Method for mass-culturing mesenchymal stem cells

Abstract

The present invention relates to: a method for mass-culturing mesenchymal stem cells, comprising a step of gradually increasing the P / V value of a bioreactor in a specific range; mesenchymal stem cells prepared using the method; and a use thereof. The mass-culturing method can increase the proliferation rate of stem cells while maintaining the characteristics thereof, and thus can be effectively used in related technical fields such as the treatment of autoimmune diseases, inflammatory diseases, or hair loss by using mesenchymal stem cells.

Description

[Technical Field]

[0001] The present disclosure relates to a massive culture method of mesenchymal stem cells, mesenchymal stem cells isolated by the method, and a pharmaceutical composition comprising the same as an active ingredient. [Background Art]

[0002] Stem cells are undifferentiated cells that are self-renewable and may differentiate into various types of cells by appropriate bio-signals and external stimulation. The possibility that the stem cells can be used as cell therapeutics for various diseases has been found through various studies. Among them, adult stem cells are divided into hematopoietic stem cells and mesenchymal stem cells, and the mesenchymal stem cells may be isolated from various tissues of the human body, such as umbilical cord blood, bone marrow, adipose tissue, skeletal muscle, peripheral blood, and the like. Recently, cell therapeutics using stem cells has attracted considerable attention in the fields of regenerative medicine and cell-based therapy for the purpose of treating various diseases. However, in the case of mesenchymal stem cells (MSCs), it is known that 1 to 5 million cells per kg of body weight are required per injection to exhibit a therapeutic effect, and at least 0.3 to 5 x 108 cells are required per injection for adults weighing 70 kg. Therefore, in order to commercialize stem cell therapeutics, it is necessary to secure massive manufacturing technology that can simultaneously satisfy both the quantity and quality of cells (Olsen, T.R. et al. (2018) Front. Med. 5, 178).

[0003] A monolayer cell culture system commonly used in cell culture is a 2-dimensional culture method, and has the advantages of being relatively easy to culture and having excellent cell viability, which has been widely used in the field of biology. However, the monolayer cell culture system has limitations in implementing 3-dimensional microenvironmental conditions such as human tissue, and thus cannot maintain specific cell functions for a long time, and is not suitable for massive cell culture because the number of cells per culture area is significantly limited. When massive culture is performed using 2-dimensional culture production, the production time becomes longer as the amount of cells and media increases, and production capacity may also decrease. The problems may be partially resolved by input of production manpower, but cause an increase in the unit production cost due to increased labor costs, so that there is a limitation that price competitiveness may decrease after product development in the future.

[0004] As a new culture system capable of complementing the limitations of such a monolayer cell culture system, a 3-dimentional culture system is attracting attention. The advantage of the 3-dimentional culture system is maximizing the efficacy of stem cells by culturing cells in a microenvironment similar to an in vivo environment, thereby implementing a cell growth mechanism more precisely. In addition, a massive culture system using a 3-dimentional bioreactor based on microcarriers provides a high surface area / volume ratio at which cells may adhere and proliferate, which is suitable for massive culture of cells. A massive culture method using a 3D bioreactor first starts with small-scale culture to stabilize cell lines and gradually scales up to a larger-scale bioreactor to increase the capacity. Since the scale-up process of such a bioreactor requires precise environmental control because cells may be killed or deformed due to rapid environmental changes, and requires setting of reasonable standards for culture conditions that can be expected to have the same effect even through the scale-up process.

[0005] Under this background, the present inventors confirmed an effect of increasing the proliferation rate while maintaining the characteristics of mesenchymal stem cells when a P / V value of the bioreactor is gradually increased within a specific range in a massive culture method 2 of mesenchymal stem cells and then completed the present disclosure. [Disclosure] [Technical Problem]

[0006] An object of the present disclosure is to provide a massive culture method of mesenchymal stem cells comprising: a) injecting a medium comprisingmicrocarriers into a bioreactor; b) inoculating mesenchymal stem cells (MSCs) into the bioreactor of step a); c) replacing the medium 3 days after inoculation; and d) harvesting cells 5 to 6 days after inoculation, in which a power per unit volume (P / V) value gradually increases from 0.30 to 0.40 W / m3 to 0.85 to 0.95 W / m3 from the inoculation time to the harvest time.

[0007] Another object of the present disclosure is to provide mesenchymal stem cells prepared by the massive culture method.

[0008] Yet another object of the present disclosure is to provide a pharmaceutical use of the mesenchymal stem cells, and more particularly, a pharmaceutical composition comprisingthe mesenchymal stem cells as an active ingredient for preventing or treating autoimmune diseases, inflammatory diseases, or hair loss, a use of the mesenchymal stem cells for preventing or treating autoimmune diseases, inflammatory diseases, or hair loss, or a method for preventing or treating autoimmune diseases, inflammatory diseases, or hair loss, including administering the mesenchymal stem cells. [Technical Solution]

[0009] The present disclosure will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present disclosure can also be applied to each other description and embodiment. That is, all combinations of various elements disclosed herein fall within the scope of the present disclosure. In addition, it cannot be considered that the scope of the present disclosure is limited by the specific descriptions to be described below.

