Apoptosis-induced stem cells, method for producing the same, and composition for preventing or treating inflammatory disease or renal disease containing the same

Abstract

The present invention relates to apoptosis-induced stem cells, a method for producing the same, and a composition containing the same for preventing or treating inflammatory diseases or renal diseases. The cells according to one aspect of the present invention and a composition containing the same as an active ingredient are characterized by having an increased IL-10 expression level without containing a cryoprotectant, and therefore have the effect of being useful for preventing or treating inflammatory diseases or renal diseases.

Description

[Technical Field]

[0001] The present invention relates to stem cells in which apoptosis has been induced, a method for producing the same, and a pharmaceutical composition containing the same for preventing or treating inflammatory diseases or renal diseases. [Background technology]

[0002] Stem cell therapeutics are medicines that are administered to patients with diseases using stem cells as raw materials. However, because these stem cells and stem cell therapeutics are made from living cells, they have the disadvantages of being sensitive to the environment and having a short shelf life.

[0003] Therefore, cryopreservation is essential for long-term storage while maintaining stem cell properties. Cryopreservation of cells involves the formation of intracellular ice crystals, which can damage cells. Therefore, cryoprotectants are commonly used. However, there are few studies on cryopreservation methods that efficiently preserve stem cells for long periods and are harmless to the human body, and no standardization has been established. Previously, substances such as dimethyl sulfoxide (DMSO) have been widely used as cryoprotectants for the cryopreservation of various somatic cells, including stem cells, reproductive cells such as fertilized eggs, ova, and sperm, and blood. However, DMSO inhibits cell survival and growth after freezing and thawing, making its incorporation into culture media undesirable. Furthermore, when attempting to directly transplant frozen stem cells, its injection into the body is highly cytotoxic. Furthermore, cryoprotectants containing heterologous or animal-derived components, such as fetal bovine serum (FBS) and human serum, pose the risk of viral and prion infections and immune reactions. Furthermore, they are unsuitable for the preservation of cell therapy agents used for therapeutic purposes.

[0004] Apoptosis is a type of programmed cell death observed in multicellular organisms. Apoptosis refers to the death of a cell caused by changes in its morphology and internal biochemical processes. When adult stem cells are administered into the body, apoptosis is known to be induced within one hour. Furthermore, most of the administered stem cells are eliminated from the body within two to three days.

[0005] Under these circumstances, there is a need for a safe, cryopreserved stem cell therapeutic agent that can replace the currently available stem cell therapeutic agents. Summary of the Invention [Problem to be solved by the invention]

[0006] One aspect of the present invention is to provide isolated cells in which apoptosis has been induced by freezing, in which the expression level of interleukin-10 (IL-10) has increased by 1.5 times or more compared to the cells before freezing.

[0007] Another aspect of the present invention is to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases, comprising, as an active ingredient, apoptosis-induced cells or a culture medium of said cells, in which the expression level of interleukin-10 (IL-10) in cells in which apoptosis has been induced by freezing has increased by 1.5 times or more compared to the cells before freezing.

[0008] Another aspect of the present invention is to provide a method for preventing or treating an inflammatory disease, comprising the step of administering the cells or a culture medium of the cells to an individual in need thereof.

[0009] Another aspect of the present invention is to provide a use of the cells or a culture medium of the cells for the manufacture of a pharmaceutical composition for the prevention or treatment of an inflammatory disease.

[0010] Another aspect of the present invention is to provide a use of said cells or a culture medium of said cells for use in the prevention or treatment of inflammatory diseases.

[0011] Yet another aspect of the present invention is to provide a method for producing an apoptosis-induced cell-containing composition, comprising the steps of: supporting isolated cells in a solution containing a blood substitute to produce a cell-containing composition; freezing the cell-containing composition to produce an apoptosis-induced cell-containing composition; and thawing the apoptosis-induced cell-containing composition. [Means for solving the problem]

[0012] One aspect of the present invention is to provide isolated apoptosis-induced cells in which the expression level of interleukin-10 (IL-10) in cells in which apoptosis has been induced by freezing is increased by 1.5 times or more compared to the cells before freezing.

[0013] In one embodiment, the cells may be stem cells.

[0014] The stem cells may be totipotent stem cells, pluripotent stem cells or multipotent stem cells.

[0015] The stem cells may be any one or more of the group consisting of embryonic stem cells (ESCs), adult stem cells (ASCs), and induced pluripotent stem cells (iPSCs).

[0016] The induced pluripotent stem cells, also known as induced totipotent stem cells, refer to cells with pluripotency obtained by reverse differentiation of differentiated cells (e.g., somatic cells). The induced pluripotent stem cells can differentiate into various organ cells. The induced pluripotent stem cells are obtained by reprogramming differentiated cells with reverse differentiation inducers.

[0017] The adult stem cells may be derived from any one or more tissues of the group consisting of umbilical cord blood, fat, nerve, bone marrow, peripheral blood, muscle, liver, skin, placenta, amniotic membrane, and umbilical cord.

[0018] The adult stem cells may be any one or more types selected from the group consisting of hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), neural stem cells (NSCs), cardiac stem cells (CSCs), intestinal stem cells (ISCs), muscle stem cells (MuSCs), hair follicle stem cells (FSCs), and endothelial progenitor cells (EPCs).

[0019] In one embodiment, the cells may be lineage-restricted progenitor cells.

[0020] The lineage-restricted progenitor cells may be any one or more types of the group consisting of hematopoietic progenitor cells, mesenchymal progenitor cells, neural progenitor cells, cardiac progenitor cells, intestinal progenitor cells, adipose progenitor cells, muscle progenitor cells, hair follicle progenitor cells, and endothelial progenitor cells.

[0021] In one embodiment, the cells may be differentiated somatic cells.

[0022] The differentiated somatic cells may be any one or more types of cells selected from the group consisting of hematopoietic cells, mesenchymal cells, nerve cells, cardiac cells, intestinal cells, adipocytes, muscle cells, hair follicle cells, and vascular endothelial cells.

[0023] As used herein, the term "stem cell" refers to a cell that has the ability to proliferate under given circumstances without substantial differentiation, and that has the ability or potential to differentiate into a more specialized or differentiated phenotype under specific circumstances.

