Method for preventing animal diseases and composition for preventing animal diseases

Abstract

Provided are a method for preventing animal diseases and composition for preventing animal diseases. This method for preventing animal diseases is characterized by comprising a step for administering mesenchymal stem cells or a composition containing mesenchymal stem cells to an animal. The mesenchymal stem cells are preferably adipose-derived stem cells, and the animal is preferably a young animal.

Description

Method for preventing disease in animals and composition for preventing disease in animals

[0001] The present invention relates to a method for preventing disease in animals and a composition for preventing disease in animals.

[0002] Dogs and cats have been kept as pets since ancient times and are irreplaceable to humans. While the average lifespan of animals has increased in recent years, the number of animals suffering from illnesses during their lives has increased, resulting in the increasing medical expenses borne by pet owners. Putting aside the medical expenses, it is the common wish of pet owners that their animals live long, healthy lives without suffering from illness.

[0003] In order to maintain the health of animals, it is important to manage their physical condition through daily diet and exercise, and to respond quickly to any illnesses, but there are limits to disease prevention through such physical condition management.

[0004] Therefore, there is a need for a means to prevent animals from contracting the disease.

[0005] Recently, in the field of veterinary medicine for small animals such as dogs and cats, clinical studies using mesenchymal stem cells have been reported for the treatment of keratoconjunctivitis sicca (KCS), inflammatory bowel disease (IBD), and osteoarthritis in dogs, and non-viral stomatitis in cats (Non-Patent Documents 1 to 5). However, these reports concern the treatment of diseases, and do not describe the prevention of diseases.

[0006] Perez-Merino, E.M., Uson-Casaus J.M., Zaragoza-Bayle C., Duq ue-Carrasco, J., Marinas-Pardo L., Hermida-Prieto M., Barrera-Chacon, R., Gu altieri,M.(2015) Safety and efficacy of allogeneic adipose tissue-derived me senchymal s tem cells for treatment of dogs with inflammatory bowel disease: Clinical and la boratory outcomes. Vet J. 206(3):385-390.Villatoro, A.J., Fernandez, V., Claros, S., Rico-Llanos, G.A., B ecerra, J., Andrades, J.A.(2015) Use of adipose-derived mesenchymal ste m cellsin keratoconjunctivitis sicca in a canine model.Biomed Res Int. 201 5;2015:527926.Harman, R., Carlson, K., Gaynor, J., Gustafson, S., Dhupa S.,Clement, K., Hoelzler, M., McCarthy, T., Schwartz, P., Adams, C. (2016) A P rospective, randomized, masked, and placebo-controlled efficacy study of int raarticular allogeneic adipose stem cells for the treatment of osteoarthriti s in dogs. Fr ont Vet Sci. 16;3:81.Arzi,B., Mills-Ko, E., Verstraete, F.J., Kol, A., Walker, N.J ., Badgley,M.R., Fazel, N., Murphy, W.J., Vapniarsky, N., Borjesson, DL(2015)Therapeutic efficacy of fresh, autologous mesenchymal stem cells for severe singref tractory Gingivostomatitis in Cats. Stem Cells Transl Med. 5(1):75-86.Hoffman AM, Dow, SW(2016) Concise Review: Stem Cell ls U CompanionAnimal Disease Models. Stem Cells. 34(7):1709-1729.

[0007] As described above, there is a need for a method and a prophylactic agent for preventing animal diseases. Therefore, an object of the present invention is to provide a method for preventing animal diseases and a composition for preventing animal diseases.

[0008] As a result of extensive research to solve the above problems, the present inventors discovered that administering mesenchymal stem cells to animals makes it possible to prevent diseases in animals, and thus completed the present invention.

[0009] That is, the present invention relates to the following [1] to

[16] . [1] A method for preventing disease in an animal, comprising the step of administering to an animal mesenchymal stem cells or a composition containing mesenchymal stem cells. [2] A method for preventing disease in an animal according to [1], wherein the mesenchymal stem cells are adipose-derived stem cells. [3] A method for preventing disease in an animal according to [1], wherein the animal is a juvenile animal. [4] A method for preventing disease in an animal according to [3], wherein the juvenile animal is 4 to 10 weeks old. [5] A method for preventing disease in an animal according to [1], wherein the animal is an elderly animal. [6] A method for preventing disease in an animal according to [5], wherein the elderly animal is 7 years of age or older. [7] A method for preventing disease in an animal according to [1], wherein the mesenchymal stem cells are allogeneic adipose-derived stem cells from the animal. [8] A method for preventing disease in an animal according to [1], wherein the animal is a dog or a cat. [9] The method for preventing animal disease according to [8], which is a method for preventing digestive system diseases in dogs, skin diseases in dogs, digestive system diseases in cats, ear diseases in cats, urinary system diseases in cats, or respiratory system diseases in cats.