[0010] In order to achieve the object, an aspect of the present disclosure provides a massive culture method of mesenchymal stem cells comprising : a) injecting a medium comprisingmicrocarriers into a bioreactor; b) inoculating mesenchymal stem cells (MSCs) into the bioreactor of step a); c) replacing the medium 3 days after inoculation; and d) harvesting cells 5 to 6 days after inoculation, in which a power per unit volume (P / V) value gradually increases from 0.30 to 0.40 W / m3 to 0.85 to 0.95 W / m3 from the inoculation time to the harvest time.

[0011] The present inventors have devised a massive culture method capable of minimizing environmental changes that occur during a scale-up process of a bioreactor by delicately controlling the P / V value, in order to overcome the limitations of a conventional massive culture method of mesenchymal stem cells using a bioreactor. Since the mesenchymal stem cells isolated using the method show an excellent proliferation rate while maintaining the characteristics of stem cells, the mesenchymal stem cells can be very usefully used for the treatment of various diseases, such as cell therapeutics that require a large amount of mesenchymal stem cells.

[0012] In the present disclosure, the “mesenchymal stem cells (MSCs)” refer to adult stem cells that may be isolated from a biologically derived tissue and may differentiate into at least one mesenchymal cell of bone, cartilage, and fat.

[0013] In the present disclosure, the “biologically derived tissue” refers to a biological source, i.e., tissue obtained from an organism, such as animal or human subjects. The biologically derived tissue of the animal or human subject refers to an isolated tissue that comprises cells of the animal or human subject. The biologically derived tissue of the animal or human subject may comprise cells of one or more types of tissue. The biologically derived tissue may be derived from at least any one tissue selected from the group consisting of, for example, umbilical cord, umbilical cord blood, bone marrow, fat, muscle, synovium, bone tissue, nerve, skin, amniotic membrane, placenta, Wharton’s jelly, amniotic fluid, peripheral blood, dental pulp, fallopian tube, 4 corneal stroma, lung, and fetal liver, and specifically may be derived from bone marrow or adipose tissue, but is not limited thereto.

[0014] In one embodiment, the mesenchymal stem cells may be mesenchymal stem cells isolated by a subfractionation culture method comprising, but not limited to, a) culturing a biologically derivedtissue in a first vessel to harvest a supernatant; b) transferring the supernatant of the first vessel to a second vessel; c) culturing cells present in the second vessel to harvest a supernatant; and d) repeating steps b) and c) at least once.

[0015] The subfractionation culture method of the mesenchymal stem cells refers to culturing cells isolated from a floating layer of a suspension comprising cells or tissues, unlike a conventional primary culture method that directly attaches and cultures a suspension comprising cells or tissues. The subfractionation culture method is simple, economical, and may isolate mesenchymal stem cells while minimizing the possibility of contamination, and is efficient for clinical trials and cell therapeutic production. In addition, the subfractionation culture method has an effect of continuously maintaining the characteristics of stem cells by isolating the stem cells according to a difference in specific gravity.

[0016] In the present disclosure, the “inoculation time (or indicated as D0)” refers to the time when cells are first inoculated into a bioreactor for massive culture of cells. In the present disclosure, day 1 of inoculation, day 2 of inoculation, day 3 of inoculation, day 4 of inoculation, day 5 of inoculation, and day 6 of inoculation refer to 1 day (or indicated as D1), 2 days (or indicated as D2), 3 days (or indicated as D3), 4 days (or indicated as D4), 5 days (or indicated as D5), and 6 days (or indicated as D6) after the inoculation time, respectively. In the present disclosure, the harvest time refers to the time when the massive culture of cells is completed and the entire cells proliferated in the bioreactor are separated from the microcarriers and recovered.

[0017] Each step of the massive culture method of the mesenchymal stem cells of the present 5 disclosure will be specifically described as follows.

[0018] Step a) is a step of injecting the medium comprising microcarriers into the bioreactor, which is a preparation step for massive culture.

[0019] In the present disclosure, the “microcarrier” refers to a microparticle on which cells adhere to the surface to survive, grow, and proliferate. The microcarrier serves as a support on which cells may adhere and proliferate within the bioreactor, and may provide a sufficient surface area on which cells may be cultured, thereby maximizing the cell culture area. A material of which cells can adhere to the surface of the microcarrier may be selected from dextran, gelatin, collagen, polystyrene, polyethylene, polyacrylamide, glass, or cellulose, and is not particularly limited in type as long as the material is a material on which cells can adhere, survive, grow, and proliferate.

[0020] The surface of the microcarrier may further comprise a cell adhesive material. The cell adhesive material may bind with a transmembrane protein of the cell to allow cells to stably adhere to and proliferate on the surface of the microcarrier. The cell adhesive material may be selected from a peptide comprisingan acrylic polymer, gelatin, collagen, fibronectin, chitosan, polydopamine, L-lysine, vitronectin, a peptide comprisingRGD, an acrylic polymer, lignin, cationic dextran, or derivatives thereof, and is not particularly limited in type as long as the material is a material to which cells can adhere.

[0021] The average diameter of the microcarrier may be 50 ^m to 500 gm, and specifically, 100 gm to 300 gm. When the average diameter of the microcarrier is less than 50 gm, the cell culture efficiency decreases, which is not preferable, and when the average diameter exceeds 500 gm, the proliferation rate decreases due to a decrease in the interaction between adherent cells, which is not preferable.