[0024] In one embodiment, the term "progenitor cell" or "stem cell" refers to a generalized maternal cell whose progeny typically specialize in different directions by differentiation, acquiring distinct characteristics, as occurs, for example, in the progressive diversification of embryonic cells and tissues. Cell differentiation is a complex process that typically occurs through many cell divisions. Differentiated cells can themselves be derived from pluripotent cells, which in turn are derived from pluripotent cells. While each of these pluripotent cells is considered a stem cell, the range of cell types each can generate can vary considerably. Also, some differentiated cells have the ability to generate cells of higher developmental potential. Such ability may be natural or may be artificially induced upon treatment with various factors. In many biological cases, stem cells can be "pluripotent" because they can generate progeny of more than one distinct cell type, but this is not required for "stemness."

[0025] As used herein, the term "differentiated cell" refers to a cell that is further downstream in the developmental pathway than the cell for which it is being considered. Thus, stem cells can differentiate into lineage-restricted progenitor cells (e.g., myocyte progenitors), which in turn can differentiate into other types of progenitor cells further downstream in the pathway (e.g., myocyte precursors), and into subsequent terminally differentiated cells, e.g., myocytes, which may or may not have the capacity to play a characteristic role in a given tissue type and further proliferate.

[0026] In one embodiment, the cells may be frozen and then thawed. The freezing refers to a process of preserving cells at a low temperature for long-term storage while maintaining the characteristics of the cells. The thawing refers to a process of restoring frozen cells to active cells at a relatively high temperature for use as intended.

[0027] The cells may be viable cells in which apoptosis has not been induced, with 75% or more of the entire population being Annexin V and PI negative before freezing. This means that at least 75% or more of the entire population is Annexin V and PI negative. More specifically, at least 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 100%, 75-99%, 80-99%, 90-99%, 75-95%, 85-95%, 75-90%, or 85-90% of the entire population may be Annexin V and PI negative.

[0028] The detection of Annexin V and PI can be derived from flow cytometry or FACS (fluorescence activated cell sorting) analysis of the cells.

[0029] The flow cytometry analysis can be single or multiplexed flow cytometry analysis, which can be a technique that uses single or multiple lasers to perform multiparameter analysis of single cells. Each cell can be analyzed by the scattering of visible light or the emission of fluorescence that occurs as each laser passes quickly through the cell. The fluorescence can be introduced through transfection and expression of a conventional fluorescent protein (e.g., GFP), fluorescently conjugated antibodies, or staining with fluorescent dyes.

[0030] The FACS is a type of flow cytometry analysis, and may be a type of flow cytometry analysis that has an added function of classifying heterogeneously mixed cells based on their respective light scattering or fluorescence characteristics.

[0031] Herein, the parameter in flow cytometry may be Annexin V or PI (propidium iodide).

[0032] As used herein, the term "positive," in relation to an apoptotic marker, means that the marker is present in a greater amount or at a higher concentration than other non-apoptotic cells as a reference. That is, a cell is positive for a marker if the marker is present inside or on the cell, allowing the cell to be distinguished from one or more other cell types. It also means that the cell has a sufficient amount of the marker to produce a detectable signal, e.g., a flow cytometric signal, that is greater than background.

[0033] In the present invention, the term "negative" in relation to an apoptotic marker can mean that there is no difference when the marker is compared to other non-apoptotic cells as a reference, i.e., the marker is not detectable compared to background values.

[0034] Apoptosis can be the death of a cell due to changes in its morphology and internal biochemical processes. This includes cell swelling and cracking, changes in the cell membrane, chromatin condensation, chromosome cleavage, and macrophage phagocytosis. It can be divided into early and late apoptosis stages. In the early stage of apoptosis, cells maintain their morphology but undergo membrane filtration, exposing phosphatidylserine (PS) to the outside of the cell. This can refer to a complex cascade process. In the late stage, cell shrinking and membrane blebbing are observed.

[0035] In one embodiment, the apoptosis marker may be Annexin V or PI (propidium iodide). Annexin V may bind to PS (phosphatidylserine) on the cell membrane of apoptotic cells, and PI may bind to intracellular DNA in cells at the late stage of apoptosis. The apoptosis markers may be derived through fluorescence analysis of Annexin V and PI. The fluorescence analysis may be performed by flow cytometry.

[0036] In one embodiment, negative Annexin V and negative PI indicate normal cells, positive Annexin V and negative PI indicate early stage apoptotic cells, positive Annexin V and positive PI indicate late stage apoptotic cells, and negative Annexin V and positive PI indicate necrotic cells.

[0037] The freezing may be carried out at a temperature of -130°C or -70°C or lower for 6 months or less, or 6 months or more. More specifically, the freezing may be carried out at a temperature of -60 to -80°C, -65 to -80°C, -70 to -80°C, -130°C or lower, or -196°C for 1 to 12 months, 6 to 12 months, 12 months or more, 1 to 24 months, 24 months or more, 12 to 36 months, 36 months or more, 6 months or more, 1 hour to 6 months, 12 hours to 6 months, 1 day to 6 months, 15 days to 6 months, 1 to 6 months, 3 to 6 months, 6 months or less, 6 months, 5 months, 4 months, 1 hour to 3 months, 12 hours to 3 months, 1 day to 3 months, 15 days to 3 months, It may be performed for 1 to 3 months, 3 months or less, 3 months, 1 hour to 2 months, 12 hours to 2 months, 1 day to 2 months, 15 days to 2 months, 2 months or less, 2 months, 1 hour to 1 month, 12 hours to 1 month, 1 day to 1 month, 15 days to 1 month, 1 month or less, 1 month, 1 hour to 15 days, 12 hours to 15 days, 1 to 15 days, 15 days or less, 15 days, 1 hour to 10 days, 12 hours to 10 days, 1 to 10 days, 10 days or less, 10 days, 1 hour to 5 days, 12 hours to 5 days, 1 to 5 days, 5 days or less, or 5 days.

[0038] The thawing can be carried out at 0 to 5°C for 10 minutes to 5 hours. More specifically, it can be performed at a temperature of 0 to 10°C, 0 to 2°C, 0 to 4°C, 0 to 6°C, 0 to 8°C, 2 to 3°C, 2 to 5°C, 2 to 8°C, 3 to 5°C, 3 to 7°C, or 4 to 5°C for 5 minutes to 5 hours, 10 minutes to 5 hours, 15 minutes to 5 hours, 30 minutes to 5 hours, 1 to 5 hours, 3 to 5 hours, 4 to 5 hours, 5 to 30 minutes, 10 to 30 minutes, 15 to 30 minutes, 5 to 20 minutes, 10 to 20 minutes, 15 to 20 minutes, 5 minutes to 1 hour, 10 minutes to 1 hour, 15 minutes to 1 hour, 30 minutes to 1 hour, 5 minutes to 3 hours, 10 minutes to 3 hours, 30 minutes to 3 hours, 1 to 3 hours, 5 minutes to 4 hours, 10 minutes to 4 hours, 30 minutes to 4 hours, 1 to 4 hours, or 3 to 4 hours.

