Method for producing serum for culturing mammalian cells

Abstract

Provided is a method for producing blood serum that exhibits improved cell proliferation activity.　A method for producing blood serum for culturing mammalian cells, the method including: maintaining blood at a temperature of 15-25°C, the blood having been harvested from a first mammal; extending the maintenance of said temperature to within 48 hours; and preparing blood serum from said blood.

Description

[Technical field]

[0001] The present invention relates to a method for producing serum for culturing mammalian cells.The present invention also relates to a method for producing a medicament comprising a culture of mammalian cells. [Background technology]

[0002] In recent years, research and development of regenerative medicine for the central nervous system has been conducted. One example of regenerative medicine using stem cells is cell therapy, which replenishes damaged cells. In cell therapy for brain and nerve diseases, cells capable of differentiating into nervous system cells are extracted from tissue and cultured, and the culture is then transplanted into an individual (Patent Document 1).

[0003] When using cell therapy to treat acute disorders such as spinal cord injury caused by accidents, it is important to rapidly and massively grow cells provided by a donor (donor cells). Regarding cell growth outside of the body, it has been reported that growth-promoting factors (e.g., serum) can be added to the medium in which donor cells are cultured in order to improve cell survival and proliferation rates.

[0004] In the field of cell therapy, serum is often used to efficiently grow desired cells. The serum used in the manufacture of medicines for cell therapy is desirably autologous serum derived from the subject, which has little effect on the subject, since it is introduced into the body when the medicine containing cultured cells is applied to the body of the subject. However, human serum alone does not provide the desired cell growth promoting activity, so fetal bovine serum (FBS) or other growth factors are added (Patent Document 2 and Patent Document 3).

[0005] Medical facilities where blood, which is the raw material for autologous serum, is collected from subjects rarely have the equipment to prepare serum in sufficient quantity and quality for the production of pharmaceuticals for cell therapy, and the serum is usually transported to a manufacturing facility for medical cell cultures that meets specified standards (for example, a cell processing center (CPC) that meets GMP standards) and then produced there. In this case, the blood needs to be transported from the medical facility to the manufacturing facility, and depending on the location, this may require a long transportation period, which may lead to a deterioration in the quality of the blood. In order to prevent the deterioration of blood quality due to long transportation periods, a method of adding a preservative to the blood has been proposed (Patent Document 4). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. WO02 / 00849 A1 Brochure [Patent Document 2] Japanese Patent Application Publication No. 10-179148 [Patent Document 3] JP 2003-235548 A [Patent Document 4] JP 2015-151334 A Summary of the Invention [Problem to be solved by the invention]

[0007] Even if a desired cell proliferation-promoting activity can be obtained by adding a proliferation-promoting factor derived from another organism, such as FBS, to a culture medium containing human serum, the safety testing of the proliferation-promoting factor derived from another organism is time-consuming and costly. Therefore, there has been a demand in the art for a method for producing serum that exhibits a desired cell proliferation activity without adding a proliferation-promoting factor derived from another organism.

[0008] In cell therapy using autologous cells grown in a medium containing autologous serum, the amount of blood that can be collected at one time is limited from the viewpoint of maintaining the health of the subject. For this reason, there has been a demand in this field for a method for producing serum exhibiting the desired cell proliferation activity from a collectable amount of blood.

[0009] Although the addition of a preservative can provide stability for long-term transportation, in the case of pharmaceuticals for cell therapy, such preservatives are also introduced into the body of the subject. Therefore, from the viewpoint of safety, there has been a demand for a method for producing a serum that exhibits the desired cell proliferation activity without using a preservative. [Means for solving the problem]

[0010] The inventors have discovered that by maintaining blood taken from an individual at a temperature within a predetermined range and maintaining the blood at that temperature for a period of time within a predetermined period of time, serum prepared from the blood exhibits the desired cell proliferation activity, and have completed the present invention.

