JP2003267801A - Composition for preservative and preservative of cell or organ of animal containing the same composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a polyphenol-containing preservative capable of storing cells or organs of animals for a long period without freezing, giving a preservative solution with low turbidity and pale color, scarcely causing precipitation and having a definite preservation effect. <P>SOLUTION: The composition for preservative is obtained by purifying green tea polyphenol so as to contain ≥90 mass% epigallocatechin gallate concentration as an active ingredient and ≤5 mass% gallocatechin gallate concentration as impurity. The purpose is attained by using the composition for preservative as a preservative for cells or organs of animals. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a preservative composition and a preservative for animal cells or organs. For more information,
The present invention relates to animal cells, organs for transplantation, and preservatives for blood, blood cells, platelets, etc., protein stabilizers, or preservatives for preventing, treating, and improving organ disorders after organ transplantation.

[0002]

2. Description of the Related Art Conventionally, to preserve cells, a cryopreservation method at an extremely low temperature of -196 ° C. has been used, and when necessary, these frozen cells are rapidly thawed to obtain living cells. However, the survival rate after freezing and thawing depends on the cell type and the skill of the experimenter, but the survival rate of normal useful cells other than cancer, such as islets of Langerhans of the pancreas and hepatocytes, is extremely low, at most 10- 30%.

Further, since blood cell components and platelets cannot be cryopreserved, their effective period is very short, 12 to 72 hours.

[0004] The number of cases of organ transplantation is increasing with the recent progress of surgical procedures and immunosuppressive agents, but in the present situation where the preservation method of organs is undeveloped, it is removed from the donor (donor). The organ must be delivered to the recipient (recipient) in a short time and transplanted. However, donors and recipients are often far away, and for long-distance rapid transportation, we have to rely on special and high-cost transportation such as a helicopter. Often cannot be provided to the recipient. Therefore, the development of organ preservation methods is extremely important for the spread of organ transplantation.

At present, as the organ preservation methods, the cryopreservation method for the purpose of suppressing the metabolism of organs and the perfusion preservation method for the purpose of maintaining the metabolism are applied, and various preservation solutions used for them have been developed. Clinically applied.

Eurocolins solution has been used for the initial transplantation, but the organ preservation period is 2 even for the liver.
It is less than 4 hours, and there has been a demand for a storage solution that can be stored for a longer period. However, recently, a group of the University of Wisconsin in the United States used UW (Univ.
ersityof Wisconsin, Wahlbe
rg, J.I. A. , Et al. , Transplant
ation, 43, pp. 5-8,1987) was developed, and it was confirmed that it is effective not only as a preservation solution for pancreas but also as a preservation solution for liver and kidney.

However, the preservation solution such as the UW solution is still unsatisfactory such that the preservation time is less than 40 hours, and the viability of the organ can be preserved for a long time, resulting in a better organ preservation effect. The development of promising preservatives is strongly desired.

What is common to various organs is that free radicals generated at the time of ischemia and resumption of blood flow are triggered to promote lipid peroxidation of biological membranes, resulting in biological membrane damage. Dysfunction occurs. Therefore,
If the cell damage due to the production of lipid peroxide can be prevented, it will be possible to develop a more efficient storage solution.

[0009] In recent years, many studies have been reported on the effect of free radicals on the living body, and it is becoming clear about antioxidants. The main well-known antioxidants have been superoxide dismutase (SOD) as an enzyme system, and vitamin E, vitamin C, daltathione, carotenoids as a non-enzyme system.
There are flavonoids, sugars, iron chelating agents, uric acid, albumin and the like.

Although many studies on tissue growth control and organ preservation using these antioxidants are not known,
Recently, it has been reported that polyphenols exhibit antioxidative action, antibacterial action, and anticancer action.

Therefore, the present inventors have found that green tea polyphenol can dormant various animal cells and freely control the growth, and developed a preservative that extends the conventional storable time by at least 2 times or more. (JP 2000-3
44602).

[0012]

DISCLOSURE OF THE INVENTION The inventors have conducted various studies to put the above-mentioned polyphenol-containing preservative into practical use, but the preservative effect is not constant, the turbidity of the preservative is high, the pigment is colored, and the precipitate is not formed. Defects such as easy occurrence were found. As a result of examining these defects in more detail, it was found that the purity of the green tea polyphenol used was a problem.
That is, conventional polyphenols contain 8 kinds of catechins, and additionally contain caffeine, chlorophyll, proteins, sugars and minerals. Therefore, as long as the natural green tea polyphenol is used as it is, the above-mentioned drawbacks occur because of the presence of various contaminating components and the composition ratio of various polyphenols being not constant. Also,
Although it contains epigallocatechin gallate as the main active ingredient, its effect is low because it consists of various catechin analogues, and the most problematic is that the preservative effect is not constant.

