Purified concentrate of culture supernatant of mesenchymal stem cells or precursor cells derived therefrom, and method for producing the same.

Abstract

To provide: a purified concentrate of a culture supernatant, which has high effectiveness and high safety; and a method for producing the purified concentrate.SOLUTION: The present invention discloses a purified concentrate of a culture supernatant of a mesenchymal stem cell or a progenitor cell derived therefrom. The purified concentrate contains at least one component selected from IGFBP, HGF, VEGF, PDGF, EGF, KGF(FGF-7), PDGFR, TGFα and TGFβ secreted from a mesenchymal stem cell or a progenitor cell derived therefrom. From the purified concentrate, lactic acid and ammonia have been removed, which are metabolic waste products from the mesenchymal stem cell or the progenitor cell derived therefrom.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a purified concentrate of the culture supernatant of mesenchymal stem cells or precursor cells derived therefrom, which is useful in regenerative medicine. The invention further relates to a method for producing the above-mentioned purified concentrate. [Background technology]

[0002] Cells, both in vivo and in culture environments, are known to release physiologically active substances that help maintain the normal function of themselves and surrounding cells. In particular, somatic stem cells and progenitor cells release cell-specific physiologically active substances (cytokines, growth factors, angiogenic factors, exosomes, enzymes, extracellular matrix, etc.) that play an important role in organ regeneration and homeostasis maintenance.

[0003] Stem cells exist in various locations throughout the body, including bone marrow, fat, and skin. Stem cells present in subcutaneous adipose tissue (adipose-derived stromal / stem cells, ASCs) have been reported to have properties almost identical to those of bone marrow-derived stem cells. Compared to bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells have several advantages: they are present in larger quantities per tissue, simple methods for isolation, proliferation, and preservation from tissue have been established, they produce larger amounts of various factors (such as hepatocyte growth factor and vascular endothelial growth factor), they have high immunosuppressive capacity, and they are less burdensome for patients during collection and have less impact on the patient's long-term health and lifespan after collection.

[0004] Regarding conventional stem cell therapy, it has been noted that the effects of humoral factors secreted by stem cells are more significant than the engraftment, differentiation, and function of the cells themselves. Stem cell culture supernatants are beginning to be used as injectables, topical preparations, and cosmetics for purposes such as tissue repair and cosmetic applications.

[0005] Patent Document 1 describes a method for producing a lens hardening inhibitor or therapeutic agent, which includes the steps of contacting mesenchymal stem cells with the inner surface of a hollow fiber membrane, perfusing the lumen and lumen of the hollow fiber membrane with cell culture medium to culture the mesenchymal stem cells, and recovering the cell culture medium that has been perfusing the lumen of the hollow fiber membrane.

[0006] Patent Document 2 describes a cell culture apparatus comprising: a culture vessel for containing a cell suspension containing cells; a first filter section having a first filter membrane for performing a membrane separation treatment on the cell suspension withdrawn from the culture vessel; a first circulation channel for returning components blocked by the first filter membrane to the culture vessel; a second filter section having a second filter membrane for performing a membrane separation treatment on components of the cell suspension that have permeated through the first filter membrane; a second circulation channel for returning components that have permeated through the second filter membrane to the culture vessel; and a recovery channel for recovering components blocked by the second filter membrane. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2019-172607 [Patent Document 2] International Publication No. WO2018 / 159847 [Overview of the project] [Problems that the invention aims to solve]

[0008] Currently, the culture supernatant used clinically is either the stock solution or a diluted solution obtained by collecting the supernatant from the incubator after cell culture and removing any stem cells mixed in the supernatant by centrifugation or filtration as needed. Such culture supernatants have low concentrations of physiologically active substances such as hepatocellular growth factor (HGF), insulin-like growth factors (IGFs), and vascular endothelial growth factor (VEGF), and also contain metabolic waste products released by cells (lactic acid, ammonia, etc.), thus not exhibiting their maximum effectiveness. If culture supernatant containing metabolic waste products is used directly in treatment, it may harm tissues and cells. The problem to be solved by this invention is to provide a purified concentrate of culture supernatant that has high efficacy and high safety, and a method for producing the same. [Means for solving the problem]

[0009] The inventors diligently conducted research to solve the above problems. First, they found that the amount of physiologically active substances produced in the culture supernatant of mesenchymal stem cells or their precursor cells is affected by oxygen concentration, environmental stress, cell density (confluence) during culture, culture period, culture medium composition, gravity, and atmospheric pressure. Focusing on these culture conditions, they established an optimal culture protocol to obtain a highly functional culture supernatant stock solution. On the other hand, they confirmed that harmful substances contained in the culture supernatant can be removed by a purification process using an appropriate protocol. They demonstrated that this purification process can achieve both the removal of harmful substances and the concentration of useful physiologically active substances. The present invention was completed based on these findings.

[0010] In other words, the present invention provides the following invention. <1> A purified concentrate of a culture supernatant of mesenchymal stem cells or progenitor cells derived therefrom, which contains at least one or more of IGFBP, HGF, VEGF, PDGF, EGF, KGF (FGF-7), PDGFR, TGFα, and TGFβ secreted by the mesenchymal stem cells or progenitor cells derived therefrom, and from which lactic acid and ammonia, which are metabolic waste products by the mesenchymal stem cells or progenitor cells derived therefrom, have been removed. <2> The purified concentrate according to <1>, for use as a therapeutic agent for regenerative medicine, an immunosuppressant, an anti-inflammatory agent, or an anti-fibrotic agent. <3> The purified concentrate according to <1> or <2>, which contains all of IGFBP, HGF, VEGF, PDGF, EGF, KGF (FGF-7), PDGFR, TGFα, and TGFβ secreted by mesenchymal stem cells or progenitor cells derived therefrom. <4> The purified concentrate according to any one of <1> to <3>, in which the concentration of at least one or more of IGFBP, HGF, VEGF, PDGF, EGF, KGF (FGF-7), PDGFR, TGFα, and TGFβ is concentrated 1.2 times or more as compared with the culture supernatant before concentration. <5> The purified concentrate according to any one of <1> to <4>, in which the mesenchymal stem cells or progenitor cells derived therefrom are adipose-derived mesenchymal stem cells or adipose-derived vascular endothelial progenitor cells. <6> The purified concentrate according to any one of <1> to <5>, in which the ammonia concentration is 20 μg / dL or less and the HGF concentration is 50000 pg / mL or more. <7> A culture supernatant acquisition step of obtaining a culture supernatant by culturing mesenchymal stem cells or progenitor cells derived therefrom in a culture solution; a concentration step of concentrating useful components from the culture supernatant A purification step of removing metabolic waste products from the concentrated culture supernatant; A method for producing the purified concentrate according to any one of <1> to <6>, which includes these steps. <8> The method according to <7>, in which in the culture supernatant acquisition step, the culture supernatant is obtained from a culture of mesenchymal stem cells or progenitor cells derived therefrom in a proliferative phase or a confluent phase state. <9> The method according to <7> or <8>, in which the metabolic waste products removed in the purification step are lactic acid and ammonia. <10> The method according to any one of <7> to <9>, wherein the purification process is performed by ultrafiltration. <11> The method according to <10>, wherein the ultrafiltration is performed using a filtration membrane with a molecular weight cut-off of 2 kDa to 30 kDa.

