Rapid layer-separation culturing method for mesenchymal stem cells
Patent Information
- Application Number
- AU2025237538
- Authority / Receiving Office
- AU · AU
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2024-12-06
- Filing Date
- 2025-03-11
- Publication Date
- 2026-10-08
Smart Images
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Abstract
Description
[TECHNICAL FIELD]
[0001] The present disclosure relates to a rapid layer-separation culturing method for mesenchymal stem cells, mesenchymal stem cells isolated by the method, and a pharmaceutical composition comprising the same as an active ingredient. [BACKGROUND ART]
[0002] Mesenchymal stem cells (MSCs) are adult stem cells capable of differentiating into various types of cells and have attracted considerable attention as potential therapeutic agents in the field of regenerative medicine because of their ability to provide growth factors and regulate immune responses.
[0003] MSCs may be isolated from various tissues, such as bone marrow, adipose tissue, and umbilical cord blood, and conventional methods for isolating MSCs have mainly employed a method of recovering a fraction containing mesenchymal stem cells by density-gradient centrifugation (J Cell Physiol. 1998 176:57-66; J Clin Invest. 2003 111:71-79). However, mesenchymal stem cells isolated by density-gradient centrifugation exhibited heterogeneous differences in regenerative capacity and clinical outcomes, and a clinically effective MSC population could not be obtained because of its low abundance. The present inventors developed a layer-separation culturing method to overcome these limitations of conventional MSC isolation methods (Korean Patent Nos. 10-0802011 and 10-1769551). However, the manufacturing process was cumbersome because culture had to be performed while transferring a supernatant several times, and the process was disadvantageous in terms of time and cost for obtaining MSCs in large quantities because it involved obtaining colonies derived from single cells.
[0004] Under these circumstances, the present inventors developed a rapid layer-separation culturing method suitable for the mass production of MSCs and completed the present disclosure by confirming that MSCs isolated by the method exhibit high proliferative and differentiation capacities while showing considerably high homogeneity. [DISCLOSURE] [TECHNICAL PROBLEM]
[0005] An object of the present disclosure is to provide a layer-separation culturing method for mesenchymal stem cells, including: culturing, in a culture vessel for 25 minutes to 3 hours, a mixture in which a biological sample containing mesenchymal stem cells and a medium are mixed with each other at a volume ratio of 1 : 8 to 1 : 25; transferring a supernatant of the culture vessel to a new vessel; and adherently culturing cells present in the new vessel.
[0006] Another object of the present disclosure is to provide mesenchymal stem cells produced by the layer-separation culturing method.
[0007] Yet another object of the present disclosure is to provide medicinal uses of the mesenchymal stem cells, and specifically, to provide a pharmaceutical composition for preventing or treating an autoimmune disease, an inflammatory disease, or hair loss, including the mesenchymal stem cells as an active ingredient; a use of the mesenchymal stem cells for preventing or treating an autoimmune disease, an inflammatory disease, or hair loss; or a method for preventing or treating an autoimmune disease, an inflammatory disease, or hair loss, including administering the mesenchymal stem cells. [TECHNICAL SOLUTION]
[0008] The present disclosure will be described in detail below. Each description and embodiment disclosed in the present disclosure may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present disclosure fall within the scope of the present disclosure. In addition, the scope of the present disclosure should not be construed as being limited by the specific descriptions set forth below.
[0009] The present inventors developed a layer-separation culturing method to overcome the limitations of conventional methods for isolating MSCs by density-gradient centrifugation. However, the conventional layer-separation culturing method had disadvantages in that the manufacturing process was complicated and required considerable time and cost because the supernatant had to be transferred several times and a step of obtaining colonies derived from single cells was included. Accordingly, the present inventors devised a rapid layer-separation culturing method in which a biological sample containing mesenchymal stem cells and a medium are mixed with each other at a specific ratio and cultured for a predetermined period, and a supernatant containing mesenchymal stem cells is then transferred only once to a new culture vessel and subjected to adherent culture. The mesenchymal stem cells isolated using the method maintain high purity and functionality while exhibiting excellent proliferative capacity. Thus, MSCs may be obtained in large quantities by a simple method and may be highly useful for treating various diseases using MSCs.
[0010] Layer-separation culturing method for Mesenchymal Stem Cells
[0011] One aspect of the present disclosure for achieving the above object provides:
[0012] (a) culturing, in a culture vessel for 25 minutes to 3 hours, a mixture in which a biological sample containing mesenchymal stem cells and a medium are mixed with each other at a volume ratio of 1 : 8 to 1 : 25;
[0013] (b) transferring a supernatant of the culture vessel to a new vessel; and
[0014] (c) adherently culturing cells present in the new vessel, thereby providing a layer separation culturing method for mesenchymal stem cells.
[0015] In the present disclosure, the term “layer-separation culturing method” refers to culturing a suspended layer of a suspension containing cells or tissue, unlike a conventional primary culture method in which the suspension containing cells or tissue is directly subjected to adherent culture. In particular, the rapid layer-separation culturing method of the present disclosure is characterized in that the supernatant from the mixture of the biological sample and the medium is passaged only once and subjected to adherent culture.
[0016] In the present disclosure, step (a) is a step of culturing the biological sample containing mesenchymal stem cells, in which the biological sample and the medium are mixed with each other while adjusting the ratio therebetween and cultured for a predetermined period, or the mixture mixed at a specific ratio is cultured while adjusting the culture time thereof.
[0017] In the present disclosure, the term “mesenchymal stem cell” refers to an adult stem cell capable of differentiating into at least one mesodermal cell selected from bone, cartilage, and adipose cells.