In view of the above-mentioned circumstances, the present invention provides a polyphenol-containing preservative capable of preserving animal cells or organs for a long time without freezing, and the preservative solution has low turbidity, light color, and precipitation. The present invention is intended to provide a preservative that hardly occurs and has a constant preservative effect.

[0014]

[Means for Solving the Problems] In the process of studying the practical use of a preservative containing polyphenol, the present inventors purified epigallocatechin gallate to a high purity among the components of green tea catechin which is an active ingredient. It has been found that it has a more constant cell preservation effect by being used as such, and based on this finding, the present invention has been completed.

That is, the preservative composition of the present invention is characterized by containing epigallocatechin gallate as an active ingredient in an amount of 90% by mass or more.

In the preservative composition of the present invention, it is preferable that the content of gallocatechin gallate as an impurity is 5% by mass or less.

The preservative for animal cells or organs of the present invention comprises 0.01 to 0.2% by mass of the above preservative composition.
It is characterized by containing.

According to the present invention, it is possible to provide a preservative for animal cells or organs which has a low turbidity of the preservative solution, is light in color, hardly causes precipitation, and has a constant preservative effect.

The animal cell or organ preservative of the present invention further comprises a stem cell, a skin cell, a mucous cell, a hepatocyte, a pancreatic islet cell, a nerve cell, a chondrocyte, which is isolated from a human or animal tissue. It is preferably animal cells including endothelial cells, epithelial cells, osteocytes, muscle cells, or livestock and fish sperms, eggs or fertilized eggs.

In the animal cell or organ preservative of the present invention, the organ is preferably skin, blood vessel, cornea, kidney, heart, liver, umbilical cord, intestine, nerve, lung, placenta or pancreas.

[0021] Then, the method for applying to the preservation of animal cells or organs of the present invention is characterized in that the above-mentioned preservative is used for the prevention, treatment and amelioration of organ damage after organ transplantation.

According to the invention, not only can the cells or organs of the animal be stably preserved for a long period of time, but they can also be useful for the prevention, treatment and amelioration of organ disorders after organ transplantation.

Further, the method for obtaining a composition for a preservative from the green tea polyphenol of the present invention is characterized by using a column chromatography method.

According to the above invention, it is possible to obtain a composition for a preservative having a high purity of epigallocatechin gallate and a low concentration of impurities affecting the effect of the preservative.

The preservative for a protein solution of the present invention is characterized by containing 0.005 to 0.08% by mass of the above-mentioned preservative composition to stabilize the protein.

The blood, blood cell or platelet preservative of the present invention is characterized by containing 0.005 to 0.1% by mass of the above-mentioned preservative composition.

Further, the preservative for microbial cells of the present invention is characterized by containing 0.005 to 0.04% by mass of the above-mentioned preservative composition.

According to the above invention, it is possible to provide a preservative capable of stably storing a protein solution, blood, blood cells, platelets, or microbial cells for a long time.

[0029]

BEST MODE FOR CARRYING OUT THE INVENTION Preferred embodiments of the present invention will be described below.

As a preferred embodiment of the preservative of the present invention,
The composition for a preservative using epigallocatechin gallate of the present invention as an active ingredient is added to various culture solutions or organ preservation solutions generally used for cell culture. As the culture solution and the preservation solution for organ preservation used in the present invention, known ones are used, for example, Euro-Collins solution (Euro-Col).
Lins solution, Squifflet, J. P. , Eta
l. , Transplant Proc. , 13,69
3, 1981), UW liquid and the like can be used.

The epigallocatechin gallate as a preservative for cells and organs of the present invention can be used with a purity of 90% by mass or more, but a purified product with a purity of 95% or more is more suitable. Of course, it is more effective if the purity is higher.

When the preservative of the present invention is used for the preservation of animal cells, it is preferable that epigallocatechin gallate 90% by mass is contained in various culture solutions and preservation solutions for organ preservation which have been used for various cell cultures. The preservative composition was
01-0.2 mass% addition, more preferably 0.02-
0.1 mass% is added.

For example, when the preservative of the present invention is used for organ preservation, the above-mentioned preservative composition is further added to Eurocolins solution or UW solution which has been clinically used as a preservation solution for organs for transplantation. Add 0.01 to 0.2% by mass.
A more preferable concentration is 0.02 to 0.1% by mass.

