JPH11292701A - Cell protecting liquid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a cell protecting liquid stable for a long period of time, giving less damage to cells and organs and having a good taking rate by adding β-hydroxylactic acid in a specific concentration to an electrolyte solution containing sodium ion, potassium ion and phosphate ion. SOLUTION: This cell protecting liquid is obtained by adding 0.2-5 mmol/L β-hydroxylactic acid to an electrolyte solution containing sodium ion, potassium ion and phosphate ion. Preferably, the liquid is at pH 6.5-9.0, shows 250-400 mOsm osmotic pressure and 1.5-5 cp viscosity, and contains an organic acid (ion) and/or carbonic acid (ion), 2-10% hydroxyethyl starch having 150,000-900,000, preferably 400,000-900,000 average molecular weight. Also, the ratio of potassium ion to sodium ion is preferably 0.3-10, more preferably 0.3-0.8.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cell protection solution, particularly to a cell protection solution used for organ transplantation.

[0002]

2. Description of the Related Art Cells, in particular, liver, kidney, pancreas, heart,
At the time of transplanting an organ such as a lung, the removed organ needs to be kept fresh until transplantation, and the organ preservation solution plays an important role.

[0003] In the initial transplantation, Eurocollins solution was used, but in this case, the organ can be stored for less than 24 hours even in the case of a liver. An efficient preservation solution has been demanded.

Recently, a group at the University of Wisconsin has developed UW solution as a preservative solution for pancreas transplantation (Japanese Patent Publication No.
No. 68082, JP-B 8-22801). This UW solution was useful not only for preserving the pancreas but also as a preserving solution for the liver and kidney, and the liver could be stored for 24 hours.

[0005] However, the UW solution is unstable at room temperature in a solution state, and needs to be stored in a cold place, and is expensive. In addition, since UW liquid has high viscosity,
Difficulty in rapid infusion and the need to rinse the solution from the graft immediately prior to reperfusion because the electrolyte composition is based on the intracellular fluid composition, which also impairs organ viability . Also, hyperosmotic pressure can cause reperfusion injury due to rapid changes around the graft. An extracellular fluid-type organ preservation solution obtained by improving such a UW solution has also been proposed (JP-A-6-566).
No. 01, No. 9-328401).

[0006]

SUMMARY OF THE INVENTION In view of such problems of the conventional products, the present inventors have found that they are more stable in a solution state for a longer period of time, can store organs for a longer period of time, We studied the development of an inexpensive organ preservation solution that does not require rinsing, has less dysfunction after transplantation, and has a good survival rate.

[0007]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that by coexisting a certain amount of beta-hydroxybutyric acid (β-hydroxybutyric acid), damage to cells and organs is reduced, and The present inventors have found that the engraftment rate of is improved, and completed the present invention.

That is, the present invention provides: 0.2-5 mmol / L in the electrolyte solution containing sodium ion, potassium ion and phosphate ion
1. A cytoprotective solution characterized by adding beta hydroxybutyric acid of pH 6.5-8.0 and osmotic pressure of 250-4
2. the cell protective solution according to the above 1, which is 00 mOsm / L; Organic acid (ion) or (and) carbonate (ion)
3. The cell preservation solution according to 1 or 2 above, 4. the cytoprotective solution of the above 1-3, containing 2-10% of hydroxyethyl starch having an average molecular weight of 150,000 to 900,000; 5. The cell protective solution according to the above 1-4, wherein the viscosity is 1.5-5 cp. 6. the cytoprotective solution of the above 1-5, which contains 2-3.5% of hydroxyethyl starch having an average molecular weight of 150,000 to 900,000; 7. the cell protective solution according to 1-6 above, wherein the hydroxyethyl starch has an average molecular weight of 400,000 to 900,000; The molar ratio of potassium ion to sodium ion is 0.
8. the cell protective solution according to the above 1-7, which is 3-10; The molar ratio of potassium ion to sodium ion is 0.
9. the cell protective solution according to the above 1-8, which is 3-0.8; 2-3.5% hydroxyethyl starch having an average molecular weight of 400,000 to 900,000, phosphoric acid 12-28 mmol / L, gluconic acid 45-135 mmol / L, beta-hydroxybutyric acid 0.2-10 mmol / L and mannitol 20-60 mmol / L, the molar ratio of potassium ion to sodium ion is 0.2-0.8, and p
H is 7.0-8.0 and osmotic pressure is 280-330m
10. a cell protective solution having an Osm / L and a viscosity of 1.8 to 2.5 cp; 2-7% hydroxyethyl starch having an average molecular weight of 150,000-350,000, phosphoric acid 12-28 mmol / L, gluconic acid 45-135 mmol / L, beta-hydroxybutyric acid 0.2-10 mmol / L, and mannitol 20- 60 mmol / L, the molar ratio of potassium to sodium ions is 1-10, and the pH value is 6.5-
11. a cytoprotective solution having a pH of 8.0, an osmotic pressure of 280-330 mOsm / L and a viscosity of 1.5-5 cp; 12. the cell protective solution according to 1-11, which has been gas-replaced with a mixed gas containing 20 to 40% of carbon dioxide; The cell protective liquid according to the above item 1-12, which has been subjected to activated carbon treatment and bubbling treatment with carbon dioxide gas.