[0022] In the present disclosure, a culture medium used for culturing a biologically derived tissue comprisingmesenchymal stem cells means a medium for maintaining or culturing a cell population 6 comprisingnutrients that maintain the cell viability of mesenchymal stem cells and support proliferation, and may be used without limitation by those skilled in the art as long as such functions can be achieved.

[0023] The culture medium used in the present disclosure comprises a basic medium. The basic medium is any basic medium suitable for culturing animal or human cells, particularly mesenchymal stem cells. The basic medium typically comprises a plurality of ingredients required to support the maintenance of cultured cells. A combination of suitable ingredients may be easily formulated by an expert in consideration of the following contents. In addition, the basic medium comprises a nutrient solution comprising general standard cell culture ingredients, for example, amino acids, vitamins, lipid supplements, mineral salts, carbon energy sources, and buffers.

[0024] The basic medium may be obtained commercially and may be, for example, one or more selected from the group consisting of RPMI, DMEM (Dulbecco’s modified eagles media), MEM (minimum essential media), KO-DMEM (knockout-DMEM), G-MEM (Glasgow’s minimum essential media), BME (basal medium eagle), DMEM / Ham’s F12, advanced DMEM / Ham’s F12, IMDM (Iscove’s modified Dulbecco’s media), Ham’s F-10, Ham’s F-12, medium 199, and knockout serum replacement xenofree medium, but is not limited thereto. The medium of the present disclosure may be prepared by adding the basic medium with antibiotics, serum (or serum substitute), etc., or may be a commercially available complete medium (e.g., RoosterNourishTM-MSC-XF, etc.), and a type thereof is not particularly limited within a range capable of maintaining / proliferating a population of mesenchymal stem cells.

[0025] In the present disclosure, the “bioreactor” refers to a cell culture device that reproduces a chemical reaction occurring in a living organism in vitro to enable mass production of cells. A microcarrier-based bioreactor provides a system that can mass-culture cells by creating a large 7 culture area and a specialized microenvironment, and can produce cells of a certain quality by controlling culture parameters. The culture parameters are culture conditions that may affect cell culture, and may comprise a microcarrier density, an inoculated cell density, a culture temperature, a stirring speed, an osmotic pressure, pH, a dissolved oxygen content, a carbon dioxide partial pressure, and a culture period, and these conditions may affect intracellular metabolism and activity.

[0026] The bioreactor may be selected from a vertical-wheel bioreactor, a stirred suspension bioreactor, a rotating wall vessel, a microbioreactor, a parallel plates bioreactor, and a hollow-fiber bioreactor, and may be specifically a vertical-wheel bioreactor, but any bioreactor capable of cell culture that is commonly used in the art may be used without limitation. The P / V value, which is a key parameter in the present disclosure, may be applied regardless of a type of bioreactor, and an equivalent effect may be expected.

[0027] The density of the microcarrier in step a) may be 10 g / L to 50 g / L, and preferably 20 g / L to 40 g / L. When the density of the microcarrier is less than 10 g / L, the surface area to which cells may adhere decreases, and when the density of the microcarrier is more than 50 g / L, the production efficiency decreases, which is not preferable.

[0028] Step b) is a step of inoculating the mesenchymal stem cells (MSCs) into the bioreactor of step a).

[0029] The mesenchymal stem cells inoculated into the bioreactor in step b) adhere to the surface of the microcarrier in the bioreactor and proliferate, and the mesenchymal stem cells adhering to the microcarrier may be cultured in a suspended state in the medium through stirring of the bioreactor after the inoculation time. In addition, the method may comprise stopping stirring for a certain period of time (e.g., 5 to 60 minutes) after the inoculation time in order to attach the mesenchymal stem cells to the microcarrier.

[0030] At the inoculation time, the density of the mesenchymal stem cells may be 8,000 cells / mL to 30,000 cells / mL, and excellent proliferation ability is exhibited at such an inoculation density. Specifically, the density of the mesenchymal stem cells at the inoculation time may be 10,000 cells / mL to 25,000 cells / mL, but is not limited thereto. When the density of the mesenchymal stem cells is less than 8,000 cells / mL, the final cell harvest amount is significantly reduced, which is not preferable, and when the density of the mesenchymal stem cells is more than 30,000 cells / mL, the proliferation ability decreases and thus the production efficiency decreases, which is not preferable.

[0031] Step c) is a step of replacing the medium 3 days after inoculation, and the method and timing of supplying the medium are very important for cell proliferation and survival in massive culture.

[0032] Step c) may further comprise a step of replacing the medium 4 days after inoculation.

[0033] Step c) may further comprise a step of replacing the medium 5 days after inoculation.

[0034] Step c) may be replacing 60% to 90% of the total medium volume in the bioreactor, specifically, 75% to 85% of the total medium volume, but is not limited thereto.

[0035] Step c) provides an effect of increasing the cell proliferation rate by removing the medium in which materials necessary for cell growth are consumed by cell proliferation after the inoculation time, injecting a fresh medium to supplement materials necessary for cell growth, and removing toxic metabolites, proliferation inhibitors, waste products, etc. that may be secreted by the cells.

