WO2013047665A1 - Agent de conservation pour la conservation basse température de substances biologiques, et procédé de conservation de substances biologiques à basses températures - Google Patents

Agent de conservation pour la conservation basse température de substances biologiques, et procédé de conservation de substances biologiques à basses températures Download PDF

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WO2013047665A1
WO2013047665A1 PCT/JP2012/074907 JP2012074907W WO2013047665A1 WO 2013047665 A1 WO2013047665 A1 WO 2013047665A1 JP 2012074907 W JP2012074907 W JP 2012074907W WO 2013047665 A1 WO2013047665 A1 WO 2013047665A1
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cell
cells
biological material
preservative
solution
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Japanese (ja)
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加藤　文法
清三 藤川
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石原産業株式会社
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B4/00—General methods for preserving meat, sausages, fish or fish products
    • A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
    • A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
    • A23B4/20—Organic compounds; Microorganisms; Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B7/00—Preservation or chemical ripening of fruit or vegetables
    • A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
    • A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
    • A23B7/154—Organic compounds; Microorganisms; Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3481—Organic compounds containing oxygen
    • A23L3/349—Organic compounds containing oxygen with singly-bound oxygen
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3481—Organic compounds containing oxygen
    • A23L3/3499—Organic compounds containing oxygen with doubly-bound oxygen
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3544—Organic compounds containing hetero rings
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3562—Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a preservative for cryopreserving a biological material, and a preservation solution for cryopreserving a biological material containing the preservative. Furthermore, the present invention relates to a method for preserving biological materials at low temperatures.
  • the cells have different ion compositions inside and outside the cell membrane, and the difference in the distribution of ions having this charge brings about a potential difference.
  • the intracellular potential is negative with respect to the outside of the cell (membrane potential), and this membrane potential exists as a basic principle common to living organisms regardless of animals or plants.
  • the regulation mechanism of membrane potential is indispensable for life support and cell function, and its failure directly leads to life or cell death.
  • ion pumps and ion channels on the inner and outer membranes there are various ion pumps and ion channels on the inner and outer membranes, and ion balance is constantly adjusted.
  • the most important regulatory mechanism includes a sodium pump (Na + -K + ATPase) in animal cells and a proton pump (H + -ATPase) in plant cells.
  • Na + -K + ATPase sodium pump
  • H + -ATPase proton pump
  • These ion pumps are membrane proteins that actively transport specific ions using ATP energy. If ATP is depleted for some reason or the ambient temperature deviates from the optimum range, the function of the ion pump will be reduced or stopped.
  • the sodium pump mainly pumps out three intracellular sodium ions each time, and conversely, two potassium ions are pumped into the cell from the outside. Therefore, normally, the intracellular potassium concentration is high (sodium concentration is low) and the extracellular concentration is high (sodium concentration is low).
  • the temperature of the cell falls below a certain temperature, the function of the sodium pump declines, so that sodium cannot be pumped out of the cell, and the intracellular sodium concentration increases.
  • the intracellular osmotic pressure rises, and the influx of water molecules causes the cells to swell and eventually lead to cell rupture (cell damage).
  • the above mechanism is considered to be one of the main causes of cell damage when organs for transplantation are cryopreserved at the time of organ transplantation in the medical field.
  • the basic composition of the electrolyte is intracellular low sodium and high potassium.
  • Organ preservation solutions have been developed. Typical examples are Euro Collins (EC) solution and UW (University Wisconsin) solution. Compared with conventional extracellular (high sodium, low potassium) preservatives mainly of Ringer's solution, these can greatly extend the organ preservation period and are clinically applied as major organ preservation solutions in Japan and overseas. ing.
  • these intracellular storage solutions have a risk of reversing and causing cytotoxicity when the storage temperature rises. In addition, these are not applicable to all tissues and organs, and further improvement in performance is expected, including further extension of the storage period.
  • ES cells embryonic stem cells
  • iPS cells induced pluripotent stem cells
  • MEF embryonic fibroblast
  • bovine in vitro fertilization technology made it possible to produce high quality in vitro fertilized embryos from ovaries derived from slaughterhouses.