[0039] In one embodiment, the freezing may be performed without a cryoprotectant.

[0040] The cryoprotectant may be used to minimize cell damage due to ice crystal formation during cell cryopreservation, and may refer to compounds such as DMSO (dimethyl sulfoxide), glycerol, 1,2-propanediol, sucrose, methanol, glucose, proline, galactose, lactose, glycine betaine, or fructose, or substances containing animal-derived components such as fetal bovine serum or human serum.

[0041] The cryoprotectants mentioned above interfere with cell survival and growth after freezing and thawing, making them undesirable for contamination of the culture medium. Furthermore, when attempting to directly transplant frozen stem cells, they can be highly cytotoxic when injected into the body. Furthermore, cryoprotectants containing animal-derived components may pose a risk of infection with viruses, prions, etc., or of causing immune reactions.

[0042] The freezing without a cryoprotectant may be performed using a conventional infusion solution, which may be a blood substitute, a blood substitute, or an artificial blood.

[0043] The blood substitute, blood substitute, or artificial blood may refer to a solution used as a substitute for blood, or may refer to a preparation used for blood supplementation, plasma supplementation, osmotic pressure maintenance, etc. The terms "blood substitute," "blood substitute," and "artificial blood" may be used interchangeably.

[0044] The blood substitute, blood substitute or artificial blood may be saline, 0.9% saline, 0.9% saline, 9% saline, 9% saline, sterile saline, Hartmann, Hartmann's solution, Hartmandex, Hartmandex solution, Hartmanndi, Hartmanndi solution, 5% dextrose saline, sodium chloride solution, 3% sodium chloride solution, 40% sodium chloride injection, 0.45% sodium chloride, 5% dextrose and 0.45% sodium chloride solution, sodium chloride dextrose solution, sodium chloride dextrose 1:3 solution (SD1:3), sodium chloride dextrose 1:4 solution (SD1:4), sodium lactate sodium chloride dextrose 1:2:3 solution (LSD1:2:3), potassium chloride solution, 20% potassium chloride injection, 5% dextrose and potassium sodium solution, Fatimasol solution, Plasma Solution A solution, Plascon solution, Halfsol sol solution and Plaju OP solution.

[0045] The blood substitute, blood substitute, or artificial blood may contain one or more components selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, sodium lactate, glucose, dextrose (D-glucose), sodium gluconate, and sodium acetate.

[0046] Specifically, saline infusion can include sodium chloride, Hartmann's solution can include sodium chloride, potassium chloride, calcium chloride, and sodium lactate, and Hartmandex infusion can include sodium chloride, potassium chloride, calcium chloride, sodium lactate, and dextrose (or glucose).

[0047] The concentrations of the above components in the blood substitute, blood substitute, or artificial blood can be adjusted appropriately depending on the purpose. Specifically, saline infusion can contain 9 g / L sodium chloride, Hartmann's solution can contain 6 g / L sodium chloride, 300 mg / L potassium chloride, 200 mg / L calcium chloride, and 3.1 g / L sodium lactate, and Hartmandex solution can contain 6 g / L sodium chloride, 300 mg / L potassium chloride, 200 mg / L calcium chloride, 6.2 g / L sodium lactate, and 50 g / L dextrose (or glucose).

[0048] In one embodiment, the solution can refer to a solution containing the cells and a conventional transfusion or blood substitute.

[0049] In one embodiment, the cells may be subjected to apoptosis induction after freezing, the apoptosis may be induced by the infusion, or the apoptosis may be induced in 90% or more of the cells by the infusion.

[0050] The cells in which apoptosis is induced after freezing may be those in which apoptosis is induced in 75% or more of the cells in the entire population. This means that, as a result of flow cytometry of the cells, at least 75% or more of the entire population is positive for annexin V. More specifically, at least 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 75-99%, 80-99%, 100%, 90-99%, 75-95%, 85-95%, 75-90%, or 85-90% of the entire population may be positive for annexin V.

[0051] The Hartmandex infusion solution can induce the most early stage apoptosis, which means that the early stage apoptotic cells largely maintain their original functions, and the early stage apoptotic cells can be stored for a longer period of time than the late stage apoptotic cells.

[0052] The cells in which apoptosis is induced after freezing may refer to cells in which apoptosis is induced after the thawing of frozen cells.

[0053] In one embodiment, the cells in which apoptosis is induced after freezing may further increase the expression of an immune or anti-inflammatory related substance after freezing, which may be IL-10.

[0054] In one embodiment, the cells in which apoptosis is induced after freezing may further increase the expression of an immune or anti-inflammatory related substance upon thawing after freezing, and the immune or anti-inflammatory related substance may be IL-10.

[0055] IL-10 can regulate the proliferation and differentiation of various immune cells such as T cells, B cells, NK cells, antigen-presenting cells, MAST cells, and granulocytes, and can play a role in suppressing inflammatory responses or regulating immune activation.

[0056] The expression of IL-10 may be increased by 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.8-fold or more, or 2.0-fold or more relative to non-frozen cells. More specifically, 1.5 to 2.5 times, 1.2 to 5.0 times, 1.5 to 5.0 times, 2.0 to 5.0 times, 2.5 to 5.0 times, 3.0 to 5.0 times, 1.2 to 4.0 times, 1.5 to 4.0 times, 2.0 to 4.0 times, 2.5 to 4.0 times, 3.0 to 4.0 times, 1.2 to 3.0 times, 1.3 to 3.0 times, 1.5 to 3.0 times, 1.8 to 3.0 times, 2.0 to 3.0 times, 2.2 to 3.0 times, 2.5 to 3.0 times, 1.2 to 2.7 times, 1.3 to 2.7 times, 1.5 to 2.7 times, 1.8 to 2.7 times, 2.0 to 2.7 times, 2.2 to 2.7 times, 2.5 to 2.7 times, 1.2 to 2.5 times, 1.3 to The increase may be 2.5 times, 1.5 to 2.5 times, 1.8 to 2.5 times, 2.0 to 2.5 times, 2.2 to 2.5 times, 1.2 to 2.2 times, 1.3 to 2.2 times, 1.5 to 2.2 times, 1.8 to 2.2 times, 2.0 to 2.2 times, 1.2 to 2.0 times, 1.3 to 2.0 times, 1.5 to 2.0 times, 1.8 to 2.0 times, 1.2 to 1.9 times, 1.3 to 1.9 times, 1.5 to 1.9 times, 1.8 to 1.9 times, 1.2 to 1.8 times, 1.3 to 1.8 times, 1.5 to 1.8 times, 1.2 to 1.7 times, 1.3 to 1.7 times, 1.5 to 1.7 times, 1.2 to 1.5 times, or 1.3 to 1.5 times.