[0036] Step d) is a step of harvesting cells 5 to 6 days after inoculation. Through the massive culture method of the present disclosure, clinical-grade mesenchymal stem cells may be harvested in large quantities.

[0037] The massive culture method of the mesenchymal stem cells of the present disclosure is 9 particularly characterized by gradually increasing a power per unit volume (P / V) value from 0.30 to 0.40 W / m3 to 0.85 to 0.95 W / m3 from the inoculation time to the harvest time.

[0038] If the P / V value at any time from the inoculation time to the harvest time is less than 0.30, toxic metabolites, growth inhibitors, waste products, etc. that may be secreted by the cells may accumulate, and oxygen delivery to cells may not be performed well, which may inhibit cell proliferation. When the P / V value at any time from the inoculation time to the harvest time is more than 0.95, a physical stimulation received by cells may increase, which may damage cells, and inhibit cell proliferation.

[0039] In the present disclosure, the “P / V (power per unit volume) value” means the amount of power per unit volume in a bioreactor culture vessel, and represents power density. In deriving the P / V value, a first variable “P (power)” means the power of the bioreactor and may be derived by the following Equation 1. In deriving the P / V value, a second variable “V (volume)” means the capacity of the bioreactor.

[0040] [Equation 1] E F Xd              RPM P - — -----mxgx2nxrx

[0041] {         1                                       r)li

[0042] (F = force [N], E = energy [J], T = time [s], d = distance [m], m = mass [kg], g = acceleration of gravity [m / s2], r = spindle radius, RPM = rotations per minute [rotations / min])

[0043] In the present disclosure, the P / V value means the amount of energy transferred from a stirrer in the bioreactor to a liquid in the vessel, and may also be represented as the size of stress or stimulation received by the cells present in the bioreactor. Since the effect of the stirring speed on the cells may vary depending on various factors such as a culture scale, a cell type, a medium composition, and the like, the stirring speed may not be an appropriate parameter for massive culture, but the P / V value, which is a parameter used in the present disclosure, is an energy value 10 applied to the cells and a parameter that is less affected by an external environment.

[0044] In one embodiment, the P / V values at the inoculation time to 3 days after inoculation may be 0.30 to 0.40 W / m3, specifically 0.32 to 0.38 W / m3, and more specifically 0.34 to 0.36 W / m3, but are not limited thereto.

[0045] In another embodiment, the P / V values at 3 days after inoculation to 5 days after inoculation may be 0.40 to 0.50 W / m3, specifically 0.41 to 0.49 W / m3, more specifically 0.42 to 0.48 W / m3, but are not limited thereto.

[0046] In another embodiment, the P / V values at 5 days after inoculation to the harvest time may be 0.85 to 0.95 W / m3, specifically 0.87 to 0.93 W / m3, more specifically 0.89 to 0.91 W / m3, but are not limited thereto.

[0047] The capacity of the bioreactor may be 0.3 to 20 L. For example, the capacity of the bioreactor may be 0.5 L, 3 L, or 15 L, but is not limited thereto.

[0048] When the mesenchymal stem cells are cultured according to the massive culture method of the mesenchymal stem cells of the present disclosure, significant mesenchymal stem cell proliferation rates were observed all in bioreactors with capacities of 0.5 L, 3 L, and 20 L.

[0049] In the present disclosure, “scale-up” refers to a process of gradually increasing the amount of cells by repeating a process of transferring cells to a culture vessel or culture system with a larger surface area than the previous culture vessel during a cell subculture process, and is an important process in the cell massive culture process. In the present disclosure, the scale-up may refer to subculturing from a monolayer culture to a bioreactor having a capacity of 0.3 L to 20 L, subculturing from a bioreactor having a capacity of 0.5 L to a bioreactor having a capacity of 1 L to 20 L, or subculturing from a bioreactor having a capacity of 3 L to a bioreactor having a capacity of 5 L to 20 L.

[0050] In the present disclosure, the “culture vessel” may refer to a place where cell growth, 11 proliferation, growth, division, and an increase in cellular ingredients, an increase in cell-derived ingredients, etc. occur, and refers to a place where a chemical reaction and / or a biological reaction involving an organism or a biochemically active ingredient derived from the organism occurs.

[0051] Another aspect of the present disclosure provides mesenchymal stem cells prepared by the massive culture method.

[0052] Yet another aspect of the present disclosure provides a pharmaceutical composition for preventing or treating autoimmune diseases, inflammatory diseases, or hair loss, comprising the mesenchymal stem cells as an active ingredient.

[0053] Yet another aspect of the present disclosure provides a method for treating autoimmune diseases, inflammatory diseases, or hair loss, comprising administering the composition to a subject in need thereof.

[0054] Yet another aspect of the present disclosure provides a pharmaceutical composition for preventing or treating autoimmune diseases, inflammatory diseases, or hair loss, or a use of the composition for preventing or treating autoimmune diseases, inflammatory diseases, or hair loss.

[0055] The mesenchymal stem cells are as described above.