[0018] In the present disclosure, the term “biological sample” refers to a sample obtained from a biological source, that is, an organism such as an animal or human subject. The biological sample of an animal or human subject refers to an isolated sample containing cells of the animal or human subject. The biological sample of an animal or human subject may include cells of one or more tissue types. The biological sample may be derived from, for example, one or more tissues selected from the group consisting of bone marrow, adipose tissue, umbilical cord blood, amniotic fluid, Wharton’s jelly, placenta, peripheral blood, skin, dental pulp, fallopian tube, corneal stroma, lung, muscle, and fetal liver, and may specifically be derived from bone marrow or adipose tissue, but is not limited thereto.
[0019] In the present disclosure, a culture medium used to culture a biological sample containing mesenchymal stem cells means a medium for maintaining or culturing a cell population, which contains nutrients that maintain the viability of the mesenchymal stem cells and support proliferation thereof.
[0020] The culture medium used in the present disclosure includes a basal medium. The basal medium may be any basal medium suitable for culturing animal or human cells, particularly mesenchymal stem cells. The basal medium typically contains a number of components required to support the maintenance of cultured cells. An appropriate combination of components may be readily formulated by a person skilled in the art in view of the disclosure below. The basal medium also includes a nutrient solution containing conventional standard cell-culture components, for example, amino acids, vitamins, lipid supplements, inorganic salts, a carbon energy source, and a buffer.
[0021] The basal medium is commercially available and may be, for example, one or more selected from the group consisting of RPMI, DMEM (Dulbecco’s Modified Eagle’s Medium), MEM (Minimum Essential Medium), KO-DMEM (KnockOut-DMEM), G-MEM (Glasgow’s Minimum Essential Medium), BME (Basal Medium Eagle), DMEM / Ham’s F12, Advanced DMEM / Ham’s F12, IMDM (Iscove’s Modified Dulbecco’s Medium), 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 an antibiotic, serum (or a serum substitute), and the like to the basal medium, or may be a commercially available complete medium (e.g., RoosterNourish™-MSC-XF). The type of medium is not particularly limited as long as the medium may maintain and / or proliferate a mesenchymal stem cell population.
[0022] In one specific aspect, the mixture of step (a) may be prepared by mixing a biological sample containing mesenchymal stem cells and a medium at a volume ratio of 1 : 8 to 1 : 25. More specifically, the mixing ratio may be 1 : 12 to 1 : 22, and still more specifically 1 : 13 to 1 : 15, but is not limited thereto.
[0023] In one specific aspect, the mixture of step (a) may be cultured in a culture vessel for 25 minutes to 3 hours. More specifically, the culture time may be 30 minutes to 3 hours, and still more specifically 1 hour to 3 hours, but is not limited thereto.
[0024] In one example, step (a) may include culturing, in a culture vessel for 2 hours, a mixture in which a biological sample containing mesenchymal stem cells and a medium are mixed with each other at a volume ratio of 1 : 14.
[0025] In the present disclosure, step (b) is a step of transferring the supernatant obtained in step (a) to a new vessel, and step (c) is a step of adherently culturing the cells present in the new vessel. Compared with a conventional layer-separation culturing method requiring repeated transfer of the supernatant, the method is simplified and may reduce time and cost and minimize the effects of external stress.
[0026] In one specific aspect, the mesenchymal stem cells of step (c) may be cultured through passages 1 to 10, and their proliferative and differentiation capacities are maintained during the culture period.
[0027] In the present disclosure, the mesenchymal stem cells are positive for CD73, CD90, and CD105 markers, in which specifically 80% or greater, more specifically 90% or greater, and still more specifically 95% or greater thereof are positive for each of the CD73, CD90, and CD105 markers.
[0028] In the present disclosure, the mesenchymal stem cells are negative for CD14, CD34, and CD45 markers, in which specifically less than 3%, more specifically less than 2.5%, and still more specifically less than 2% thereof are positive for each of the CD14, CD34, and CD45 markers.
[0029] In one example, the mesenchymal stem cells may be detected at 95% or greater for the CD73, CD90, and CD105 markers and may be detected at less than 2% for the CD14, CD34, and CD45 markers.
[0030] In the present disclosure, the mesenchymal stem cells may reduce the secretion levels of the inflammatory cytokines IFN-y and IL-17 and increase the secretion level of the antiinflammatory cytokine IL-10.
[0031] In the present disclosure, the term “inflammatory cytokine” refers to a protein that is activated in the immune system and regulates inflammation and immune responses. In particular, the mesenchymal stem cells of the present disclosure may control inflammatory responses by reducing the secretion levels of inflammatory cytokines. The inflammatory cytokine whose secretion level is reduced by the mesenchymal stem cells of the present disclosure may be, for example, one or more selected from the group consisting of TNF-a, IFN-y, IL-1a, IL-10, IL-6, IL-17, IL-20, IL-22, IL-23, CXCL1, and CCL20, and may specifically be IFN-y and IL-17.
[0032] In the present disclosure, the term “anti-inflammatory cytokine” refers to a protein that contributes to suppressing and regulating inflammatory responses. In particular, the mesenchymal stem cells of the present disclosure may suppress inflammation and minimize tissue damage by regulating or stimulating the secretion of anti-inflammatory cytokines. The anti-inflammatory cytokine whose secretion level is increased by the mesenchymal stem cells of the present disclosure may be, for example, one or more selected from the group consisting of TNF-0, IL-4, IL-10, and IL-13.
[0033] In the present disclosure, the term “immunoregulatory factor” refers to an inhibitory factor that suppresses inflammation or an immune response. The mesenchymal stem cells of the present disclosure may increase the expression of one or more immunoregulatory factors selected from the group consisting of the immunoregulatory factors IDO, ICOSL, TSG6, CXCL9, CXCL10, VCAM1, and bFGF.
