WO2002001952A1

WO2002001952A1 - Preservation fluid for cells and tissues

Info

Publication number: WO2002001952A1
Application number: PCT/JP2001/005509
Authority: WO
Inventors: Hiromi Wada; Kenji Ohnaka
Original assignee: Hiromi Wada; Kenji Ohnaka
Priority date: 2000-07-05
Filing date: 2001-06-27
Publication date: 2002-01-10
Also published as: AU2001267836A1; JPWO2002001952A1; JP4908718B2

Abstract

A preservation fluid for cells and tissues, containing a polyphenol as the active ingredient. The fluid may further contain trehalose. The preferable osmotic pressure range is 270 to 450 Osm/l and the preferable pH range is 7 to 8. The preservation fluid is superior to those of the prior art in protective action and can maintain the structures and functions of cells and tissues for a prolonged period.

Description

Akira Cell and tissue preservation solution Technical field

The present invention relates to a liquid for preserving cells and preserving organs, limbs, skin and other tissues. Background art

When transplanting organs, keep the transplanted organs removed from the donor until surgery. Also, when anastomosis is performed on the limbs such as fingers and arms that have been cut due to an accident, the limbs should be preserved until surgery. Thus, in the field of biochemistry or the field of medicine, there are cases where cells and tissues such as organs, limbs and skin are preserved. When preserving cells and tissues, preserve them in a cell and tissue preservation solution so that their structure and function are not impaired.A conventional cell or tissue preservation solution is, for example, Eurocolins solution. (Euro-Collins solution) and UW solution (University of Wisconsin solution).

Euro-cold liquid is a solution containing potassium chloride, potassium dihydrogen phosphate, dicalcium hydrogen phosphate, sodium hydrogen carbonate and glucose. Although this preservation solution can be used for kidneys that have high function retention, it does not provide sufficient protection for organs such as the lungs, and has a short function maintenance period. It is a solution containing sodium, rafinose, and hydroxethyl starch as a colloid osmotic agent, and also contains adenosine and insulin. With this preservation solution, the protective effect is increased and the function maintenance period is prolonged, but it is pharmaceutically unstable.

In addition to these, as a conventional preservation solution, disclosed in JP-A-6-4 / 801 Storage solution (hereinafter referred to as ET-Kyot ο solution). This preservation solution has a better protective effect on cells and tissues, has a longer function-maintaining period, and is more pharmaceutically stable than Euro-Rinz solution.

An object of the present invention is to provide a cell / tissue preservation solution that has a better protective effect than conventional preservation solutions and can maintain its structure and function for a long period of time. Disclosure of the invention

As a result of intensive studies, the present inventors were able to complete the cell / tissue preservation solution of the present invention containing polyphenol as an active ingredient. Since polyphenol has an antioxidant effect, the preservation solution of the present invention has a high protective effect on cells and tissues. Therefore, according to the preservation solution of the present invention, the structure and function of cells and tissues can be maintained for a long period of time.

In the preservation solution of the present invention, examples of polyphenols include catechin, epicatechin, gallocatechin, pigallocatechin, rutin, chromogen, kercetin, anthocyanin, flavonoid and the like. These compounds can be extracted from plants such as tea, buckwheat, coffee, evening onion, and purple potato. Further, these compounds may be chemically synthesized. The preferred concentration of polyphenol is from 0.01 mM to 2000 mM, more preferably from 0.1 mM to 200 mM, particularly preferably from 0.1 mM to 10 mM, cells and tissues. In order to further enhance the protective effect against trehalose, the preservation solution of the present invention may contain trehalose. Trehalose includes ο ;, α-trehalose, HI, β-trehalose, and] 3, j3-trehalose, and any of them may be used. Preferably, naturally occurring α ,,-trehaus is used. Preferred trehalose concentrations are from 50 mM to 240 mM. In the preservation solution of the present invention, the osmotic pressure is preferably in the range of 270 to 450〇sm / 1 in order to prevent cells and tissues from expanding or contracting during storage. Further, in order to prevent acid degradation of the cells, it is desirable that the pH is in the range of 7-8. In order to keep the osmotic pressure and pH of the storage solution of the present invention within these ranges, it is preferable to add an appropriate osmotic agent or electrolyte.

