to the well. The six-well plates were incubated at atmospheric pressure and 4°C for 24 to 48 hours. At the end of the preservation period, the cold preservative solution in each well was collected in a 15-mL centrifuge tube (Corning, Corning, NY). One milliliter of a 0.02% EDTA solution containing 0.05% trypsin was added to each well, and the plates were incubated at 37°C for 5 minutes. Then 2 mL of a 10% fetal calf serum–Dulbecco's modified Eagle medium solution was added to each well to inactivate trypsin, and the cultured cells were detached from the well with a pipette. The detached cells were collected in the above mentioned 15-mL centrifuge tube and centrifuged at 1,500 rpm for 5 minutes; the supernatant was discarded. Five hundred microliters of phosphate-buffered saline solution was added to each tube, and the mixture was thoroughly mixed with a pipette. Then 500 µL of phosphate-buffered saline solution containing 0.3% trypan blue was added to each tube and the number of cells was determined with a hemocytometer under a light microscope. At least 300 cells were counted in each specimen, and the viability was defined as the ratio of the number of viable cells to the total number of cells. Three wells were used to test each solution in one experiment, and each experiment was repeated three times.

MTT Assay
F-2 cells, 5 x 105/mL 10% fetal calf serum-supplemented Dulbecco's modified Eagle medium were plated in 96-microwell plates (Costar) and incubated until confluence in a humidified atmosphere of 5% carbon dioxide at 37°C. Then the culture medium was discarded. Each well was washed twice with each test solution, and 100 µL of the solution was added to the well. The 96-microwell plates were incubated at atmospheric pressure and 4°C for 24 to 168 hours. The MTT stock solution was prepared as follows: 25 mg of MTT (Sigma Chemical Co, St. Louis, MO) was dissolved in 5 mL of phosphate-buffered saline solution (5 mg of MTT/mL of phosphate-buffered saline solution) sterilized with a 0.45-µm filter (Millipore Co, Bedford, MA), and stored at 4°C [8]. At the end of the incubation period, the preservative solution was discarded, and 50 µL (10 µg of MTT/50 µL of total medium volume) of the MTT stock solution freshly diluted to 1:25 with Dulbecco's modified Eagle medium (serum free) was added to each well. Five wells without F-2 cells were prepared to serve as background samples. Then the plates were incubated in a humidified atmosphere of 5% carbon dioxide at 37°C for 4 hours [7, 8]. Solubilization of formazan was performed by adding 150 µL of dimethyl sulfoxide (Nacalai Tesque Inc, Kyoto, Japan) with the use of a multichannel pipette. The absorbance of each well at 570 nm was measured with 630 nm as a reference in a scanning microplate spectrophotometer (Immunoreader NJ-2001, Nippon InterMed Inc., Tokyo, Japan). The viability of the cells in each well was calculated by subtracting the absorbance of the background from that of a well. Five wells were used to test each solution in one experiment, and each experiment was repeated three times.

The statistical analysis was performed by the two-tailed unpaired t test. A p value less than 0.05 was considered to be significant. All data are presented as mean and standard error of the mean.


   Results
Top
 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
Comment
 References
 
Trypan Blue Exclusion
The viability after 24 hours of cold storage was 56.2 ± 1.9% in ET-K, 73.7 ± 2.3% in IT-K, 39.9 ± 4.9% in EC, and 62.8 ± 2.3% in UW (ET-K or UW versus EC, p < 0.05; IT-K versus EC, p < 0.01). The viability after 48 hours of cold storage was 49.5 ± 4.7% in ET-K, 59.5 ± 0.7% in IT-K, 29.2 ± 2.5% in EC, and 55.3 ± 7.6% in UW (ET-K or UW versus EC, p < 0.05; IT-K versus EC, p < 0.01) (Fig 1Go). The viability in the EC group was significantly lower than that of the ET-K, IT-K, or UW group. After the 24 hours of storage, the viability in the IT-K group was significantly higher than in the ET-K group; however, the differences among the ET-K, IT-K, and UW groups after 48 hours of storage were not significant.



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  		Fig 1. . In vitro viability of murine endothelial cells, preserved in each solution at 4°C, was assessed by trypan blue exclusion and expressed as a percentage of viable cells. Values shown represent means ± standard error (*p < 0.05 versus Euro-Collins solution; **p < 0.01 versus Euro-Collins solution; EC = Euro-Collins solution; ET-K = ET-Kyoto solution; IT-K = IT-Kyoto solution; UW = University of Wisconsin solution.)