When used as a preservative for blood, blood cells and platelets, a preservative solution containing commercially available glucose, phosphate or the like added in advance or a solution added at a later date, or a polyoxyethylene-based preservative solution. The preservative composition of the present invention is added in an amount of 0.005 to 0.1% by mass to a liquid to which a nonionic surfactant has been added in advance or a liquid added at a later date.

When this preservative is used as a stabilizer for a protein solution, 0.005 to 0.08% by mass of a preservative composition is further added to a preservative solution containing a commercially available fatty acid ester.

Further, when the preservative is used for preventing, treating or ameliorating organ disorders after organ transplantation, the preservative composition of the present invention is added to a commercially available preservative in an amount of 0.01 to 0.2. It can be used by adding mass%. The preservative can be administered by ordinary intravenous, oral, nasal, suppository or transdermal administration methods. Further, the dose is not particularly limited depending on the age and symptoms of the patient.

Other antioxidants such as SOD, vitamins E and C, and glutathione can also be used in combination with these culture solutions and preservation solutions.

As a condition for using the preservative of the present invention, the application temperature is preferably 0 to 37 ° C. Comparing the preservative of the present invention with those on the market, the application temperature is relatively high, no freezing is required, the service life is relatively long, and the application to organ transplantation / surgery is excellent.

The cells in the present invention include stem cells, skin cells, mucosal cells, hepatocytes, pancreatic islet cells, nerve cells, chondrocytes, endothelial cells, epithelial cells, bone cells, muscle cells isolated from human or animal tissues. And animal cells or livestock and fish sperms, eggs or fertilized eggs.

Further, the organ of the present invention includes skin, blood vessels,
It includes the cornea, kidney, heart, liver, umbilical cord, intestine, nerve, lung, placenta or pancreas.

Since the 1980s, many researchers have found that green tea polyphenol and its main component, epigallocatechin gallate, are excellent in various physiologically active actions such as anticancer action, antioxidant action, and antibacterial / antiviral action. Have been reported by. Although studies on the effect of epigallocatechin gallate on cell growth have been reported, most of them are for suppressing the growth of cancer cells.

Therefore, as in the present invention, it was not possible to find a phenomenon that "the animal cells hibernate under body temperature conditions" in which various normal animal cells were used and the cell growth was freely controllable. . Furthermore, it was naturally not discovered that normal cell proliferation / division can be resumed after hibernation and various functions can be expressed.

The fact that the growth of various animal cells can be freely controlled in this way is not only a new breakthrough in basic research on cell engineering, but also cell preservation, blood component preservation, and long-term preservation. This is an epoch-making invention that enables even time organ preservation.

The composition for preservatives of animal cells or organs of the present invention and the preservatives are not limited to the above-mentioned embodiments, and various modifications can be made without departing from the scope of the present invention. Of course, it can be added.

[0045]

The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto.

Example 1 For the preparation of the composition for a preservative of the present invention, green tea polyphenol TP-6 obtained by a commonly used extraction method was used.
0 (manufactured by Pharma Foods Co., Ltd., having a green tea polyphenol content of 60% or more) was used. Since TP-60 contains a large amount of chlorophyll, chlorophyll is first removed. Add 1 liter of water and 1 liter of alcohol to 1000 g of TP-60, and from 10 minutes to 30
Heat and dissolve for 1 minute, and leave the solution in the refrigerator overnight.
After confirming that a precipitate had formed after 24 hours, it was filtered at low temperature and dried to obtain 810 g of dechlorophyll.

Next, chlorophyll-removed catechin fraction (TP
-DCh), epigallocatechin gallate was highly purified by column chromatography. 810 of TP-DCh
g was packed in a low pressure column of Diaion HP-20, chromatographic separation was carried out with an aqueous solution of 20% ethanol, and separation was performed while monitoring with UV to obtain an 80% epigallocatechin gallate fraction. Next, Toyopearl HW-40C
The column method was used to perform chromatographic separation of the 80% epigallocatechin gallate fraction with an aqueous 40% methanol solution to obtain 210 g of a fraction containing 90% or more epigallocatechin gallate.

This product was separated by a Shimadzu SCL10A high performance liquid chromatograph. J'spere OD
SM-80 (150 × 4.6 mm ID) column, mobile phase methanol / 0.05% phosphoric acid aqueous solution = 20/8
0, column temperature 30 ° C., detection wavelength 280 nm showed that the purity was 97% or more as shown in FIG. Thus, a high-purity product (hereinafter referred to as EGCg-90) containing 90% or more of epigallocatechin gallate was obtained from TP-60.
It could be obtained with a yield of 1%.