[10] A composition for preventing animal disease, characterized by comprising animal-derived mesenchymal stem cells.

[11] The composition for preventing animal disease according to

[10] , wherein the mesenchymal stem cells are adipose-derived stem cells.

[12] The composition for preventing animal disease according to

[10] , which is administered to young animals or old animals.

[13] The composition for preventing animal disease according to

[10] , which is a prophylactic drug.

[14] The composition for preventing animal disease according to

[10] , wherein the animal is a dog or a cat.

[15] The composition for preventing animal diseases according to

[14] , which is a composition for preventing digestive system diseases in dogs, a composition for preventing skin diseases in dogs, a composition for preventing digestive system diseases in cats, a composition for preventing ear diseases in cats, a composition for preventing urinary system diseases in cats, or a composition for preventing respiratory system diseases in cats.

[16] Mesenchymal stem cells or a composition comprising mesenchymal stem cells for use in preventing digestive system diseases or skin diseases in dogs.

[0010] According to the present invention, it is possible to provide a method for preventing disease in animals and a composition for preventing disease in animals.

[0011] 1 is a graph showing the results of Example 1. FIG. 2 is a graph showing the results of Example 1. FIG. 3 is a graph showing the results of Example 1. FIG. 4 is a graph showing the results of Example 1. FIG. 5 is a graph showing the results of Example 2. FIG. 6 is a graph showing the results of Example 2.

[0012] <Disease Prevention Method for Animals> The disease prevention method for animals of the present invention is characterized by comprising the step of administering mesenchymal stem cells or a composition containing mesenchymal stem cells to an animal. In the present invention, disease prevention in animals means preventing an animal from contracting a disease, and prevention means reducing the possibility of the animal contracting a disease. Furthermore, the diseases targeted for disease prevention in the present invention are not limited to specific diseases. Preferably, the disease prevention method suppresses or reduces the risk of the animal contracting a disease during its lifetime. By implementing the disease prevention method for animals of the present invention, the possibility of an animal contracting a disease can be reduced, which is expected to lead to an increase in lifespan and healthy lifespan, a reduction in insurance payments, and ultimately a reduction in insurance premiums.

[0013] [Subjects for Administration] In the present invention, the animals to which mesenchymal stem cells or compositions containing mesenchymal stem cells are administered are not particularly limited except for humans, and mammals such as dogs and cats are preferred. Young dogs or cats, or elderly dogs or cats, are more preferred. Administration to young dogs or cats provides a high disease prevention effect because the dogs or cats will live longer thereafter. Furthermore, young and elderly dogs and cats have less physical strength than adult dogs and cats, making them more likely to contract a disease, or more likely to develop a severe disease if they do contract one. Therefore, the disease prevention method of the present invention is expected to be highly effective.

[0014] As the young dog or cat, a dog or cat immediately after birth, or a dog or cat within 12 weeks of age, within 10 weeks of age, or within 8 weeks of age is preferred. Furthermore, as the young dog or cat, a dog or cat within 4 to 10 weeks of age is preferred, and a dog or cat within 6 to 8 weeks of age is more preferred.

[0015] As elderly dogs or cats, dogs or cats aged 6 years or older are preferred, dogs or cats aged 7 years or older are more preferred, and dogs or cats aged 8 years or older are even more preferred.

[0016] The animals to be administered are preferably animals that are not yet overtly affected by the disease or animals that are in a healthy state. It is also preferable to target animals that are at high risk of the disease due to their genetic background. For example, genetic testing of animals can be performed, and the disease prevention method of the present invention can be used on animals that are at high risk of the disease but have not yet developed the disease.

[0017] [Mesenchymal stem cells] Mesenchymal stem cells (MSCs) are somatic stem cells present in the living body of mammals. They can be easily cultured outside the body to expand the cell number, and they also have effects such as angiogenic, immunosuppressive, and anti-inflammatory properties. Therefore, clinical research and trials are being conducted on them as immunosuppressants and therapeutic agents for intractable autoimmune diseases, and they are attracting attention as an important field of cell therapy.

[0018] Mesenchymal stem cells are a type of adult stem cell and have been reported to be found in bone marrow, fat, umbilical cord, synovium (tissue surrounding joints), etc. The mesenchymal stem cells used in the present invention preferably have the following characteristics: canine mesenchymal stem cells (1) are positive for the cell surface antigens CD44, CD73, CD90, and / or CD105, and negative for CD14, CD19, CD34, CD45, and / or MHC Class II; (2) adhere to plastic under standard culture conditions, exhibit a fibroblast-like morphology, and proliferate; and (3) have the potential to differentiate into adipocytes, osteoblasts, and chondroblasts. Feline mesenchymal stem cells (1) are positive for CD44 and / or CD29, (2) are negative for CD34 and / or MHC Class II, and (3) have the potential to differentiate into adipocytes, osteoblasts, and chondroblasts.