[0057] In one embodiment, the cells in which apoptosis is induced after freezing may be effective in preventing or treating inflammatory diseases or renal diseases.

[0058] The inflammatory disease may be any one selected from the group consisting of renal failure, nephritis, gastritis, inflammatory bowel disease, inflammatory respiratory disease, hepatitis, gastric ulcer, asthma, dermatitis, and enteritis, but is not limited thereto.

[0059] As used herein, the term "treat" means curing an inflammatory disease, renal disease, etc. in a shorter time than natural healing. The treatment may include improvement and / or alleviation of the inflammatory disease, renal disease, etc. Furthermore, the treatment may mean curing and / or alleviating symptoms induced by the inflammatory disease, renal disease, etc.

[0060] The term "prevention" as used herein refers to a reduction in the occurrence of pathological cells or the degree of cell damage or loss in an individual. Prevention may be complete or partial. In this case, it may refer to a phenomenon in which the occurrence of pathological cells or abnormal immune activity in an individual is reduced compared to when the composition for preventing or treating an inflammatory disease or renal disease is not used.

[0061] In one embodiment, the cells in which apoptosis is induced after freezing may have a distribution pattern similar to that of non-frozen cells after transplantation, and may also have a cellular change pattern and migration pattern similar to that of non-frozen cells after transplantation.

[0062] In one embodiment, the cells in which apoptosis has been induced after freezing may have a therapeutic effect that is no different from that of non-frozen cells after transplantation into the body.

[0063] Another embodiment provides a composition for ameliorating, preventing, or treating inflammatory or renal diseases.

[0064] In one embodiment, the composition may contain, as an active ingredient, apoptosis-induced cells or a culture medium of such cells in which the expression level of interleukin-10 (IL-10) in cells in which apoptosis has been induced by freezing has increased by 1.5 times or more compared to the cells before freezing.

[0065] In another embodiment, a pharmaceutical composition for preventing or treating inflammatory diseases is provided, which comprises, as an active ingredient, apoptosis-induced cells in which the expression level of interleukin-10 (IL-10) has increased by 1.5 times or more compared to the cells before freezing, or a culture medium of such cells.

[0066] Yet another embodiment provides a method for preventing or treating an inflammatory disease, comprising administering the cells or a culture medium of the cells to an individual in need thereof.

[0067] Another embodiment provides the use of the cells or the culture medium thereof for the manufacture of a pharmaceutical composition for the prevention or treatment of an inflammatory disease.

[0068] Another embodiment provides a use of the cells or a culture medium of the cells for the prevention or treatment of inflammatory diseases.

[0069] The composition may contain, as an active ingredient, cells in which apoptosis has been induced after freezing.

[0070] The cells in which apoptosis was induced after freezing were as described above.

[0071] The inflammatory disease may be any one selected from the group consisting of renal failure, nephritis, gastritis, inflammatory bowel disease, inflammatory respiratory disease, hepatitis, gastric ulcer, asthma, dermatitis, and enteritis.

[0072] More specifically, the inflammatory disease includes renal failure, nephritis, glomerulonephritis, gastritis, inflammatory bowel diseases (IBD), hepatitis, inflammatory respiratory disease, gastric ulcer, irritable bowel syndrome, Behcet's disease, enteritis, Crohn's serum disease, asthma, dermatitis, ulcerative colitis, vascular ulcers, and the like. colitis, mucositis, stomatitis, periimplantitis, periodontitis, pulpitis, gingivitis, pneumonia, dermatitis, atopic dermatitis, contact dermatitis, CREST syndrome, dermatitis herpetiformis, dermatomyositis, systemic scleroderma, erythema nodosum, Henoch-Schönlein purpura, hidradenitis serum suppurativa, lichen protoplanus, Majeed syndrome, Schnitzler syndrome pulsyndrome, psoriasis, eczema, acne, mouth inflammationThe inflammation may be any one selected from the group consisting of ulcers, uveitis, pharyngitis, tonsillitis, otitis including otitis media, psoriatic arthritis, synovitis, meningitis, encephalitis, Bickerstaff's encephalitis, encephalomyelitis, spondylitis (National Dylitis), osteomyelitis, Guillain-Barré syndrome, myelitis, neuromyelitis optica, cystitis, or acute inflammation at the site of infection or wound.

[0073] More specifically, the kidney disease includes chronic renal failure, acute renal failure, glomerulonephritis, interstitial nephritis, diabetic nephropathy, glomerulosclerosis, renal fibrosis, Alport syndrome, IDDM nephritis, mesangial focal proliferative glomerulonephritis, focal proliferative glomerulonephritis, crescentic glomerulonephritis, renal interstitial fibrosis, focal segmental glomerulosclerosis, membranous nephropathy, minimal change disease, pauci-immune rapidly progressive glomerulonephritis, IgA nephropathy, polycystic kidney disease, Dent's disease, and the like. disease), nephrocytinosis, Heymann nephritis, polycystic kidney disease, nephrotic syndrome, renal ischemia, podocytopathy or podocytic disorder, proteinuria, pre-eclampsia, renal lesions, collagen vascular disease, benign orthostatic (postural) proteinuria, IgM nephropathy, amino aciduria, Fanconi syndrome, hypertensive nephrosclerosis, hemoglobinuria, myoglobinuria, Wegener's granulomatosis, reduced glomerular filtration rate (GFR), nephroangiosclerosis, lupus nephritis, proximal tubular dysfunction, acute renal transplant rejection, chronic renal transplant rejection, and non-IgA mesangial focal proliferative glomerulonephritis.

[0074] The term "containing as an active ingredient" means that the cells of the present specification are added to the extent that the aforementioned effect can be achieved, and also means that various ingredients are added as accessory ingredients for drug delivery, stabilization, etc., and the cells are formulated in various forms.