The cell protection solution of the present invention contains sodium ions and potassium ions, and may further contain, if necessary, cations such as magnesium ions and calcium ions. In addition, as anions, phosphate ions including primary phosphate ions and secondary phosphate ions, as necessary, halogen ions such as chloride ions, carbonate ions including bicarbonate ions, and lactic acid, acetic acid, Also included are those obtained from organic acids such as propionic acid, citric acid, lactobionic acid and gluconic acid, and these form an electrolyte solution.

[0010] The cell protective solution of the present invention exerts a cytoprotective effect regardless of whether it is an extracellular solution having a high sodium ion concentration or an intracellular solution having a high potassium ion concentration. Can be. Therefore, the molar ratio of potassium ion to sodium ion is 0.2-0.
8, preferably an extracellular fluid type in the range of 0.3-0.5, or an intracellular fluid type with this ratio of 1-10. Such an electrolyte solution is prepared at a predetermined pH of 6.5 to 8.0, preferably 7.0- with an alkali or an acid comprising a cation or an anion as described above.
Adjust to 8.0. The osmotic pressure is 250-
It can be adjusted to 340 mOsm / L by a known means.

The cell protective solution of the present invention has an average molecular weight of 15
It is preferable to contain 2 to 10% of -900,000 hydroxyethyl starch. It is desirable that these do not substantially contain, for example, those having a molecular weight of 50,000 or less, preferably 30-9.
It is preferable that the distribution is as narrow as possible, ie, 100,000, more preferably, 600,000 to 800,000. In the latter case, for example, those substantially free of those having a molecular weight of 200,000 or less are preferred. Hydroxyethyl starch is usually 2-10%, preferably 2-10%.
It is preferable to use a concentration of -3.5%.

[0012] In addition, saccharides having a relatively small molecular weight such as mannitol, trehalose, xylose, glucose, lactose and raffinose can be added in an appropriate amount. Further, if necessary, adenosine, allopurinol, glutathione, dexamethasone, and the like can be appropriately added.

Although the osmotic pressure can be set to a desired osmotic pressure, it is preferably in the range of 280-330 mOsm / L, and 280-310 mOsm / L and 290-3.
It is particularly preferred that it is in the range of 00 mOsm / L. The viscosity can be adjusted to a desired viscosity.
Preferably, the range is 2.5 cp. 1.9-2.
More preferably, the range is 2 cp.

The organ preservation solution thus obtained is preferably treated with an activated carbon added or activated carbon filter or the like. In addition, the stability can be maintained for a long time by bubbling the preservation solution with carbon dioxide gas as necessary. Further, the carbon dioxide concentration is 20-40%, preferably 20%.
Filling with -30% of the inert gas can prevent the composition from changing.

The thus obtained organ preservation solution of the present invention can be stored more stably for a long period of time by storing it in a glass bottle or bag and sterilizing it.

[0016]

EXAMPLES [Experimental Example 1] 1) Preparation of test solution Glucose-free Hanks buffer solution (p.
H7.4) [136.8 mmol of sodium chloride, 0.95 mmol of potassium chloride, 1.26 mmol of calcium chloride, 0.44 mmol of dibasic potassium phosphate, 0.16 mmol of monosodium phosphate, 4 sodium bicarbonate .17 mmol and magnesium sulphate 0.83 mmol], β-hydroxybutyric acid (BHB)
0.3 and 1.0 mmol and 5.6 mmol glucose as control were added.