[0019] Mesenchymal stem cells can be collected from bone marrow, but they can also be collected from adipose tissue. Subcutaneous fat has the following advantages over bone marrow: (1) tissue collection is relatively easy, and (2) the amount of mesenchymal stem cells contained per unit volume of tissue is several hundred times higher than that of bone marrow. Because it is easy to secure the number of cells, subcutaneous fat is a preferred resource for mesenchymal stem cells. Therefore, in the present invention, it is preferable to use adipose-derived stem cells as mesenchymal stem cells. Adipose-derived stem cells may contain cells other than mesenchymal stem cells.

[0020] [Adipose-derived stem cells] Adipose-derived stem cells (ASCs) are somatic stem cells contained in adipose tissue. For example, they can be collected from subcutaneous adipose tissue. In the present invention, adipose-derived stem cells from the same species as the animal to be administered are preferably used. That is, canine adipose-derived stem cells are preferably administered to dogs, and feline adipose-derived stem cells are preferably administered to cats. Either autologous or allogeneic adipose-derived stem cells may be used, but considering the cost and effort involved in regular administration, allogeneic adipose-derived stem cells are preferred. When using autologous adipose-derived stem cells, stem cells are isolated from the adipose tissue of the animal to be administered, cultured, and then administered to the animal. When using allogeneic adipose-derived stem cells, pre-prepared adipose-derived stem cells can be used.

[0021] Adipose-derived stem cells can be prepared by harvesting and culturing them using known methods. For example, in the case of allogeneic transplantation, subcutaneous fat is harvested from a healthy dog ​​under anesthesia using surgical instruments, and the wound is sutured closed. The harvesting site is the abdomen. Adipose-derived stem cells are then recovered from the harvested subcutaneous fat by enzyme treatment, and cultured for several days, for example, 5 to 10 days, preferably 6 to 8 days, and more preferably about 7 days, and then cryopreserved to obtain adipose-derived stem cells. They are thawed appropriately before use when administered.

[0022] For canine adipose-derived stem cells, surface antigen markers of CD90 ≧85% and CD44 ≧85% are preferred, with adipose-derived CD45 ≦3% and MHCII ≦3% being preferred. For feline adipose-derived stem cells, CD44 ≧85% or CD29 ≧20% are preferred, with adipose-derived CD45 ≦3% and MHCII ≦3% being preferred. Here, "-%" refers to the positivity rate of a specific marker in cells contained in the cell population, which can be measured by known methods. If these markers are confirmed, it is not necessary to confirm the above-mentioned preferred mesenchymal stem cell markers.

[0023] When administering allogeneic adipose-derived stem cells, immunosuppressants can be used in combination, but they can also be administered without the use of immunosuppressants. When not using immunosuppressants, it is preferable to check the presence or absence of markers that affect immune responses, such as MHC class II. For example, if the MHC class II positivity rate of the adipose-derived stem cells to be administered exceeds 3%, it is highly likely that they will cause an immune rejection reaction, so it is preferable to avoid administering them to animals.

[0024] [Preparation of adipose-derived stem cells] An example of a procedure for preparing adipose-derived stem cells (procedures such as culturing, selection, recovery, freezing, and thawing) is described below. Specifically, one embodiment of a process for isolating and obtaining adipose-derived stem cells from adipose tissue contained in a dog is shown. The basic procedure is similar for cats and other animals. However, the present invention should not be construed as being limited thereto.

[0025] (1) Preparation of cell population from adipose tissue Adipose tissue is collected by means of resection or the like under anesthesia. The collected adipose tissue is exposed to 70% ethanol for a short period of time to clean and sterilize bacteria, viruses, etc. adhering to the tissue, immersed in a buffer solution or culture medium, and then subjected to the following enzyme treatment. Sterilization treatment also includes methods using known disinfectants such as 10% iodine solution.

[0026] The enzymatic treatment involves enzymatically digesting the adipose tissue with an enzyme solution of collagenase Type I (0.1 to 5 mg / mL) for 30 to 120 minutes at the optimum temperature for the enzyme, for example, 37°C, to obtain a cell population (solution) containing adipose-derived stem cells. The enzymes used to decompose the adipose tissue include trypsin, dispase, and other known methods using commercially available digestive enzymes for adipose tissue.