[0034] Mesenchymal Stem Cells and Uses Thereof
[0035] Another aspect of the present disclosure provides mesenchymal stem cells produced by the layer-separation culturing method.
[0036] Yet another aspect of the present disclosure provides a pharmaceutical composition for preventing or treating an autoimmune disease, an inflammatory disease, or hair loss, including the mesenchymal stem cells as an active ingredient.
[0037] Yet another aspect of the present disclosure provides a method for treating an autoimmune disease, an inflammatory disease, or hair loss, including administering the composition to a subject in need thereof.
[0038] Yet another aspect of the present disclosure provides a pharmaceutical composition for use in preventing or treating an autoimmune disease, an inflammatory disease, or hair loss, or a use of the composition for preventing or treating an autoimmune disease, an inflammatory disease, or hair loss.
[0039] The mesenchymal stem cells are as described above.
[0040] In the present disclosure, the autoimmune or inflammatory disease may include any disease for which the mesenchymal stem cells may exhibit preventive or therapeutic efficacy. Examples thereof may include one or more 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, polymyositis, lupus, rheumatoid arthritis, chronic thyroiditis, chronic obstructive pulmonary disease, Meniere’s syndrome, vitiligo, Behget’s disease, fibrositis, Sjogren’s syndrome, silicosis, asbestosis, atopic dermatitis, aphthous stomatitis, Addison’s disease, poststreptococcal glomerulonephritis, inflammatory bowel disease, Wilson’s disease, graft-versus-host disease, certain acquired aplastic anemias, primary cirrhosis, 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 erythematosis, but are not limited thereto.
[0041] In the present disclosure, the term “treatment” refers to intervention intended to alter the natural course of an individual or cell having a disease, and may be performed during the progression of a pathological condition or to prevent the pathological condition. Desired therapeutic effects include alleviating symptoms of the disease, reducing all direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or temporarily relieving the disease condition, inducing remission, or improving prognosis. In particular, in the present disclosure, the term includes any action that improves the course of an autoimmune disease, an inflammatory disease, or hair loss through administration of the composition including the mesenchymal stem cells as an active ingredient. In addition, the term “prevention” refers to any action that suppresses or delays the onset of a disease through administration of the mesenchymal stem cells. When the mesenchymal stem cells of the present disclosure are used for therapeutic or preventive purposes, they are administered to a subject in a therapeutically effective amount.
[0042] In the present disclosure, the term “therapeutically effective amount” indicates an effective amount of the mesenchymal stem cells. Specifically, the “therapeutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. An effective dose level may be determined according to factors including the type and severity of the subject, age, sex, type of disease, activity of the drug, sensitivity to the drug, administration time, administration route and excretion rate, treatment period, and concurrently used drugs, as well as 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 another therapeutic agent and may be administered sequentially or simultaneously with a commercially available therapeutic agent. In addition, the pharmaceutical composition may be administered in a single or multiple doses. In consideration of all of the above factors, it is important to administer an amount that may achieve a maximum effect with a minimum amount without side effects, and the amount may be readily determined by a person skilled in the art. The dosage 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 combination with a known compound having an effect of preventing, ameliorating, or treating symptoms of an autoimmune disease, an inflammatory disease, or hair loss.
[0043] The mesenchymal stem cells according to the present disclosure or the composition including the same may be administered by intravenous administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, rectal administration, topical administration, intranasal administration, or the like, but is not limited thereto. [ADVANTAGEOUS EFFECTS]
[0044] The layer-separation culturing method of the present disclosure can rapidly proliferate mesenchymal stem cells from a biological sample while maintaining high purity through a simplified manufacturing process and thus can be usefully applied in relevant technical fields, such as the treatment of an autoimmune disease, an inflammatory disease, or hair loss using mesenchymal stem cells. [DESCRIPTION OF DRAWINGS]
[0045] FIG. 1 schematically illustrates a layer-separation culturing process for isolating mesenchymal stem cells from a biological sample.
[0046] FIG. 2 shows morphological changes in bone marrow-derived mesenchymal stem cells according to the volume ratio of bone marrow to medium.
[0047] FIG. 3 shows morphological changes in bone marrow-derived mesenchymal stem cells according to the culture time of a mixture of bone marrow and medium.
[0048] FIG. 4 shows morphological changes in adipose-derived mesenchymal stem cells according to culture vessels.
[0049] FIG. 5 shows the degree of differentiation of bone marrow-derived mesenchymal stem cells into adipocytes according to the volume ratio of bone marrow to medium or the culture time of the mixture.
[0050] FIG. 6 shows the degree of differentiation of bone marrow-derived mesenchymal stem cells into osteocytes according to the volume ratio of bone marrow to medium or the culture time of the mixture.
[0051] FIG. 7 shows the degree of differentiation of bone marrow-derived mesenchymal stem cells into chondrocytes according to the volume ratio of bone marrow to medium or the culture time of the mixture.
[0052] FIG. 8 shows the degree of differentiation of adipose-derived mesenchymal stem cells into adipocytes, osteocytes, and chondrocytes according to culture vessels.
[0053] FIG. 9 is a graph showing the results of a mixed lymphocyte reaction (MLR) assay in which PBMCs and adipose-derived mesenchymal stem cells were co-cultured at ratios of 1 : 4, 1 : 10, and 1 : 20, respectively, to evaluate immunogenicity.
[0054] FIG. 10 is a graph showing the results of measuring secretion levels of inflammatory and anti-inflammatory cytokines by co-culturing PBMCs and adipose-derived mesenchymal stem cells at ratios of 1 : 4, 1 : 10, and 1 : 20, respectively, under PHA stimulation conditions.