2) Test Method The vascular endothelial cells were obtained from porcine thoracic aorta, a new chemistry experiment course 10 published by Tokyo Kagaku Dojinsha, a method for separating and culturing vascular endothelial cells of blood vessels and animals (pp. 51-55, 1993). For isolation and culture. After culturing the endothelial cells, they were washed twice with glucose-free Hanks' buffer (m-Hank's). Next, a medium containing each test solution (see Tables 1 and 2)
Was added to the cultured cells, placed in a desiccator ventilated with 4% oxygen-containing gas, and kept at 37 ° C. for 2 hours (low oxygen group). Then, it was moved to a 5% carbon dioxide incubator kept at 37 ° C. and reoxygenated (reoxygenation group).

The cytotoxicity was measured by collecting the medium added to each group and measuring the lactate dehydrogenase activity leaked into the medium. The lactate dehydrogenase activity when the endothelial cells were solubilized with a surfactant was defined as the total lactate dehydrogenase activity, and the percentage (%) of the activity was calculated as the cytotoxicity.

[0019]

[Table 1]

[Table 2] 3) Results The cytotoxicity after hypoxia for 2 hours is shown in FIG. 1, and the cytotoxicity when reoxygenation is further performed is shown in FIG.

Example 1 Potassium gluconate 43.5
Mmol, 46.5 mmol of sodium gluconate, 10 mmol of sodium bicarbonate, 1.2 mmol of beta-hydroxybutyric acid, 6.5 mmol of sodium dihydrogen phosphate, 18.5 mmol of sodium monohydrogen phosphate, 40 mmol of mannitol and average 30 g of hydroxyethyl starch having a molecular weight of 700,000 was dissolved and the pH was adjusted to 7.4 to obtain 1 liter of an aqueous solution. The molar ratio of potassium ion to sodium ion was 0.435, the osmotic pressure was 325 mOsm / L, and the viscosity was 2.30 cp.

Example 2 In 1 liter, 1 mmol of beta-hydroxybutyric acid and 90 g of sodium gluconate were added.
Containing 10 mmol of sodium hydrogencarbonate, 6.5 mmol of potassium dihydrogen phosphate, 30 mmol of potassium monohydrogen phosphate, 40 mmol of mannitol, and 3% concentration of hydroxyethyl starch having an average molecular weight of 600,000, The ratio of potassium ion to sodium ion is 0.66
5. An organ preservation solution having a pH value of 7.4, an osmotic pressure of 325 mOsm / L, and a viscosity of 2.30 cp.

Example 3 Potassium dihydrogen phosphate 6.5
Mmol, 18.5 mmol potassium hydrogen phosphate, 90 mmol sodium gluconate, 0.8 mmol beta hydroxybutyric acid, 10 mmol sodium bicarbonate, 40 mmol mannitol and 30 g hydroxyethyl starch with an average molecular weight of 600,000, pH 7.
Adjusted to 32 to obtain 1 liter of aqueous solution.

This aqueous solution has a molar ratio of potassium ion to sodium ion of 0.435 and an osmotic pressure of 291 mOs.
It is an organ preservation solution having a m / L and a viscosity of 2.30 cp.

Example 4 10 mmol of potassium dihydrogen phosphate, 35 mmol of potassium monohydrogen phosphate, 90 mmol of sodium gluconate, beta hydroxybutyric acid 2
Mmol, 10 mmol sodium bicarbonate, 50 mmol trehalose, 30 g hydroxyethyl starch with an average molecular weight of 600,000, 0.72 mmol allopurinol,
4 milligrams of dexamethasone was blended per liter and the pH was adjusted to 7.32.

This compounding agent has a molar ratio of potassium ion to sodium ion of 0.80 and an osmotic pressure of 300 mOsm.
/ L, an organ preservation solution having a viscosity of 2.30 cp.

Example 5 10 mmol of sodium gluconate, 86.5 mmol of potassium gluconate, 10 mmol of sodium bicarbonate, 6.5 dipotassium dihydrogen phosphate
Mmol, 18.5 mmol potassium hydrogen phosphate, 1 mmol sodium beta-hydroxybutyrate, 60 g hydroxyethyl starch with an average molecular weight of 600,000, 90 mmol mannitol, 100 mg allopurinol,
4 mg of dexamethasone was blended per liter.