[0056] In the present disclosure, the autoimmune or inflammatory disease may comprise all diseases that may exhibit preventive or therapeutic efficacy by the mesenchymal stem cells. For example, the autoimmune or inflammatory disease may be at least one selected from the group consisting of IgA nephropathy, ankylosing spondylitis, psoriasis, polyarteritis nodosa, arthritis, ulcerative colitis, Graves’ hyperthyroidism, Guillain-Barre syndrome, multiple sclerosis, multiple myeloma, multiple myositis, lupus, rheumatoid arthritis, chronic thyroiditis, chronic obstructive pulmonary disease, Meniere’s syndrome, leukoplakia, Behcet’s disease, fibromyalgia, Sjogren’s syndrome, silicosis, asbestosis, atopic dermatitis, aphthous stomatitis, Addison’s disease, poststreptococcal glomerulonephritis, inflammatory enterocolitis, Wilson’s disease, graft-versus-12 host disease, some acquired aplastic anemias, primary cirrhotic autoimmune encephalomyelitis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, endometriosis, relapsing polychondritis, systemic scleroderma, type 1 diabetes, myasthenia gravis, asthma, pemphigus, pancreatitis, temporal arteritis, Crohn’s disease, Fanconi syndrome, sepsis, dermatomyositis, Hashimoto’s thyroiditis, hyperthyroidism, and erythema multiforme, but is not limited thereto.

[0057] As used herein, the term “treatment” refers to intervening to alter a natural process of individuals or cells having diseases, which may be performed while a pathological condition is in progress or to prevent the pathological state. A desired therapeutic effect comprises alleviation of the symptoms of a disease, reduction of all direct or indirect pathological consequences according to a disease, prevention of metastasis, reduction in disease progression rate, alleviation or temporary relief of a disease condition, remission, or improvement of a prognosis. In particular, in the present disclosure, the treatment comprises all actions of improving the course of autoimmune diseases, inflammatory diseases, or hair loss by administering a composition comprising mesenchymal stem cells as an active ingredient. In addition, the term “prevention” refers to all actions of inhibiting or delaying the onset of a disease by administering the mesenchymal stem cells. When the mesenchymal stem cells of the present disclosure are used for treatment or prevention, the mesenchymal stem cells are administered to a subject in a therapeutically effective amount.

[0058] The term “therapeutically effective amount” used herein refers to an effective amount of mesenchymal stem cells. Specifically, the “therapeutically effective amount” refers to an amount enough to treat diseases at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level may be determined according to factors comprising a kind of subject, the severity, age, sex, a type of disease, the activity of a drug, the sensitivity to a drug, a time of administration, a route of administration, an excretion rate, duration of treatment, and agents to be 13 simultaneously used, and other factors well-known in the medical field. The pharmaceutical composition of the present disclosure may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and sequentially or simultaneously administered with commercially available therapeutic agents. In addition, the pharmaceutical composition may be administered singly or in multiple. It is important to administer an amount capable of obtaining a maximum effect with a minimal amount without side effects in considering all the factors, and the amount thereof may be easily determined by those skilled in the art. The dose of the pharmaceutical composition of the present disclosure may be determined by a specialist according to various factors such as the condition, age, sex, and complications of a patient. The therapeutic composition according to the present disclosure may be administered in parallel with known compounds having an effect of preventing, improving, or treating symptoms of autoimmune diseases, inflammatory diseases, or hair loss.

[0059] The mesenchymal stem cells according to the present disclosure or the composition comprisingthe mesenchymal stem cells may be administered by intravenous administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, rectal administration, topical administration, intranasal administration, etc., but it is not limited thereto.

[0060] The embodiments of the present disclosure can be modified in various other forms, and the scope of the present disclosure is not limited to embodiments to be described below. Further, the embodiments of the present disclosure are provided for more completely describing the present disclosure to those having average knowledge in the art. Furthermore, throughout the present specification, “comprising” a component does not exclude other components unless specifically stated otherwise, but means further comprising other components. [Advantageous Effects]

[0061] According to the present disclosure, the massive culture method can minimize 14 environmental changes that may occur during a scale-up process of a bioreactor, thereby maintaining the characteristics of mesenchymal stem cells and increasing the proliferation rate, and thus can be usefully used in related technical fields, such as massive production of stem cells and treatment of autoimmune diseases, inflammatory diseases, or hair loss using the stem cells. [Description of Drawings]

[0062] FIG. 1 is a schematic diagram schematically illustrating a scale-up process in a massive culture method of stem cells according to some embodiments of the present disclosure.

[0063] FIG. 2 shows the shapes of cells from an inoculation time to a harvest time in a massive culture method of mesenchymal stem cells according to some embodiments of the present disclosure.

[0064] FIG. 3 is a schematic diagram schematically illustrating an overall process in a massive culture method of stem cells according to some embodiments of the present disclosure.

[0065] FIG. 4 is a graph showing the number of cells proliferated according to changes in microcarrier density in a massive culture method of stem cells according to the present disclosure.

[0066] FIG. 5 is a graph showing the number of cells proliferated according to changes in inoculated cell density in a massive culture method of stem cells according to the present disclosure.

[0067] FIG. 6 is a graph showing the number of cells proliferated according to changes in a medium replacement condition in a massive culture method of stem cells according to the present disclosure.