  • bovine in vitro fertilization technology made it possible to produce high quality in vitro fertilized embryos from ovaries derived from slaughterhouses.
  • eggs for bovine in vitro fertilization are collected from bovine living bodies, there is a problem that viability cannot be maintained unless they are cultured immediately under appropriate conditions after being collected from bovine living bodies.
  • Patent Document 1 discloses that immature eggs collected by using a medium containing a protein synthesis inhibitor typified by cycloheximide is stored at room temperature and in the air. A method is disclosed.
  • Patent Document 2 discloses a method for preserving bovine ovary by immersing bovine ovary in a preservation solution and preserving it at 10 to 20 ° C.
  • Patent Document 3 discloses a method for preserving a biological material by adding a preservation solution containing one or more polyphenols to the biological material and cooling.
  • catechins are disclosed as polyphenols. Only.
  • Patent Document 4 discloses a method of refrigerated storage of cells at a temperature at which water does not crystallize, for example, a temperature around 4 ° C., by adding an enkephalin derivative to the cell culture medium.
  • Patent Document 5 discloses a medical polyphenol solution containing polyphenol and 0.0001 to 0.05% by weight of ascorbic acid or a metal salt of ascorbic acid for use as a cell preservative, tissue preservative, or the like. It is disclosed that decomposition is suppressed and generation of hydrogen peroxide is suppressed. In Examples, only epigallocatechin gallate (EGCg) is disclosed as a polyphenol.
  • Patent Document 6 discloses a composition for a preservative containing 90% by mass or more of epigallocatechin gallate as an active ingredient, and the effect of preserving cells is more constant by using purified epigallocatechin gallate with high purity. It is described that it can be.
  • Patent Document 7 discloses a flavonoid glycoside having an ability to promote the supercooling ability of an aqueous solution.
  • an antifreeze liquid that can be used at about -15 ° C with water. Therefore, it is described that biological materials and the like can be stored in the antifreeze liquid.
  • the following patent documents 8 to 10 also report on the method of using the supercooling promoting substance flavonoid glycoside disclosed in Patent Document 7.
  • Patent Document 8 discloses a beverage that can maintain a supercooled state containing the flavonoid glycoside.
  • Patent Document 9 discloses a cryopreservation solution in which the above flavonoid glycoside is contained in a vitrification solution, which is less toxic than conventional vitrification solutions and increases the viability of cells and the like by storage. It describes what you can do.
  • Patent Document 10 discloses that by using an organ preservation solution containing the flavonoid glycoside, it is possible to preserve an animal organ at a temperature of 0 ° C. or lower without freezing. Has been.
  • Patent Documents 1 to 6 do not substantially disclose the use of a compound having a flavonoid glycoside structure in a solution for preserving cells and organs.
  • Patent Documents 7 and 10 disclose that flavonoid glycosides are used for storing cells and organs at 0 ° C. or lower. However, storage under such severe conditions reduces cell viability. It becomes a factor to make.
  • the present invention relates to a preservative for cryopreservation of a biological material containing a compound having a flavonoid glycoside structure, a preservation solution for cryopreservation of a biological material containing the preservative, and a compound having a flavonoid glycoside structure
  • An object of the present invention is to provide a method for preserving biological materials at low temperatures using the
  • the present inventors have found that storing a cell at a temperature lower than 0 ° C. using a specific compound having a flavonoid glycoside structure increases the cell viability and provides a protective effect against cold injury. Obtained.
  • the present invention has been completed based on these findings, and provides the following preservatives, preservatives, and storage methods.
  • R 1 and R 2 are —H or a glucose residue, at least one is a glucose residue, and R 3 to R 9 are the same or different and represent —H, —OH or an alkoxy group
  • (I-2) The preservative according to (I-1), wherein the flavonoid glycoside compound is at least one selected from kaempferol-7-glucoside and quercetin-3-O-glucoside.