[0055] FIG. 11 is a graph showing the results of measuring expression levels of immunoregulatory factors in adipose-derived mesenchymal stem cells isolated from lipoaspirates from two donors. [BEST MODE]
[0056] Hereinafter, the configuration and effects of the present disclosure will be described in more detail through examples. These examples are provided only to illustrate the present disclosure, and the scope of the present disclosure is not limited by the examples.
[0057] The present inventors sought to improve a conventional layer-separation culturing method that required considerable time and cost and involved a cumbersome manufacturing process. Accordingly, the present inventors devised a rapid layer-separation culturing method capable of rapidly producing mesenchymal stem cells in large quantities by culturing, for a predetermined period, a mixture in which a biological sample and a medium are mixed with each other at a specific ratio, transferring only the supernatant to a new culture vessel, and then adherently culturing the cells present in the new vessel. Through a simplified manufacturing process, the rapid layer-separation culturing method may proliferate multipotent mesenchymal stem cells in large quantities while maintaining high purity without external stress.
[0058] As shown in FIG. 1, the conventional method required transferring the supernatant several times and obtaining colonies derived from single cells and thus involved a complicated and cumbersome manufacturing process (D3 to D5). In contrast, in the rapid layer-separation culturing method of the present disclosure (adherent culture at D2), tissue isolated from a living body and a medium are mixed with each other while adjusting the ratio therebetween, the mixture is cultured for a predetermined period, only the supernatant thereof is then transferred to a new vessel, and the cells are adherently cultured. Accordingly, mesenchymal stem cells proliferated in large quantities may be obtained while retaining their original characteristics through a simplified manufacturing process.
[0059] Example 1. Preparation of Bone Marrow-Derived Mesenchymal Stem Cells
[0060] After the hip area of a bone-marrow donor was anesthetized with a local anesthetic, a needle was inserted into the hip bone to collect bone marrow. For each 1 mL of bone marrow, 5 mL to 20 mL of RoosterNourish™-MSC-XF medium (RoosterBio, KT-016) was added, and the resulting mixture was placed in a 100 mm culture vessel and cultured in a 37°C, 5% CO2 incubator for 30 minutes to 18 hours. After the culture, 10 mL to 12 mL of the supernatant was transferred to a new 100 mm culture vessel. After 15 mL of fresh medium was supplemented in the new culture vessel, the cells were cultured in a 37°C, 5% CO2 incubator and observed at intervals of 24 hours. The cells were cultured while replacing the medium once every 3 days until fibroblast-like cells were detected under an optical microscope.
[0061] After 6 to 8 days of culture, when the cells had grown to a confluence of 80% to 90% in the culture vessel, subculture was performed (passage 0). After treatment with a 0.5% TrypLE solution, the cells were plated at 2,000 cells / cm2 and obtained after 4 to 5 days of culture (passage 1).
[0062] Example 2. Preparation of Adipose-Derived Mesenchymal Stem Cells Isolated from Lipoaspirate
[0063] A lipoaspirate was obtained with consent from a healthy female donor.
[0064] Fourteen milliliters of RoosterNourish™-MSC-XF medium (RoosterBio, KT-016) were added to 1 mL of the lipoaspirate, and the resulting mixture was placed in a 100 mm culture vessel and cultured in a 37°C, 5% CO2 incubator for 2 hours. After 2 hours, 10 mL to 12 mL of an upper fatty layer containing adipose tissue was transferred to a new 100 mm culture vessel. After 15 mL of fresh medium was supplemented in the new culture vessel, the cells were cultured in a 37°C, 5% CO2 incubator and observed at intervals of 24 hours. The cells were cultured while replacing the medium once every 3 days until fibroblast-like cells were detected under an optical microscope.
[0065] After 6 to 8 days of culture, when the cells had grown to a confluence of 80% to 90% in the culture vessel, subculture was performed (passage 0). After treatment with a 0.5% TrypLE solution, the cells were plated at 2,000 cells / cm2 and obtained after 4 to 5 days of culture (passage 1).
[0066] Example 3. Evaluation of Morphological Changes in Mesenchymal Stem Cells
[0067] Example 3-1. Bone Marrow-Derived Mesenchymal Stem Cells
[0068] Morphological changes in bone marrow-derived mesenchymal stem cells prepared according to the ratio of the biological sample to the medium and the culture time of the mixture were observed under a microscope, and the results are shown in FIGS. 2 and 3. A typical fibroblast morphology was observed at both passage 1 and passage 2 (FIGS. 2 and 3).
[0069] Example 3-2. Adipose-Derived Mesenchymal Stem Cells
[0070] Morphological changes in adipose-derived stem cells according to subculture were observed under a microscope, and the results are shown in FIG. 4. A mixture of various cells, including adipose cells and red blood cells, was observed at passage 0, whereas a change to a typical stem-cell morphology was observed at passage 2.
[0071] Example 4. Evaluation of Proliferative Capacity of Mesenchymal Stem Cells
[0072] Example 4-1. Bone Marrow-Derived Mesenchymal Stem Cells
[0073] The proliferation rates of bone marrow-derived stem cells prepared according to the ratio of the biological sample to the medium were evaluated by comparing population doubling time (PDT) and population doubling level (PDL), respectively. PDL and PDT were calculated using the following equations.
[0074] PDL = 3.322 x log(number of harvested cells / number of seeded cells)
[0075] PDT = culture period x log2 (log(number of obtained cells / initial number of cells))
[0076] The cells were seeded into a culture vessel at a density of 2,000 cells / cm2, and the number of cells was measured at each subculture. The results are shown in Table 1 below.