[0027] The cell population containing adipose-derived stem cells is fractionated by centrifugation. The enzyme-treated solution is dispensed into a 50 mL centrifuge tube and centrifuged at 750 to 1500 G (1.0 G = 9.80665 m / s ２ ) and recover a cell population containing adipose-derived stem cells from the sediment fraction at the bottom of the tube. The centrifugal acceleration may be changed depending on the amount of adipose tissue. The sediment fraction is added to a new centrifuge tube containing D-PBS and washed to remove remaining oil and fat components and other impurities. The solution containing adipose-derived stem cells is filtered through a cell strainer with a pore size of 70 to 100 μm to remove ECM, undegraded adipose tissue, and other components from the adipose tissue. The buffers, filtration filters, and devices used in this section may also include other known buffers, filtration membranes, and devices.

[0028] (2) Selective culture of adherent fibroblast-like cells and recovery of cells (P0) The solution containing the cells is centrifuged at 750 to 1500 G, and the precipitated fraction at the bottom of the tube is recovered as a cell population containing adipose-derived stem cells. An appropriate medium is added to this and the cells are suspended, and then the cells are collected by centrifugation at 225 cm ２The suspension is transferred to a culture flask and cultured for 7 to 10 days, with repeated washing and medium changes every 3 to 4 days. The culture environment in the culture incubator is 37°C with a carbon dioxide concentration of 5%. A typical culture medium for animal cell culture can be used. Examples include Dulbecco's Modified Eagle's Medium (DMEM) (Fujifilm Wako Pure Chemical Corporation, etc.), α-MEM (Fujifilm Wako Pure Chemical Corporation, etc.), DMED:Ham's F12 mixed medium (1:1) (Fujifilm Wako Pure Chemical Corporation, etc.), and Ham's F12 Medium (Fujifilm Wako Pure Chemical Corporation, etc.). Serum added to the culture medium can be derived from fetal bovine serum (FBS), human serum, sheep serum, etc. The amount of serum or serum substitute added may be, for example, within the range of 5% (v / v) to 30% (v / v) of the medium during culture.

[0029] To recover the proliferated cells, the recovery procedure follows the standard method for detaching cells adhered to the bottom of the flask, for example, by detaching the cells after enzymatic treatment (trypsin or dispase treatment). The detached cells are suspended in a medium containing fetal bovine serum to inhibit trypsin activity, and then centrifuged at 500-1500 G to remove medium components. The cell population is then washed again with D-PBS and used as a suspension of adipose-derived stem cells (P0) at Passage 0.

[0030] (3) Selective culture of adipose-derived stem cells and collection of cells (P1) 3-5 × 10 adipose-derived stem cells of P0 were collected. ４ cells / cm ２ The cells were prepared at a concentration of 225 cm. ２ The suspension is transferred to a culture flask and cultured for 6 to 10 days, with repeated washing and medium changes every 3 to 4 days. The culture environment in the incubator is 37°C with a carbon dioxide concentration of 5%. As with P0 cell culture, a normal animal cell culture medium can be used for the culture medium.

[0031] The proliferated cells (P1) can be recovered by conventional methods for detaching cells adhered to the bottom of a flask, for example, by detaching the cells after enzymatic treatment (trypsin or dispase treatment). The detached cells are suspended in a medium containing FBS to inhibit trypsin activity, then washed with D-PBS and centrifuged at 500-1500 G to wash away the contained medium components. The cell population is then washed again with D-PBS and used as a mesenchymal stem cell suspension for Passage 1 (P1).

[0032] (4) Cell Cryopreservation Method P0 and P1 cells were cryopreserved at 1 × 10 ６ ~1 x 10 ７ The cells are suspended in a cryopreservation solution at a concentration of 1000 cells / mL, dispensed into cryotubes, and slowly frozen in a slow freezer at -80°C. A cryopreservation solution typically used for freezing animal cells can be used. Commercially available cell freezing solutions containing or not containing dimethyl sulfoxide, such as CELL BANKER I (TAKARA), COS BANKER (Cosmo Bio Co., Ltd.), and Bambanker (Nippon Genetics), can also be used. The concentration of dimethyl sulfoxide (DMSO) contained in the freezing solution is, for example, 0% (v / v) to 10% (v / v), preferably 1% to 10%, and more preferably 5 to 10%. The freezing solution may contain another cryoprotectant instead of DMSO.

[0033] (5) Cell Thawing Method The frozen P0 and P1 cells are thawed according to standard cell thawing methods. The thawed cell suspension is centrifuged at 400 to 1500 G, and after removing the cell freezing solution, a buffer such as D-PBS is added to the cell mass, followed by repeated washing and centrifugation. Methods for thawing frozen tubes include rapid thawing using, for example, an automatic thawing device, the Thawstar Cell Freezing and Thawing Station (Biocision).