  • the biological material is egg cells, fertilized egg cells, sperm cells, embryonic stem cells, iPS cells, adult stem cells, tissue stem cells, fibroblasts, feeder cells, vascular endothelial cells, bone marrow cells, immune cells, hepatocytes , Kidney cells, neurons, pancreatic cells, smooth muscle cells, cardiomyocytes, myoblasts, corneal cells, retinal cells, chondrocytes, cartilage progenitor cells, synovial cells, synovial stem cells, osteoblasts, odontocytes
  • (I-5) The preservative according to (I-1) or (I-2), wherein the biological material is beef, pork, chicken, fish, shellfish, cereals, vegetables or fruits, or flowers as food.
  • (I-6) A cryoprotective agent comprising a flavonoid glycoside compound represented by the formula (I).
  • R 1 and R 2 are —H or a glucose residue, at least one of which is a glucose residue, and R 3 to R 9 are the same or different and represent —H, —OH or an alkoxy group
  • a biological material is immersed in a solution containing the flavonoid glycoside compound represented by the formula (1)], and the solution is kept at a low temperature higher than 0 ° C. and lower than 20 ° C.
  • III-2 The method according to (III-1), wherein the flavonoid glycoside compound is at least one selected from kaempferol-7-glucoside and quercetin-3-O-glucoside.
  • the biological material is egg cell, fertilized egg cell, sperm cell, embryonic stem cell, iPS cell, adult stem cell, tissue stem cell, fibroblast, feeder cell, vascular endothelial cell, bone marrow cell, immune cell, hepatocyte , Kidney cells, neurons, pancreatic cells, smooth muscle cells, cardiomyocytes, myoblasts, corneal cells, retinal cells, chondrocytes, cartilage progenitor cells, synovial cells, synovial stem cells, osteoblasts, odontocytes , Periodontal ligament cells, oral mucosa cells, mesenchymal stem cells, adipocytes, adipose stem cells, ovary, semen, blood, blood cells or platelets, according to any one of (III-1) to (III-3) .
  • the survival rate of the biological material such as cells can be increased, and a low-temperature damage protection effect can be obtained. Therefore, since it is possible to preserve the biological material without exposing the biological material to a harsh condition of 0 ° C. or lower, further improvement in the survival rate of the biological material such as cells and organs can be expected. Therefore, the present invention can be expected to be applied in fields such as organ transplantation, blood transfusion medicine, regenerative medicine, livestock breeding, and fresh food.
  • FIG. 5 is a graph showing the relationship between the amount of compound (2), catechins and gallic acid added in Test Example 3 and the number of viable cells (added compound final concentration: 100 ⁇ g / ml, 10 ⁇ g / ml, 1 ⁇ g / ml).
  • the preservative for low temperature storage of the biological material of the present invention is represented by the formula (I):
  • R 1 and R 2 are —H or a glucose residue, at least one is a glucose residue, and R 3 to R 9 are the same or different and represent —H, —OH or an alkoxy group It is characterized by including the flavonoid glycoside compound represented by these.
  • the biological material storage method of the present invention is characterized in that the biological material is immersed in a solution containing the flavonoid glycoside compound and the solution is kept at a low temperature.
  • R 3 and R 4 are preferably the same or different and are —H or —OH.
  • the alkoxy group is preferably an alkoxy group having 1 to 6 carbon atoms, more preferably an alkoxy group having 1 to 3 carbon atoms, and particularly preferably —OCH 3 .
  • the alkyl part of the alkoxy group may be linear or branched. Examples of the alkoxy group having 1 to 6 carbon atoms include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentyloxy, isopentyloxy, hexyloxy and the like.
  • Examples of the flavonoid glycoside compound represented by the formula (I) include kaempferol-7-Glucoside, quercetin-3-O-Glucoside, and the like. It is done.
  • the above-mentioned flavonoid glycoside compound can be chemically synthesized by a known method, and since it is contained in organisms such as plants, it can also be obtained by extraction from these by a known method. Moreover, it is also possible to obtain with a commercial item.
  • low temperature means a temperature higher than 0 ° C. and lower than 20 ° C.