[0068] FIG. 7 is a graph showing the number of cells proliferated according to changes in a P / V value in a massive culture method of stem cells according to the present disclosure. [Modes]

[0069] Hereinafter, configurations and effects of the present disclosure will be described in more detail through Examples. These Examples are only illustrative of the present disclosure, and the scope of the present disclosure is not limited by these Examples.

[0070] Example 1. Isolation and culture of mesenchymal stem cells

[0071] Mesenchymal stem cells used in Example of the present disclosure were harvested by the following method. The hip area of a bone marrow donor was anesthetized with a local anesthetic, and then an injection needle was inserted into the hip bone to collect bone marrow. 10 ml of DMEM (GIBCO-BRL, Life-technologies, MD, USA) comprising 20% FBS and 1% penicillin / streptomycin and 1 ml of human bone marrow were added to a 100 mm culture vessel and cultured for 2 hours in a cell incubator at 37°C and 5 % CO2.  After culturing, the culture vessel was tilted slightly to one side to prevent the cells attached to the bottom from being detached, and only the supernatant in the culture vessel was transferred to a new vessel as much as possible.

[0072] After repeating the same process once more, the harvested culture medium was transferred to a new culture vessel (Becton Dickinson) and cultured at 37°C for 2 hours. The supernatant was transferred to a new culture vessel, and after 24 hours, the supernatant was transferred to a new vessel again, and after 24 hours, the supernatant was transferred to a new vessel again. Finally, after 48 hours, the supernatant was transferred to a new vessel and then it was visually confirmed that the remaining cells were attached to the bottom of the culture vessel and grew. It can be inferred that cells that have reached this step through the previous several subfractionation steps are cells with a significantly smaller specific gravity than other cells. After about 3 to 5 days, the cells formed single clones, and these single clones were isolated by trypsin treatment and then transferred to a 6-well culture vessel with a cell number of 102 to 6 x 102 per well. After culturing for 4 to 5 days in a 37°C, 5% CO2 cell incubator, when the cells grew about 80%, the cells were treated with 0.25% trypsin / 1 mM EDTA (GIBCO-BRL), harvested, and then transferred to a 75 cm2 culture vessel and subcultured. The harvested subfractionation bone marrow-derived mesenchymal stem cells (cMSC) were resuspended in cell freezing media (FBS with 10% DMSO), 16 placed in a cryotube, and stored in a liquid nitrogen tank at -80°C.

[0073] In order to be used in the stem cell massive culture experiment according to the present disclosure, cMSCs at passage 3 that had been frozen were thawed at 37°C and cultured. The cMSCs were cultured in a 175 cm2 culture plate in a RoosterNourishTM-MSC-XF (Roosterbio, KT-016) medium at a density of 2,500 cells / cm2 and cultured in a cell incubator at 37°C and 5% CO2 for 4 days. The cultured cells were detached by trypsin treatment and seeded in a larger cell culture plate, approximately 6,000 cm2 CS-10 chamber (CellSTACK culture 10 chamber, Corning), at a density of 2,500 cells / cm2 and cultured for 4 days under the same conditions above. The harvested cells were used in the experiment (FIG. 1).

[0074] Example 2. Massive culture of mesenchymal stem cells

[0075] The mesenchymal stem cells harvested and stabilized in Example 1(cMSCs) were mass-cultured using the following method (FIGS. 2 and 3). The bioreactor for massive culture was used with a vertical-wheel bioreactor, PBS-15 (PBSbiotech).

[0076] 1) Two days before cell inoculation, nitrogen, oxygen, and carbon dioxide gas supply lines were connected and opened, and probes of a dissolved oxygen sensor and a pH sensor were connected and calibrated. The DO probe, the pH probe, a thermal well, and a dip tube were connected to a single-use vessel and then connected to the bioreactor. Approximately 70 v / v% of RoosterNourishTM-MSC-XF (Roosterbio, KT-016) medium was added to the vessel, and the rotation speed was increased to equilibrate the medium.

[0077] 2) One day before cell inoculation, a portion of the medium in the bioreactor was collected, the pH was measured, and the device was set up to start cell culture by calibrating the probe of each sensor.

[0078] 3) At an inoculation time (indicated as D0), polystyrene microcarriers coated with synthetic peptide acrylate (Corning, 3781, Low concentration synthemax II, diameter 125 to 212 17 pm) were injected into the vessel of the bioreactor where the medium equilibrium was prepared, and then mesenchymal stem cells were inoculated. The microcarrier density, mesenchymal stem cell inoculation density, and bioreactor P / V value were adjusted according to each experimental condition.

[0079] 4) At a harvest time, in order to harvest cells whose culture was completed, first, the rotation was stopped for 10 to 30 minutes to settle all cells in the vessel, and then the supernatant was removed as much as possible and washed with PBS at least twice.

[0080] 5) After removing PBS and adding and reacting TrypLE (CTS™ TrypLE™ Select Enzyme, Thermofisher), the microcarriers and the cells were separated by increasing the rotation speed.

[0081] 6) When the separation of the microcarriers and the cells was confirmed, the microcarriers were removed using a cell harvester (Harvestainer™ Microcarrier Separation System, Thermofisher) and the separated cells were collected in a harvesting vessel to finally harvest the cells.

[0082] 7) The finally obtained cells were counted using an automated cell counter (NucleoCounter® NC-202).