[0011] A first aspect of the present invention provides a method for producing serum for culturing mammalian cells, comprising maintaining blood collected from a first mammal at a temperature of 15° C. to 25° C. for no more than 48 hours, and preparing serum from the blood. A second aspect of the present invention provides a method for producing a pharmaceutical comprising a culture of mammalian cells, comprising culturing mammalian cells in a medium, the medium comprising serum for culturing the mammalian cells, the serum being prepared from blood collected from a first mammal at a temperature of 15° C. to 25° C. for no more than 48 hours. Effect of the Invention

[0012] The present invention provides a method for producing a serum exhibiting improved cell proliferation activity, and a method for producing a medicament, which comprises proliferating mammalian cells using the serum produced by the method. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is a bar graph showing the cell proliferation rate obtained using serum prepared from blood that had been incubated at 10° C., 20° C., or 25° C. for a given period of time. [Diagram 2] FIG. 2 is a bar graph showing the cell proliferation rate obtained using serum prepared from blood that had been incubated at 15° C., 20° C., or 25° C. for the indicated times. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] As used herein, "blood" refers to a body fluid flowing through a mammalian body, and containing blood cell components such as red blood cells, white blood cells, and platelets, and plasma. The blood collected from a mammal may be, but is not limited to, venous blood, arterial blood, or peripheral blood. In one embodiment, the blood collected from a mammal is peripheral blood. The blood can be collected from a mammal according to, but is not limited to, a known method. In one example, the blood can be collected using a syringe needle and a blood collection container. In one embodiment, the blood for preparing serum does not contain a preservative.

[0015] As used herein, a "blood collection container" refers to a container capable of aseptically holding blood collected from a mammal. The blood collection container may be, but is not limited to, a container known in the art of blood collection and commercially available. The blood collection container may be, for example, a blood collection bag or a blood collection tube. In one example, the blood collection container may be a blood-containing device as described in U.S. Patent Application Publication No. US2013 / 0130382, the contents of which are incorporated herein by reference.

[0016] As used herein, "serum" refers to a liquid portion separated from a clot formed by coagulation of mammalian blood. Serum can be produced from blood collected from a mammal using a known method, without being limited thereto. For example, serum can be used by adding it to a medium for culturing mammalian cells using a dispenser or the like.

[0017] As used herein, the term "blood clot" refers to a structure formed during the process of clotting of mammalian blood. Blood clots include, but are not limited to, blood cell components and fibrin (also called "fibrin"). In one example, a blood clot can be formed by leaving mammalian blood in a sterile container at room temperature (e.g., 22°C to 27°C) without the addition of an anticoagulant according to a known method.

[0018] As used herein, "serum for culturing mammalian cells" refers to serum obtained from blood collected from a mammal and maintained at a temperature of 15°C to 25°C for 48 hours or less. Maintaining the blood at a temperature of 15°C to 25°C may mean, but is not limited to, placing a container containing the blood inside a specific temperature in the range of 15°C to 25°C or a container or device capable of adjusting the temperature to a specific temperature range, or a combination thereof. The blood maintained at a temperature of 15°C to 25°C may be, but is not limited to, left stationary or transported.

[0019] In this specification, the term "temperature-adjustable container or device" refers to a container or device that has a function of adjusting the temperature inside the container or device to a predetermined temperature range. The temperature-adjustable container or device may be, but is not limited to, a container or device that is covered with a heat insulating material and is not easily affected by the surrounding environmental temperature. The heat insulating material may be, for example, polystyrene foam, hard urethane foam, or vacuum insulation material (for example, a structure in which a glass wool core material or a carbon material is wrapped in a gas barrier film and the inside of the film packaging is made vacuum and sealed), or a combination thereof. The temperature-adjustable container may include, but is not limited to, a heat storage material, a heat generating agent, a cold storage material, a cold packing agent, or a temperature adjustment unit (for example, a small cooling device equipped with a Peltier element controlled by a thermostat) or a combination thereof inside the container. The temperature-adjustable device may include, but is not limited to, a temperature adjustment unit (for example, a small cooling device equipped with a Peltier element controlled by a thermostat), and in some cases a heat storage material or a cold storage material. The temperature-controllable device may be, for example, a means of transportation equipped with a temperature control function (for example, a car, a ship, or an airplane). The combination of a temperature-controllable container and a device may be, but is not limited to, a combination of a container containing blood in a temperature-controllable container, and the container in the temperature-controllable device.