  • the lower limit of the low temperature in the present invention is preferably 0.01 ° C. or higher, more preferably 0.1 ° C. or higher.
  • the upper limit of the low temperature in the present invention is preferably 15 ° C. or less, more preferably 10 ° C. or less.
  • a low temperature injury means a cell injury caused by a low temperature
  • a low temperature injury protection effect means an effect of protecting cells from the low temperature injury. Therefore, taking this meaning into consideration, the preservative of the present invention can also be referred to as a low temperature damage protective agent.
  • the biological material used in the present invention is not limited as long as the effects of the present invention are obtained, but any biological material can be used.
  • animal or plant cells cultured cell sheets, tissues, organs, organs And individuals.
  • the tissue, organ and individual may be derived from any animal or plant. Mammals (humans, monkeys, cows, pigs, horses, dogs, cats, rabbits, mice, rats, etc.) are desirable as animals targeted by the present invention.
  • Animal cells include egg cells, fertilized egg cells, sperm cells, ES cells, iPS (induced pluripotent stem) cells, adult stem cells, hematopoietic stem cells, tissue stem cells, fibroblasts, feeder cells, bone marrow (stem) cells, dental pulp (stems) ) Cells, immune cells, hepatocytes, kidney cells, pancreas cells, blood cells (blood cells) (red blood cells, white blood cells and platelets), cardiomyocytes, osteoblasts, neurons, vascular endothelial cells, smooth muscle cells, bone cells, rupture Osteocytes, chondrocytes, cartilage progenitor cells, adipocytes, adipose stem cells, epithelial cells, endothelial cells, muscle cells, epidermal cells, myoblasts, corneal cells, retinal cells, synovial cells, synovial stem cells, odontocytes , Periodontal ligament cells, oral mucosal cells, mesenchymal stem cells and
  • An immune cell means a cell involved in an immune response, and examples of such a cell include T cell, B cell, NK cell, NKT cell, monocyte, dendritic cell, macrophage, eosinophil, Examples include neutrophils and basophils.
  • animal organs and organs include ovary, semen, blood, skin, blood vessels, diaphragm, cornea, kidney, heart, brain, liver, eyeball, spleen, lung, intestine, nerve, placenta, umbilical cord, and retina.
  • the biological material used in the present invention may be meat, animals and plants used as food, and examples include beef, pork, chicken, fish, shellfish, cereals, vegetables, fruits and the like. .
  • Other biological materials include flower buds, which are ornamental plants.
  • a known additive may be appropriately blended in the preservative of the present invention.
  • the preservation solution for low temperature preservation of the biological material of the present invention is characterized by containing the above preservative.
  • the flavonoid glycoside compound (I) when the biological material is stored at a low temperature, the flavonoid glycoside compound (I) is usually used as a solution.
  • Solvents that dissolve the flavonoid glycoside compound (I) are not particularly limited, but include, for example, physiological saline, buffer solutions (PBS, Tris buffer solution, Hepes buffer solution, MOPS buffer solution, PIPES buffer solution, etc.), cells Examples include culture solutions (RPMI1640, DMEM, etc.), organ preservation solutions (EC solution, UW solution, etc.), modena solution, and the like.
  • the concentration of the flavonoid glycoside compound (I) in the preservation solution of the present invention is usually 0.001 to 1000 ⁇ g / ml, preferably 0.01 to 100 ⁇ g / ml, more preferably 0.1 to 10 ⁇ g / ml.
  • other conventional components such as buffers, antibiotics, antibacterial agents, antioxidants, serum, saccharides, lipids, vitamins, proteins, peptides, amino acids, pH indicators, chelating agents, An osmotic pressure regulator and the like can also be included.
  • the biological material can be stored by immersing the biological material in a solution containing the flavonoid glycoside compound (I).
  • the solution may be cooled to a low temperature before immersing the biological material, or may be cooled to a low temperature after immersing the biological material.
  • the solution containing the biological material is cooled to a low temperature, it is kept at a low temperature.
  • it is not always necessary to maintain a constant temperature, and the temperature may be outside the low temperature range for a short time.