Osmotic agents include collagen osmotic agents such as hydroxyxethyl starch and dextran starch. The hydroxyxethyl starch preferably has a degree of substitution in the range of 0.4 to 0.8 and an average molecular weight of 2000 to 900, and more preferably an average molecular weight of 3 to 900. It is from 5,000 to 800,000. The electrolyte may be a sodium or potassium salt of an organic acid, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium dihydrogen phosphate, dihydrogen phosphate. Examples thereof include lithium, sodium hydrogen phosphate, sodium potassium hydrogen phosphate, sodium hydrogen carbonate, potassium hydrogen carbonate and sodium carbonate. Organic acids include dalconic acid, lactic acid, acetic acid, propionic acid, / 3-hydroxybutyric acid and cunic acid. The preferred composition of the preservation solution of the present invention is as follows.

Polyphenol 0.011 to 2000 mM Trehalose 50 to 240 mM

Na + 10 ~ 140mM

K + 4 ~ 140mM

H 2 PO 4 or HPO 4 12 to 65 mM C 1 —, HCO ₃ —, CO ₃ , organic acid or organic acid anion 15 to 150 mM hydroxyxethyl starch 1 to 80 g / l The more preferred composition of the preservation solution of the present invention is as follows.

Polyphenol 10-200mM Tre-Yachiguchi 100-210mM

Na ⁺ 20-120mM

K ⁺ 20-130mM

H ₂ PO 4-or _{HPO 4 - 20~ 60mM CI -,} HC 0 3 -, CO -, organic acid or organic acid Anion 20 ~ 1 0 mM arsenate Dorokishechiru starch. 20 to 40 g / l In addition to these, for example M g ⁺⁺ and ^{Ca ÷ +} may be contained in an amount of 1 to 1 O mM each. In addition, other additives such as cell activators such as AT'P, vasodilators such as prostaglandins, and antibiotics can be added. However, it is desirable not to add unstable compounds such as insulin in order to stabilize the formulation. The method of using the preservation solution of the present invention is not particularly limited. For example, cells or tissues may be immersed in the preservation solution of the present invention and stored at a low temperature or frozen. BEST MODE FOR CARRYING OUT THE INVENTION

Example 1

In 80 O ml of distilled water at about 50 ° C, 1 g (3.45 mmol) of catechin extracted from tea, a, α-torenodulose 41 g (120 mmo1), Hydroxyshetyl starch (average molecular weight: 420,000, degree of substitution: 0.55) 30 g, sodium dalconate 21.8 1 g (100 mmol), dihydrogen phosphate After dissolving 0.085 g (6.5 mmol) of potassium and 3.22 g (18.5 mmol) of dipotassium hydrogen phosphate, distilled water was added to make a total of 1 g. The volume was set to 00 ml. This is immediately filtered and placed in a glass bottle. The mixture was sealed and steam-sterilized to obtain a stock solution having an osmotic pressure of 370 mOsm / lpH7.40. One test example 1 —

The organ protection effect of the preservation solution obtained in Example 1 (Example 1 solution) was examined using the following rat lung extracorporeal perfusion model. For comparison, a preservation solution (ET-Kyoto solution) and an euro-colin solution prepared in the same manner as in Example 1 except that catechin was not added were similarly examined. In this study, 30 male Lewis rats (weighing 300 g to 350 g) were randomly divided into three groups (groups I, II, and III), each with 10 rats. ET-Kyoto solution was used for the group I, Example 1 solution was used for the group II, and gulp-cold solution was used for the group 111.