Example 2 Mouse fibroblast L-929 Fibroblast was prepared from Eagle's MEM (containing 60 mg / l of kanamycin, Eagl).
e'sMEM maintaining kanamyci
n) and 10% fetal bovine serum (fetal bovin)
e serum; FBS, M .; A. Bioproduct
T., Marylard, USA). The cell density was adjusted to 1.76 × 10 ⁵ cells / mL, and serum culture (serummediu) was used as a control group.
m) only (1 group), 3 in the culture system as a TP-60 addition group
A cell growth test was carried out by adding TP-60 at a concentration of 00 mg / L (group 2), and adding EGCg-90 at a concentration of 100 mg / L to the culture system as a group to which the composition of the present invention was added (group 3). As a result, in the control group (group 1), proliferation rapidly started after 2 days and the cell number was 1.4 after 5 days.
It increased twice, but TP-60 addition group (2 groups) and EGC
In the g-90-added group (3 groups), no growth was observed at all for one week and the animals were dormant. However, the medium (media)
m) was replaced and only serum culture was resumed, and one week after that, the number of cells in the TP-60 addition group (2 to 1 group) was 2.6 times, and the EGCg-90 addition group (3 group). It increased to 3.0 times in 1 group). Also, this sleep is 3
I also confirmed that it will last for months. Thus EGCg-
It was confirmed that the 90-added group (3 group) had the same dormant effect even though the concentration was lowered, and the subsequent cell proliferation property was higher than that of the TP-60-added group (2 group).

Example 3 Porcine hepatocytes were inoculated into culture blood coated with type I (type I) collagen at 2.1 × 10 5 cells and cultured. The control group (4 groups) contained Dulbecco's modified eagle culture once every 3 days (fetal bovine serum 100 mg / L, penicillin 50000 unit / L, streptomycin 100 mg / L, EGF 0.5 mg / mL, insulin 0.25 mg / L. Dulbecco. 'smod
if Eagle's medium; DMEM,
containing bovine serum 10
0 mg / L, Penicillin 50000 unit /
L, stereptomycin 100 mg / L, EG
F 0.5 mg / mL, insulin 0.25 mg /
L) was exchanged and 300 was added to the culture system as a TP-60 addition group.
Add TP-60 at a concentration of mg / L (group 5) and EGCg-90 at a concentration of 150 mg / L to the culture system as a group to which epigallocatechin gallate was added (group 6) to increase hepatocyte proliferation ability and hepatocyte function. Compared. Similar to mouse fibroblasts, cells proliferated in the control group (group 4), and in the TP-60-added group (group 5) and EGCg-90-added group (6 group), no proliferation was observed after one week, and sleep was observed. I was doing, but then me
Replacing the dim restarted proliferation. As a result of checking D-glucose as a hepatocyte function and lidocaine clearance (Lidocaine clearance), hepatocytes added with TP-60 or EGCg-90 and awakened for 1 week were similar to those in the control group. In the EGCg-90-added group, a similar effect of maintaining cell function was observed in the EGCg-90-added group, although the concentration was lower than that in the TP-60-added group.

Example 4 Rat Pancreas Langerhans Island About 2,000 Langerhans islets (La Mr. Island) were inoculated from the pancreas of Wistar rats (body weight: 380 g), and 200 of them were cultured. The control group (7 group) is RPMI culture 1640 (containing L-glutamine, without glucose, RPMI Medium
1640, Gibco BRL, lot no. 1019
650, with L-glutamine, with
out glucose, LIFETECHNOLOG
(Made by IES) is used and Fetal Bovin
e Serum (Lot # 29110643, CASE
RAINTERNATIONAL INC. CANAD
A company) 10% glucose 1 g /
L, and Antibiotic-Antimycotic Lot No. 1013807, LIFETECHNOLO
Each system to which GIES) was added was cultured at 37 ° C. for 1 week. As a TP-60 addition group, 0.1% by mass concentration of TP-60 was added to the culture system (8 groups), and EGCg-90 was added.
As the addition group, 0.04% by mass concentration of epigallocatechin gallate was added to the culture system (9 groups), and the observation and function of the La Islet tissue were compared. In the control group (7 group), 50% of Lai Island was destroyed after 2 days, and 100% of Lai Island had been dead after 4 days. On the other hand, in the TP-60-added group (8 group) and the EGCg-90-added group (9 group), even after 1 week, no destruction of Laijima island was observed and 100% hibernation was performed. After hibernation for 1 week, TP-60 or E
Insulin-EIATES was tested by washing La Islet with a culture system containing no GCg-90.
T GLAZYME, Wako Pure Chemical Industries, Ltd.),
When the insulin treatment function was checked by the one-step enzyme immunoassay, it showed the same normal function as that of La Islet immediately after seeding from the rat pancreas, and in the EGCg-90 addition group (9 group), the TP-60 addition group. Compared with (Group 8), although the concentration was low, the same or higher tissue hibernation effect and function maintenance effect were observed.