[0020] The blood collected from a mammal may be kept at a temperature within a specific range, including but not limited to, a temperature range of 15°C to 24°C, a temperature range of 15°C to 23°C, a temperature range of 15°C to 22°C, a temperature range of 15°C to 21°C, a temperature range of 15°C to 20°C, a temperature range of 16°C to 25°C, a temperature range of 16°C to 24°C, a temperature range of 16°C to 23°C, a temperature range of 16°C to 22°C, a temperature range of 16°C to 21°C, a temperature range of 16°C to 20°C, a temperature range of 17°C to 25°C, a temperature range of 17°C to 24°C, a temperature range of 17°C to 23°C, a temperature range of 17°C to 22°C, or a temperature range of 16°C to 25°C. The temperature is controlled within a container or device, or a combination thereof, capable of controlling the temperature to a temperature range of 17°C to 21°C, a temperature range of 17°C to 20°C, a temperature range of 18°C ​​to 25°C, a temperature range of 18°C ​​to 24°C, a temperature range of 18°C ​​to 23°C, a temperature range of 18°C ​​to 22°C, a temperature range of 18°C ​​to 21°C, a temperature range of 18°C ​​to 20°C, a temperature range of 19°C to 25°C, a temperature range of 19°C to 24°C, a temperature range of 19°C to 23°C, a temperature range of 19°C to 22°C, a temperature range of 19°C to 21°C, or a temperature range of 19°C to 20°C.

[0021] In one embodiment, blood collected from a mammal is placed inside a temperature-controllable container or device, or a combination thereof, to a specific temperature, for example, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, or 25°C. The specific temperature means a temperature within the error range of the temperature-controllable container. In one example, when the error range of the temperature-controllable container or device, or a combination thereof, is ±0.5°C, the specific temperature of 20°C means 19.5°C to 20.5°C.

[0022] In one embodiment, blood drawn from the mammal is placed inside a temperature controllable container or device or combination thereof to provide a temperature range of 17°C to 23°C, 18°C ​​to 22°C, or 19°C to 21°C.

[0023] The "maintenance time" or "maintenance time" at a particular temperature described herein means the period from the time when blood taken from a mammal is placed in a temperature-controllable container or device or a combination thereof to the time when it is removed from the container or device or a combination thereof. The maintenance time is not particularly limited as long as it is within 48 hours, and may be, for example, within 45 hours, within 40 hours, within 35 hours, within 30 hours, or within 24 hours. The temperature during the time when the temperature is maintained at a particular temperature can be confirmed, without being limited thereto, by placing a temperature recording device together with the blood taken from the mammal.

[0024] In one embodiment, the time taken for blood drawn from a mammal to be placed in a temperature-controllable container or device, or a combination thereof, is within 1 hour, within 0.5 hours, within 0.3 hours, within 15 minutes, within 10 minutes, within 5 minutes, or within 3 minutes from the time blood is drawn from the mammal.

[0025] In one embodiment, the step of preparing serum includes a step of leaving the blood of a mammal taken out of a temperature-controllable container or device or a combination thereof to stand, separating the blood into a clot and a liquid portion, and recovering the liquid portion. The step of leaving the blood to stand may be, but is not limited to, maintaining the blood at room temperature (e.g., 21°C to 27°C). The time of the leaving step may be, but is not limited to, performed until the formation of a clot can be confirmed, or may be a preset time (any time from 0.5 hours to 2 hours). In one example, the step of leaving the blood to stand may be further added if the formation of a clot cannot be confirmed after the blood taken out of a temperature-controllable container or device or a combination thereof is kept at room temperature for a preset time (e.g., 2 hours). The confirmation of the formation of a clot may be, but is not limited to, performed visually.

[0026] The serum can be collected from the clot by a known method, but is not limited thereto. The serum can be collected, for example, by transferring the liquid portion separated from the clot to another container using a tool such as a pipette, by precipitating the clot by centrifugation and transferring the supernatant to another container using a tool such as a pipette, or by filtering the mixture of the clot and the liquid portion through a filter to collect the serum as a filtrate.

[0027] In one embodiment, the method for producing serum further comprises carrying out one or both of a test for serum tumor markers and a test for infectious agents on a part of the blood sample collected from the mammal or a part of the prepared serum. In one embodiment, the method for producing serum further comprises carrying out one or both of a test for serum tumor markers and a test for infectious agents on a part of the blood sample collected from the mammal. These tests can be performed by known measurement methods for each marker or factor, and can be determined as positive or negative by comparing with known appropriate values ​​or standard values ​​for each marker or factor.