[0083] Example 3. Confirmation of stem cell proliferation rate according to microcarrier density

[0084] First, in order to confirm the stem cell proliferation rate according to microcarrier density, the mesenchymal stem cells harvested from Example 1 were cultured in the same manner as Example 2 according to conditions of Table 1. At D3 and D4, 80% of the total volume of the medium was replaced with an equal amount of fresh medium. After the inoculation time, some of the cells were sampled every day to count the cells and the cell proliferation rate was confirmed. The results were shown in FIG. 4.

[0085] [Table 1] Experimental Group Inoculated cell density [cells / mL] Microcarrier density [g / L] Medium replacement condition Bioreactor capacity [L] C1 22,500 20 80% medium replacement at D3, D4 3 C2 22,500 40 80% medium replacement at D3, D4 3 C3 22,500 40 Simultaneous addition of sodium bicarbonate at D0 and 80% medium replacement at D3, D4 3

[0086]

[0087] As a result of confirming the cell proliferation rate according to the density of the microcarrier, when the density of the microcarrier was 20 g / L to 40 g / L, excellent cell proliferation rates were observed at D5. No difference was observed depending on whether sodium bicarbonate was added.

[0088] Example 4. Confirmation of stem cell proliferation rate according to density of inoculated cells

[0089] Next, in order to confirm the stem cell proliferation rate according to a density of inoculated cells, the mesenchymal stem cells harvested from Example 1 were cultured in the same manner as Example 2 according to conditions of Table 2. The medium used was RoosterNourishTM-MSC-XF (Roosterbio, KT-016), and 80% of the total volume of the medium was replaced with an equal amount of fresh medium at D3 and D4. After the inoculation time, some of the cells were sampled every day to count the cells and the cell proliferation rate was confirmed. At D5, the culture was terminated, and the entire cells were harvested and counted. The results were shown in FIG. 5.

[0090] [Table 2] Experimental Group Inoculated cell density [cells / mL] Microcarrier density [g / L] Medium replacement condition Bioreactor capacity [L] C2 45,000 20 80% medium 0.5 replacement at D3, D4 C3 22,500 20 80% medium replacement at D3, D4 0.5

[0091]

[0092] As a result of confirming the cell proliferation rate according to the density of inoculated cells, it was confirmed that when the density of inoculated cells was 22,500 cells / mL, the cell harvest amount was significantly higher than when the density of inoculated cells was 45,000 cells / mL.

[0093] Example 5. Confirmation of stem cell proliferation rate according to medium replacement conditions

[0094] In order to confirm the stem cell proliferation rate according to medium replacement conditions, the mesenchymal stem cells harvested from Example 1 were cultured in the same manner as Example 2 according to conditions of Table 3. The medium used was RoosterNourishTM-MSC-XF (Roosterbio, KT-016). The medium was replaced by setting the P / V value of the bioreactor to 0, stopping the rotation for about 20 minutes, and removing about 80% of the supernatant using a bioreactor pump when the cells were settled, and then injecting an equal amount of fresh medium using the pump. The medium was supplemented by injecting a certain volume of fresh medium into the vessel during culturing using the bioreactor pump. After the inoculation time, some cells were sampled daily and counted, and at D5, the culture was terminated, and the entire cells were obtained and counted. The results were shown in FIG. 6.

[0095] [Table 3] Experimental Group Inoculated cell density [cells / mL] Microcarrier density [g / L] Medium replacement condition Bioreactor capacity [L] C1 45,000 20 80% medium replacement at D3, D4 0.5 C2 22,500 20 80% medium replacement at D3, D4 0.5 C3 22,500 20 Medium supplement at D3 and 80% medium replacement at D4 0.5 C4 22,500 20 80% medium replacement at D3 0.5

[0096]

[0097] As a result of confirming the cell proliferation rate according to the medium replacement condition, when 80% of the medium was replaced at D3 or D3 and D4, an excellent cell proliferation rate was observed compared to the condition where the medium was supplemented. This indicates that the medium replacement condition affects the cell harvest amount, and that replacing 80% of the medium at D3 may achieve excellent effects in terms of production efficiency and cost. Meanwhile, through the experiment, it was confirmed once again that the cell proliferation rate decreased when the cells were inoculated at the level of 45,000 cells / mL.

[0098] Example 6. Confirmation of stem cell proliferation rate according to P / V value change conditions

[0099] In order to confirm the stem cell proliferation rate according to a P / V value change, the stem cells of Example 1 were cultured in the same manner as Example 2 according to conditions of Table 4. The microcarrier density of each experimental group was 20 g / L, the medium used was RoosterNourishTM-MSC-XF (Roosterbio, KT-016), and 80% of the total volume of the medium was replaced with an equal amount of fresh medium at D3, D4 and D5. After the inoculation time, some of the cells were sampled every day to count the cells and the cell proliferation rate was confirmed. The results were shown in FIG. 7.