[0077] [Table 1]
[0078] Bone marrow : Medium (mL) Passage 0 Passage 1 Passage 2 Culture period No. of cells («105) No. of harvested cells ki Iture period PDL PDT PDUPDT Expected no. of cells, passages 0-1 No. o1 harvested cells Culture period PDL PDT PDUPDT Expected no. of cells, passages 0-2 1:5 9 3.17.E+06 5.72.E+06 4 4.03 0.99 4.06 5.09.E+07 2.56.E+07 5 6.19 0.81 7.67 3.74.E+09 1:10 9 4.04.E+06 1.10.E+07 4 4.98 0.80 6.20 1.28.E+08 3.14.E+07 5 6.49 0.77 8.41 1.15.E+10 1:14 9 4.21.E+06 1.43.E+07 4 5.35 0.75 716 1.71.E+08 3.40.E+07 5 6.60 0.76 8.72 1.66.E+10 1:20 9 5.79. E+06 1.14.E+07 4 5.02 0.80 631 1.89.E+08 3.06.E+07 5 6.45 0.78 832 1.65.E+10
[0079] As shown in the table above, it was confirmed that the amount of bone marrow-derived mesenchymal stem cells obtained using the mixtures in which the biological sample and the medium were mixed at volume ratios of 1 : 10 to 1 : 20 was at least approximately three times the amount obtained when the biological sample and the medium were mixed at a volume ratio of 1 : 5.
[0080] In the same manner as described above, the proliferation rates of the mesenchymal stem cells prepared according to the culture time of the mixture were measured, and the results are shown in Table 2 below.
[0081] [Table 2]
[0082] Mixture culture time Passage 0 Passage 1 Passage 2 Culture period No. of cells (x10s) No. of harvested cells 'n Iture period PDL PDT PDL / PDT Expected no. of cells, passages o-l No. of harvested cells Culture period PDL PDT PDL / PDT Expected no. of cells, passages 0-2 30 min 9 5.26.E+06 8.25.E+06 4 4.53 0.89 5.16 1.25.E+08 2.98.E+07 5 6.41 0.78 8.22 1.08.E+10 2 hr 9 4.21.E+06 1.43.E+07 4 5.35 0.75 7.16 1.71.E+08 3.40.E+07 5 6.60 0.76 8.72 1.66. E+10 6 hr 9 1.94.E+06 8.49.E+06 4 4.60 0.87 5.29 4.69. E+07 2.97.E+07 5 6.41 0.78 8.21 3.98.E+09 18 hr 9 1.97.E+06 1.44.E+07 4 5.36 0.75 7.17 8.01.E+07 1.56 E+07 5 5.48 0.91 6.01 3.58.E+09
[0083] As shown in Table 2, when the mixture of the biological sample and the medium was cultured for 30 minutes to 2 hours, approximately three times as many mesenchymal stem cells could be obtained as when the mixture was cultured for 6 hours or 18 hours.
[0084] Example 4-2. Adipose-Derived Mesenchymal Stem Cells
[0085] In the same manner as described above, the cells were seeded into a culture vessel at a density of 2,000 cells / cm2, and the number of cells was measured at each subculture to compare PDT and PDL, respectively. The results are shown in Table 3 below.
[0086] [Table 3] Condition Passage 0 Passage 1 Passage 2 Culture period No. of cells (-101) Density cells, cm1. 175T (ea) No. of harvested cells Curtate period Fold increase POL PDT Expected no. of ceas. passages 0-1 175T (ea) No. at harvested cells Cufluir period Fold increase PDL PDT Expected no. of ceils, passages 0-2 Corning #1 8 2.96.E+06 2000 8 9.70.E+06 4 64.67 6.02 0.67 1.91 E+08 547 417.E+06 5 27.80 4.80 1.04 5.32.E + 09 Corning #2 8 3.04.E+06 2000 9 9.05.E+06 4 60.33 5.92 0.68 1.83.E+08 524 3.87.E+06 5 25.80 469 1 07 4.73.E+09 NEST #1 8 2.62.E+06 2000 7 9.38.E+06 4 62.50 5.97 067 1.64.E+08 468 3.93.E + 06 5 26.20 471 1 06 4.29.E + 09 NEST #2 8 2.18.E+06 2000 6 1.06.E+07 4 70.33 6.14 0.65 1.53.E+08 438 4 11.E+06 5 27.40 4.78 1.05 4.20.E+09 As shown in the table above, no significant difference according to the culture vessel was
[0087]
[0088] identified. The average PDL value was measured as 6.00 at passage 1 and 4.70 at passage 2. The average PDT values were 0.67 (passage 1) and 1.05 (passage 2), confirming that a large number of cells could be obtained within a short period.
[0089] Example 4-3. Evaluation of Sizes of Bone Marrow- and Adipose-Derived Mesenchymal Stem Cells
[0090] In addition, the diameters of bone marrow- and adipose-derived mesenchymal stem cells were measured. For the bone marrow-derived mesenchymal stem cells, the diameters of the mesenchymal stem cells obtained according to the ratio of the biological sample to the medium and the culture time of the mixture were compared. The results are shown in Table 4 below.
[0091] [Table 4]
[0092] Condition Cell diameter (pm) Passage 0 Passage 1 Passage 2 Bone marrow : Medium volume ratio (culture time: 2 hr) 1:5 mL 14,4 14.7 14.8 1:10 mL 14,6 14.6 14.6 1:14 mL 14.2 14.1 14.4 1:20 mL 14 6 14.5 14.3 Mixture culture time (bone marrow : medium volume ratio 1:14 mL) 30 min 14.3 14.6 14.4 6 hr 14.5 14.3 14.3 18 hr 15.5 15.6 16.1 DISH (adipose-derived) Corning #1 15.4 14.9 16.2 Corning #2 15.5 14.9 16.1 NEST #1 15.2 14.8 16.0 NEST #2 15.5 14.7 16.3
[0093] Example 5. Analysis of Surface Antigen Expression in Mesenchymal Stem Cells
[0094] Example 5-1. Bone Marrow-Derived Mesenchymal Stem Cells
[0095] Fluorescence-activated cell sorting (FACS) was used to evaluate the expression characteristics of surface marker antigens in bone marrow-derived stem cells prepared according to the ratio of the biological sample to the medium and the culture time of the mixture.