[0034] [Composition containing mesenchymal stem cells] The composition containing mesenchymal stem cells is the same as the disease preventive composition of the present invention described below.

[0035] [Administration Method] The method of administering mesenchymal stem cells is not particularly limited and known methods can be used. From the viewpoint of reducing the burden on animals, intravenous drip, intravenous injection, subcutaneous injection, or intramuscular injection is preferred. Subcutaneous injection or intramuscular injection is more preferred than intravenous drip because it allows for a shorter administration time. The present invention relates to a preventive method and preventive composition, which is preferably administered before an animal becomes afflicted with a disease. Therefore, it is not administered directly to the affected area. The administration site is preferably from the head and neck to the trunk, limbs, or back muscles (around the latissimus dorsi or lumbar muscles) of the animal, and more preferably the legs, buttocks, or abdomen of the animal.

[0036] [Dosage] The preferred dosage of mesenchymal stem cells is 1 x 10 per administration. ４ pieces / kg (weight) ~1×10 ８ 1 x 10 ５ pieces / kg ~ 1×10 ７ Pieces / kg (body weight).

[0037] [Administration Schedule] In the preventive method of the present invention, adipose-derived stem cells are administered once or multiple times. A single administration in infancy is expected to provide a long-term disease preventive effect thereafter. Periodic administration is also preferred. Periodic administration is expected to continuously suppress disease onset. For example, administration schedules include administration once a year, administration once every few months, preferably once every 3 months to a year, or once every 6 months to a year.

[0038] <Disease preventive composition> The disease preventive composition of the present invention is characterized by containing mesenchymal stem cells, and is used to prevent diseases in animals such as dogs and cats. That is, the disease preventive composition of the present invention is a composition for use in preventing diseases in animals such as dogs and cats.

[0039] The disease-preventing composition of the present invention contains mesenchymal stem cells, preferably adipose-derived stem cells, as an active ingredient. The composition is preferably a cell suspension. The concentration of mesenchymal stem cells in the composition is preferably 1 x 10 ４ cells / mL or more, and more preferably 1 x 10５ 5000 / mL or more, more preferably 4 x 10 ６ The disease preventive composition of the present invention may contain, in addition to the active ingredient, a solvent such as water, and a secondary ingredient such as a buffer solution.

[0040] Examples of the buffer solution include Ringer's solution, L-sodium lactate Ringer's solution, 5% glucose-lactated Ringer's solution, acetated Ringer's solution, 5% glucose-acetated Ringer's solution, Dulbecco's phosphate buffered saline (D-PBS), saline, Good's buffer, Hank's balanced salt solution, phosphate buffer (PBS), imidazole buffer, triethanolamine hydrochloride buffer (TEA), and combinations thereof.

[0041] The disease preventive composition of the present invention may also contain nutrients and chelating agents. Furthermore, the disease preventive composition of the present invention is preferably substantially free of medium components for animal cells, organic solvents, and dextran.

[0042] The disease preventive composition of the present invention comprises mesenchymal stem cells, preferably adipose-derived stem cells, from animals such as dogs and cats suspended in a buffer solution, and the concentration of mesenchymal stem cells in the composition is 1×10 ５ Preferably, the concentration of ascorbic acid in the buffer solution is 1 mmol / L to 150 mmol / L, and the concentration of ethylenediaminetetraacetic acid or citric acid in the buffer solution is 1 mmol / L to 100 mmol / L, and the buffer solution contains at least one of ascorbic acid, ethylenediaminetetraacetic acid, and citric acid, but does not contain any sugars other than trehalose and does not contain 2% or more of trehalose.

[0043] The disease preventive composition of the present invention may be stored frozen or kept at 0 to 10°C.

[0044] [Dosage Form] The disease preventive composition of the present invention is preferably a prophylactic drug. The dosage form of the prophylactic drug is not particularly limited, and any known dosage form may be used. Examples include dosage forms such as a cell suspension, an injection solution, an infusion solution, and a capsule. A cell suspension in which cells are suspended in a solvent is preferred, and an injection solution or an infusion solution made of a cell suspension is more preferred.