[0075] In other embodiments, the composition may be a pharmaceutical composition or a cellular therapeutic agent.

[0076] The pharmaceutical composition or cell therapy agent may further contain pharmaceutically acceptable diluents, carriers, and / or additives, such as sterile water, saline infusion, conventional buffers (such as phosphate, citric acid, and other organic acids), stabilizers, salts, antioxidants (such as ascorbic acid), surfactants, suspending agents, isotonicity agents, or preservatives. For local administration, it is also preferable to combine the composition or cell therapy agent with organic materials such as biopolymers, or inorganic materials such as hydroxyapatite, specifically, collagen matrices, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers, and chemical derivatives thereof.

[0077] When the cell therapy agent or pharmaceutical composition according to one embodiment is prepared in a dosage form suitable for injection, the cell aggregates may be dissolved in a pharmaceutically acceptable carrier or frozen in a dissolved solution state.

[0078] The pharmaceutical composition can be formulated into an oral or parenteral dosage form. The oral dosage form can be granules, powders, liquids, tablets, capsules, dry syrups, or a combination thereof. The parenteral dosage form can be an injection.

[0079] Yet another embodiment provides a method for producing an apoptosis-induced cell-containing composition, comprising the steps of: supporting isolated cells in a blood substitute-containing solution to produce a cell-containing composition; freezing the cell-containing composition to produce an apoptosis-induced cell-containing composition; and thawing the apoptosis-induced cell-containing composition.

[0080] The cell-containing composition may be effective in preventing or treating inflammatory diseases or renal diseases.

[0081] The cell-containing composition can be included as an active ingredient in a pharmaceutical composition or a cell therapy agent.

[0082] The cells, freezing, thawing, solution, cells in which apoptosis has been induced after freezing, inflammatory diseases, renal diseases, pharmaceutical compositions, and cell therapeutic agents are the same as those described above.

[0083] In one embodiment, the cells in the step of producing the cell-containing composition may be Annexin V and PI negative, and the cells in the step of thawing may be Annexin V positive.

[0084] The positive and negative results for Annexin V and PI are the same as those described above. Negative results for both Annexin V and PI may indicate normal cells, positive results for Annexin V may indicate cells in the apoptotic stage, and positive results for both Annexin V and PI may indicate cells in the late apoptotic stage.

[0085] Yet another embodiment provides a method for preventing, ameliorating, or treating a condition in an individual, comprising administering or administering to the individual in need thereof an effective amount of the composition described above.

[0086] The condition of the individual may be a condition associated with an inflammatory disease or a renal disease, the inflammatory disease or renal disease being similar to those described above.

[0087] The pharmaceutical composition or cell therapy agent can be administered to a patient in a therapeutically effective amount or a pharmaceutically effective amount.

[0088] The terms "administering," "introducing," and "implanting" are used interchangeably and can refer to the placement of a composition according to an embodiment into an individual by a method or route that results in at least partial localization of the composition according to an embodiment at a desired site. Administration can be by any suitable route that delivers at least a portion of the cells or cellular components of a composition according to an embodiment to a desired location within a living individual. Following administration to an individual, the survival period of the cells can be as short as a few hours, e.g., 24 hours to a few days, or as long as several years. The pharmaceutical composition can be administered to a patient in a therapeutically or pharmaceutically effective amount.

[0089] The term "therapeutically effective amount" or "pharmaceutically effective amount" refers to an amount of a cell population or composition effective in preventing or treating an inflammatory disease or renal disease, sufficient to treat the disease without causing side effects, at a reasonable benefit / risk ratio applicable to any medical treatment. The level of the effective amount depends on factors including the patient's health condition, the type and severity of the disease, drug activity, drug sensitivity, administration method, administration time, administration route and excretion rate, treatment duration, co-administered or concomitant drugs, and other factors well known in the medical field.

[0090] Administration can be by methods known in the art. Administration can be by any means, including direct administration to an individual, such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal, or subcutaneous administration. Administration can be systemic or local.

[0091] The pharmaceutical composition or cell therapy agent may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Taking all of these factors into consideration, it is important to administer an amount that will provide the maximum effect with the minimum amount without side effects, and this can be easily determined by those skilled in the art.

[0092] The dosage of the pharmaceutical composition or cell therapy agent according to one embodiment is 1.0 x 10 based on adherent cells. 3 ~1.0×10 10 cells / kg (body weight) or individual, or 1.0 x 10 7 ~1.0×10 8 The dosage may be determined by cell / kg (body weight) or individual. However, the dosage may vary depending on factors such as the formulation method, administration method, patient age, body weight, sex, pathological condition, food intake, administration time, administration route, excretion rate, and reaction sensitivity, and those skilled in the art will be able to appropriately adjust the dosage taking these factors into consideration. The number of administrations may be one or more than two times within the range of clinically acceptable side effects, and the administration site may be one or more than two sites. For non-human animals, the dosage may be the same as that for humans per kg or per individual, or may be calculated by converting the dosage based on, for example, the volume ratio (e.g., average value) of organs (e.g., heart) between the target animal and humans. Target animals for treatment in one embodiment include humans and other mammals of interest, specifically humans, cats, dogs, monkeys, mice, rats, rabbits, sheep, cattle, horses, pigs, etc. [Effects of the Invention]

[0093] The cells according to one embodiment of the present invention and a composition containing the cells as an active ingredient are characterized by having an increased expression level of IL-10 without containing a cryoprotectant, and therefore have the effect of being useful for the prevention or treatment of inflammatory diseases or renal diseases. [Brief explanation of the drawings]

[0094] [Figure 1] Figure 1 is a diagram showing the degree of apoptosis in stem cells according to one embodiment. Fresh: negative control group, cryoprotectant: positive control group, saline infusion, Hartmann and Hartmanndex: experimental groups. [Figure 2] 2 is a diagram showing the level of IL-10 expression in stem cells according to one embodiment, where viable cells (fresh): control group, apoptotic cells (Hartmundex): experimental group. [Figure 3] 3 is a diagram showing changes in stem cells in vivo when they are administered to mice according to one embodiment: Viable cells (fresh): control group, apoptotic cells (Hartmundex): experimental group. [Figure 4] 4 is a diagram showing changes in stem cells in internal organs when they are administered to mice according to one embodiment of the present invention: Viable cells (fresh): control group, Apoptotic cells (Hartmundex): experimental group. [Figure 5] 5 shows the recovery of mice after administration of stem cells according to one embodiment: Model: negative control group, viable cells (fresh): positive control group, apoptotic cells (Hartmundex): experimental group. [Figure 6] 6 shows histological changes in the kidney and changes in the values ​​of renal failure after administration of stem cells to mice according to one embodiment. Model: negative control group, viable cells (fresh): positive control group, apoptotic cells (Hartmundex): experimental group. DETAILED DESCRIPTION OF THE INVENTION

[0095] Preferred examples will be disclosed below to aid in understanding the present invention. However, the following examples are provided merely to facilitate understanding of the present invention, and the content of the present invention is not limited to the following examples. These examples can be modified in various ways, and the embodiments are not limited to the examples disclosed below, but can be embodied in various forms.