[0096] Microtubes were prepared, and 1 x 105 to 2 x 105 cells were dispensed into each tube. After centrifugation at 500 g and 4°C for 5 minutes, the supernatant was discarded. One hundred microliters of FACS buffer were added to each microtube to suspend the cells, and 1 pL each of a CD14 antibody (BD), a CD34 antibody (BD), a CD45 antibody (BD), a CD73 antibody (BD), a CD90 antibody (BD), and a CD105 antibody (Bio-Rad) was added. The cells were stained at 4°C for 30 minutes to 1 hour under light-blocking conditions and analyzed using a FACSVerse instrument (BD Biosciences).
[0097] The results are shown in Tables 5 and 6 below. The mesenchymal stem cells prepared according to the volume ratio of the biological sample to the medium exhibited positive immunological characteristics of 99% or greater for all of CD73, CD90, and CD105, which are mesenchymal stem cell surface marker antigens. Likewise, when the mixture was cultured for 30 minutes or 2 hours, the mesenchymal stem cells exhibited positive immunological characteristics of 99% or greater. However, when the mixture was cultured for a prolonged period of 6 hours or more, the homogeneity of the mesenchymal stem cells was found to decrease.
[0098] The cells exhibited negative immunological characteristics of less than 2% for all of CD14, a monocyte surface marker antigen, and CD34 and CD45, hematopoietic stem cell surface marker antigens.
[0099] [Table 5]
[00100] Bone marrow : Medium volume ratio (mL) Purity markers (%) Identification markers (%) CD14 CD34 CD45 CD73 CD90 CD105 1:5 1.16 0.32 0.36 99.90 99.80 99.80 1:10 1.93 0.31 0.35 99.00 99.75 99.90 1:14 1.40 0.44 0.32 99.05 99.95 99.80 1:20 1.30 0.43 0.41 99.50 99.85 99.80
[00101] [Table 6]
[00102] Mixture culture time Purity markers (%) Identification markers (%} CD14 CD34 CD45 CD73 CD90 CD105 30 min 1.91 0.31 0.27 99.00 99.75 99.80 2 hr 1.40 0.44 0.32 99.05 99.95 99.80 6 hr 1.32 0.28 0.25 96.60 99.90 99.70 18 hr 1.11 0.35 0.32 97.50 99.85 99.90
[00103] Example 5-2. Adipose-Derived Mesenchymal Stem Cells
[00104] Microtubes were prepared, and 1 x 105 to 2 x 105 cells were dispensed into each tube. After centrifugation at 500 g and 4°C for 5 minutes, the supernatant was discarded. One hundred microliters of FACS buffer were added to each microtube to suspend the cells, and 1 pL each of a CD14 antibody (BD), a CD34 antibody (BD), a CD45 antibody (BD), a CD73 antibody (BD), a CD90 antibody (BD), and a CD105 antibody (Bio-Rad) was added. The cells were stained at 4°C for 30 minutes to 1 hour under light-blocking conditions and analyzed using a BD FACSVerse instrument.
[00105] As a result, the cells exhibited positive immunological characteristics for all of CD73, CD90, and CD105, which are mesenchymal stem cell surface marker antigens, and negative immunological characteristics for all of CD14, a monocyte surface marker antigen, and CD34 and CD45, hematopoietic stem cell surface marker antigens. No significant variation according to the culture vessel was observed. The results are shown in Table 7 below.
[00106] [Table 7]
[00107] Marker expression (%) Corning NEST #1 #2 #1 #2 Passage 0 Passage 1 Passage 2 Passage 0 Passage 1 Passage 2 Passage 0 Passage 1 Passage 2 Passage 0 Passage 1 Passage 2 Purity markers CD14 1.28 1.25 0.20 0.85 2.25 0.61 2.24 0.44 0.51 2.40 1.17 0.44 CD34 1.04 0.79 0.53 0.54 1.11 0.76 0.66 0.96 1.03 1.40 0.94 0.95 CD45 0.56 0.30 0.21 0.34 1.09 0.37 0.38 0.59 0.64 0.54 0.62 0.49 Identification markers CD73 99.60 99.50 97.70 99.40 99.70 98.30 99.80 99.90 99.40 99.50 99 90 99.30 CD90 99.70 99.90 100.00 99.10 99.80 99.90 99.60 100.00 100.00 99.70 99.90 99.80 CD105 99.70 96.00 98.30 99.70 99.60 99.60 99.60 99.40 99.50 99.70 99.40 99.80
[00108] Example 6. Evaluation of Differentiation Capacity of Mesenchymal Stem Cells
[00109] Example 6-1. Bone Marrow-Derived Mesenchymal Stem Cells
[00110] To evaluate the differentiation capacity of bone marrow-derived mesenchymal stem cells prepared according to the ratio of the biological sample to the medium and the culture time of the mixture, differentiation into adipocytes, osteocytes, and chondrocytes was induced, and the differentiation capacity was evaluated as follows. The results are shown in FIGS. 5 to 7.
[00111] 1) Capacity for Differentiation into Adipocytes
[00112] To evaluate the capacity of the bone marrow-derived stem cells to differentiate into adipocytes, 5 x 104 cells were seeded in a 4-well plate and cultured in a 37°C, 5% CO2 incubator for 1 to 2 days. Differentiation was initiated when cell confluence reached 90%. A differentiation medium (PromoCell, Cat. No. C-28016) was replaced once every 3 to 4 days, and differentiation was performed for 7 to 14 days until lipid droplets were visually observed. After completion of differentiation, whether differentiation had occurred was evaluated by Oil Red O staining.