First, a rat is placed in an air-collecting bottle containing 3 m 1 of enfluran (sold by Dainippon Pharmaceutical Co., Ltd., trade name: ET-REN (registered trademark)), and the rat is inhaled and anesthetized. 1 ml was injected intraperitoneally. The trachea was incised, the force was introduced, and the ventilator was connected and ventilated. After laparotomy and thoracotomy, 0.3 ml of heparin was injected into the peritoneal vena cava. Next, another force puncture was inserted into the pulmonary artery from the right ventricle, the inner jacket was removed while removing air, and a tube was connected to the force neurite via a three-way cock to remove air. The inferior vena cava and aorta were cut, and the left atrial appendage and right ventricle were incised. Thereafter, 1 ml of the preservative solution (containing prostaglandin E1) was slowly flushed from the tube. In addition, 50 ml of the stock solution (containing 5 wg of prostaglandin El) was flushed using a 20 cm drop. Subsequently, the cardiopulmonary block was removed while towing each cannula inserted into the trachea and right ventricle. Then, the extracted cardiopulmonary block was immersed in a Petri dish containing a preservation solution, and stored at 4 ° C. After storage for 15 hours, the right lung was removed from the cardiopulmonary block, and the remaining left heart lung was connected to a perfusion circuit. The perfusion circuit is housed in a box with a temperature of 37 ° C and a humidity of 100%, and a fresh cardiopulmonary block of the rat is also connected to this circuit. As perfusate, fresh mixed venous blood of 30 ml obtained from three rats (100 U / heparin) was used, and the port flow rate was 4 ml / min. Perfused underneath for 60 minutes. During perfusion, the left lung after storage for 15 hours was ventilated with 100% oxygen gas under the conditions of a tidal volume of 3 ml and a ventilation rate of 60 times / min. The cardiopulmonary block was ventilated with a mixed gas consisting of 4% oxygen, 8% carbon dioxide and 88% nitrogen under the conditions of a tidal volume of 3 ml and a ventilation rate of 60 / min. As a result, the oxygen and carbon dioxide concentrations in the perfusate were almost constant. Perfusion was stopped if exudate from pulmonary edema leaked from the tube.

At 10 minutes and 60 minutes after the start of perfusion, the shunt rate, mean pulmonary artery pressure, and maximum airway pressure in the left heart of the heart were examined. Furthermore, the wet-dry weight ratio of the left heart lung after perfusion for 60 minutes or after cessation of perfusion was determined, and the degree of the occurrence of pulmonary edema was examined from this. Table 1 shows the results.

, 1 group (n = 10) Π group (n-10) ΠΙ group (n = 10) Shunt rate 10 minutes 21.5 persons 6.1 7.8 ± 0.9 66.5 ± 5.0

(%)

60 min 46.2+ 3.2 10.8 ± 1.9 73.5 ± 8.1 Mean pulmonary artery pressure 10 min 24.5 ± 3.1 11.8 0.9 31.5 ± 5.7

(m m H g)

60 minutes 27.2 ± 2.3 12.8 ± 2.1 43.7+ 6.5 ΐ¾ Internal pressure 10 minutes 13.5 person 3.1 9.8 ± 0.9 21.6 ± 2.7 Im m Hg)

60 min 20.2 2.5 2.5 10.8 ± 1.9 38.5 ± 3.2 Wet / dry weight ratio 9.5+ 1.1 5.8 ± 0.9 15.5 ± 2.5 As shown in Table 1, all items were significantly lower in the order of Group II, Group I, and Group III. That is, the organ protecting effect of the preservation solution was higher in the order of Example 1, the ET-Kyoto solution, and the Euro-colin solution. This result indicates that catechin, a polyphenol, was contained in the solution of Example 1 but not in the ET-Kyoto solution and the urea-colin solution, so that organs high in polyphenol were found. Indicates a protective effect. In addition, trehalose was contained in the solution of Example 1 and the ET-Kyoto solution but not in the Eurokorin solution, indicating that trehalose also has an organ protective effect. From the above, it was revealed that the preservation solution containing polyphenol has a high organ protection effect, and the preservation solution containing polyphenol and trehalose has a higher organ protection effect.

—Example 2—

After dissolving 0.2 g (0.69 mmo 1) of catechin extracted from tea in 3 O ml of a commercially available cell cryopreservation solution (Cell lvation solution) called Cellvation ™ (manufactured by Funakoshi), then Ce The total volume was adjusted to 500 ml by adding 11 va-tion solution. As a result, a freezing stock solution containing polyphenol was obtained.

