WO2013047665A1 - Preservative for low-temperature preservation of biological materials, and method for preserving biological materials at low temperatures - Google Patents

Abstract

Disclosed are a preservative for the low-temperature preservation (low temperature signifies temperatures higher than 0°C but not more than 20°C) of biological materials, said preservative containing a flavonoid glycoside compound represented by formula (I) [in the formula, R¹ and R² are each -H or a glucose residue, with at least one being a glucose residue, and R³ to R⁹ are the same or different, and are each -H, -OH or an alkoxy group]; a preservation solution for the low-temperature preservation of biological materials, said preservation medium containing said preservative; and a method for preserving biological materials characterized by immersing a biological material in a solution containing a flavonoid glycoside compound represented by formula (I), and maintaining said solution at a low temperature that is higher than 0°C but not more than 20°C.

Description

Preservative for cryopreservation of biological material and method for preserving biological material at low temperature

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. Usually, 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.

For this reason, 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. 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.

In physiological cells, under physiological conditions, 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). When 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. As the sodium concentration rises, 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. However, 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.

In recent years, the development of regenerative medicine has been remarkable, and embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) are expected to be used for medical applications. Co-culture in embryonic fibroblast (MEF) cell feeder cell layer. The long-term storage of these MEF cells is generally carried out in a -80 ° C deep freezer or in liquid nitrogen, but in short-term storage, it is not always preferable to freeze the cells. If possible, it is expected that its application range will be expanded.

On the other hand, bovine in vitro fertilization technology made it possible to produce high quality in vitro fertilized embryos from ovaries derived from slaughterhouses. However, with the start of the BSE test at the slaughterhouse, it is difficult to take out the collected ovaries until the test result is found to be negative. Therefore, it is necessary to preserve the ovaries in a form that minimizes the effect on survival from the time of slaughtering to the production of in vitro fertilized eggs. In addition, although 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.

Even in the field of livestock breeding, various problems still remain regarding the low-temperature storage of ovaries (egg cells), fertilized eggs, sperm, etc. It will be possible to greatly contribute to the development of the breeding industry.

As a method for coping with such a problem, 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.

Further, techniques for preserving cells and organs as described above are disclosed in, for example, the following Patent Documents 3 to 6.

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. In Examples, 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. By using this supercooling promoting substance, 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.

Furthermore, 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.

Japanese Patent Laid-Open No. 2001-89302 Japanese Patent Laid-Open No. 5-112401 Special Table 2007-519712 JP 2002-335954 A JP 2006-188436 A JP 2003-267801 A International Publication No. 2008/007684 JP 2009-219394 JP 2009-219395 A JP 2009-221128 JP

However, 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.

Accordingly, 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.

(I) Preservative
(I-1) Formula (I):

[Wherein, 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 Is a preservative for cryopreservation of a biological material containing a flavonoid glycoside compound represented by the formula (low temperature is higher than 0 ° C. and 20 ° C. or lower).
(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.
(I-3) The preservative according to (I-1) or (I-2), wherein the biological material is an animal or plant cell, a cultured cell sheet, a tissue, an organ or an organ.
(I-4) 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 The preservation according to any one of (I-1) to (I-3), which is a periodontal ligament cell, oral mucosa cell, mesenchymal stem cell, adipocyte, adipose stem cell, ovary, semen, blood, blood cell or platelet Agent.
(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).

(II) Stock solution
(II-1) A preservation solution for low-temperature preservation of biological material containing the preservative according to any one of (I-1) to (I-5).

(III) Storage method
(III-1) Formula (I):

[Wherein 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.
(III-3) The method according to (III-1) or (III-2), wherein the biological material is an animal or plant cell, a cultured cell sheet, a tissue, an organ or an organ.
(III-4) 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) .
(III-5) The method according to (III-1) or (III-2), wherein the biological material is beef, pork, chicken, fish, shellfish, cereals, vegetables or fruits, or flowers as food.

By storing the biological material at a low temperature higher than 0 ° C. by the preservative and method of the present invention, 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.

It is a graph which shows the standard line of the number of living cells and a light absorbency. 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 μg / ml, 10 μg / ml, 1 μg / ml).

Hereinafter, the present invention will be described in detail.

The preservative for low temperature storage of the biological material of the present invention is represented by the formula (I):

[Wherein, 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.

In the present invention, 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.

As used herein, a low temperature injury means a cell injury caused by a low temperature, and 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. For example, 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 the like. 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.

Examples of 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.

In addition, 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.

In addition to the flavonoid glycoside compound (I), 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. In the present invention, 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 μg / ml, preferably 0.01 to 100 μg / ml, more preferably 0.1 to 10 μg / ml. In the preservation solution of the present invention, 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.