[00113] As a result, as shown in FIG. 5, neutral lipid accumulation was observed after adipocyte differentiation was induced in all of the mesenchymal stem cells prepared according to the ratio of the biological sample to the medium and the culture time of the mixture.
[00114] 2) Capacity for Differentiation into Osteocytes
[00115] To evaluate the capacity of the bone marrow-derived stem cells to differentiate into osteocytes, 5 x 104 cells were seeded in a 4-well plate and cultured in a 37°C, 5% CO2 incubator for 1 to 2 days. Differentiation was initiated when cell confluence reached 90%. A differentiation medium (PromoCell, Cat. No. C-28013) was replaced once every 3 to 4 days, and differentiation was performed for 10 to 14 days. After completion of differentiation, whether differentiation had occurred was evaluated by Alizarin Red S staining.
[00116] As a result, as shown in FIG. 6, extracellular calcium was observed after osteocyte differentiation was induced in all of the mesenchymal stem cells prepared according to the ratio of the biological sample to the medium and the culture time of the mixture.
[00117] 3) Capacity for Differentiation into Chondrocytes
[00118] To evaluate the capacity of the bone marrow-derived stem cells to differentiate into chondrocytes, 2 x 105 cells were prepared in a 15 mL tube and centrifuged at 500 g and 4°C for 5 minutes. After the cell pellet was identified, the cells were cultured in a 37°C, 5% CO2 incubator while the tube cap was left slightly open. After one day of culture in the CO2 incubator, when the pellet that had settled at the bottom of the 15 mL tube formed a spheroid, the medium was replaced with a differentiation medium (PromoCell, Cat. No. C-28012). The differentiation medium was replaced once every 3 to 4 days, and differentiation was performed for 21 days. After completion of differentiation, the pellet was sectioned to a thickness of 8 um using a cryotome, and whether differentiation had occurred was evaluated by Safranin O staining.
[00119] As a result, as shown in FIG. 7, stained aggrecan was observed after chondrocyte differentiation was induced in all of the mesenchymal stem cells prepared according to the ratio of the biological sample to the medium and the culture time of the mixture.
[00120] Example 6-2. Adipose-Derived Mesenchymal Stem Cells
[00121] To evaluate the differentiation capacity of adipose-derived mesenchymal stem cells at passages 1 and 2, differentiation into adipocytes, osteocytes, and chondrocytes was induced, and the differentiation capacity was evaluated as follows. The results are shown in FIG. 8.
[00122] 1) Capacity for Differentiation into Adipocytes
[00123] To evaluate the capacity of the adipose-derived stem cells to differentiate into adipocytes, 5 x 104 cells were seeded in a 4-well plate and cultured in a 37°C, 5% CO2 incubator for 1 to 2 days. Differentiation was initiated when cell confluence reached 90%. A differentiation medium (PromoCell, Cat. No. C-28016) was replaced once every 3 to 4 days, and differentiation was performed for 7 to 14 days until lipid droplets were visually observed. After completion of differentiation, a red color indicating neutral lipid accumulation was observed by Oil Red O staining, demonstrating that the adipose-derived mesenchymal stem cells of the present disclosure may differentiate into adipocytes.
[00124] 2) Capacity for Differentiation into Osteocytes
[00125] To evaluate the capacity of the adipose-derived stem cells to differentiate into osteocytes, 5 x 104 cells were seeded in a 4-well plate and cultured in a 37°C, 5% CO2 incubator for 1 to 2 days. Differentiation was initiated when cell confluence reached 90%. A differentiation medium (PromoCell, Cat. No. C-28013) was replaced once every 3 to 4 days, and differentiation was performed for 10 to 14 days. After completion of differentiation, extracellular calcium was observed by Alizarin Red S staining, demonstrating that the adipose-derived mesenchymal stem cells of the present disclosure may differentiate into osteocytes.
[00126] 3) Capacity for Differentiation into Chondrocytes
[00127] To evaluate the capacity of the adipose-derived stem cells to differentiate into chondrocytes, 2 x 105 cells were prepared in a 15 mL tube and centrifuged at 500 g and 4°C for 5 minutes. After the cell pellet was identified, the cells were cultured in a 37°C, 5% CO2 incubator while the tube cap was left slightly open. After one day of culture in the CO2 incubator, when the pellet that had settled at the bottom of the 15 mL tube formed a spheroid, the medium was replaced with a differentiation medium (PromoCell, Cat. No. C-28012). The differentiation medium was replaced once every 3 to 4 days, and differentiation was performed for 21 days. After completion of differentiation, the pellet was sectioned to a thickness of 8 pm using a cryotome, and stained aggrecan was observed by Safranin O staining, demonstrating that the adipose-derived mesenchymal stem cells of the present disclosure may differentiate into chondrocytes.
[00128] Example 7. Analysis of Immunoregulatory Capacity of Adipose-Derived Mesenchymal Stem Cells
[00129] Example 7-1. Mixed Lymphocyte Reaction (MLR) Assay
[00130] To evaluate the immunoregulatory capacity of adipose-derived mesenchymal stem cells through a mixed lymphocyte reaction (MLR) assay, peripheral blood mononuclear cells (PBMCs) from two different donors were mixed to induce T-cell activation by an antigen (allogeneic MLR), and immunogenicity was then measured through co-culture with the mesenchymal stem cells.
[00131] Human PBMCs and adipose-derived mesenchymal stem cells stained with CFSE and eFluor670, respectively, were co-cultured at ratios of 1 : 4, 1 : 10, and 1 : 20, respectively, and cultured for 8 days. The cells were measured by flow cytometry using a FACSVerse instrument (BD Biosciences), and the results are shown in FIG. 9.