[0052]

As described above, according to the composition for a preservative of the present invention, a preservative having a low turbidity of the preservative, a light color, and less precipitation is provided, and the preservative has a constant preservative effect. be able to.

The animal cell or organ preservative of the present invention enables more stable preservation of animal hepatocytes, pancreatic islet cells and fertilized eggs without freezing for a long period of time. From this, it can be used for cell engineering or tissue engineering for producing useful substances such as cytokines produced by animal cells. In addition, by adding high-purity epigallocatechin gallate to the conventional organ preservation solution, the preservation period of organs for transplantation, such as liver, kidney, pancreas, cornea and skin, nerves, and umbilical cord, can be significantly extended. Can be made. And when comparing the preservative of the present invention with those on the market, the application temperature is relatively high and does not require refrigeration, the service life is relatively long, and the adaptation to organ transplantation / surgery is excellent. There is.

Furthermore, according to the preservative for blood, blood cells or platelets of the present invention, even blood, blood cells or platelets can be stored for a long period of time, and high-purity epigallocatechin gallate can be added to conventional platelet storage solutions. Therefore, it can be used as a platelet preservation solution in which plasma is replaced.

According to the protein solution preservative of the present invention, various kinds of protein solutions and epigallocatechin gallate are allowed to coexist in the system so that they can be used in various fields such as food, clinical, clinical diagnosis and medical devices. The storage stability of a protein (enzyme, antibody, antigen, immunologically active substance, physiologically active peptide, etc.) in a solution or in a dry state can be improved.

[Brief description of drawings]

FIG. 1 is a diagram showing a chemical structural formula of epigallocatechin gallate.

FIG. 2 is an analysis chart when the purity of epigallocatechin gallate (EGCg) is analyzed by high performance liquid chromatography in Example 1, and a pie chart showing the purity of epigallocatechin gallate.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Kim Takehisa             24 Koshinishi-cho, Kichijoin Ishihara-do, Minami-ku, Kyoto-shi, Kyoto Prefecture             No. 5 within Pharma Foods Laboratories, Inc. (72) Gen Inoue             13 29, Uji Mausoleum, Uji City F-term (reference) 4B065 AA90X BD12 BD34                 4C087 AA10 BB34 DA10 NA03 NA06                 4H011 CA01 CB02 CB05 CB06 CB07                       CB08 CB14 CD02 CD06 DA13

Claims (10)

[Claims] 1. A composition for a preservative, which comprises 90% by mass or more of epigallocatechin gallate as an active ingredient. 2. Impurity of gallocatechin gallate is 5
The composition for a preservative according to claim 1, wherein the composition is at most mass%. 3. A preservative for animal cells or organs, which comprises 0.01 to 0.2% by mass of the preservative composition according to claim 1 or 2. 4. The cells include stem cells, skin cells, mucous cells, hepatocytes, pancreatic islet cells, nerve cells, chondrocytes, endothelial cells, epithelial cells, bone cells, muscle cells isolated from human or animal tissues. The animal cell or preservative for animal cells or organs according to claim 3, which is a sperm, egg or fertilized egg of livestock and fish. 5. The preservative for animal cells or organs according to claim 3 or 4, wherein the organ comprises skin, blood vessel, cornea, kidney, heart, liver, umbilical cord, intestine, nerve, lung, placenta or pancreas. 6. A method of applying the preservative according to any one of claims 3 to 5 to the preservation of animal cells or organs for the prevention, treatment and amelioration of organ disorders after organ transplantation. 7. The method for obtaining the composition for a preservative according to claim 1 or 2, from a green tea polyphenol, which is characterized by using a column chromatography method. 8. A preservative for a protein solution, which comprises 0.005 to 0.08% by mass of the composition for a preservative according to claim 1 or 2 and stabilizes a protein. 9. A preservative for blood, blood cells or platelets, which contains 0.005 to 0.1% by mass of the composition for preservative according to claim 1 or 2. 10. A preservative for microbial cells containing the preservative composition according to claim 1 or 2 in an amount of 0.005 to 0.04% by mass.