  • the solution containing the flavonoid glycoside compound (I) of the present invention it is possible to significantly increase the survival rate of biological materials by storing cells at a temperature lower than 0 ° C. can get.
  • the present invention is advantageous in that the biological material is stored without exposing the biological material to a harsh condition of 0 ° C. or lower.
  • the preservation solution of the present invention having the characteristics as described above is a preservation solution for an isolated organ in the field of organ transplantation, a preservation solution for blood cell components in the field of transfusion medicine, ES cells, iPS cells, tissue stem cells and feeder cells in the field of regenerative medicine.
  • preservation solution for ovary, egg cells, fertilized eggs and sperm cells in the field of livestock breeding, preservation solution for fruits and vegetables (vegetables and fruits) in the field of fresh food, fresh fish and meat, and in the field of ornamental plants
  • Application as a preservation solution is expected.
  • Test example 1 For the compounds of the present invention, the protective effect against cold injury of cells was evaluated by the following method.
  • RPMI-1640 (SIGMA, No.R8758) containing 10% fetal calf serum (Thermo, No.SH3D396.03) in 5% carbon dioxide gas, 37 ° C incubator (manufactured by Sanyo Electric Co., Ltd., MCO-17AIC) (10% FBS-RPMI)
  • Human promyelocytic leukemia cell line HL-60 cultured in culture medium was collected, centrifuged at 4 ° C, 1,000 rpm, 5 minutes (TOMY, EIX-135), and the supernatant was It was removed and resuspended in physiological saline to 2 ⁇ 10 6 cells / ml.
  • 0.25 ml of this cell suspension and 0.25 ml of the test compound were mixed in a 2 ml sterilized microtube.
  • the test compound was dissolved in 100 mg / ml with dimethyl sulfoxide (Nacalai Tesque, No. 13407-45) (DMSO) before the test, and then in physiological saline (Otsuka Pharmaceutical Co., Ltd. Was diluted 500 times and prepared as a 0.2 mg / ml physiological saline solution.
  • the mixture of the cell suspension and the test compound was allowed to stand for about 24 hours in a cooling vessel (No. SC-DF25, manufactured by TWINBIRD) set at 4 ° C. Thereafter, 2.5 ⁇ ml of 10% FBS-RPMI culture solution was added and centrifuged at 4 ° C. and 1,000 rpm for 5 minutes. After removal of the supernatant, it is suspended in 2.5 ml of 10% FBS-RPMI culture solution, added to a 96-well microplate (IWAKI, No. 3860-096) at 0.1 ml / well, 5% carbon dioxide, 37 ° C. The cells were cultured for about 24 hours in an incubator.
  • a cooling vessel No. SC-DF25, manufactured by TWINBIRD
  • Test example 2 For test compound (2) (final concentration: 100 ⁇ g / ml, 10 ⁇ g / ml, 1 ⁇ g / ml or 0.1 ⁇ g / ml), the temperature during low-temperature storage was set to ⁇ 5 ° C., 0 ° C., 4 ° C. or 10 ° C. Except for this, the same test method as in Test Example 1 was followed. As a result, as shown in Table 2, the low temperature failure protection effect was confirmed at all storage temperatures.
  • Test example 3 When stored at low temperature, the compounds shown in Table 1 (2), catechins (catechin, epicatechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate) and gallic acid (100 ⁇ g / ml, 10 ⁇ g each) The same test as in Test Example 1 was conducted except that / ml or 1 ⁇ g / ml (final concentration) was added.
  • polyphenols are generally known to have antioxidant ability, but a compound having high antioxidant ability does not necessarily have a high low-temperature damage protection effect. Absent. Since there is no clear correlation between the antioxidant ability and the low temperature injury protection effect, it can be seen that the low temperature injury protection effect of the compound of the present invention is not simply due to the antioxidant ability of polyphenols.