[0028] The serum tumor marker is, for example, one or more selected from the group consisting of ferritin, CEA, AFP, BFP, CA125, CA15-3, CA19-9, CA72-4, STN, DUPAN-2, SLX, ST-439, SPAN-1, SCC, PSA, G-seminoprotein, TPA, Cyfra, PAP, NSE, C-peptide, PIVKA, Pro-GRP, HCGB, elastase, β2 microglobulin, S-NTX, anti-p53 antibody, and HER2. The infectious agent is, for example, one or more selected from the group consisting of HIV, ATL, HBV, HCV, Treponema pallidum, and human parvovirus B19.

[0029] In one embodiment, the method for producing serum may be continued if all serum tumor markers tested are negative and all infectious agents tested are negative, in another embodiment, the method for producing serum may be discontinued if any one serum tumor marker tested is positive or any one infectious agent tested is positive.

[0030] As used herein, "mammal" refers to any individual mammal. The mammal may be, but is not limited to, a human or a non-human mammal. The non-human mammal may be, but is not limited to, a rodent such as a mouse, rat, guinea pig, or hamster, a non-human primate such as a chimpanzee, an even-toed ungulate such as a cow, goat, or sheep, an odd-toed ungulate such as a horse, or a pet such as a rabbit, dog, or cat. In one embodiment, the mammal is a horse, a pet, a non-human primate, or a human. In one embodiment, the mammal is a human.

[0031] The "first mammal" in the context of blood collection to produce serum, the "second mammal" in the context of mammalian cells, and the "third mammal" in the context of pharmaceutical administration may be the same individual or different individuals. When the first mammal, the second mammal, and the third mammal are different individuals, the first mammal, the second mammal, and the third mammal may be the same biological species (homogeneous) or different biological species (heterogeneous). In a preferred embodiment, the first mammal, the second mammal, and the third mammal are the same species. In a more preferred embodiment, the first mammal, the second mammal, and the third mammal are the same individual.

[0032] As used herein, "stem cell" refers to a cell that has both the ability to self-renew and the ability to differentiate. As used herein, "self-renewal ability" refers to the ability to continuously produce stem cells with the same abilities as itself as at least one daughter cell through cell division. As used herein, "differentiation ability" refers to the ability to divide into specialized cells different from itself.

[0033] The stem cells may be, but are not limited to, pluripotent stem cells that can differentiate into multiple types of cells belonging to each of the three germ layers, endoderm, ectoderm, and mesoderm, multipotent stem cells that can differentiate into multiple types of cells limited to one germ layer, and unipotent stem cells that can differentiate into only one type of cell. The pluripotent stem cells may be, for example, embryonic stem cells or recombinant cells that have been given pluripotency from somatic cells by genetic engineering (induced pluripotent stem cells). The pluripotent stem cells may be, for example, hematopoietic stem cells or mesenchymal stem cells. The unipotent stem cells may be, for example, germline stem cells. Stem cells may be obtained from biological samples derived from mammals using known methods, or may be commercially available.

[0034] As used herein, "mesenchymal stem cells" are non-hematopoietic, adhesive cells that have the ability to self-replicate and differentiate into connective tissue cells such as bone cells, chondrocytes, and adipocytes, as well as into nerve cells, cardiac muscle cells, and liver cells. It is known that mesenchymal stem cells are present in bone marrow fluid in very small amounts (0.001 to 0.01% of the cell population having a nucleus).

[0035] As used herein, the term "biological sample" refers to a portion of a mammalian tissue or body fluid. The biological sample may be, but is not limited to, a portion of a tissue or body fluid taken from the mammal, or may be a portion that has been appropriately treated as necessary (e.g., removal of unnecessary components, purification of specific cell fractions, enzymatic treatment, preservation treatment). The body fluid may be, for example, bone marrow fluid, blood (peripheral blood or umbilical cord blood), or lymphatic fluid. The tissue may be, for example, muscle tissue, bone tissue, skin, lymphatic tissue, vascular, or digestive tissue, or an embryo (e.g., a mammalian embryo other than a human embryo).

[0036] The biological sample can be obtained from a mammal according to a known method. When the biological sample is bone marrow fluid, the biological sample can be collected, for example, by anesthetizing the second mammal from which the bone marrow fluid is collected (local or general anesthesia), inserting a needle into the sternum or ilium, and aspirating with a syringe. When the biological sample is umbilical cord blood, the biological sample can be collected, for example, by inserting a needle directly into the umbilical cord and aspirating with a syringe.