In the present invention, the biological material can be stored by immersing the biological material in a solution containing the flavonoid glycoside compound (I). In this case, 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. Once the solution containing the biological material is cooled to a low temperature, it is kept at a low temperature. However, it is not always necessary to maintain a constant temperature, and the temperature may be outside the low temperature range for a short time.

By using 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.

Hereinafter, examples will be given to explain the present invention in more detail. However, the present invention is not limited to these examples.

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 μml 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. Subsequently, 10 μl / well of WST-8 solution (Nacalai Tesque, Cell Count Reagent SF, No. 07553-44) was added, and further cultured for 3 hours in an incubator, and then the absorbance was measured at a wavelength of 450 nm. (WAKO, SPECTRA MAX250). From this absorbance value, the number of viable cells was calculated using the standard line (FIG. 1) of the number of cells and the absorbance prepared in advance.

As a result, as shown in Table 1, in the case of no addition, cells were allowed to stand at 4 ° C for 24 hours (cold injury), resulting in the number of viable cells from the initial number of cells from 20,000 to 500 / well. Decreased to. On the other hand, when the compound (1) and (2) were added to the cells at a concentration of 100 μg / ml during low-temperature storage, a low-temperature injury protective effect was observed. The number of viable cells at this time was 26.5 to 62.8 times higher than that of the physiological saline alone without the compound.

Test example 2
For test compound (2) (final concentration: 100 μg / ml, 10 μg / ml, 1 μg / ml or 0.1 μg / 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 (100μg / ml, 10μg each) The same test as in Test Example 1 was conducted except that / ml or 1 μg / ml (final concentration) was added.

The results are shown in FIG. From these results, it can be seen that catechins and gallic acid cannot obtain a low-temperature damage protection effect at 1 μg / ml or less, whereas the compounds of the present invention can provide a low-temperature damage protection effect even at 1 μg / ml. I understand.

In addition, as shown in Table 3 below, 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. As a result, as shown in Table 4, 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)
Measurement of the protective effect of the compound of the present invention against cold injury of mouse fetal fibroblast (MEF) cells (DS Pharma Biomedical, Primary Mouse Embryo Fibroblast, No. R-PMEF-HL) was evaluated by the following method.

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 μg / ml, 1 μg / ml, 0.1 μg / ml, or 0.01 μg / 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). Subsequently, the supernatant was removed and replaced with a culture solution for MEF cells. After culturing in an incubator for about 24 hours, 10 μl / well of WST-8 solution was added and further cultured for 2 hours. Absorbance was measured at a wavelength of 450 nm, and the number of viable cells was calculated from the previously prepared standard line of cell number and absorbance (viable cell number = 15506 × absorbance, R ² = 0.996).

As a result, as shown in Table 5, when the cells were stored at 4 ° C. for 3 days (cold injury), the number of viable cells was 5,000 to 349 from the initial number of cells / well when not added in physiological saline. It decreased to 1026 cells / well in the culture medium for MEF cells. On the other hand, by adding compound (2) to cells at a concentration of 0.01 to 10 μg / ml during low-temperature storage, the number of viable cells is maintained at a maximum of 15.0 times higher than when no compound is added in physiological saline, The low temperature injury protection effect of the compound of the present invention was confirmed.

The 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)
Measurement of the protective effect of the compound of the present invention against cold injury of Hs68 human normal diploid fibroblasts (human foreskin fibroblast, ATCC, No. CRL-1635) was evaluated by the following method.

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 μg / ml, 10 μg / ml, 1 μg / ml, or 0.1 μg / 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). Subsequently, the supernatant was removed and replaced with a culture medium for Hs68 cells. After culturing in an incubator for about 24 hours, 10 μl / well of WST-8 solution was added and further cultured for 2 hours. Absorbance was measured at a wavelength of 450 nm, and the number of viable cells was calculated from the previously prepared standard number of cells and absorbance (viable cell number = 7103 × absorbance, R ² = 0.998).

As a result, as shown in Table 6, when the cells were stored at 4 ° C. for 3 days (cold injury), the number of viable cells was increased from the initial number of 10,000 cells / well to 203 when no addition was made in physiological saline. It decreased to 75 cells / well in the culture medium for Hs68 cells. On the other hand, by adding the compound (2) to the cells at a concentration of 0.1 to 100 μg / ml during low-temperature storage, the number of viable cells is maintained up to 52.9 times higher than when no compound is added in physiological saline, The low temperature injury protection effect of the compound of the present invention was confirmed.