[0045] [Diseases] The disease targeted for disease prevention in the present invention is not limited to a specific disease. Preferably, the disease is prevented or reduced in animals such as dogs and cats from contracting any disease during their lifetime. Examples of the types of diseases targeted in the present invention include skin diseases, otic diseases, musculoskeletal diseases, ophthalmological diseases, digestive system diseases, systemic diseases, urinary system diseases, hepatic, biliary, and pancreatic diseases, circulatory system diseases, nervous system diseases, respiratory system diseases, dental and oral diseases, endocrine system diseases, reproductive system diseases, and blood and hematopoietic system diseases. Preferred are digestive system diseases, skin diseases, otic diseases, urinary system diseases, and respiratory system diseases. More preferred are digestive system diseases or skin diseases in dogs, and digestive system diseases, otic diseases, urinary system diseases, and respiratory system diseases in cats. Examples of skin diseases include dermatitis, atopic dermatitis, and pyoderma. Examples of otic diseases include otitis externa and otitis media. Examples of musculoskeletal diseases include patellar luxation and herniated disc. Examples of ophthalmic diseases include conjunctivitis, eye discharge, keratitis, corneal ulcer / erosion, epiphora, cataracts, and glaucoma. Examples of digestive diseases include gastritis, enteritis, gastroenteritis, vomiting, diarrhea, and bloody stool. Examples of systemic diseases include loss of energy and collapse. Examples of urinary diseases include cystitis and urolithiasis. Examples of hepatobiliary and pancreatic diseases include biliary sludge and chronic renal failure. Examples of circulatory system diseases include valvular disease and cardiomyopathy. Examples of nervous system diseases include epilepsy and seizures. Examples of respiratory system diseases include coughing, rhinitis, tracheal collapse, and bronchial stenosis. Examples of dental and oral diseases include periodontal disease and stomatitis. Endocrine system disorders include, for example, hypothyroidism and diabetes. Reproductive system disorders include, for example, mammary tumors and balanitis. Blood and hematopoietic system disorders include, for example, lymphatic tissue tumors and thrombocytopenia.

[0046] The subjects to which the disease preventive composition of the present invention is administered are the same as those in the disease preventive method described above.

[0047] Abbreviations and trade names in the examples have the following meanings: PBS: D-PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd.) Sodium ascorbate: L(+)-sodium ascorbate (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0048] Example 1 Preparation of Canine Adipose-Derived Stem Cells (1) Preparation of Cell Population from Adipose Tissue Adipose tissue was collected from the subcutaneous fat of healthy dogs. The collected adipose tissue was sterilized for a short time with 70% ethanol and then enzymatically digested with a collagenase Type I enzyme solution (0.1-5 mg / mL) at 37°C for 60 minutes to obtain a cell population (solution) containing adipose-derived stem cells.

[0049] To fractionate a cell population containing adipose-derived stem cells, the enzyme-treated solution was dispensed into a 50 mL centrifuge tube and centrifuged at 750-1500 G, and the precipitated fraction at the bottom of the tube was collected as a cell population. The precipitated fraction was then transferred to a new centrifuge tube containing PBS and rinsed to remove any remaining oils and fats and other impurities. The extracellular matrix and undegraded adipose tissue in the adipose tissue were then removed by filtration through a cell strainer with a pore size of 70 μm, preparing a cell-containing solution.

[0050] (2) Cultivation and recovery of adipose-derived stem cells The solution containing the cells was centrifuged at 1500 G, and the precipitated fraction at the bottom of the tube was recovered as a cell population containing adipose-derived stem cells. An appropriate medium was added to this and the cells were suspended, and then the cells were collected at 225 cm ２ The suspension was transferred to a culture flask and cultured for 7 to 10 days, with repeated washing and medium changes every 3 to 4 days. Culture was carried out at 37°C and a carbon dioxide concentration of 5%. The culture medium used was Mesenchymal Stem Cell Growth Medium Bullet Kit™ (MSCGM, Lonza).

[0051] To recover the proliferated cells, the recovery procedure followed the standard method for detaching cells adhered to the bottom of the flask, and the cells were recovered by reacting them with 0.05% (v / v) trypsin at 37°C for 5 minutes. The detached cells were suspended in a medium containing fetal bovine serum (FBS, Yakensha Co., Ltd. (product of HiClone Co., Ltd.)) and then centrifuged at 500-1500 G to remove the trypsin. The cells were then suspended in medium to obtain a P0 adipose-derived stem cell suspension.

[0052] (3) Cultivation and collection of adipose-derived stem cells. P0 mesenchymal stem cells were cultured at 225 cm ２ The suspension was transferred to a culture flask and cultured for 7 to 10 days, with repeated washing and medium changes every 3 to 4 days. Culture was carried out at 37°C and a carbon dioxide concentration of 5%. The culture medium used was D-MEM (Fujifilm Wako Pure Chemical Corporation) or similar medium supplemented with FBS. After reaction with trypsin in the same manner as for the recovery of P0 above, centrifugation was carried out to obtain a P1 adipose-derived stem cell suspension.