[0037] As used herein, "mammalian cells" refers to cells derived from a mammal that can be grown by cell culture. Mammalian cells may be, but are not limited to, cells present in a biological sample taken from the mammal, or may be recombinant cells of mammalian origin produced by genetic engineering. Mammalian cells may be, for example, cells that can be cultured attached to a culture vessel, or cells that can be cultured in a suspended state. In one example, mammalian cells may be cells present in a biological sample taken from a mammal, and may be, for example, somatic cells or stem cells. Somatic cells may be, for example, one or more of osteoblasts, fibroblasts, epithelial cells, and vascular endothelial cells. Stem cells include, for example, at least one type of mesenchymal stem cell.

[0038] In one embodiment, the mammalian cell is a stem cell. In one embodiment, the mammalian cell is a mesenchymal stem cell. In a preferred embodiment, the mammalian cell is a human mesenchymal stem cell. In a more preferred embodiment, the mammalian cell is a human mesenchymal stem cell derived from bone marrow. Bone marrow-derived mesenchymal stem cells can be obtained by, but are not limited to, proliferating mesenchymal stem cells present in bone marrow fluid collected from humans through cell culture.

[0039] In the present specification, a "medicine" including a culture of mammalian cells means a composition used to treat a disease or disorder for which cell therapy can be effective. The medicine satisfies various standards required for medical products, such as standards for impurities derived from the manufacturing process. The medicine may include, but is not limited to, a medicament that is pharma- ceutical acceptable diluent, excipient, and / or substrate depending on the application. The medicine may be, but is not limited to, an injection form (liquid) and an implant form (e.g., sheet form). The method of delivering the medicine to the treatment site is appropriately determined depending on the form of the medicine. The delivery method of the medicine may be, for example, local implantation by surgical means, intravenous administration, lumbar puncture administration, local injection administration, subcutaneous administration, intradermal administration, intraperitoneal administration, intramuscular administration, intracerebral administration, intraventricular administration, or intravenous administration. In one embodiment, the medicine is in an injection form and is administered intravenously. The injection form of the medicine may be, for example, for bolus administration or drip administration.

[0040] The medicament can be used for, but is not limited to, tissue repair or tissue regeneration.The medicament can be used for treating neurological disease.Neurological disease includes, but is not limited to, central and peripheral demyelinating disease, central and peripheral degenerative disease, stroke (including cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage), brain tumor, dementia and other high-level functional disorders, psychiatric disease, epilepsy, traumatic neurological disease (including head trauma, cerebral contusion, spinal cord injury) and spinal cord infarction.

[0041] The medicament is not limited to the treatment of neurological diseases, but can be used for the treatment of diseases related to abnormalities in tissues such as cardiac muscle, joints, cartilage, liver, bone marrow, etc. In one embodiment, the medicament can be used for treating spinal cord injury, stroke, dementia, or spinal cord infarction, for example.The medicament can be useful for the above-mentioned diseases, as disclosed in, for example, US Patent Application Publication No. US2010 / 254953 (the contents of which are incorporated herein by reference).

[0042] The mammalian cell culture used in the pharmaceutical can be produced by culturing mammalian cells in a medium containing serum prepared by the method for producing serum described herein, according to a known cell culture method, such as that disclosed in JP 2012-100662 A (the contents of which are incorporated herein by reference).

[0043] As used herein, "medium" refers to a composition that allows mammalian cells to survive and divide in vitro. The medium contains serum prepared by the method for producing serum described herein. The medium can be produced using a basal medium for cell culture that contains amino acids, vitamins, and electrolytes as main components. The basal medium can be, but is not limited to, Dulbecco's modified Eagle's medium (DMEM), NPBM, or αMEM. In one embodiment, the medium is DMEM. The medium can be prepared according to known methods and is commercially available.

[0044] The medium contains, but is not limited to, 1 to 20% by volume, preferably 5 to 15% by volume, more preferably 8 to 12% by volume of serum prepared by the method for producing serum described herein in the basal medium. The medium may contain, but is not limited to, antibiotics (e.g., penicillin, streptomycin, etc.) commonly used in the field of cell culture, either alone or in combination. In one example, the medium may contain 0.5 to 2% by volume, preferably 0.8 to 1.2% by volume of penicillin and streptomycin, respectively, relative to the medium. In one embodiment, the medium contains an anticoagulant in an amount of less than 0.02 U / mL relative to the volume of the medium.