[0096] [Example 1] Preparation of stem cells Adult stem cells were prepared as follows. First, using an optical microscope, adult stem cells were confirmed to have proliferated to approximately 80–90% on the bottom of a T75 flask, and then the culture medium was removed. 5 ml of Ca / Mg-free DPBS was added to each T75 flask, and then 3 ml of trypsin-EDTA (0.5%) was added. The flask was shaken side-to-side to evenly distribute the cells across the bottom. The flask was then incubated in a 37°C, 5% CO2 incubator for 3 minutes. After 35 washes, 10 ml of Ca / Mg-free DPBS was added to each flask. The cells were then collected and transferred to a 50 ml tube and centrifuged at 500 g for 5 minutes. After centrifugation, the cell pellet was confirmed, the supernatant was removed, and the cell pellet was loosened by tapping and resuspended in Ca / Mg-free DPBS. 10 μl of the cell suspension was mixed with trypan blue to determine the total cell count. After measuring the cell number, the mixture was centrifuged at 500 g for 3 minutes, the cell pellet was confirmed, and the supernatant was removed.

[0097] Frozen adult stem cells were prepared as follows: the cell pellet was diluted with cryoprotectant, saline infusion, Hartmann infusion, and Hartmanndex infusion (1.0 × 10 per vial). 6 The cells were resuspended in 1 ml of PBS (prepared so that 1 ml of cells would fit in 1 ml of PBS), and then 1 ml of the cell suspension was placed in each of the prepared 1.5 ml tubes. The tubes were then capped and stored in an ultra-low temperature freezer at -80°C or below for one month. The frozen vials were thawed at 4°C for 15 minutes and used in Experiments 1, 2, and 3.

[0098] [Example 2] Induction of a mouse model of renal failure and administration of stem cells to the mice For the induction of renal failure animal models, renal failure animal models were prepared. A mouse model of renal failure was induced using cisplatin and lipopolysaccharide (LPS). Cisplatin (12 mg / kg) was administered intraperitoneally on days 0 and 7, and LPS (5 mg / kg) was administered intraperitoneally on days 1, 4, 8, and 11.

[0099] To confirm the therapeutic effect of the cells on renal failure, the cells were administered to the animal model prepared above. Specifically, the mice were placed in a mouse fixation frame and fixed, and then the tails were immersed in warm water to dilate the blood vessels. The stem cells prepared in Example 1 were then intravenously administered into the mouse tails using an insulin syringe.

[0100] [Experimental Example 1] Confirmation of apoptosis of stem cells after freezing using infusion Apoptosis of the stem cells in Example 1 was confirmed by analyzing Annexin V and PI (Propidium Iodide) levels across non-frozen adult stem cells (negative control group), adult stem cells treated with a cryoprotectant (positive control group), and adult stem cells treated with no cryoprotectant but with a cryoprotectant (experimental group).

[0101] The stem cells of Example 1 were cultured at 2×10 5 After preparing the cells at a volume of 100 μl, the cells were stained with Annexin V and PI at 4°C for 15 minutes and then analyzed by flow cytometry.

[0102] Figure 1 shows the degree of apoptosis in stem cells according to one embodiment: Fresh: negative control group, cryoprotectant: positive control group, saline, Hartmann and Hartmanndex: experimental groups.

[0103] As shown in Figure 1, more than 80% of the unfrozen adult stem cells (negative control group) survived, and more than 70% of the adult stem cells frozen using a cryoprotectant (positive control group) survived after freezing and thawing.

[0104] In contrast, in the experimental groups in which no cryoprotectant was used and infusion was administered according to one embodiment, apoptosis of 90% or more was induced in all experimental groups. Saline infusion induced the most late apoptosis, while Hartmandex infusion induced the least late apoptosis and the most early apoptosis.

[0105] The above results show that in one embodiment of stem cells treated with no cryopreservative and with a solution, apoptosis is induced in most cells when frozen, and in particular, the initial apoptosis induction effect is greatest when Hartmandex infusion is used.

[0106] In the following Examples and Experimental Examples, adult stem cells treated with Hartmandex infusion, which has the greatest early apoptosis-inducing effect, were used as the experimental group.

[0107] [Experimental Example 2] IL-10 expression in fresh cells (viable cells) and Hartmandex (apoptotic cells) after freezing and thawing (apoptotic cells) The level of IL-10 expression was confirmed in the stem cells of Example 1. The level of IL-10 expression was analyzed in non-frozen adult stem cells (control group) and adult stem cells infused with Hartmandex without using any cryoprotectant (experimental group).

[0108] One vial each of the unfrozen adult stem cells (control group) and the adult stem cells treated with Hartmandex infusion (experimental group) prepared in Example 1 was thawed at 4°C and centrifuged to isolate the cells. The isolated cells were lysed in a lysis solution to prepare a protein mixture, and the protein concentration was measured. Equal amounts of the protein mixture were subjected to SDS (sodium dodecyl sulfate) electrophoresis to separate the proteins by size on the gel and then transferred to a PVDF membrane. The membrane was reacted with IL-10 and β-actin antibodies with fluorescent probes, and bands were identified using detection reagents. IL-10 bands were then quantified relative to the β-actin bands to compare IL-10 expression.

[0109] Figure 2 shows the level of IL-10 expression in stem cells according to one embodiment: Viable cells (fresh): control group, Apoptotic cells (Hartmundex): experimental group.

[0110] As shown in Figure 2, the adult stem cells treated with Hartmandex infusion without using any cryoprotectant (experimental group) showed a statistically significant increase in the level of IL-10 expression compared to the non-frozen adult stem cells (control group).