Example 6 9 mmol of sodium hydrogen carbonate, 19 mmol of potassium chloride, 10 mmol of potassium dihydrogen phosphate, 30 mmol of potassium monohydrogen phosphate, 0.8 mmol of sodium beta-hydroxybutyrate, 4.3% of mannitol, 6% of hydroxyethyl starch having an average molecular weight of 450,000 was blended per liter.

Example 7 50 g of hydroxyethyl starch having an average molecular weight of 300,000, 35.83 g of lactobionic acid, 0.5 g of sodium beta-hydroxybutyrate, 3.4 g of potassium monobasic phosphate, magnesium sulfate · 7 water 1.23 g of the sump, 17.83 g of raffinose pentahydrate, 1.34 g of adenosine, 0.136 g of allopurinol and 0.922 g of glutathione are mixed per liter, and sodium hydroxide and potassium hydroxide are mixed. And adjusted to pH 7.4.

Example 8 Potassium dihydrogen phosphate 4.5
Mmol, 18.5 mmol of potassium hydrogen phosphate, 100 mmol of sodium gluconate, 0.8 mmol of beta-hydroxybutyric acid, 40 mmol of mannitol, and 30 g of hydroxyethyl starch having an average molecular weight of 700,000. To 1 liter of aqueous solution.

This aqueous solution has a molar ratio of potassium ion to sodium ion of 0.415 and an osmotic pressure of 291 mOs.
It is an organ preservation solution having m / L and a viscosity of 2.1 cp.

[0031]

The cytoprotective solution obtained according to the present invention is not only stable for a long period of time, but also causes less damage to cells and organs and has a good survival rate.

[0032]

[Brief description of the drawings]

FIG. 1. Cytotoxicity after 2 hours of hypoxia

FIG. 2: Cytotoxicity rate when reoxygenation is performed after 2 hours of hypoxia

Claims (13)

[Claims] 1. An electrolyte containing sodium ions, potassium ions and phosphate ions contains 0.2-5 mmol
/ L beta-butyric acid. 2. The composition has a pH of 6.5 to 8.0 and an osmotic pressure of 2
2. The cytoprotective solution according to claim 1, wherein the cytoprotective solution is 50 to 400 mOsm / L. 3. The cell protective solution according to claim 1, which contains an organic acid (ion) or (and) carbonate (ion). 4. The composition according to claim 1, wherein said starch contains 2 to 10% of hydroxyethyl starch having an average molecular weight of 150,000 to 900,000.
-3 cytoprotective solution. 5. The cell protective solution according to claim 1, wherein the viscosity is 1.5-5 cp. 6. The cell protection solution according to claim 1, which contains 2-3.5% of hydroxyethyl starch having an average molecular weight of 150,000 to 900,000. 7. The hydroxyethyl starch having an average molecular weight of 40
7. The cell protective solution according to claim 1, wherein the cell protective solution is -900,000. 8. The method according to claim 1, wherein the molar ratio of potassium ion to sodium ion is 0.3-10.
Cell protection solution. 9. The method according to claim 1, wherein the molar ratio of potassium ion to sodium ion is 0.3-0.8.
8 cell protection solution. 10. Hydroxyethyl starch having an average molecular weight of 400,000 to 900,000 is 2-3.5%, and phosphoric acid is 12-28 mm.
ol / L, 45-135 mmol / L of gluconic acid, 0.2-10 mmol / L of beta-hydroxybutyric acid and 20-60 mmol / L of mannitol, and the molar ratio of potassium ion to sodium ion is 0.2-0.
8, the pH value is 7.0-8.0, and the osmotic pressure is 2
80-330 mOsm / L, and the viscosity is 1.8-2.
A cell protective solution having a molecular weight of 5 cp. 11. Hydroxyethyl starch having an average molecular weight of 150,000 to 350,000 is 2-7%, and phosphoric acid is 12 to 28 mmol /
L, 45-135 mmol / L of gluconic acid, 0.2-10 mmol / L of beta-hydroxybutyric acid and 20-60 mmol / L of mannitol, the molar ratio of potassium ion to sodium ion is 1-10, and p
H value is 6.5-8.0 and osmotic pressure is 280-330
A cell protection solution having a mOsm / L and a viscosity of 1.5-5 cp. 12. The cell protection solution according to claim 1, wherein the gas is replaced with a mixed gas containing 20 to 40% of carbon dioxide. 13. The cell protection solution according to claim 1, wherein the cell protection solution has been treated with activated carbon and subjected to bubbling treatment with carbon dioxide gas.