The 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

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.

Suspend cells in culture medium for HUVEC (TOYOBO, No.CA211K500), seed in 96-well microplate to 5 × 10 ³ cells / 0.1 ml / well, and in 5% carbon dioxide in a 37 ° C incubator. Cultured for 3 hours. Thereafter, the supernatant was removed, and compound (2) (final concentration final concentration 100 μg / ml, 10 μg / ml, 1 μg / ml, or 0.1 μg / ml) adjusted with physiological saline was added to the cells at 0.1 ml / well. did. The 96-well microplate was stored in a cooling container set at 4 ° C. for 3 days (72 hours). Subsequently, the supernatant was removed and replaced with a culture solution for HUVEC. After culturing for about 24 hours in an incubator, 10 μl / well of WST-8 solution was added and further cultured for 2 hours. Absorbance was measured at a wavelength of 450 nm, and the number of viable cells was calculated from the previously prepared standard line of cell number and absorbance (viable cell number = 13545 × absorbance + 165, R ² = 0.999).

As a result, as shown in Table 7, when the cells were stored at 4 ° C. for 3 days (cold injury), the number of viable cells was 5,000 to 740 from the initial number of cells / well when not added in physiological saline. It decreased to 244 cells / well in the culture medium for HUVEC. On the other hand, by adding compound (2) to cells at a concentration of 0.1 to 100 μg / ml during low-temperature storage, the number of viable cells is maintained up to 6.5 times higher than when no compound is added in physiological saline, The low temperature injury protection effect of the compound of the present invention was confirmed.

Test Example 8 (Cold storage of mES cells)
Measurement of the protective effect of the compound of the present invention against cold injury of mouse embryonic stem (mES) cells (RIKEN, No. AESO125) was evaluated by the following method.

In a 96-well microplate pretreated with 0.1% gelatin solution for ES cells (DS Pharma Biomedical, No.R-ES-006B), 1.5 × 10 ⁴ / 0.1 mES cells suspended in ES cell medium It seed | inoculated so that it might become ml / well, and it culture | cultivated for 24 hours in the incubator of 5% carbon dioxide gas and 37 degreeC. Thereafter, the supernatant was removed, and compound (2) (final concentration 10 μg / ml, 1 μg / ml, 0.1 μg / ml, or 0.01 μg / ml) adjusted in concentration 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 2 days. Subsequently, the supernatant was removed and replaced with a medium for ES cells. After culturing in an incubator at 5% carbon dioxide and 37 ° C. for about 24 hours, 10 μl / well of WST-8 solution was added and further cultured for 3 hours. Absorbance was measured at a wavelength of 450 nm, and the number of viable cells was calculated from the previously prepared standard line of cell number and absorbance (viable cell number = 16275 × absorbance, R ² = 0.993).

As a result, as shown in Table 8, when the cells were stored at 4 ° C. for 3 days (cold injury), the number of viable cells in the case of no addition in physiological saline was 15,000 cells / well to 137 It decreased to 592 cells / well in the ES cell medium. On the other hand, by adding compound (2) to cells at a concentration of 0.01 to 10 μg / ml during low-temperature storage, the number of viable cells can be maintained up to 34.5 times higher than when no compound is added in physiological saline, The low temperature injury protection effect of the compound of the present invention was confirmed.

The 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
Β-2 mercaptoethanol for ES cells, No, R-ES-007E (MEAA) (invitrogen, No.11140) 1 ml
LIF (Leukemia Inhibitory Factor from mouse, SIGMA, No.L5158, 10μg / 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 .

Thereafter, the supernatant was removed, and compound (2) (final concentration 100 μg / ml, 10 μg / ml, 1 μg / ml, or 0.1 μg / ml) adjusted in concentration with physiological saline was added to cells at 0.1 μml / well. The 96-well microplate was stored in a cooling container set at 4 ° C. for 1 day (24 hours). Subsequently, the supernatant was removed and replaced with a medium for culturing liver cells. After culturing in an incubator for about 24 hours, 10 μl / well of WST-8 solution was added and further cultured for 2 hours. Absorbance was measured at a wavelength of 450 nm, and the number of viable cells was calculated from the standard line of the number of cells and the absorbance prepared in advance.

As a result, as shown in Table 9, when the cells were stored at 4 ° C. for 1 day (cold injury), the number of viable cells increased from initial 30,000 cells / well to 1216 when no addition was made in physiological saline. It decreased to 1640 cells / well in the medium for liver cells (Incubation Medium). On the other hand, by adding compound (2) to cells at a concentration of 0.1 to 100 μg / ml during low-temperature storage, the number of viable cells is maintained at a maximum of 12.6 times higher than when no compound is added in physiological saline, The low temperature injury protection effect of the compound of the present invention was confirmed.