[0045] The above-mentioned basal medium may be supplemented with a proliferation factor and / or a differentiation inducer, if necessary. The proliferation factor and differentiation inducer are selected according to the desired direction and level of differentiation, the required proliferation rate, etc. Examples of the proliferation factor and differentiation inducer include, but are not limited to, vitamins such as ascorbic acid and nicotinamide, neurotrophic factors such as NGF and BDNF, bone morphogenetic factors such as BMP, epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor, and cytokines such as IL-2. In one embodiment, the medium does not contain serum prepared from blood of a different species (heterologous species) from the serum prepared by the method for producing serum described herein.

[0046] Mammalian cells can be cultured under conditions known in the field of cell culture. Mammalian cells can be cultured under conditions including, but not limited to, 37°C and 5% CO2. Mammalian cells can be cultured using culture vessels of any size and shape. The size or shape of the mammalian cell culture can be adjusted depending on the size or shape of the cell attachment surface of the culture vessel. Mesenchymal stem cell culture is disclosed, for example, in JP 2012-100662 A (the contents of which are incorporated herein by reference).

[0047] As used herein, "cells are not substantially in contact with anticoagulant" means that the amount of anticoagulant used at any time point throughout the entire culture period from collection of mammalian cells is substantially reduced. In one example, it means that anticoagulant is added to a container (such as a blood collection tube) for collection of mammalian cells to the extent that the inner wall is wetted with anticoagulant solution, or that no anticoagulant is added at all, or that the anticoagulant in the sample is substantially removed when culture is started. In order to grow mammalian cells more quickly and in large quantities, it is preferable that the amount of anticoagulant added when collecting a biological sample is small.

[0048] In one embodiment, the amount of anticoagulant added to the biological sample collected from the second mammal (i.e., pre-added to the container in which the collected biological sample is contained) is less than 0.2 U / mL relative to the volume of the biological sample.

[0049] As used herein, the term "anticoagulant" refers to a substance that, when present in a biological sample or culture medium, binds to the cell surface, interacts with a protein having anticoagulant activity present in the extracellular matrix, and inhibits adhesion between cells and the extracellular matrix, between cells, or between cells and a substrate. Anticoagulants include, for example, heparin and heparin derivatives (e.g., glycosaminoglycans obtained by desulfating the 6-position of D-glucosamine, which constitutes heparin), or salts thereof.

[0050] Embodiments of the present invention may be, for example, but are not limited to, the following: [Item 1] A method for producing serum for culturing mammalian cells, comprising maintaining blood collected from a first mammal at a temperature of 15°C to 25°C for up to 48 hours, and preparing serum from the blood. [Item 2] The method of producing according to Item 1, wherein the first mammal is a human. [Item 3] The manufacturing method described in Item 1 or Item 2, further comprising subjecting a portion of the blood sample or a portion of the prepared serum sample to either or both of a test for serum tumor markers and a test for infectious agents. [Item 4] The method according to any one of Items 1 to 3, wherein the mammalian cells are derived from a second mammal of the same species as the first mammal. [Item 5] The method according to any one of Items 1 to 4, wherein the mammalian cells are derived from the same individual as the first mammal. [Item 6] The method according to any one of Items 1 to 5, wherein the mammalian cells are stem cells. [Item 7] The method according to any one of Items 1 to 6, wherein the mammalian cells are bone marrow-derived mesenchymal stem cells.

[0051] [Item 8] A method for producing a pharmaceutical comprising a culture of mammalian cells, comprising culturing the mammalian cells in a medium, the medium containing serum for culturing the mammalian cells, the serum being prepared from blood collected from a first mammal and maintained at a temperature of 15°C to 25°C for up to 48 hours. [Item 9] The method according to Item 8, wherein the first mammal is a human. [Item 10] The method according to item 8 or 9, wherein the serum has been subjected to either or both of a test for a serum tumor marker and a test for an infectious agent. [Item 11] The method according to any one of Items 8 to 10, wherein the medium contains the serum at 1 to 20% per volume of the medium. [Item 12] The method according to any one of Items 8 to 11, wherein the medium contains an anticoagulant in an amount of less than 0.02 U / mL relative to the volume of the medium. [Item 13] The method according to any one of Items 8 to 12, wherein the mammalian cells are derived from a second mammal of the same species as the first mammal. [Item 14] The method according to any one of Items 8 to 13, wherein the mammalian cells are derived from the same individual as the first mammal. [Item 15] The method according to any one of Items 8 to 14, wherein the mammalian cells are stem cells.