Advantages of the Invention

[0011] According to the method of the present invention, the growth ability of cells cultured in vitro can be increased by about 2 times compared to the culture supernatant recovered by the conventional recovery method. That is, according to the present invention, a culture supernatant that is safe and has a high regenerative therapy effect can be supplied. The purified concentrate of the culture supernatant of the present invention can be mass-produced and does not contain cells, so it can also be used as a product derived from cells of other sources.

Brief Description of the Drawings

[0012] [Figure 1] [[ID=1⑧]]Figure 1 shows the effect of inhibitors on ASC cell proliferation activity (Mean±SD, n=4, WST8 assay). [Figure 2] Figure 2 shows the effect of lactate elimination from CM using an ultrafiltration membrane (Mean±SD, n=3). [Figure 3] Figure 3 shows the results of examining oxygen culture conditions for accelerating cell growth speed. [Figure 4] Figure 4 shows the ASC seeding density and the amount of HGF secreted into the culture supernatant (n=1). [Figure 5] Figure 5 shows the confluency of ASC and the amount of HGF. [Figure 6] Figure 6 shows the types of growth factors secreted into CM. [Figure 7] Figure 7 shows the culture supernatant recovery and medium exchange schedule in Example 5. ​​​​​​ [Figure 10] Figure 10 shows the results of measuring ammonia concentrations in the culture supernatant, concentrate, and waste liquid after concentration. [Figure 11] Figure 11 shows the results of measuring the effect of culture supernatant, concentrate, and waste liquid after concentration on the proliferation ability of hASCs (cell proliferation experiment). [Figure 12] Figure 12 shows the results of measuring the effects of culture supernatant and purified concentrate on the proliferation of human dermal fibroblasts and normal human epidermal keratinocytes (cell proliferation experiment). [Figure 13] Figure 13 shows the results of measuring the effect of culture supernatant and purified culture supernatant concentrate on the ability of vascular endothelial cells to form lumen (endothelial cell lumen formation test). [Figure 14] Figure 14 shows the results of evaluating the wound healing-promoting effect in type 2 diabetic mice. [Figure 15] Figure 15 shows the results of measuring the effects of platelet lysis on cell proliferation and HGF production in ASC. [Figure 16] Figure 16 shows the results of measuring the effect of various growth factors on HGF production capacity in ASC. [Figure 17] Figure 17 shows the results of measuring the effects of culture supernatant and purified concentrate on inflammatory cytokine production. [Modes for carrying out the invention]

[0013] The embodiments of the present invention will be described in detail below. [1] Purified concentrate of the culture supernatant of mesenchymal stem cells or their progenitor cells This invention relates to a purified concentrate obtained by recovering, purifying, and concentrating the culture supernatant of mesenchymal stem cells or precursor cells derived therefrom that have been cultured in vitro. The culture supernatant of mesenchymal stem cells or precursor cells derived therefrom contains physiologically active substances such as growth factors, cytokines, exosomes, angiogenic factors, and enzymes secreted from the cells. These physiologically active substances play an important role in restoring the function of cells damaged by injury and aging, and in the regeneration of tissues and organs, and can be used as regenerative medicine to treat various intractable diseases.

[0014] Specifically, the present invention relates to a purified concentrate of the culture supernatant of mesenchymal stem cells or progenitor cells derived therefrom, which contains at least one of the following secreted by mesenchymal stem cells or progenitor cells: IGFBP (IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, etc.), HGF, VEGF (VEGF-A, etc.), PDGF (PDGF-AA, PDGF-AB, PDGF-BB, etc.), EGF, KGF (FGF-7), PDGFR (PDGFR-α, PDGFR-β, etc.), TGFα, and TGFβ, and from which lactic acid and ammonia, which are metabolic waste products of mesenchymal stem cells or progenitor cells derived therefrom, have been removed.

[0015] The purified concentrate of the present invention may contain at least one of IGFBP, HGF, VEGF, PDGF, EGF, KGF (FGF-7), PDGFR, TGFα, and TGFβ, but preferably contains at least two, and more preferably contains all of the above.

[0016] In the purified concentrate of the present invention, it is preferable that the concentration of at least one (preferably at least two, and more preferably all of the above) of IGFBP, HGF, VEGF, PDGF, EGF, KGF (FGF-7), PDGFR, TGFα, and TGFβ is concentrated to 1.2 times or more compared to the culture supernatant before concentration, and the concentration ratio may be 1.5 times or more, 2 times or more, 3 times or more, 5 times or more, 10 times or more, 15 times or more, 20 times or more, 25 times or more, or 30 times or more.

[0017] In the purified concentrate of the present invention, the ammonia concentration is preferably 20 μg / dL or less, more preferably 15 μg / dL or less, and even more preferably 10 μg / dL or less. The HGF concentration is preferably 50,000 pg / mL or more, and preferably 50,000 pg / mL to 2,500,000 pg / mL. The HGF concentration may also be 100,000 pg / mL or more, or 200,000 pg / mL or more.

[0018] Examples of mesenchymal stem cells or their derived progenitor cells include adipose-derived stem cells, bone marrow-derived stem cells, umbilical cord blood-derived stem cells, or progenitor cells derived therefrom. Examples of adipose-derived stem cells include vascular endothelial (progenitor) cells.

[0019] Adipose-derived stem cells are multipotent cells derived from fat that can differentiate into adipocytes, osteoblasts, chondrocytes, myofibroblasts, osteocytes, muscle cells, or nerve sheath cells. The proportion of adipose-derived stem cells can be identified as CD31-negative and CD90-positive cells. Furthermore, adipose-derived stem cells can be identified as CD45-negative, CD44-positive, CD29-positive, and CD13-positive. These markers can be measured by FACS (fluorescence-activated cell sorting).

[0020] Endothelial (progenitor) cells are cells that make up the inner surface of blood vessels and are in contact with the lumen through which blood circulates. The term "endothelial (progenitor) cells" encompasses both endothelial cells and endothelial progenitor cells. Endothelial (progenitor) cells retain the ability to divide and proliferate. Endothelial (progenitor) cells can be identified using CD45 negativity and CD31 positivity as indicators, and can also be identified as CD146 positivity and CD144 positivity.

[0021] [2] Method for producing a purified concentrate of the culture supernatant of mesenchymal stem cells or precursor cells derived therefrom The present invention relates to a method for producing a purified concentrate of the culture supernatant of mesenchymal stem cells or progenitor cells derived therefrom, comprising a step of obtaining a culture supernatant by culturing mesenchymal stem cells or progenitor cells derived therefrom in a culture medium; a concentration step of concentrating the culture supernatant; and a purification step of removing metabolic waste products from the concentrated culture supernatant.