 
MTT Assay
The absorbance after 48 hours of cold storage was 0.366 ± 0.0066 in ET-K, 0.358 ± 0.0044 in IT-K, 0.336 ± 0.011 in EC, and 0.362 ± 0.0019 in UW (ET-K or UW versus EC, p < 0.05). The absorbance after 96 hours of cold storage was 0.335 ± 0.0036 in ET-K, 0.306 ± 0.0042 in IT-K, 0.250 ± 0.012 in EC, and 0.316 ± 0.0078 in UW (ET-K versus IT-K or EC, p < 0.01). The absorbance after 120 hours of cold storage was 0.303 ± 0.0038 in ET-K, 0.269 ± 0.0034 in IT-K, 0.186 ± 0.011 in EC, and 0.265 ± 0.0066 in UW (ET-K versus UW, p < 0.05; ET-K versus IT-K, p < 0.01; ET-K, IT-K, or UW versus EC, p < 0.01) (Fig 2Go). After 48 hours, absorbance in the ET-K and UW groups was significantly higher than in the EC group. After 96 hours, absorbance in the ET-K group was significantly higher than in the IT-K group. After 120 hours, absorbance in the ET-K group was significantly higher than in the UW group. During 24 to 168 hours of storage, there was no significant difference in absorbance between the IT-K and UW groups.



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  		Fig 2. . In vitro viability of murine endothelial cells, preserved in each solution at 4°C, was assessed by MTT assay. Values shown represent means ± standard error. (*p < 0.01 versus Euro-Collins solution; **p < 0.01 versus Euro-Collins solution, IT-Kyoto solution; ***p < 0.01 versus Euro-Collins solution, IT-Kyoto solution, and University of Wisconsin solution; EC = Euro-Collins solution; ET-K = ET-Kyoto solution; IT-K = IT-Kyoto solution; UW = University of Wisconsin solution.)

 

   Comment
Top
 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
 Comment
References
 
Currently, intracellular fluid-type solutions are used most frequently to preserve donor organs. The major differences between the ET-K and IT-K solutions and EC or UW solution are that the former contain trehalose instead of glucose or raffinose and gluconate instead of chloride ion or lactobionate. Moreover, ET-K, IT-K, and UW solutions contain hydroxyethyl starch, whereas the EC solution does not. Trehalose has been reported to stabilize the cell membrane at a low temperature by binding to the polar head of membrane phospholipid [9]. Ischemia-induced intracellular acidosis could encourage an extracellular chloride ion to enter the cell with water molecules, causing intracellular edema [10]. Von Oppell and colleagues [11] reported the superiority of gluconate in extracellular fluid-type solutions. Hydroxyethyl starch can create colloidal osmotic pressure, preventing the expansion of extracellular space [12]. On the basis of these findings, ET-K and IT-K solutions were developed. Their components are almost identical except for their ion concentrations (see Table 1Go).

Many reports have indicated that the extracellular fluid-type of solution is superior to its intracellular fluid-type counterpart for lung preservation [13]. Although prostaglandin E1 or prostacyclin is currently used as a vasodilator to prevent pulmonary vasoconstriction caused by intracellular fluid-type solutions [14], we have shown that an ET-K solution without prostaglandin E1 is more effective in preserving canine lung than are IT-K or EC solutions containing prostaglandin E1 [3]. In the present study the MTT assay proved that in ET-K solution cell viability is greater than in IT-K solution.

The proof of the definite usefulness of a storage solution requires the examination of its ability to preserve organs of animal models. However, it is expensive and time consuming to conduct animal experiments, and it is sometimes difficult to analyze the results because many unidentified factors can influence them. On the other hand, rapid screening is possible with the use of cultured cells, and it is much easier to analyze and readjust the composition of factors as one improves the preservation solution. Therefore, many studies have been conducted on human [11], rat [15], rabbit [16], pig [17], and bovine [18] primary culture cells. Endothelial cells [17] and alveolar type II cells [15] are used for research on lung preservation. An endothelial cell was chosen in this study because it is the only tissue that comes in contact with a storage solution for transplant organs and because lethal interstitial lung edema is induced by damaged endothelial cells. Little work has been done using murine endothelial cell lines, probably because there have been few reports of immortal human endothelial cell lines, and it is possible that a transformed cultured cell line may not reflect the response of in vivo endothelial cell as much as nonspecific cytotoxic response. The advantages of the use of a cell line are that a large quantity of cells can be obtained at a low price and highly reproducible data can be obtained because their characteristics are identical. The F-2 cell line used in this study requires no growth factors and is easily maintained with high growth activity [7].