[0052] [Item 16] The method according to any one of Items 8 to 15, wherein the mammalian cells are bone marrow-derived mesenchymal stem cells. [Item 17] The method according to any one of Items 8 to 16, wherein the mammalian cells are derived from the same individual as a third mammal in need of administration of the pharmaceutical agent. [Item 18] A manufacturing method according to any one of Items 8 to 17, wherein the mammalian cells are collected from a second mammal without substantially coming into contact with the anticoagulant, with the amount of anticoagulant added to the biological sample collected from the second mammal being less than 0.2 U / mL relative to the volume of the biological sample. [Item 19] The method according to any one of Items 12 to 18, wherein the anticoagulant is heparin, a heparin derivative, or a salt thereof. [Item 20] The method according to any one of Items 8 to 19, wherein the medicament is for treating spinal cord injury, stroke, dementia, or spinal cord infarction. [Item 21] The production method according to any one of Items 8 to 20, further comprising recovering the mammalian cells cultured in the medium.

[0053] Specific examples will be described below, which are intended to illustrate preferred embodiments of the present invention and are not intended to limit the invention described in the appended claims in any way.

[0054] [Serum preparation] (control) Peripheral blood was collected from volunteers in a plastic centrifuge tube. The collected blood was left to stand at room temperature to separate the blood clot from the liquid portion. The blood clot was precipitated by centrifugation at room temperature at 2380×g, and the supernatant was placed in a sterilized plastic container to prepare serum.

[0055] (Comparative Examples 1 to 3) Serum was prepared in the same manner as in the control, except that the collected blood was left to stand at 10° C. for 9 hours (Comparative Example 1), 24 hours (Comparative Example 2), or 48 hours (Comparative Example 3).

[0056] (Examples 1 to 3) Serum was prepared in the same manner as the control, except that the collected blood was allowed to stand at 20° C. for 9 hours (Example 1), 24 hours (Example 2), or 48 hours (Example 3). (Examples 4 to 6) Serum was prepared in the same manner as in the control, except that the collected blood was allowed to stand at 25° C. for 9 hours (Example 4), 24 hours (Example 5), or 48 hours (Example 6).

[0057] When serum was prepared from the blood of Comparative Examples 1 to 3, the time required to separate the blood clot from the liquid portion tended to be longer than the time required when serum was prepared from the blood of Examples 1 to 6.

[0058] [Preparation of medium] Medium A was prepared by mixing DMEM (88 v / v%), L-glutamine solution (1 v / v%), penicillin-streptomycin (1 v / v%), and each serum (10 v / v%), followed by sterilization by filtration through a 0.2 μm filter.

[0059] [Mammalian cell culture] Mesenchymal stem cells (Lonza) were added to medium A, which had been kept at 37°C in advance, and 2.0*10 5 The cells were seeded so that there were 100 cells. The cells were cultured at 37°C and 5% CO2 to allow them to attach to the culture dish substrate, after which the medium was aspirated to remove floating components. The culture dishes were washed with phosphate-buffered saline. After washing, medium A was added and the cells attached to the culture dish substrate were further cultured. After one of the dishes reached approximately 80% confluence, they were subcultured. For subculture, the cultured cells were collected from the dishes using a detachment solution consisting of trypsin and EDTA (ethylenediaminetetraacetic acid), suspended in medium A, and then subcultured at 2.0*10 5The cells were seeded onto a 100 mm culture dish so that each cell numbered 1.

[0060] As described above, the cells seeded in the culture dishes were cultured at 37°C and 5% CO2. After one of the dishes reached approximately 80% confluence, the cultured cells were harvested and the cell numbers were counted (passage 2).