[0022] Adipose-derived stem cells or their progenitor cells can be obtained from adipose tissue. Adipose tissue can be obtained, for example, by surgical excision from humans, or other mammals or birds. Examples of other mammals include dogs, cats, cattle, horses, pigs, goats, sheep, monkeys, ferrets, rabbits, mice, rats, gerbils, guinea pigs, and hamsters. Examples of birds include chickens. Local anesthesia may be used during surgical excision. Adipose tissue can also be obtained by aspiration by inserting a cannula into the subcutaneous adipose tissue of the abdomen, thighs, buttocks, or the entire body. The amount of adipose tissue obtained is, for example, 0.1g to 1000g, preferably 1g to 500g, 1g to 100g, 2g to 50g, or 2g to 40g, but is not limited to these amounts.

[0023] It is preferable to visually confirm that the obtained adipose tissue is free from neoplastic lesions and contamination. The adipose tissue may also be confirmed to be negative for HBV, HCV, HIV, HTLV-1, and TPHA / RPR. The adipose tissue may also be confirmed to have mycoplasma at a chromosome level of less than 128x (PA method) and herpes simplex at a chromosome level of less than 320x (CF method).

[0024] The method for preparing adipose-derived stem cells can be carried out by known methods and is not particularly limited. For example, aspirated adipose tissue can be allowed to stand, and after separating the oil layer, fat layer, and aqueous layer, the oil and aqueous layers can be removed to recover only the fat layer. The obtained fat layer can then be subjected to enzymatic treatment. Alternatively, the fragmented adipose tissue can be directly attached to a culture dish or the like to promote the proliferation and adherent culture of adipose-derived cells.

[0025] It is preferable to warm the adipose tissue before enzymatic treatment in a 37°C water bath at room temperature for 5 to 15 minutes.

[0026] Enzyme treatment can be carried out by adding an appropriate amount of enzyme reaction solution to the fat layer in the tube, fixing the tube to a constant temperature shaker, and shaking it. The temperature of the enzyme treatment is not particularly limited as long as the enzyme reaction proceeds, but is generally 25°C to 50°C, preferably 30°C to 45°C, for example 37°C. The shaking may be reciprocating or oscillating. In the case of reciprocating shaking, the reciprocating shaking speed is not particularly limited, but is generally 10 rpm to 300 rpm, preferably 50 rpm to 200 rpm, for example 120 rpm. The reaction time is not particularly limited, but is generally 10 minutes to 3 hours, preferably 15 minutes to 1 hour, for example 30 minutes.

[0027] As the enzyme, it is preferable to use at least collagenase. Collagenase is a recombinant protein produced by animal tissue cells, inflammatory cells, tumor cells, or bacteria such as Clostridium histolyticum, or artificially produced by genetic engineering technology. It is an enzyme that breaks down type I, type II, and type III collagen. Additionally, neutral proteases, thermolysin, trypsin, dispase, etc., may be added.

[0028] The final concentration of collagenase in the enzyme-treated solution is preferably 0.02% to 2%, more preferably 0.1% to 1%, even more preferably 0.1% to 0.5%, even more preferably 0.1% to 0.4%, even more preferably 0.1% to 0.3%, and most preferably 0.2%.

[0029] The enzyme treatment solution used for the enzyme treatment may also preferably contain DNase I in addition to collagenase. When DNase I is used, the concentration of DNase I in the enzyme treatment solution is preferably 100 to 10,000 U / mL, more preferably 200 to 5,000 U / mL, and even more preferably 500 to 2,000 U / mL.

[0030] The enzyme treatment solution used for the enzyme treatment preferably also contains CaCl2. The concentration of CaCl2 in the enzyme treatment solution is preferably 0.01 mM to 10 mM, more preferably 0.1 mM to 5 mM, and even more preferably 1 mM to 4 mM, for example, 3 mM.

[0031] The enzyme treatment solution used for the enzyme treatment is preferably a buffer solution, and more preferably HBSS (Hanks' Balanced Salt Solution).

[0032] The mixture of the fat layer and enzyme solution can be reacted by rotating it in a centrifuge tube at a temperature suitable for the enzymatic reaction (for example, 30°C to 45°C, preferably 35-40°C). After the reaction, further centrifugation at 800G allows separation into three layers: an oil layer, a fat layer, an aqueous layer, and a precipitate layer (cell layer). By removing the oil layer, fat layer, and aqueous layer, only the precipitate layer (cell layer) can be obtained. By suspending the precipitate layer in a suitable physiological saline solution, a suspension of adipose-derived cells (stromal vascular fraction, SVF) can be obtained.

[0033] In this invention, the culture supernatant is obtained by culturing mesenchymal stem cells or precursor cells derived therefrom in a culture medium.

[0034] The culture medium is not particularly limited as long as it is a medium capable of culturing mesenchymal stem cells or precursor cells derived therefrom, and examples include EGM-2 (Lonza), αMEM, Dulbecco's modified Eagle medium (DMEM), Dulbecco's modified Eagle medium / Ham F-12 mixed medium (DMEM / F12), and RPMI1640. To these culture media, various additives applicable to normal cell culture may be added, such as serum, various vitamins, various antibiotics, various hormones, and various growth factors. Particularly preferred as the culture medium are DMEM / F12, or EGM-2, EGM-2MV (both Lonza).

[0035] Culturing is preferably carried out in a culture vessel such as a flask at 5% CO2 and 37°C. The culture medium can be changed, for example, every two days. Culturing can be carried out at an oxygen concentration of 1-21%. Particularly preferred is culturing at an oxygen concentration of 2-6%. The culture period is not particularly limited, but for example, culture can be performed for 1 to 14 days. After culturing for 1 to 6 days, the cells may be subcultured and cultured again for, for example, 3 to 6 days. The number of subcultures and cultures is not particularly limited.

[0036] The seeding of cell suspensions is not particularly limited, but one example is 5.1 × 10 4 Viable nucleated cells / cm 2 The culture medium volume is 0.25 mL / cm³. 2 This can be done to a certain extent. Once subculturing becomes stable, for example, 4000 viable nucleated cells / cm³ 2 The culture medium volume is 0.25 mL / cm³. 2 It can be done this way.

[0037] In the process of obtaining the culture supernatant, the culture supernatant can be obtained from a culture of mesenchymal stem cells or precursor cells derived therefrom that are in the proliferative or confluent phase.

[0038] The culture supernatant can be prepared by methods known in the art. For example, the culture supernatant can be obtained by centrifuging the obtained culture medium and passing it through a filter (or strainer) of an appropriate pore size. Here, centrifugation can be carried out, for example, at 300 to 1200 × g for 5 to 20 minutes.