  • Test example 4 Solvents to which the compounds to be added during low-temperature storage are added from physiological saline, phosphate buffered saline (PBS, SIGMA No. D 8537), EC solution (composition K 2 HPO 4 ; 7.4 g / L, KH 2 PO 4 2.05 g / L, KCl; 1.12 g / L, NaHCO 3 ; 0.84 g / L, glucose; 35 g / L), UW solution (Biaspan; manufactured by Astellas Pharma Inc.), RPMI1640 culture solution (RPMI), or 10% FBS -The same test as in Test Example 1 was performed by changing to the RPMI culture solution.
  • PBS phosphate buffered saline
  • EC solution composition K 2 HPO 4 ; 7.4 g / L, KH 2 PO 4 2.05 g / L, KCl; 1.12 g / L, NaHCO 3 ; 0.84 g / L, glucose; 35
  • the low temperature damage protection effect was confirmed in all the solvents used. That is, the solvent of the compound of the present invention is not limited to physiological saline, and it has been confirmed that it exhibits a low-temperature damage protection effect even in widely used solvents such as physiological buffers, organ preservation solutions, and cell culture solutions. It was done.
  • Test Example 5 Cold storage of MEF cells
  • MEF mouse fetal fibroblast
  • the cells were suspended in a culture medium for MEF cells, seeded in a 96-well microplate at 5 ⁇ 10 3 cells / 0.1 ml / well, and cultured in an incubator at 37 ° C. with 5% carbon dioxide. Thereafter, the supernatant was removed, and a test compound (final concentration 10 ⁇ g / ml, 1 ⁇ g / ml, 0.1 ⁇ g / ml, or 0.01 ⁇ g / ml) whose concentration was adjusted with physiological saline was added to the cells at 0.1 ml / well.
  • the 96-well microplate was stored in a cooling container set at 4 ° C. for 3 days (72 hours).
  • composition of the culture solution for MEF cells used in this test example is as follows. Composition of MEF cell culture medium (100 ml) Iscove's Modified Dulbecco's Medium (IMDM) (invitrogen, No. 12440) 88 ml MEM Non-Essential Amino Acids Solution 10 mM (100X), liquid (MEAA) (invitrogen, No.11140) 1 ml Penicillin-Streptomycin-Glutamine (100X), liquid (invitrogen, No.10378) 1 ml Fetal Bovine Serum (FBS) 10 ml
  • Test Example 6 Hs68 cell cryopreservation
  • Hs68 human normal diploid fibroblasts human foreskin fibroblast, ATCC, No. CRL-1635
  • the cells were suspended in a culture medium for Hs68 cells, seeded in a 96-well microplate so as to be 1 ⁇ 10 4 cells / 0.1 ml / well, and cultured for 2 hours in an incubator at 5% carbon dioxide and 37 ° C. Thereafter, the supernatant was removed, and compound (2) (final concentration 100 ⁇ g / ml, 10 ⁇ g / ml, 1 ⁇ g / ml, or 0.1 ⁇ g / ml) whose concentration was adjusted with physiological saline was added to the cells at 0.1 ml / well.
  • the 96-well microplate was stored in a cooling container set at 4 ° C. for 3 days (72 hours).
  • composition of the culture solution for Hs68 cells used in this test example is as follows. Composition of culture medium for Hs68 cells (100 ml) Dulbecco's Modified Eagles Medium (DMEM) (SIGMA, No.D5791) 90 ml Fetal Bovine Serum (FBS) 10 ml
  • DMEM Dulbecco's Modified Eagles Medium
  • FBS Fetal Bovine Serum
  • Test Example 7 (HUVEC low temperature storage) Measurement of the protective effect of the compound of the present invention against low temperature injury of normal human umbilical vein endothelial cells (HUVEC) (Toyobo Co., Ltd., No.GCA200K05N) was evaluated by the following method.
  • Test Example 8 Cold storage of mES cells
  • composition of the ES cell medium used in this test example is as follows. Composition of mES cell culture medium (100 ml) Stem Medium (DS Pharma Biomedical, No.DSRK100) 99 ml ⁇ -2 mercaptoethanol for ES cells, No, R-ES-007E (MEAA) (invitrogen, No.11140) 1 ml LIF (Leukemia Inhibitory Factor from mouse, SIGMA, No.L5158, 10 ⁇ g / ml) 0.1 ml
  • Test Example 9 Cold preservation of rat liver cells
  • Measurement of the protective effect of the compound of the present invention against low-temperature injury of rat liver cells was evaluated by the following method.