The cell protecting action of the preservation solution obtained in Example 2 (Example 2 solution) in cryopreservation was examined as follows. For comparison, the cell protection effect of the Cellvation solution was also examined. First, a commercially available cell type of dog kidney cells called MDCK (produced by Dainippon Pharmaceutical Co., Ltd., original ATTC strain number CCL-134, survival rate 95%) was prepared, and this was collected at 600 to 800 rpm. Centrifuged for 10 minutes. Subsequently, the supernatant was removed, and a preservation solution was added to the supernatant so as to be 1 × 10 ⁶ to 1 × 10 ⁷ ce 11 s / m 1, followed by gentle suspension. The suspension was dispensed in 1.5 ml portions into 10 plastic vials, each vial was sealed and left at room temperature for 25 minutes. Thereafter, each vial was placed in an insulated container and left in a freezer at 170 ° C. for 2 hours. Next, each vial was transferred to a liquid nitrogen vapor phase, allowed to stand for 24 hours, and then transferred to a liquid nitrogen liquid phase and stored frozen as it was. After 30 days, each vial was removed and quickly thawed in the 37 water bath. After thawing, each vial was wiped with 70% ethanol and left at room temperature for 70 minutes. Then, the cells in each vial were counted to determine the cell viability.

As a result, the viability of the cells cryopreserved with the Ce11 variation solution was 83% on average, whereas the viability of the cells cryopreserved with the Example 2 solution was 90% on average. Example 2 The survival rate in solution 2 was significantly higher. Thus, it was found that the preservation solution containing polyphenol had a high cytoprotective effect. Example 3—

The protective effect of polyphenol on oxidative stress associated with ischemia-reperfusion of organs was examined in a cell culture model using alveolar epithelial cells. The experimental method and results are shown in the following cell culture experiments 1-3.

(1.1) This experiment demonstrated that green tea against IL-8 produced by oxidative stress from alveolar epithelial cells The purpose of this study is to examine the inhibitory effect of polyphenol.

(1-2) Cells used: Alveolar epithelial cell line A549

(1 * 3) Green Tea Polyphenol (Pharma Foods Co., Ltd.) Composition

Weight ratio (¾)

Catechin C15H1406 3.2

Epicatechin C15H1406 7.7

Gal! Ocatechin C15H1407 5.5

Ep i ga 11 oca tech in C15H1407 7.2

Catechin gal late C22H18010 3.4

Epicatechin gal late C22H18010 11.6

Gal locatechin gal late C22H18011 12.9

E igal locatechin gal late C22H18011 26.9

(1 · 4) Method

A549 cells were cultured in a 24-wel I dish at a concentration of lxl0 ⁵ cells / ml, and after 16 hours, the medium was replaced with a green tea polyphenol-containing medium (green tea polyphenol concentration: 0-0.4 mM) and cultured for 2 hours. . After that, the cells were stimulated with (final concentration 400 iM) and inflammatory cytokine TNF-H (final concentration 20 nM), and the culture medium was collected 1, 3, and 6 hours later. IL-8 concentration in the medium was measured using IL-8 ELISA kit: Pharmingen, OptEIA Human IL-8 Set).

(1 · 5) result

H _z 0 ₂ and TNF-o; IL- 8 produced from Α549 cells by stimulation increases, this increased production were dose-dependently inhibited by green tea polyphenols (Tables 2 and 3). These results clearly indicate that green tea polyphenols suppress IL-8 production from alveolar epithelial cells associated with oxidative stress. Table 2 TNF-α stimulation group: (mean SD, n = 9, * p <0.05 vs Omg / ml group)

Table 3

H _₂ 0 ₂ stimulation group: (mean soil SD, n = 9, * p <0.05 vs Omg / ml group)

Cell culture experiment 2

(2 · 1) In this experiment, the activation of two mogen activa ted protein kinases, which play an important role in regulating IL-8 production, namely jun N-terminal kinase (JNK) and p38 ( The purpose of this study is to examine the effect of green tea polyphenol on phosphorylation.