[0111] The above results indicate that the cryoprotectant-free and infused stem cells according to one embodiment have an increased level of IL-10 expression compared to non-frozen stem cells.

[0112] This means that cryopreservative-free and infusion-treated stem cells according to one embodiment are more effective in regulating the proliferation and differentiation of various immune cells, such as T cells, B cells, NK cells, antigen-presenting cells, MAST cells, and granulocytes, and in suppressing inflammatory responses or regulating immune activation, compared to non-frozen stem cells.

[0113] [Experimental Example 3] Confirmation of the therapeutic effect of apoptosis-induced stem cells on renal failure [Experimental Example 3.1] Changes in distribution over time after implantation The changes in distribution over time were observed after transplantation of the stem cells from Example 1 into mice. The distribution of stem cells in the mouse body and internal organs was observed for 24 hours after transplantation using non-frozen adult stem cells (control group) and adult stem cells treated with Hartmandex infusion without cryoprotectant (experimental group).

[0114] Specifically, non-frozen adult stem cells (control group) and adult stem cells without cryoprotectant and with Hartmandex infusion (experimental group) were prepared using the method described in Example 1 above.

[0115] The cells were stained with CellVue NIR815, which expresses GFP, and after transplantation, the cell fluorescence was photographed using a Pearl Trilogy device. The cells were stained using NIR815 dye at 2 × 10 cells per well before the Hartmann infusion solution of Example 1 was administered. -6 The tissue was stained with M for 5 minutes and prepared for transplantation with Hartmann's solution.

[0116] The stem cells were transplanted using the method described in Example 2. After transplantation, the fluorescence inside the mouse body and in the internal organs, namely the heart, lungs, liver, spleen, and kidneys, was measured using a Pearl Trilogy device.

[0117] 3 shows the changes in stem cells in vivo when they are administered to mice according to one embodiment: Viable cells (fresh): control group, Apoptotic cells (Hartmundex): experimental group.

[0118] 4 shows changes in internal organs caused by stem cells administered to mice according to one embodiment of the present invention. Viable cells (fresh): control group, apoptotic cells (Hartmundex): experimental group.

[0119] As shown in Figure 3, fluorescence appeared in the mice one hour after transplantation of both unfrozen adult stem cells (control group) and adult stem cells administered with Hartmandex infusion without cryoprotectant (experimental group). In both groups, the amount of fluorescence expressed was lower 24 hours after administration than it was one hour after administration. There was no significant difference in the amount of fluorescence expressed in the mice between the control and experimental groups.

[0120] As shown in Figure 4, the fluorescence in the internal organs was greatest in the liver for both adult stem cells (control group) and adult stem cells treated with Hartmandex infusion without cryoprotectant (experimental group). The amount of fluorescence expression in the kidney was greater in the experimental group than in the control group. There was no significant difference in the pattern of fluorescence changes in the internal organs of the control and experimental groups.

[0121] The above results indicate that the stem cells treated with no cryoprotectant and no cryoprotectant according to one embodiment show no difference in the pattern of change in total stem cell volume or migration pattern after stem cell transplantation compared to non-frozen stem cells.

[0122] Furthermore, the cryoprotectant-free and cryoprotectant-treated stem cells according to one embodiment of the present invention have a greater therapeutic effect on the kidney than non-frozen stem cells.

[0123] This means that cryoprotectant-free and cryoprotectant-treated stem cells according to one embodiment have no significant differences in stem cell migration, differentiation, apoptosis, treatment of inflammatory diseases, treatment of renal failure or treatment of renal diseases compared to non-frozen stem cells.

[0124] [Experimental Example 3.2] Recovery of mice after transplantation into a mouse model of renal failure After transplanting the stem cells of Example 1 into the mice of Example 2, the recovery of the mice was confirmed. Comparative experiments were conducted on mouse weight, kidney weight, kidney length, and BUN, an indicator of renal failure, using model mice (negative control group), non-frozen adult stem cells (positive control group), and adult stem cells treated with no cryoprotectant and Hartmandex infusion (experimental group).

[0125] The kidneys of the three groups were also analyzed histologically. Specifically, model mice (negative control group) were prepared using the method described in Example 2, while non-frozen adult stem cells (positive control group) and adult stem cells treated with no cryoprotectant and Hartmandex infusion (experimental group) were prepared using the method described in Example 1.

[0126] The renal failure mouse model was induced and stem cells were administered to the mice by the method described in Example 2.

[0127] Mouse weight, kidney weight, kidney length, and BUN were measured using the following methods. The mice were weighed using a scale and sacrificed using a CO2 chamber. The abdomen of the mice was opened with scissors, blood was collected from the femoral vein using a 1 cc syringe, and the kidneys were then removed. The kidney weight was measured using a precision scale, and kidney length was measured using a ruler.

[0128] The collected blood was centrifuged at 3000 rpm for 15 minutes, and only the plasma was transferred to a new tube, after which the BUN results were obtained using a Toshiba Accute device.

[0129] Histological analysis of the kidneys was performed using H&E and MT staining methods. The kidneys were fixed in 4% paraformaldehyde for 3 days and then prepared into paraffin blocks. The sections were then cut at 4 μm, mounted on slides, and subjected to H&E staining using hematoxylin and eosin stains. For Masson's Trichrome (MT) staining, the paraffin blocks were cut at 4 μm, mounted on slides, and stained with hematoxylin, Biebrich Scarlet Acid Fuchsin, and aniline blue stains.

[0130] 5 shows the recovery of mice following administration of stem cells according to one embodiment: Model: negative control group, viable cells (fresh): positive control group, apoptotic cells (Hartmundex): experimental group.

[0131] Figure 6 shows the histological changes in the kidney and the changes in the numerical values ​​of renal failure when stem cells were administered to mice according to one embodiment. Model: negative control group, viable cells (fresh): positive control group, apoptotic cells (Hartmondex): experimental group.

[0132] As shown in Figure 5, both the non-frozen adult stem cells (positive control group) and the adult stem cells (experimental group) that were not treated with cryoprotectant and used Hartmandex infusion restored mouse weight, kidney weight, kidney length, and BUN to normal levels after transplantation compared to the model mice (negative control group).

[0133] As shown in FIG. 6, after transplantation, the kidney tissue and histological renal failure values ​​in both the positive control group and the experimental group returned to normal levels compared to the negative control group. There was no significant difference between the positive control group and the experimental group in terms of the recovery of the above indicators to normal levels.