  • Frozen rat hepatocytes (derived from Biopredic International, Batch No. HEP134026, Sprague Dawley 7-week-old male rat) were thawed in a water bath and added to a cell thawing medium kept at 37 ° C. to obtain a cell suspension. . After centrifugation at 1,000 ° C. for 1 minute at 4 ° C., the supernatant is removed and resuspended in a cell seeding medium, and a collagen-coated 96-well microplate (Biopredic) is added to 3 ⁇ 10 4 cells / 0.1 ml / well. International, Collagen type 1 coated multi-well plate, No.PLA136), cultured for 5 hours in an incubator at 5% carbon dioxide, 37 ° C, replaced with liver cell culture medium, and further cultured for about 20 hours .
  • Cell thawing medium Thawing Medium without glucose (No.MIL261)
  • Cell seeding medium Seeding Medium (No.MIL212)
  • Liver cell culture medium Incubation Medium (No.214-100M)
  • Test Example 10 (Cryogenic preservation of mouse skin tissue section) Measurement of the protective effect of the compound of the present invention against cold injury of mouse skin tissue sections was evaluated by the following method.
  • BALB / cAnNCrlCrlj female, purchased from Nihon Charles River Co., Ltd. was exsanguinated under anesthesia, the tail was removed, and the entire skin tissue was peeled off. This was cut into 4 to 4.5 mm square skin tissue sections using a scalpel. Each section was submerged in physiological saline containing 0.5 ⁇ ml / well (24-well culture plate) physiological saline or compound (2) previously cooled to 4 ° C. The test group in which the skin tissue section was immersed in 0.5% ml / well (24-well culture plate) of physiological saline containing 0.2% Tween 20 was used as a positive control (tissue damage rate 100%).
  • the 24-well culture plate was stored in a cooling container set at 4 ° C. for 7 days, and then a part of the supernatant was sampled from each well and diluted 10-fold with physiological saline. 0.05 ml of the diluted solution was added to the well of another 96-well microplate, and the lactate dehydrogenase (LDH) activity in the supernatant was measured using LDH-Cytotoxic Test wako [Wako 299-50601].
  • LDH lactate dehydrogenase
  • Tissue failure rate (%) (SN) / (PN) x 100 (Formula I)
  • S is the absorbance in the specimen
  • N is the absorbance in the negative control
  • P is the absorbance in the positive control.
  • Test Example 11 Cold preservation of mouse intestinal tissue section
  • Measurement of the protective effect of the compound of the present invention against cold injury in mouse intestinal tissue sections was evaluated by the following method.
  • the 24-well culture plate was stored in a cooling container set at 4 ° C. for 3 days, and then a part of the supernatant was sampled from each well and diluted 100-fold with physiological saline. 0.05 ml of the diluted solution was added to the wells of another 96-well microplate, and LDH activity was measured by the same method as in Example 10. As a negative control, the same LDH measurement operation was performed using 0.05 ml of physiological saline instead of the 100-fold diluted solution.
  • S is the absorbance in the specimen
  • N is the absorbance in the negative control
  • P is the absorbance in the positive control.

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Abstract

La présente invention concerne un agent de conservation pour la conservation basse température (basse température signifie des températures supérieures à 0 °C mais inférieures à 20 °C) de substances biologiques, ledit agent de conservation contenant un composé glycoside de flavonoïde représenté par la formule (I) [dans la formule, R1 et R2 sont chacun -H ou un résidu glucose, l'un des deux au moins étant un résidu glucose, et R3 à R9 sont identiques ou différents, et sont chacun un groupe -H, -OH ou alcoxy] ; une solution de conservation pour la conservation basse température de substances biologiques, ledit milieu de conservation contenant ledit agent de conservation ; et un procédé de conservation de substances biologiques caractérisé par l'immersion d'une substance biologique dans une solution contenant un glycoside de flavonoïde représenté par la formule (I), et par le maintien de ladite solution à une basse température supérieure à 0 °C mais pas supérieure à 20 °C.