[0061] The number of cells cultured using each serum is summarized in Table 1, and the relative values ​​based on the number of cells proliferated when cultured using the control serum are summarized in Figure 1. The relative values ​​shown in Figure 1 correspond to the proliferation rate of cells at passage 2 based on the proliferation rate of the control. [Table 1]

[0062] In the same manner as above, the collected blood was cultured and passaged using medium A containing serum prepared from blood that was not left to stand (control), serum prepared from blood that was left to stand for a predetermined time at 15°C (Examples 7 to 9), serum prepared from blood that was left to stand for a predetermined time at 20°C (Examples 10 to 12), and serum prepared from blood that was left to stand for a predetermined time at 25°C (Examples 13 to 15) (Table 2). The proliferation rates of cells at passage 2 in Examples 10 to 15, based on the proliferation rate of the control, are shown in Figure 2. [Table 2]

[0063] FIG. 1 shows that the cell proliferation rate when serum prepared from blood maintained at 10° C. (Comparative Examples 1 to 3) is used is lower than that when serum from control (no transportation) is used. FIG. 1 and FIG. 2 show that in culture using serum prepared from blood maintained at 20° C. or 25° C., the cell proliferation rate hardly changes regardless of the maintenance time (FIG. 2) or tends to decrease as the maintenance time increases (FIG. 1). FIG. 2 shows that in culture using serum prepared from blood maintained at 15° C., the cell proliferation rate tends to increase as the maintenance time increases. Based on the results shown in FIG. 1 and FIG. 2, the inventors have found that by maintaining the blood temperature at at least 15° C. to 25° C. for up to 48 hours, it is possible to prevent the decrease in cell proliferation rate caused by the serum, as seen in serum prepared from blood maintained at a relatively low temperature (10° C.).

Claims

1. A method for producing serum for culturing at least one type of mesenchymal stem cell, comprising: maintaining the blood taken from the first mammal at a temperature between 15°C and 25°C for not less than 24 hours and not more than 48 hours; separating the retained blood liquid from the blood clot; and recovering the separated liquid; The method for producing said at least one type of mesenchymal stem cell is derived from the same individual as said first mammal.

2. The method of claim 1, wherein the first mammal is a human.

3. The method for production according to claim 1 or 2, further comprising subjecting a portion of the blood sample or a portion of the produced serum sample to either or both of a test for serum tumor markers and a test for infectious agents.

4. A manufacturing method described in any one of claims 1 to 3, wherein the temperature at which the blood is maintained is not lower than 15°C and lower than 20°C.

5. The method according to any one of claims 1 to 4, wherein the at least one type of mesenchymal stem cell is a bone marrow-derived mesenchymal stem cell.

6. A method for producing a medicament comprising a culture of at least one type of mesenchymal stem cell, comprising: Culturing at least one type of mesenchymal stem cell in a medium containing serum, The serum, maintaining the blood taken from the first mammal at a temperature between 15°C and 25°C for at least 24 hours and no more than 48 hours; Separating the retained blood liquid and blood clot; and The separated liquid is collected, The method for producing said at least one type of mesenchymal stem cell is derived from the same individual as said first mammal.

7. The method of claim 6, wherein the first mammal is a human.

8. The method for producing a serum according to claim 6 or 7, wherein the serum has been subjected to either or both of a test for a serum tumor marker and a test for an infectious agent.

9. The method according to any one of claims 6 to 8, wherein the medium contains the serum in an amount of 1 to 20% per volume of the medium.

10. A manufacturing method described in any one of claims 6 to 9, wherein the temperature at which the blood is maintained is not less than 15°C and less than 20°C.

11. The method according to any one of claims 6 to 10, wherein the at least one type of mesenchymal stem cell is a bone marrow-derived mesenchymal stem cell.

12. A method for producing a culture medium described in any one of claims 6 to 11, wherein the culture medium contains an anticoagulant in an amount of less than 0.02 U / mL relative to the volume of the culture medium.

13. the at least one type of mesenchymal stem cell is derived from a biological sample taken from the same individual of the first mammal in a state substantially free of contact with an anticoagulant; The method according to any one of claims 6 to 12, wherein the biological sample in a state of being substantially free from contact with the anticoagulant contains the anticoagulant in an amount of less than 0.2 U / mL relative to the volume of the biological sample.

14. The method according to claim 12 or 13, wherein the anticoagulant is heparin, a heparin derivative or a salt thereof.

15. A manufacturing method described in any one of claims 6 to 14, wherein the pharmaceutical is for administration to the same individual as the first mammal.

16. The method according to any one of claims 6 to 15, wherein the medicament is for treating spinal cord injury, stroke, dementia, or spinal cord infarction.

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