[0039] In this invention, a concentration step is performed to concentrate useful substances in the culture supernatant, and a purification step is performed to remove metabolic waste products from the concentrated culture supernatant. The concentration step can be carried out by methods known in the art. Examples include, but are not limited to, ultrafiltration, gel filtration, and dialysis. In the purification step, the diluted concentrated culture supernatant can be further reduced or removed by using a similar filtration step to reduce lactic acid and ammonia, which are metabolic waste products. By reducing metabolic waste products to a certain concentration or below in this way, the culture supernatant can be rendered harmless.

[0040] The concentration and purification steps can preferably be carried out by ultrafiltration. In the case of ultrafiltration, it is preferable to use a filtration membrane with a molecular weight cutoff of 2kDa to 30kDa. The molecular weight cutoff of the filtration membrane may also be 2kDa to 20kDa, 2kDa to 10kDa, 2kDa to 5kDa, or 2kDa to 4kDa.

[0041] The concentration ratio of the culture supernatant is preferably 1 to 100 times, more preferably 20 to 50 times, in the liquid state at the time of transplantation. Further concentration can be expected to promote healing and suppress immunosuppression. By repeating the concentration process, it is possible to further concentrate without limit.

[0042] A key feature of this invention is that concentration and purification are performed in separate steps. The culture supernatant is collected from the flask, and cells mixed in the culture supernatant are removed by centrifugation and filtration (e.g., a 0.22 μm PES sterile filter) to obtain a cell-removed culture supernatant. The cell-removed culture supernatant is passed through an ultrafiltration membrane and concentrated, so that the concentrated cell-removed culture supernatant (indicated as CCM-pre in the examples) is collected in the ultrafiltration filter (concentration step). Next, fresh basal medium is added to the ultrafiltration filter to its maximum load, and ultrafiltration is performed again (purification step). As a result, the concentrated solution is almost entirely replaced by components of the basal medium, at least for low molecular weight substances (i.e., low molecular weight metabolites such as ammonia and lactic acid are replaced and removed by fresh basal medium), thus obtaining a purified concentrated cell-removed culture supernatant. In this invention, both the concentration step and the purification step may be repeated multiple times. Furthermore, the culture medium can be replaced with any solvent as appropriate (e.g., PBS, Ringer's solution, distilled water, etc.).

[0043] The purified concentrate of the culture supernatant obtained as described above does not contain cells, but it contains at least one of the following secreted by mesenchymal stem cells or their progenitor cells: IGFBP, HGF, VEGF, PDGF, EGF, KGF (FGF-7), PDGFR, TGFα, and TGFβ. Lactic acid and ammonia, which are metabolic waste products from mesenchymal stem cells or their progenitor cells, have been removed.

[0044] [3] Compositions for therapeutic or cosmetic purposes The purifying concentrate of the present invention can be used as a composition for the treatment of damage, degeneration, impairment, or dysfunction of biological tissues and organs, as well as for cosmetic purposes. Specifically, the purifying concentrate of the present invention can be provided as a pharmaceutical composition such as a regenerative medicine therapeutic agent, immunosuppressant, anti-inflammatory agent, or anti-fibrotic agent, or as a cosmetic composition. In particular, it can provide effects of angiogenesis, tissue regeneration, and immunosuppression. In the case of the skin, this can be expected to have cosmetic and rejuvenating effects aimed at improving wrinkles and blemishes, as well as therapeutic effects for erectile dysfunction and hair loss. The immunosuppressive effect of this composition can be widely applied to the treatment of allergic diseases such as atopic dermatitis, the treatment of various autoimmune diseases such as rheumatoid arthritis, or the treatment of cytokine storms seen in severe COVID-19.

[0045] Transplanting a purified concentrate of culture supernatant that does not contain cells avoids the risks of immune rejection and GVHD associated with cell transplantation, and simplifies the administration procedure. Furthermore, the purified concentrate of culture supernatant can be stored for a long period without deterioration by freezing in liquid form or by freeze-drying as a dried powder. In addition, while autologous transplantation is desirable for cell transplantation, requiring invasive methods to collect cells from healthy sites, this is not necessary for transplantation of purified concentrate of culture supernatant. Moreover, large doses can be administered when transplanting purified concentrate of culture supernatant. Furthermore, it is possible to use a mixture of 2-3 types of purified concentrate of culture supernatant (for example, derived from adipose stem cells and vascular endothelial cells). It is also possible to use it mixed with PRP (platelet-rich plasma), which has been reported to have a healing effect when administered to refractory skin ulcers.

[0046] By using the purified concentrate of the present invention in combination with stem cells, it is possible to efficiently regenerate various organs and tissues and enable them to engraft at the transplant site. In particular, it can promote angiogenesis and tissue regeneration and remodeling. The purified concentrate of the present invention can be used in combination with adipose tissue, adipose tissue-derived stromal vascular cells (SVF), adipose stem cells, or vascular endothelial (progenitor) cells for transplantation aimed at breast augmentation, breast reconstruction after mastectomy, and improvement of wrinkles and loss of firmness around the eyes and cheeks. The purified concentrate of the present invention can be used in combination with osteoblasts to perform transplantation for purposes such as treating fractures and bone lengthening surgery to improve short stature, bone deformities, and leg length discrepancies. The purified concentrate of the present invention, when used in combination with chondrocytes, can be used for transplantation aimed at treating diseases related to the degeneration, deformation, deformation, and other abnormalities of articular cartilage (e.g., osteoarthritis, rheumatoid arthritis, periarthritis of the shoulder, temporomandibular joint disorder, etc.). The purified concentrate of the present invention, when used in combination with smooth muscle cells, can be transplanted for the purpose of treating diseases caused by damage or abnormalities of smooth muscle cells (for example, urinary disorders (urinary incontinence, frequent urination, urinary retention, etc.), leiomyoma, leiomyosarcoma, etc.).

[0047] The composition of the present invention may be diluted with an intravenous solution, distilled water for injection, or culture medium used as a pharmaceutically acceptable medium. The intravenous solution is not particularly limited, but examples include physiological saline, 5% glucose solution, Ringer's solution, lactated Ringer's solution, acetate Ringer's solution, initiation solution (Solution 1), dehydration replacement solution (Solution 2), maintenance solution (Solution 3), postoperative recovery solution (Solution 4), etc. The number of cells used for dilution is not particularly limited, but for example, 1 × 10⁶ 3 ~1 × 10 7 It can be expressed as particles / mL.

[0048] The composition of the present invention may contain various additives to increase storage stability, sterility, isotonicity, absorbency and / or viscosity, such as emulsifiers, dispersants, buffers, preservatives, wetting agents, antibacterial agents, antioxidants, chelating agents, thickeners, gelling agents, pH adjusters, etc. Examples of thickeners include, but are not limited to, HES, dextran, methylcellulose, xanthan gum, carboxymethylcellulose, and hydroxypropylcellulose.