In this test example, the following media were used.
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 μml / 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]. That is, 0.05 ml of the coloring solution was added to 0.05 ml of the centrifugal supernatant to start the reaction, and after standing at room temperature for 45 minutes, 0.1 ml of a reaction stopping solution (0.5N hydrochloric acid solution) was mixed to stop the coloring reaction. As a negative control, the same LDH measurement procedure was performed using 0.05 ml of physiological saline instead of the 10-fold diluted solution. The tissue failure rate (%) was calculated by the following formula I.
Tissue failure rate (%) = (SN) / (PN) x 100 (Formula I)
In Formula I, S is the absorbance in the specimen, N is the absorbance in the negative control, and P is the absorbance in the positive control.

As a result, as shown in Table 10, when the mouse skin tissue section was stored at 4 ° C. for 7 days (low temperature injury), the tissue injury rate induced when no addition was made in physiological saline was 22.4%. It was. On the other hand, when the compound (2) is added to a mouse skin tissue section at a final concentration of 0.1 to 100 μg / ml during low-temperature storage, the tissue damage rate is reduced depending on the dose. A low-temperature damage protection effect was confirmed.

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.

After BALB / cAnNCrlCrlj (female, purchased from Charles River Japan Co., Ltd.) was exsanguinated under anesthesia, the small intestine was removed and a 4-4.5 mm square intestinal tissue section was obtained using a surgical scalpel. Each section was submerged in physiological saline containing 0.5 μml / well (24-well culture plate) physiological saline or compound (2) previously cooled to 4 ° C. A test group in which a mouse intestinal tissue section was immersed in 0.5% 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 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. The tissue failure rate (%) was calculated by the following formula I.
Tissue failure rate (%) = (SN) / (PN) x 100 (Formula I)
In Formula I, S is the absorbance in the specimen, N is the absorbance in the negative control, and P is the absorbance in the positive control.

As a result, as shown in Table 11, when the mouse intestinal tissue section was stored at 4 ° C. for 7 days (cold injury), the tissue damage rate induced when no addition was made in physiological saline was 88.3%. It was. On the other hand, when the compound (2) is added to the mouse intestinal tissue section at a final concentration of 0.1 to 10 μg / ml during low-temperature storage, the tissue damage rate is reduced depending on the dose, and the compound of the present invention against the mouse intestinal tissue section is reduced. A low-temperature damage protection effect was confirmed.

Claims (11)

Formula (I):

[Wherein, 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 Is a preservative for cryopreservation of a biological material containing a flavonoid glycoside compound represented by the formula (low temperature is higher than 0 ° C. and 20 ° C. or lower). The preservative according to claim 1, wherein the flavonoid glycoside compound is at least one selected from kaempferol-7-glucoside and quercetin-3-O-glucoside. The preservative according to claim 1, wherein the biological material is an animal or plant cell, a cultured cell sheet, a tissue, an organ or an organ. 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 cell, nerve Cells, pancreatic cells, smooth muscle cells, cardiomyocytes, myoblasts, corneal cells, retinal cells, chondrocytes, cartilage precursor cells, synovial cells, synovial stem cells, osteoblasts, odontocytes, periodontal ligament cells, The preservative according to claim 1, which is an oral mucosal cell, mesenchymal stem cell, adipocyte, adipose stem cell, ovary, semen, blood, blood cell or platelet. The preservative according to claim 1, wherein the biological material is beef, pork, chicken, fish, shellfish, cereals, vegetables or fruits, or flowers as food. A storage solution for low-temperature storage of biological materials containing the preservative according to claim 1. Formula (I):

[Wherein 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. The method according to claim 7, wherein the flavonoid glycoside compound is at least one selected from kaempferol-7-glucoside and quercetin-3-O-glucoside. The method according to claim 7, wherein the biological material is an animal or plant cell, a cultured cell sheet, a tissue, an organ or an organ. 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 cell, nerve Cells, pancreatic cells, smooth muscle cells, cardiomyocytes, myoblasts, corneal cells, retinal cells, chondrocytes, cartilage precursor cells, synovial cells, synovial stem cells, osteoblasts, odontocytes, periodontal ligament cells, The method according to claim 7, which is an oral mucosal cell, mesenchymal stem cell, adipocyte, adipose stem cell, ovary, semen, blood, blood cell or platelet. The method according to claim 7, wherein the biological material is beef, pork, chicken, fish, shellfish, cereals, vegetables or fruits, or flowers as food.

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