[00132] It was confirmed that the adipose-derived mesenchymal stem cells of the present disclosure inhibited T-cell proliferation by 50% or greater under all conditions and exhibited the greatest effect when the PBMCs and the adipose-derived mesenchymal stem cells were co-cultured at a ratio of 1 : 4.
[00133] Example 7-2. Analysis of Cytokine Secretion Levels
[00134] To evaluate the immunosuppressive capacity of the adipose-derived mesenchymal stem cells, T cells in PBMCs were activated under stimulation with PHA (phytohemagglutinin), an immunoregulatory active substance, and the PBMCs and the mesenchymal stem cells were then co-cultured at ratios of 1 : 4, 1 : 10, and 1 : 20, respectively, in a 24-well plate for 3 days. Thereafter, the co-culture supernatant was obtained, and the secretion levels of the inflammatory cytokines IFN-Y and IL-17 and the anti-inflammatory cytokine IL-10 were measured using IFN-y, IL-10, and IL-17 ELISA kits (RnD Systems).
[00135] As a result, as shown in FIG. 10, the secretion levels of IFN-Y and IL-17 increased upon PHA stimulation, and the secretion levels of both IFN-Y and IL-17 decreased upon treatment with the adipose-derived mesenchymal stem cells. In particular, IFN-Y decreased by 50% or greater, and the greatest effect was observed when co-culture was performed at a ratio of 1:4. The secretion level of the anti-inflammatory cytokine IL-10 was found to increase upon treatment with the mesenchymal stem cells.
[00136] Example 7-3. Analysis of Immunoregulatory Factor Expression
[00137] Adipose-derived mesenchymal stem cells prepared from lipoaspirates obtained from two donors were cultured, and total RNA was then isolated using TRIzol (Invitrogen). cDNA was synthesized from the total RNA using a PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa), and qRT-PCR was performed. Changes in the expression of IDO, ICOSL, and TSG6, which are representative immunoregulatory factors, were evaluated, and changes in the expression of CXCL9, CXCL10, VCAM1, and bFGF were additionally evaluated. The results are shown as graphs in FIG. 11.
[00138] As a result, under the TI (TNFa + IFNy) condition, high expression of the inflammatory and immunoregulatory factors was observed relative to the control group.
[00139] From the foregoing description, a person 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 features thereof. In this regard, the examples described above should be understood as illustrative in all respects and not restrictive. The scope of the present disclosure should be construed to include all changes or modified forms derived from the meaning and scope of the claims described below and equivalents thereof, rather than from the foregoing detailed description.
Claims
[CLAIMS]
1. A layer-separation culturing method for mesenchymal stem cells, comprising:(a) culturing, in a culture vessel for 25 minutes to 3 hours, a mixture in which a biological sample containing mesenchymal stem cells and a medium are mixed with each other at a volume ratio of 1 : 8 to 1 : 25;(b) transferring a supernatant of the culture vessel to a new vessel; and(c) adherently culturing cells present in the supernatant in the new vessel.
2. The layer-separation culturing method of claim 1, wherein the biological sample containing the mesenchymal stem cells is derived from a tissue selected from the group consisting of bone marrow, adipose tissue, umbilical cord blood, amniotic fluid, Wharton’s jelly, placenta, peripheral blood, skin, dental pulp, fallopian tube, corneal stroma, lung, muscle, and fetal liver.
3. The layer-separation culturing method of claim 1, wherein the mixture in step (a) is prepared by mixing the biological sample containing the mesenchymal stem cells and the medium with each other at a volume ratio of 1 : 12 to 1 : 22.
4. The layer-separation culturing method of claim 1, wherein step (a) comprises culturing the mixture in the culture vessel for 1 hour to 3 hours.
5. The layer-separation culturing method of claim 1, wherein the cells in step (c) are cultured through passages 1 to 10.
6. The layer-separation culturing method of claim 1, wherein the adherently cultured mesenchymal stem cells are positive for CD73, CD90, and CD105 markers; andthe adherently cultured mesenchymal stem cells are negative for CD14, CD34, and CD45 markers.
7. The layer-separation culturing method of claim 6, wherein 80% or greater of the mesenchymal stem cells are positive for each of the CD73, CD90, and CD105 markers.
8. The layer-separation culturing method of claim 6, wherein less than 3% of the mesenchymal stem cells are positive for each of the CD14, CD34, and CD45 markers.
9. Mesenchymal stem cells produced by the layer-separation culturing method of any one of claims 1 to 8.
10. A pharmaceutical composition for preventing or treating an autoimmune disease, an inflammatory disease, or hair loss, the composition comprising the mesenchymal stem cells of claim 9 as an active ingredient.
11. The pharmaceutical composition of claim 10, wherein the autoimmune or inflammatory disease is one or more 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, polymyositis, lupus, rheumatoid arthritis, chronic thyroiditis, chronic obstructive pulmonary disease, Meniere’s syndrome, vitiligo, Behget’s disease, fibrositis, Sjogren’s syndrome, silicosis, asbestosis, atopic dermatitis, aphthousstomatitis, Addison’s disease, poststreptococcal glomerulonephritis, inflammatory bowel disease, Wilson’s disease, graft-versus-host disease, certain acquired aplastic anemias, primary cirrhosis, 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 erythematosis.
12. Use of the mesenchymal stem cells of claim 9 in the manufacture of a medicament for use in treating or preventing an autoimmune disease, an inflammatory disease, or hair loss.
13. A method for treating or preventing an autoimmune disease, an inflammatory disease, or hair loss, the method comprising administering a therapeutically effective amount of the mesenchymal stem cells of claim 9 to a subject in need thereof.