[0049] The dosage of the composition of the present invention can be appropriately determined depending on the form of administration, method of administration, intended use, and the age, weight, and symptoms of the patient or subject. The single dose of the purified concentrate is not particularly limited, but for example, when expressed in terms of the volume of the supernatant culture used as the raw material for purification and concentration, it is 0.01 mL / kg body weight or more, 0.1 mL / kg body weight or more, or 1 mL / kg body weight or more. Also, the single dose of the purified concentrate is not particularly limited, but for example, it is 10 mL / kg body weight or less, or 5 mL / kg body weight or less.

[0050] The method of administering the composition of the present invention is not particularly limited, but may include, for example, subcutaneous injection, intra-lymph node injection, intravenous injection, intra-arterial injection, intraperitoneal injection, intrathoracic injection, or direct local injection, or direct local implantation using a catheter or the like. Furthermore, it can also be administered topically as an ointment or emulsion, as a spray lotion, or orally.

[0051] The composition of the present invention is typically administered to humans, but may be administered to other animals. Other animals include mammals and birds. Examples of mammals include dogs, cats, cattle, horses, pigs, goats, sheep, monkeys, ferrets, rabbits, mice, rats, gerbils, guinea pigs, and hamsters. Examples of birds include chickens. The present invention will be specifically described in the following examples, but the present invention is not limited to these examples. [Examples]

[0052] Things to prepare #1: HBSS without Ca++,without Mg++ (Gibco, #14175-095) #2: Crude collagenase purified from Clostridium histolyticum (Fujifilm Wako Pure Chemical Industries, #032-22364) #3: DNase1 crude purification (Worthington, #LS002139) #4: Centrifuge tube (CORNING, #431123) #5: Swing-type benchtop centrifuge (KUBOTA, #S700T) #6: Constant Temperature Shaker (Yamato Scientific, #BT100) #7: Electronic scale #8: Dropper made of silicone rubber (AS ONE, #6-356-04) #9: Cell Strainer (CORNING, 100μm; #352360, 40μm; #3552340) #10: Cell counter (Logos Biosystems, #L30001) #11: Calcium chloride (Fujifilm Wako Pure Chemical Industries, #037-24031) #12: Syringe filter 0.22μm diameter (Millipore, #2LGV-33RS) #13: Cellbanker 1 (Takara Bio, #CD011) #14: Cell culture dish (CORNING, #353025) #15: DMEM / Ham's F-12 (Fujifilm Wako Pure Chemical Industries, #048-29785) #16: Fetal Bovine Serum (FBS) #17: Penicillin-Streptomycin (100x) (Fujifilm Wako Pure Chemical Industries, #168-23191) #18: CO2 Incubator (Panasonic, #MCO-170AICUVH-PJ) #19: TrypLE Express (Gibco,#12604-021) #20: L-(+)-lactic acid (Sigma-Aldrich, #L6402) #21: 10% Ammonia Solution (Fujifilm Wako Pure Chemical Industries, #013-17505) #22: 96-well plate (PerkinElmer, SpectraPlate-96, #6005650) #23: Cell Counting Kit-8(Dojindo,#CK04) #24: Plate reader (PerkinElmer, ARVO MX) #25: Ultrafiltration membrane (Milipore, Amicon Ultra-15 10kDa, #UFC901024) #26: Lactic acid quantification kit (Abbott, GC4+) #27: Hypoxic CO2 incubator (Waken Bitech) #28: Human HGF ELISA kit (R&D systems, Quantikine 96well plate #DHG00B)<00​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​1000 units / mL DNase1 [1×10 5 units / mL stock solution (*2): 1.2 mL HBSS (#1) 58.8 mL Prepared immediately before use. When reacting with the fat layer, equal amounts of the enzyme reaction solution and the fat layer are added, so the concentrations of each component in the enzyme reaction solution are halved during the reaction.

[0056] *4: Preparation of fresh culture medium DMEM / Ham's F-12 (#15) 500mL FBS (#16) 55mL Penicillin-Streptomycin (#17) 5mL

[0057] *5: Preparation of fresh culture medium (without FBS) DMEM / Ham's F-12 (#15) 500mL Penicillin-Streptomycin (#17) 5mL

[0058] Example 1: SVF preparation The aspirated adipose tissue was allowed to stand, and the oil layer, fat layer, and aqueous layer were separated. The oil layer and aqueous layer were removed, leaving only the fat layer. The weight of an empty centrifuge tube (#4) was weighed. A 500 mL centrifuge tube was used for 50 g of fat layer. The fat layer was transferred to the centrifuge tube and weighed, and the weight of the fat layer was calculated from (centrifuge tube + fat layer) - weight of centrifuge tube. An equal volume of enzyme reaction solution (*3) was added to the fat layer. The centrifuge tube was fixed to a constant temperature shaker (#6) and reacted at 37°C, 120 rpm for 30 minutes. The tube was removed from the constant temperature shaker and centrifuged in a centrifuge (#5) at 800 G for 10 minutes. After centrifugation, the tissue separated into three layers from top to bottom: oil layer, fat layer, aqueous layer, and precipitate layer (cell layer). The oil layer, fat layer, and aqueous layer were removed with a pipette, leaving only the precipitate layer. HBSS (#1) was added to the precipitate layer to a ratio of 90 mL for 50 g of fat layer, and the cells were gently suspended and washed using a pipette. The cell suspension was passed through 100 μm and 40 μm cell strainers to remove tissue fragments. The cells were centrifuged at 800 G for 5 minutes using a centrifuge (#5). The supernatant was aspirated and resuspended in 45 mL of HBSS (#1), and centrifuged at 700 G for 5 minutes. The supernatant was aspirated and resuspended in HBSS (#1) at a ratio of 1 mL of HBSS for 5 g of fat layer. The number of nucleated viable cells was measured using a cell counter (#10). The SVF was suspended in the cryopreservation solution Cellbanker 1 (#13) and stored at cryogenic temperatures.

[0059] Example 2: Subculture of ASC Interstitial vascular cells (SVFs) were 5.1 × 10⁶ in both dormant states from cryopreservation solution and in a fresh state. 4 1 surviving nucleated cell / 0.25 mL of culture medium (*4) / cm³ 2 The cells were seeded in a culture flask (#14) and cultured in a CO2 incubator (#18) (37°C, 5% CO2, Air). The following day, the cells were washed three times with HBSS (#1) at 37°C to remove blood cells and residual tissue fragments, and fresh medium (*4) was added. The medium was changed to fresh medium every three days. On the seventh day, the cells (mainly ASCs) were dispersed for harvesting. After washing with HBS (#1) at 37°C, they were incubated in a 1×TrypLE express (#19) (1 mL TrypLE express / 15 cm) at 37°C. 2The cells were dispersed at 37°C, 5% CO2, and 5 minutes. The reaction was stopped with fresh medium (*4), and a cell pellet was obtained by centrifugation (300G, 5 minutes, 4°C). The number of nucleated cells was counted with a cell counter (#10). The cells (mainly ASCs) were divided into 4000 viable nucleated cells / 0.25 mL of medium (*4) / cm³. 2 The seeds were then seeded into a culture flask (#14) and cultured in a CO2 incubator (#18) (37°C, 5% CO2, Air). The culture medium was changed to fresh medium every three days and cultured for seven days.