[0053] (4) Cryopreservation of cells P1 cells were cryopreserved at 1 × 10 ６ ~5 x 10 ６ The cells were suspended in a cryopreservation solution at a concentration of 1000 cells / mL, dispensed into cryotubes, and slowly frozen in a slow freezer at -80°C. A commercially available cell freezing solution containing dimethyl sulfoxide (DMSO) was used as the cryopreservation solution. The cells were then stored in an ultra-low temperature freezer (-80°C to -150°C) or in liquid nitrogen (-196°C).

[0054] (5) Thawing of Cells Frozen P1 cells were thawed using an automatic cell freeze-thaw station, Thawstar (Biocision), according to standard cell thawing methods. The cells were centrifuged at 2000 G to remove the cell freezing solution. The cell population was suspended in saline and centrifuged, after which the cells were suspended in PBS containing 25 mM sodium ascorbate.

[0055] <Administration of adipose-derived stem cells to dogs> The suspension of canine adipose-derived stem cells prepared above was administered once by intramuscular injection to a total of 283 young dogs (including various breeds) aged 5 to 8 weeks. The dose was approximately 1 x 10６ The number of dogs administered adipose-derived stem cells was 1 / kg (body weight). Hereinafter, the group administered adipose-derived stem cells will be referred to as the "administration group." Additionally, 952 dogs born around the same time as the administration group but not administered the adipose-derived stem cell suspension were designated the "non-administration group." Both the administration and non-administration groups had pet insurance, and the insurance claims records were traced to confirm the amount of insurance paid to policyholders. To limit insurance claims to those due to disease, poisoning, fractures, accidents, and ingestion of foreign objects were excluded. The average amount of insurance paid to the administration group and the non-administration group per month was calculated. The results are shown in Figure 1. In Figure 1, each bar graph represents the amount of insurance paid for each month (months since the start of the experiment). For each month, the left bar represents the average amount for the non-administration group, and the right bar represents the average amount for the administration group. The line graph shows the cumulative difference between the average amount of insurance paid to the non-administration group and the average amount of insurance paid to the administration group.

[0056] The bar graph shows that the average amount of insurance claims due to illness each month was higher in the non-treated group than in the treated group, demonstrating that administering adipose-derived stem cells to dogs has a disease prevention effect. The high amount of insurance claims excluding poisoning, fractures, accidents, and ingestion of foreign objects means that there were many insurance claims due to illness. Furthermore, as is clear from the line graph showing the cumulative difference, the difference in insurance claims paid over 13 months between the treated and non-treated groups was 6,899 yen per dog.

[0057] Insurance claims due to vomiting, diarrhea, or bloody stools were collected for the above-mentioned treatment group and non-treatment group, and the average amount of insurance paid to the treatment group and the average amount of insurance paid to the non-treatment group per month were calculated. As is clear from the line graph showing the cumulative difference, the non-treatment group paid 207 yen per head more in insurance money than the treatment group over a 13-month cumulative total.

[0058] Insurance claims due to gastritis, gastroenteritis, or enteritis were collected for the above-mentioned treatment group and non-treatment group, and the average amount of insurance paid to the treatment group and the average amount of insurance paid to the non-treatment group per month were calculated. As is clear from the line graph showing the cumulative difference, the non-treatment group paid 446 yen per head more in insurance payments than the treatment group over a 13-month cumulative period.

[0059] Insurance claims due to skin diseases were collected for the above-mentioned treatment group and non-treatment group, and the average amount of insurance paid to the treatment group and the average amount of insurance paid to the non-treatment group per month were calculated. As is clear from the line graph showing the cumulative difference, the cumulative amount of insurance paid to the non-treatment group over 13 months was 1,478 yen per head more than the treatment group.

[0060] Example 2 Preparation of Feline Adipose-Derived Stem Cells (1) Preparation of a Cell Population from Adipose Tissue Adipose tissue was collected from the subcutaneous fat of a healthy cat. The collected adipose tissue was sterilized briefly with 70% ethanol and then enzymatically digested with a collagenase Type I enzyme solution (0.1-5 mg / mL) at 37°C for 60 minutes to obtain a cell population (solution) containing adipose-derived stem cells. To fractionate the cell population containing adipose-derived stem cells, centrifugation and washing were performed in the same manner as in Example 1 above to prepare a cell-containing solution. Next, (2) culturing and recovering adipose-derived stem cells, (3) culturing and recovering adipose-derived stem cells, (4) cryopreserving the cells, and (5) thawing the cells were performed in the same manner as in Example 1.