[0134] The above results indicate that there is no significant difference in the recovery of renal failure model mice after stem cell transplantation between stem cells treated with a cryoprotectant and a solution according to one embodiment, compared to non-frozen stem cells.

[0135] This means that cryoprotectant-free and cryoprotectant-treated stem cells according to one embodiment have no significant differences in stem cell migration, differentiation, apoptosis, treatment of inflammatory diseases, treatment of renal failure or treatment of renal diseases compared to non-frozen stem cells.

Claims

1. Isolated cells in which apoptosis has been induced by freezing, and in which the expression level of interleukin-10 (IL-10) has increased by 1.5 times or more compared to the cells before freezing.

2. The isolated apoptosis-induced cells according to claim 1, wherein at least 75% of the entire population is annexin V positive.

3. The cell according to claim 1 , wherein the cell is derived from any one or more of the group consisting of adult stem cells, induced pluripotent stem cells, and embryonic stem cells.

4. The cells according to claim 1, wherein the cells are derived from one or more tissues selected from the group consisting of placenta, amnion, and umbilical cord.

5. The cells of claim 1 , wherein the solution for freezing is a blood substitute.

6. The blood substitute is Saline infusion, a solution containing sodium chloride, potassium chloride, calcium chloride, and sodium lactate; 6. The cell according to claim 5, further comprising any one selected from the group consisting of a solution containing sodium chloride, potassium chloride, calcium chloride, sodium lactate, and glucose.

7. The cell according to claim 1 , wherein the cell is a cell in which apoptosis is induced after thawing a frozen cell.

8. The cells according to claim 1, wherein the cells are thawed at 0 to 5°C for 10 minutes to 5 hours.

9. 2. The isolated cell in which apoptosis has been induced according to claim 1, wherein the expression level of IL-10 has increased by 2 to 2.5 times.

10. A pharmaceutical composition for preventing or treating an inflammatory disease, comprising, as an active ingredient, apoptosis-induced cells in which the expression level of interleukin-10 (IL-10) is increased by 1.5 times or more compared to the cells before freezing, or a culture medium of said cells.

11. The inflammatory diseases include renal failure, nephritis, glomerulonephritis, gastritis, inflammatory bowel disease (IBD), hepatitis, inflammatory respiratory disease, gastric ulcer, irritable bowel syndrome, Behcet's disease, enteritis, Crohn's disease, and the like. disease, asthma, dermatitis, ulcerative colitis, vasculitis colitis), mucositis, stomatitis, peri-implantitis, periodontitis itis), pulpitis, gingivitis, pneumonia, dermatitis, atopic dermatitis dermatitis), contact dermatitis, CREST syndrome, dermatitis herpetiformis herpetiformis, dermatomyositis, systemic scleroderma, erythema nodosum, Henoch-Schönlein purpura, hidradenitis suppurativa, lichen protoplanus, Majeed syndrome, Schnitzler syndrome pulsyndrome, psoriasis, eczema, acne, mouth inflammationulcers, uveal salts (uveitis), pharyngitis (pharyngitis), tonsillitis (tonsillitis), otitis including otitis media (otitis media), arthritis (psoriatic arthritis), synovitis (synovitis), meningitis (meningitis), encephalitis (encephalitis), Bickerstaff's sequencephalitis, encephalomyelitis (encephalomyelitis), spondylitis (national dysplasia), osteomyelitis (osteomyelitis), Guillain-Barré syndrome (guillain-barré syndrome) 11. The composition according to claim 10, wherein the composition is any one selected from the group consisting of neuromyelitis, myelitis, neuromyelitis optica, cystitis, or acute inflammation at the site of infection or wound.

12. The renal diseases include chronic renal failure, acute renal failure, glomerulonephritis, interstitial nephritis, diabetic nephropathy, glomerulosclerosis, renal fibrosis, Alport syndrome, IDDM nephritis, mesangial focal proliferative glomerulonephritis, focal proliferative glomerulonephritis, crescentic glomerulonephritis, renal interstitial fibrosis, focal segmental glomerulosclerosis, membranous nephropathy, minimal change disease, pauci-immune rapidly progressive glomerulonephritis, IgA nephropathy, polycystic kidney disease, Dent's disease, and the like.

11. The pharmaceutical composition of claim 10, wherein the disease is any one or more selected from the group consisting of leukemia, cystinosis, nephrocytinosis, Heymann nephritis, polycystic kidney disease, nephrotic syndrome, renal ischemia, podocytopathy or podocytic disorder, proteinuria, pre-eclampsia, renal lesions, collagen vascular disease, benign orthostatic (postural) proteinuria, IgM nephropathy, amino aciduria, Fanconi syndrome, hypertensive nephrosclerosis, hemoglobinuria, myoglobinuria, Wegener's granulomatosis, reduced glomerular filtration rate (GFR), nephroangiosclerosis, lupus nephritis, proximal tubular dysfunction, acute renal transplant rejection, chronic renal transplant rejection, and non-IgA mesangial focal proliferative glomerulonephritis.

13. carrying the isolated cells in a blood substitute-containing solution to produce a cell-containing composition; freezing the cell-containing composition to produce an apoptosis-induced cell-containing composition; and thawing the apoptosis-induced cell-containing composition.

14. The manufacturing method described in claim 13, wherein the cells in the step of producing the cell-containing composition are at least 75% or more of the total population negative for Annexin V and negative for PI, and the cells in the step of thawing are at least 75% or more of the total population positive for Annexin V.

15. The method according to claim 13, wherein the cells are derived from any one of the group consisting of adult stem cells, induced pluripotent stem cells, and embryonic stem cells.

16. The method according to claim 13, wherein the cells are derived from one or more tissues selected from the group consisting of placenta, amnion, and umbilical cord.

17. The blood substitute is Saline infusion, a solution containing sodium chloride, potassium chloride, calcium chloride, and sodium lactate; 14. The method according to claim 13, wherein the solution contains any one selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, sodium lactate, and a solution containing glucose.

18. The method of claim 13, wherein the solution does not contain a cryoprotectant.

19. The method according to claim 13, wherein the freezing is carried out for six months or less.

20. The method according to claim 13, wherein the thawing is carried out at 0 to 5°C for 10 minutes to 5 hours.

21. The method according to claim 13, wherein the thawed cells have an increased level of IL-10 expression by 1.5 to 2.5 times compared to the cells before freezing.

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