[00100] [Table 4] Experimental Group Inoculated cell density [cells / mL] P / V value [W / m3] Bioreactor capacity [L] D0-D3 D3-D5 D5-D6 C1 22,500 0.352 0.436 0.906 0.5 C2 11,250 0.123 0.123 0.352 0.5 C3 11,250 0.352 0.436 0.906 0.5

[00101]

[00102] As a result, when the final cell numbers were compared at D6, a case of gradually 21 increasing the P / V values to 0.352, 0.436, and 0.906 (C1 and C3) had a better cell proliferation rate than a case of increasing the P / V value from 0.123 to 0.352 (C2). No difference was confirmed according to an initial inoculation cell density under the P / V value conditions of C1 and C3. This indicates that the effect of significantly increasing the final cell harvest amount during massive culture of MSCs can be achieved by delicately controlling the P / V value of the bioreactor.

[00103]

[00104] From the above description, those skilled in the art to which the present disclosure pertains will understand that the present disclosure may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For the scope of the present disclosure, it should be construed that rather than the above detailed description, all changes or modifications derived from the meaning and scope of the following claims and their equivalents are comprised in the scope of the present disclosure.

Claims

[CLAIMS]

1. A massive culture method of mesenchymal stem cells comprising:a) injecting a medium comprising microcarriers into a bioreactor;b) inoculating the mesenchymal stem cells (MSCs) into the bioreactor of step a);c) replacing the medium 3 days after inoculation; andd) harvesting cells 5 to 6 days after inoculation,wherein a power per unit volume (P / V) value gradually increases from 0.30 to 0.40 W / m3 to 0.85 to 0.95 W / m3 from an inoculation time to a harvest time.

2. The massive culture method of mesenchymal stem cells of claim 1, wherein in step d), the P / V value at the inoculation time to 3 days after inoculation is 0.30 to 0.40 W / m3.

3. The massive culture method of mesenchymal stem cells of claim 1, wherein in step d), the P / V value at 3 days after inoculation to 5 days after inoculation is 0.40 to 0.50 W / m3.

4. The massive culture method of mesenchymal stem cells of claim 1, wherein in step d), the P / V value at 5 days after inoculation to the harvest time is 0.85 to 0.95 W / m3.

5. The massive culture method of mesenchymal stem cells of claim 1, wherein step c) is replacing 60% to 90% of a total volume of the medium in the bioreactor.

6. The massive culture method of mesenchymal stem cells of claim 1, wherein a density of the microcarrier at the inoculation time is 10 g / L to 50 g / L.

7. The massive culture method of mesenchymal stem cells of claim 1, wherein a density of the mesenchymal stem cells at the inoculation time is 8,000 cells / mL to 30,000 cells / mL.

8. The massive culture method of mesenchymal stem cells of claim 1, wherein an average diameter of the microcarrier is 50 pm to 500 pm.

9. The massive culture method of mesenchymal stem cells of claim 1, wherein the mesenchymal stem cells are isolated by a subfractionation culture method comprising:a) culturing a biologically derived tissue in a first vessel to harvest a supernatant;b) transferring the supernatant in the first vessel to a second vessel;c) culturing cells present in the second vessel to harvest a supernatant; andd) repeating steps b) and c) at least once.

10. The massive culture method of mesenchymal stem cells of claim 9, wherein the biologically derived tissue is selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, synovium, bone tissue, nerve, skin, amniotic membrane, placenta, Wharton’s jelly, amniotic fluid, peripheral blood, dental pulp, fallopian tube, corneal stroma, lung, and fetal liver.

11. The massive culture method of mesenchymal stem cells of claim 1, wherein a capacity of the bioreactor is 0.3 to 20 L.

12. Mesenchymal stem cells prepared by the massive culture method of any one of claims 1to 11.

13. A pharmaceutical composition for preventing or treating autoimmune diseases, inflammatory diseases or hair loss, comprising the mesenchymal stem cells of claim 12 as an active ingredient.

14. The pharmaceutical composition of claim 13, wherein the autoimmune or inflammatory disease is at least one selected from the group consisting of IgA nephropathy, ankylosing spondylitis, psoriasis, polyarteritis nodosa, arthritis, ulcerative colitis, Graves’ hyperthyroidism, Guillain-Barre syndrome, multiple sclerosis, multiple myeloma, multiple myositis, lupus, rheumatoid arthritis, chronic thyroiditis, chronic obstructive pulmonary disease, Meniere’s syndrome, leukoplakia, Behcet’s disease, fibromyalgia, Sjogren’s syndrome, silicosis, asbestosis, atopic dermatitis, aphthous stomatitis, Addison’s disease, poststreptococcal glomerulonephritis, inflammatory enterocolitis, Wilson’s disease, graft-versus-host disease, some acquired aplastic anemias, primary cirrhotic autoimmune encephalomyelitis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, endometriosis, relapsing polychondritis, systemic scleroderma, type 1 diabetes, myasthenia gravis, asthma, pemphigus, pancreatitis, temporal arteritis, Crohn’s disease, Fanconi syndrome, sepsis, dermatomyositis, Hashimoto’s thyroiditis, hyperthyroidism, and erythema multiforme.

15. A use of the mesenchymal stem cells of claim 12 for preparation of a drug used for treatment or prevention of autoimmune diseases, inflammatory diseases or hair loss.

16. A method for treating or preventing autoimmune diseases, inflammatory diseases or hairloss, comprising administering a therapeutically effective amount of the mesenchymal stem cells of claim 12 to a subject in need thereof.

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