[0061] <Administration of adipose-derived stem cells to cats> The suspension of feline adipose-derived stem cells prepared above was administered once to a total of 7,510 young cats (including various breeds) aged 6 to 8 weeks by intramuscular injection into either the left or right thigh muscle group. The dose was approximately 1 x 10 ６The number of cats administered adipose-derived stem cells was 1 / kg (body weight). Hereafter, the group administered adipose-derived stem cells will be referred to as the "administration group." Additionally, 10,371 cats born around the same time as the administration group but not administered the adipose-derived stem cell suspension were designated the "non-administration group." Both the administration and non-administration groups were covered by pet insurance, with the contract period starting from the time of delivery to the owner (the pet insurance policyholder). Insurance payments to policyholders were confirmed by tracing pet insurance claim records. To limit insurance claims to those due to disease, poisoning, fractures, accidents, and ingestion of foreign objects were excluded. For each cat, the average insurance payments to the administration group and the non-administration group were calculated for each month from delivery to the owner (the pet insurance policyholder). The results are shown in Figure 5. For the administration group, the time between administration and delivery to the owner was within a few weeks.

[0062] Figure 5 shows that after three months of administration, the average insurance payment amount was lower in the treatment group than in the non-treatment group. This is thought to be because the administration of adipose-derived stem cells prevented the cats from contracting diseases, resulting in a reduction in pet insurance payments.

[0063] Next, we calculated the cumulative insurance amount per cat for the six-month period from the time of delivery to the owner (the pet insurance policyholder), i.e., the start of the pet insurance contract period, for the 253 cats in the treatment group that received adipose-derived stem cells between 6 and 8 weeks of age, and the 3,097 cats in the control group that were born around the same time as the treatment group but did not receive adipose-derived stem cells, in a similar manner as above. Furthermore, we selected the diseases that were the reason for the insurance claim from each insurance claim record and calculated the cumulative insurance amount for each disease. Diseases included digestive system diseases (gastritis, enteritis, gastroenteritis, vomiting, diarrhea, bloody stool), ear diseases (otitis externa, otitis media), urinary system diseases (cystitis, urolithiasis), and respiratory system diseases (cough, rhinitis, tracheal collapse, bronchial stenosis). The results are shown in Figure 6.

[0064] In Figure 6, the leftmost bar graph labeled "Overall" shows the cumulative insurance amount per pet for the six months from the time of delivery to the owner (the pet insurance policyholder), i.e., from the start of the pet insurance contract period, for both the treatment and non-treatment groups. This shows that, overall, the insurance amount for the treatment group was 26% lower than that for the non-treatment group. Next, looking at the insurance amount for each disease—digestive system disease, ear disease (otologic disease), urinary system disease, and respiratory system disease—we can see that for each disease, the treatment group had a lower insurance amount than the non-treatment group. This shows that the administration of adipose-derived stem cells has a preventive effect on feline diseases, particularly digestive system disease, ear disease, urinary system disease, and respiratory system disease, resulting in lower insurance amounts.

Claims

1. A method for preventing disease in an animal, comprising the step of administering to the animal mesenchymal stem cells or a composition containing mesenchymal stem cells.

2. The method for preventing disease in animals according to claim 1, wherein the mesenchymal stem cells are adipose-derived stem cells.

3. The method for preventing disease in animals according to claim 1, wherein the animals are young animals.

4. The method for preventing a disease in an animal according to claim 3, wherein the young animal is an animal aged 4 to 10 weeks.

5. The method for preventing disease in animals according to claim 1, wherein the animal is an elderly animal.

6. The method for preventing disease in animals according to claim 5, wherein said aged animals are animals aged 7 years or older.

7. The method for preventing disease in animals according to claim 1, wherein the mesenchymal stem cells are allogeneic adipose-derived stem cells of the animal.

8. The method for preventing disease in animals according to claim 1, wherein the animal is a dog or a cat.

9. The method for preventing animal diseases according to claim 8, which is a method for preventing digestive system diseases in dogs, skin diseases in dogs, digestive system diseases in cats, ear diseases in cats, urinary system diseases in cats, or respiratory system diseases in cats.

10. A composition for preventing disease in animals, comprising animal-derived mesenchymal stem cells.

11. The composition for preventing disease in animals according to claim 10, wherein the mesenchymal stem cells are adipose-derived stem cells.

12. The composition for preventing disease in animals according to claim 10, which is administered to young or old animals.

13. The composition for preventing a disease in an animal according to claim 10, which is a preventive drug.

14. The composition for preventing disease in animals according to claim 10, wherein the animal is a dog or a cat.

15. A composition for preventing animal diseases as described in claim 14, which is a composition for preventing digestive system diseases in dogs, a composition for preventing skin diseases in dogs, a composition for preventing digestive system diseases in cats, a composition for preventing ear diseases in cats, a composition for preventing urinary system diseases in cats, or a composition for preventing respiratory system diseases in cats.

16. Mesenchymal stem cells or a composition comprising mesenchymal stem cells for use in preventing digestive or skin diseases in dogs.

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