PCT/JP2012/074907 2011-09-29 2012-09-27 Agent de conservation pour la conservation basse température de substances biologiques, et procédé de conservation de substances biologiques à basses températures WO2013047665A1 (fr)

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WO2014010685A1 (fr) * 2012-07-11 2014-01-16 石原産業株式会社 Conservateur pour l'utilisation dans la conservation à basse température de matière biologique, et procédé de conservation de matière biologique à basse température
WO2014162910A1 (fr) * 2013-04-03 2014-10-09 石原産業株式会社 Conservateur pour la cryoconservation de matériels biologiques et procédé de conservation de matériels biologiques à basses températures
WO2015182019A1 (fr) * 2014-05-30 2015-12-03 Ｓｂｉファーマ株式会社 Solution de conservation d'organes
WO2017038805A1 (fr) * 2015-08-31 2017-03-09 石原産業株式会社 Agent de conservation pour organes ou tissus et procédé de conservation pour organes ou tissus

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WO2004019680A1 (fr) * 2002-08-30 2004-03-11 Mg Pharmacy Inc. Composition permettant de proteger un organe, un tissu ou des cellules et son procede d'utilisation
WO2006070883A1 (fr) * 2004-12-28 2006-07-06 Suntory Limited Composition de glycosides de quercetine et procede de preparation de celle-ci
WO2009109574A2 (fr) * 2008-03-06 2009-09-11 Boehringer Ingelheim International Gmbh Procédé de protection anti-inflammatoire et anti-oedémateuse d'un matériau biologique explanté jusqu'à sa transplantation chez des patients

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JPH04278044A (ja) * 1991-03-04 1992-10-02 San Ei Chem Ind Ltd 食肉の処理方法
JP2000344602A (ja) * 1999-06-02 2000-12-12 Mg Seiyaku Kk 動物の細胞または臓器の保存剤およびその保存方法。
WO2004019680A1 (fr) * 2002-08-30 2004-03-11 Mg Pharmacy Inc. Composition permettant de proteger un organe, un tissu ou des cellules et son procede d'utilisation
WO2006070883A1 (fr) * 2004-12-28 2006-07-06 Suntory Limited Composition de glycosides de quercetine et procede de preparation de celle-ci
WO2009109574A2 (fr) * 2008-03-06 2009-09-11 Boehringer Ingelheim International Gmbh Procédé de protection anti-inflammatoire et anti-oedémateuse d'un matériau biologique explanté jusqu'à sa transplantation chez des patients

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014010685A1 (fr) * 2012-07-11 2014-01-16 石原産業株式会社 Conservateur pour l'utilisation dans la conservation à basse température de matière biologique, et procédé de conservation de matière biologique à basse température
WO2014162910A1 (fr) * 2013-04-03 2014-10-09 石原産業株式会社 Conservateur pour la cryoconservation de matériels biologiques et procédé de conservation de matériels biologiques à basses températures
WO2015182019A1 (fr) * 2014-05-30 2015-12-03 Ｓｂｉファーマ株式会社 Solution de conservation d'organes
JPWO2015182019A1 (ja) * 2014-05-30 2017-04-20 Ｓｂｉファーマ株式会社 臓器保存液
CN107148215A (zh) * 2014-05-30 2017-09-08 思佰益药业股份有限公司 器官保存液
WO2017038805A1 (fr) * 2015-08-31 2017-03-09 石原産業株式会社 Agent de conservation pour organes ou tissus et procédé de conservation pour organes ou tissus
CN107949277A (zh) * 2015-08-31 2018-04-20 石原产业株式会社 脏器或组织的保存剂和脏器或组织的保存方法
US11246309B2 (en) 2015-08-31 2022-02-15 Ishihara Sangyo Kaisha, Ltd. Preserving agent for organs or tissue and preservation method for organs or tissue

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