[0060] Example 3: Inhibitors in CM <Overview> During cell culture (CM), cell-derived bioactive substances that are expected to be effective in tissue regeneration and cell culture are released. However, metabolites that inhibit cell survival and proliferation are also released from the cells. Therefore, CM typically contains both active ingredients and inhibitors. As representative metabolites, we added pure reagents of lactic acid or ammonia to fresh culture medium and confirmed their effect on ASC proliferation.

[0061] <Step 1> ASCs were cultured to a passage number (P) of 2 by the SVF preparation method described in Example 1 and the subculture of ASCs described in Example 2, and an ASC cell suspension was obtained. The number of nucleated viable cells was measured using a cell counter (#10). 4000 or 1000 ASC viable nucleated cells were seeded into a 96-well plate (#22) at a rate of 100 μL of medium (*4) per well. Two plates were prepared. Culture was started in a CO2 incubator (#18) (37°C, 5% CO2, Air). Fresh medium (*4), fresh medium containing 2,10,50 mM lactate (#20) (*4), and fresh medium containing 2,10,50 mM ammonia (#21) (*4) were prepared, and the medium was changed after 22 hours. Cell proliferation activity was confirmed using a cell counting kit (#23) 3 days after the start of culture (2 days after the medium change). The absorbance at 450 nm was measured using a plate reader (#24). The measurement results are shown in Figure 1.

[0062] Compared to fresh medium culture, cell proliferation activity was inhibited in fresh medium supplemented with lactate or ammonia. Lactate significantly inhibited culture in 50 mM lactate-fresh medium. Ammonia inhibited ASC culture from 2 mM ammonia-fresh medium, and significantly inhibited culture in 50 mM ammonia-fresh medium. The ammonia concentration in the culture supernatant (CM) collected at the confluence stage was approximately 1.428 M NH4+ (2000 μg / dL). Therefore, the ammonia concentration secreted by ASC in CM includes levels that inhibit cell proliferation, indicating that typical CM contains both physiologically active substances (beneficial) and metabolites (harmful).

[0063] <Step 2> ASCs were cultured up to a passage number (P) of 2 by the SVF preparation method described in Example 1 and the subculturing of ASCs in Example 2, and an ASC cell suspension was obtained. The number of nucleated viable cells was measured using a cell counter (#10). 4000 viable nucleated ASCs were cultured in a cell culture dish (#14) at a rate of 0.2 mL of medium (*4) per cm³. 2 The seeds were sown. CMs meeting the following three conditions were collected.

[0064] (Condition 1) CM cultured for 72 hours after being replaced with fresh medium (*4) when at 60-70% confluence. (Condition 2) CM cultured for 72 hours after being replaced with fresh medium (*4) when at 90-100% confluence. (Condition 3) CM cultured for 72 hours after being replaced with fresh medium (FBS-free) (*5) when at 90-100% confluence.

[0065] The collected CM was quickly collected in a centrifuge tube and centrifuged at 800G for 10 minutes in a swing centrifuge to precipitate cells and debris in the CM, and the supernatant was collected. The supernatant was concentrated on an ultrafiltration membrane (#25) to obtain CCM-pre. Fresh medium (FBS-free) (*5) was added to the concentrated CCM-pre on the ultrafiltration membrane to the maximum load capacity (dilution), and concentrated again. Thus, for each of the three culture conditions, CM, CCM concentrated (purified) by replacement with fresh medium, and waste of CM (flow-through from ultrafiltration of CM) were obtained. Lactic acid levels in each medium were determined using a lactate determination kit (#26). Due to the nature of the measurement kit, values ​​below 0.3 mM lactate are displayed as <0.30, and although the value is low, the actual value cannot be measured. The measurement results are shown in Figure 2.

[0066] According to the present invention, it has become clear that beneficial physiologically active substances can be concentrated while harmful low-molecular-weight metabolites can be eliminated by concentration using an ultrafiltration membrane and by substitution concentration with fresh culture medium.

[0067] <Step 3> ASCs were cultured up to a passage number (P) of 2 by the SVF preparation method described in Example 1 and the subculturing of ASCs described in Example 2, to obtain an ASC cell suspension. The number of nucleated viable cells was measured using a cell counter (#10). 4000 or 1000 viable nucleated ASCs were seeded in a 96-well plate (#22) at a rate of 100 μL of medium (*4) per well. Culture was started in a hypoxic CO2 incubator (#27) (37°C, 5% CO2) with 21%, 6%, or 1% O2. Cell proliferation activity was confirmed on day 3 and day 6 of culture using a cell counting kit (#23). Absorbance at 450 nm was measured using a plate reader (#24). The measurement results are shown in Figure 3.

[0068] In terms of oxygen environments, low oxygen levels (6%, 1%) resulted in better growth efficiency than 21% O2, with 6% O2 being particularly effective. Based on these results, we decided to prepare CM under culture conditions of 37°C, 5% CO2, and 6% O2.

[0069] Example 4: CM preparation <Overview> To obtain CMs that secrete a large amount of active ingredients, we investigated the optimal cell seeding density, culture medium volume, and culture period under oxygen concentration (6% O2) that approximates the physiological environment.

[0070] <Step 1> The SVF preparation described in Example 1 and the ASC subculture described in Example 2 were performed to culture ASCs to a passage number (P) of 1 (however, the culture oxygen concentration was changed to 6%), and a cell suspension of ASCs was obtained. The number of nucleated viable cells was measured with a cell counter (#10). Cells were seeded in a cell culture dish (#14) under the following two conditions.

[0071] (Condition 1) 2×10 4 Number of ASC viable nucleated cells / 0.2mL medium (*4) / cm 2 (Sowing density at which ASC reaches 90% confluence the day after sowing) (Condition 2) 3.5×10 4 Number of ASC viable nucleated cells / 0.2mL medium (*4) / cm 2 (Sowing density at which ASC reaches 100% confluence the day after sowing).

[0072] The following day, the culture medium was replaced with the same volume of fresh medium (*4), and three days later (four days after the start of culture), the medium was quickly collected in a centrifuge tube and centrifuged at 800G for 10 minutes using a swing centrifuge to precipitate cells and debris, and the supernatant was collected as CM. Of the growth factors secreted by ASC into the culture supernatant, the amount of HGF reported in the literature was quantified using Human HGF ELISAkit (#28). The measurement results are shown in Figure 4.