Testing methods, such as dye exclusion [18], MTT assay [19], 3H-thymidine incorporation [15], lactate dehydrogenase release [15, 16], flow cytometry analysis [20], and ultrastructural analysis [21], are used to evaluate the viability and cytotoxicity of cells. In the present study, the MTT assay and the trypan blue exclusion method were used to evaluate the viability of cells. The term viability means that the integrity of cell membrane is preserved (trypan blue exclusion method) or that the activity of mitochondria is maintained (the MTT assay). Although the trypan blue exclusion method has the advantage of being able to evaluate the viability of cells at any point of time, viable but reproductively dead cells cannot be distinguished from normal viable ones [22]. MTT is reduced by living, metabolically active cells. The amount of purple formazan generated is directly proportional to the cell number, using a homogeneous cell population. The colorimetric reaction is quantified by measuring the optical density at a wavelength of 570 nm with 630 nm as a reference using a spectrophotometer. The advantages of the MTT assay are high sensitivity, simplicity, and high reproducibility. Moreover, one can process many samples at the same time without the need for radioisotopes or expensive equipment. It has been used recently in the screening of anti-cancer agents [8]. In the present study there was a discrepancy between the results of the two methods we used. The viability of cells stored for 24 hours was significantly higher in the IT-K solution than in the ET-K solution when the trypan blue exclusion method was used. However, no significant difference was detected in the MTT assay results. Otherwise, the results of trypan blue exclusion and MTT assay were similar.

Because this study was conducted at atmospheric pressure and at low temperature, it cannot serve as an ischemia–reperfusion injury model. As a result, it can be said that the potential of many of the substances [12] that are included in the UW solution to protect cells from ischemia–reperfusion injuries, was not fully tested. However, the important point is that, although the ET-K solution does not contain any of these additives, its preservation of endothelial cells was superior to that of the UW solution.

In conclusion, ET-K solution was superior to UW solution, the IT-K and UW solutions were equal, and the ET-K and IT-K solutions were superior to EC solution in the preserving of a murine endothelial cell line at low temperature. In the future, by the addition of a variety of substances to the ET-K solution, it is thought that even better storage solutions can be developed.


   Footnotes
Top
 Footnotes
Abstract
 Introduction
 Materials and Methods
 Results
 Comment
 References
 
Address reprint requests to Dr Wada, Department of Thoracic Surgery, Chest Disease Research Institute, Kyoto University, Shogoin Sakyo-ku, Kyoto 606, Japan.


   References
Top
 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
 Comment
 References
 

  1. Crowe JH, Crowe LM. Preservation of membranes in anhydrobiotic organism: the role of trehalose. Science 1984;223:701–3.[Abstract/Free Full Text]
  2. Hirata T, Fukuse T, Liu CJ, et al. Effects of trehalose in canine lung preservation. Surgery 1994;115:102–7.[Medline]
  3. Bando T, Kosaka S, Liu CJ, et al. Effects of newly developed solutions containing trehalose on twenty-hour canine lung preservation. J Thorac Cardiovasc Surg 1994;108:92–8.[Abstract/Free Full Text]
  4. Wahlberg JA, Love R, Landegaard L, Southard JH, Belzer FO. 72-hour preservation of the canine pancreas. Transplantation 1987;43:5–8.[Medline]
  5. Hardesty RL, Aeba R, Armitage JM, Kormos RL, Griffith BP. A clinical trial of University of Wisconsin solution for pulmonary preservation. J Thorac Cardiovasc Surg 1993;105:660–6.[Abstract]
  6. Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Method 1983;65:55–63.[Medline]
  7. Toda K, Tsujioka K, Maruguchi Y, et al. Establishment and characterization of a tumorigenic murine vascular endothelial cell line (F-2). Cancer Res 1990;50:5526–30.[Abstract/Free Full Text]
  8. Alley MC, Scudiero DA, Monks A, et al. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res 1988;48:589–601.[Abstract/Free Full Text]
  9. Crowe LM, Crowe JH, Rudolph A, Wormersly C, Appel L. Preservation of freeze-dried liposomes by trehalose. Arch Biochem Biophys 1985;242:240–7.[Medline]
  10. Rink TJ. Aspects of the regulation of cell volume. J Physiol 1984;79:388–94.
  11. Von Oppell UO, Pfeiffer S, Preiss P, Dunne T, Zilla P, Reichart B. Endothelial cell toxicity of solid-organ preservation solutions. Ann Thorac Surg 1990;50:902–10.[Abstract]
  12. Belzer F. Principles of solid-organ preservation by cold storage. Transplantation 1988;45:673–6.[Medline]
  13. Yamazaki F, Yokomise H, Keshavjee SH, et al. The superiority of an extracellular fluid solution over Euro-Collins' solution for pulmonary preservation. Transplantation 1990;49:690–4.[Medline]
  14. Cooper JD. Current status of lung transplantation. Transplant Proc 1991;23:2107–14.[Medline]
  15. Maccherini M, Keshavjee SH, Slutsky AS, Patterson GA, Edelson JD. The effect of low-potassium-dextran versus Euro-Collins solution for preservation of isolated type II pneumocytes. Transplantation 1991;52:621–6.[Medline]
  16. Schmid T, Landry G, Fields BL, Belzer FO, Haworth RA, Southard JH. The use of myocytes as a model for developing successful heart preservation solutions. Transplantation 1991;52:20–6.[Medline]
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