(2 · 2) Cells used: Same as cell culture experiment 1

(2-3) Green tea polyphenol composition: same as cell culture experiment 1

(2 · 4) Method

A549 cells were cultured in a 60 mm dish at a concentration of 1 × 10 ⁶ cel ls / ml, and after 24 hours, the medium was replaced with a green tea polyphenol-containing medium (green tea polyphenol concentration: 0.½Μ) and cultured for 2 hours. Then, H ₂ Q ₂ (final concentration) Stimulation at 400 M) was performed, and after 30 minutes (p38 MAPK) and 60 minutes later UNK), the amount of protein was quantified by Western blotting, and the degree of p38 and JNK activation (phosphorylation) was examined. In preliminary experiments, it was confirmed that p38 MAPK was most strongly activated after 30 minutes and JK was activated 60 minutes after stimulation with. '

(2 · 5) result

Activation of JNK and p38 MAPK was suppressed by green tea polyphenol as shown in Table 4. Table 4

Green tea polyphenol concentration p38

100 100

0.1mM (0.05 (mg / ml) 50.6 ± 3.7 55.8 ± 7,6

0.2mU (0.1 mg / ml) 48.1 ± 3 5 39.5 ± 11.1

0.4mM (0.2mg / ml) 37.4 ± 4, 2 30.5 ± 7.8 These results suggest that suppression of IL-8 production by green tea polyphenols is related to suppression of phosphorylation of p38 and JNK . Cell culture experiment 3

(3.1) This experiment confirms the safety of green tea polyphenol against alveolar epithelial cells.

(3 * 2) Cells used: Same as cell culture experiment 1

(3 · 3) Green tea polyphenol composition: Same as cell culture experiment 1

(3 · 4) Method

Eighty-four 549 cells were cultured in a 961611 dish at a concentration of 5 10 115/1 ^ 11, and after 24 hours, green tea polypheno was added.

The medium was replaced with a medium containing green tea (green tea polyphenol concentration: 0-0.4 mM), and the cells were cultured for 8 hours. After that, trypan blue staining was performed, the total number of cells and the number of viable cells were counted, and the cell viability was calculated. (3 · 5) result As shown in Table 5, green tea polyphenol did not affect the viability of A549 cells at a concentration of 0-0.4 mM. From these results, the safety of green tea polyphenol on alveolar epithelial cells was confirmed.

Table 5

Polyphenol (m) Cell viability (¾)

0 99.6 ± 0.4

0.1 99.4 ± 0.2

0.2 99.5 ± 0.1

0.4 99.5 ± 0.2

However, cell viability (%) = (number of surviving cells / total number of cells) x 100% industrial availability

The preservation solution of the present invention has a high protective effect on cells and tissues such as organs, limbs and skin. Therefore, according to the preservation solution of the present invention, the structure and function of cells and tissues can be maintained for a long period of time.

Claims

The scope of the claims

1. Cell and tissue preservation solution containing polyphenol as an active ingredient.

2. The cell / tissue preservation solution according to claim 1, wherein the polyphenol concentration is in the range of 0.01 to 2000 mM.

3. The cell or tissue preservation solution according to claim 1, wherein the polyphenol is catechin.

4. The cell-tissue preservation solution according to any one of claims 1 to 3, which contains trehalose.

5. The cell / tissue preservation solution according to claim 4, wherein the concentration of trehalose is in the range of 50 to 24 O mM.

6. The cell / tissue preservation solution according to claim 4, wherein the trehalose is α, α-trehalose.

7. The cell / tissue preservation solution according to any one of claims 1 to 6, comprising a hydroxyxethyl starch.

8. The cell / tissue preservation solution according to any one of claims 1 to 7, wherein the osmotic pressure is in a range of 270 to 45OsmZl.

9. The cell / tissue preservation solution according to any one of claims 1 to 8, wherein the pH is in the range of 7 to 8.

10. Cell / tissue preservation solution with the following composition:

Poly phenol 0 · 01〜2000ηιΜ Trehachi loin 5 (! 〜 240mM Na ⁺ 10〜140mM

K ⁺ 4 ~ 140mM

H ₂ P 0 or HP 0 ₄ —— 12- 65ιΜ

CI -, HC_〇 _₃ -, C 0 _3, organic acids or organic acid Anion 15 ~ 15 Omm arsenide Dorokishechiru starch. 1 to 80 g / l