[0073] Condition 1 (2×10 4 (Number of individual ASCs with nucleated cells) (Seeding density at which ASCs reach 90% confluence the day after seeding) and Condition 2 (3.5 × 10⁻¹⁰ 4 There was a difference in the amount of HGF in the CM between the number of surviving nucleated ASCs (ASCs) and the seeding density at which ASCs reached 100% confluence the day after seeding. Therefore, the ASC seeding density and culture medium volume for CM preparation were determined under condition 2 (3.5 × 10⁻⁶) in a 6% O₂ environment. 4Number of ASC viable nucleated cells / 0.2mL medium (*4) / cm 2 That's what I decided.

[0074] <Step 2> The SVF preparation described in Example 1 and the ASC subculture described in Example 2 were performed to culture ASCs until the passage number (P) = 1, thereby obtaining an ASC cell suspension. The number of nucleated viable cells was measured using a cell counter (#10). The cells were cultured in a cell culture dish (#14) under condition 2 (3.5 × 10⁻¹) in a 6% O₂ environment. 4 Number of viable nucleated cells / 0.2mL medium (*4) / cm 2 ASCs were seeded in ). Culture supernatant was collected on Day 3, 6, 8, and 11, and the same amount of fresh medium (*4) was added. On Day 11 only, the cells were cultured in both the fresh medium group (*4) and the fresh medium (without FBS) group (*5). The culture supernatant was quickly collected in a centrifuge tube and centrifuged at 800G for 10 minutes in a swing centrifuge to precipitate cells and debris. The supernatant was collected as CM and stored at -80°C. The amount of HGF was quantified using the Human HGF ELISA kit (#28). The measurement results are shown in Figure 5.

[0075] The following four conditions—(1) D3-D6, (2) D8-D11 with FBS, (3) D8-D11 without FBS, and control fresh medium (without FBS) (*5)—were analyzed for the amount of secreted factors in CM using a Growth factor antibody array (#29). Images were taken using a Lumino Image Analyzer (#30). The measurement results are shown in Figure 6.

[0076] The 41 growth factors included in the antibody array are Amphiregulin, bFGF, b-NGF, EGF, EGFR, FGF-4, FGF-6, FGF-7, G-CSF, GDNF, GM-CSF, HB-EGF, HGF, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, IGF-1, IGF-1sR, IGF-2, M-CSF, M-CSF R, NT-3, NT-4, PDGFRa, PDGFRb, PDGF-AA, PDGF-AB, PDGF-BB, PLGF, SCF, SCF R, TGF alpha, TGF beta 1, TGF beta 2, TGF beta 3, VEGF-A, VEGFR2, VEGFR3, and VEGF-D.

[0077] Even at seeding densities where ASC reached 100% confluence the day after sowing, additional ASC was superimposed. HGF levels increased more in the later stages of culture (differentiation phase) than in the earlier stages (proliferation phase) (Figure 5).

[0078] An increasing trend in growth factors, indicated by arrows, was observed in the culture supernatant (CM) under each condition (Figure 6). Since the quantity and type of growth factors differ depending on the condition, CM can be recovered under the conditions required. Furthermore, these growth factors can be further concentrated by purification and concentration using an ultrafiltration membrane.

[0079] The estimated concentrations of each cytokine in the concentrate, based on a combination of antibody array and ELISA measurement results, are shown below. When concentrating the culture supernatant 30 times, in the concentrated solution, VEGF: 250 ng / mL FGF7(KGF) 12000pg / mL PDGF-AA: 50 ng / mL PDGFR-β: 125 ng / mL TGFβ: 1500 pg / mL IGFBP-6: 2000 ng / mL IGFBP-4: 350 ng / mL IGFBP-3: 170 ng / mL IGFBP-2: 270 ng / mL IGFBP-1: 40 ng / mL

[0080] Example 5: Effects of oxygen concentration and presence or absence of serum (10% FBS) on HGF production <Measurement Method / Kit> ELISA method, Human HGF Quantikine ELISA Kit(R&D Systems,#DHG00B)

[0081] <Sample to be measured> (1) Collection of culture supernatant sample hASCs at normal seeding density (2 × 10 4 cells / cm 2 The cells were seeded in flasks and cultured in incubators set to 1% O2, 6% O2, and 21% O2 at 37°C, 5% CO2, and oxygen concentrations. The schedule for culture supernatant collection and medium exchange is shown in Figure 7. After collection on D6 (day 6), the medium was changed to either a medium with serum (10% FBS) or a medium without serum.

[0082] (2) Processing of culture supernatant samples The collected culture supernatant was centrifuged to remove cell debris and other impurities, and the supernatant was stored at -80°C.

[0083] <Result> The measurement results are shown in Figure 8. Regardless of serum presence or absence, only a very small amount of HGF was detected in the culture supernatant under 1% O2 culture conditions. On the other hand, under both 6% O2 and 21% O2 culture conditions, HGF production was confirmed to be higher in culture medium containing 10% FBS compared to serum-free medium. Furthermore, ASC cultured in 6% O2 and serum-supplemented medium released even more HGF.

[0084] In the biological environment, the average oxygen concentration is said to be around 5-7%, which is a low-oxygen environment compared to the oxygen concentration in the atmosphere (normal oxygen environment). 6% O2, which is closest to the biological environment, was considered more suitable for ASC survival and also favorable conditions for HGF production. To maximize HGF release in the culture supernatant, it is presumed that it is preferable to seed a large number of hASCs so that the adhesive surface of the culture vessel becomes confluent, and to continue culturing in a medium supplemented with 10% FBS and under 6% O2 conditions.

[0085] Example 6: Quantitative measurement of HGF in culture supernatant, concentrate, and waste liquid after concentration. <Measurement Method / Kit> ELISA method, Human HGF Quantikine ELISA Kit(R&D Systems,#DHG00B)

[0086] <Sample to be measured> Culture supernatant, concentrate, and waste liquid after concentration, each in three separate mixtures [cultured under normal oxygen (21% O2) conditions].

[0087] <Method> Useful factors (e.g., HGF) produced in the culture supernatant show a positive correlation with the number of hASCs cells. Assuming that HGF concentrations would differ depending on the collection date, the one-month collection course was divided into three groups: Day 1-9, Day 10-18, and Day 19-30. Quantitative HGF analysis was performed using three sets of mixtures of the culture supernatant, concentrate, and waste liquid after concentration. Figure 9 shows the results of the quantitative analysis of HGF in the culture supernatant, concentrate, and waste liquid after concentration collected on Day 1-9.

[0088] <Result> HGF secreted from hASCs cells was detected in the culture supernatant. The HGF concentration in the concentrated solution was approximately 30 times that of the solution before concentration. Almost no residual HGF was observed in the waste solution after concentration.

[0089] <Consideration> With the filtration and concentration of the culture supernatant, an increase in useful factors was anticipated in the concentrate. This study focused on hepatocyte growth factor (HGF), the most noteworthy growth factor in the culture supernatant, and analyzed it. In the fully recovered course, the HGF concentration in the concentrate was 20 to 30 times higher than that in the culture supernatant before concentration.

[0090] Example 7: Measurement of ammonia concentration in culture supernatant, concentrate, and waste liquid after concentration. <Measurement method> Fuji Drychem NX10N (Fujifilm Medical Co., Ltd.) Fuji Drychem Slide NH3-PII (for plasma) (Fujifilm Medical Co., Ltd.)

[0091] <Sample to be measured> A mixture of culture supernatant, concentrate, and waste liquid after concentration, enough for 10 cycles (1 month). D-MEM / Ham's F-12 (10% FBS, 1% PS) is used as a control.

[0092] <Result> The quantifiable range for ammonia concentration in Fuji Drychem Slide NH3-PII (for plasma) is 10 to 500 μg / dL. Therefore, by diluting the above sample to fall within this quantifiable range and then multiplying by the dilution factor, an accurate measurement can be calculated. The measurement results are shown in Figure 10.

[0093] <Consideration> The above measurement results showed no change in ammonia concentration between the culture supernatant, concentrate, and waste liquid after concentration. However, when converted to mass, the amount of ammonia in the concentrate was approximately 1 / 30th of that in the culture supernatant. By diluting the concentrate and performing ultrafiltration, and increasing the number of times this process is performed, the ammonia concentration in the solution can be reduced to below the detection limit. For example, the concentrated solution after primary filtration is diluted by adding a basal medium (sterilized distilled water, ultrapure water, or phosphate-buffered saline, PBS, etc.) (e.g., 30-fold dilution). The diluted solution is then subjected to secondary ultrafiltration using a stirred ultrafiltration apparatus (e.g., 30-fold concentration). This process almost completely replaces low molecular weight substances (especially harmful substances such as ammonia and lactic acid) in the concentrated solution with components of the basal medium. However, since effective factors, including growth factors, have large molecular weights, most of them remain in the concentrated solution. Because the degree and number of these concentration and purification processes can be adjusted, the final degree of concentration and purification is virtually limitless.

[0094] Example 8: Effects of culture supernatant, concentrate, and waste liquid after concentration on the proliferation ability of hASCs (cell proliferation experiment) <Sample to be measured> The culture supernatant, concentrate, and waste liquid after concentration are each mixtures sufficient for 10 uses (1 month). The culture supernatant stock solution, concentrated solution and serial dilutions, and the waste liquid after concentration and serial dilutions were prepared as shown in Table 1 below. As a control, D-MEM / Ham's F-12 containing 1% PS (hereinafter abbreviated as DMEM-F12) was used, and DMEM-F12 / 10% FBS / 1% PS was used in the vehicle group.

[0095] [Table 1]

[0096] <Method> To measure cell proliferation capacity, the Cell Counting Kit-8 (CCK-8, Dojin Chemical Laboratories Co., Ltd., #CK04) was used. NADH produced by dehydrogenases in cells is reduced to the water-soluble tetrazolium salt (WST-8) by the electron transport molecule 1-Methoxy PMS, which is converted to orange formazan. The amount of this formazan dye (maximum absorption wavelength 450 nm) is proportional to the number of viable cells, and the number of viable cells can be measured by absorbance. hASCs were seeded at passage number P4 at 3000 cells / well (96-well plate) (n=4). Culture was carried out at 37°C, 5% CO2, and 21% O2. As shown in Table 1, 100 μL / well of the prepared sample was added, and the CCK-8 assay was performed on day 4 of culture. Absorbance (OD 450 nm) was measured using a microplate reader.

[0097] <Result> The measurement results are shown in Figure 11. Compared to the control group (DMEM-F12, 1%PS), the 100-fold dilution of the concentrate (CCM_1 / 100) significantly increased cell proliferation. While this proliferation-promoting ability did not differ significantly from the vehicle group (DMEM-F12 / 10%FBS / 1%PS), the concentrated solution dilutions (10-fold dilution CCM_1 / 10, 3-fold dilution CCM_1 / 3) and the undiluted concentrate significantly promoted cell proliferation. Compared to the vehicle group, the addition of a 3-fold dilution of the waste liquid after concentration (W / CCM_1 / 3) significantly suppressed cell proliferation, and the undiluted concentrate (W / CCM) drastically reduced cell proliferation.

[0098] Example 9: Effects of culture supernatant and purified concentrate on the proliferation of human dermal fibroblasts and normal human epidermal keratinocytes (cell proliferation experiment) <Reagents and Methods> Normal human dermal fibroblasts (NHDF) or normal human epidermal keratinocytes (NHEK) 1 x 10 5 Cells were seeded in 96-well plates at a concentration of cells / mL (n=4). After 24 hours, the cells were replaced with control or graded diluted purified culture supernatant concentrates.

[0099] In the NHDF, the CCM (concentrated stock solution) dilutions listed in Table 1 were prepared using either the control D-MEM / Ham's F-12 or D-MEM / Ham's F-12 with 5% FBS added. On the other hand, in the NHEK, the CCM (concentrated stock solution) dilutions listed in Table 1 were prepared using the control KGM-Gold (Lonza, #192060) medium.

[0100] To measure cell proliferation capacity, we used the Cell Counting Kit-8 (CCK-8, Dojin Chemical Research Institute Co., Ltd., #CK04). After 72 hours of incubation, the CCK-8 assay was performed, and the absorbance (OD450nm) was measured using a microplate reader.

[0101] <Result> The measurement results are shown in Figure 12. In human dermal fibroblasts, cell proliferation was confirmed to increase in a concentration-dependent manner with CCM, even in the presence of 5% FBS (left panel in Figure 12). In normal human epidermal keratinocytes, the addition of CCM was found to promote cell proliferation (right panel in Figure 12).

[0102] Example 10: Effects of culture supernatant and purified culture supernatant concentrate on vascular endothelial cell lumen formation ability (endothelial cell lumen formation test) <Measurement Sample> The culture supernatant (CM listed in Table 1) and the purified culture supernatant concentrate (CCM listed in Table 1) were prepared as shown in the table below.

[0103] [Table 2]

[0104] <Method> To evaluate the lumen-forming ability of endothelial cells, human umbilical vein endothelial cells (HUVECs) were used. HUVECs were cultured in EGM-2 medium (Lonza, #CC-4176) until they entered the logarithmic growth phase. Prior to lumen formation analysis, HUVECs were kept serum-free for 12 hours. Matrigel basement membrane matrix (Corning, #356231) was thawed overnight at 4°C and kept on ice throughout the experiment. 50 μL of thawed Matrigel was placed in each well of a 96-well plate and left in an incubator for 1 hour. After detaching the starved HUVECs using TrypLE Express, the HUVECs were pelletized. The control and experimental groups were prepared as shown in the table above, and 1 × 10⁶ HUVECs were pelleted. 5 The cells are resuspended at a density of cells / mL, and 100 μL of the cell suspension is added to each well that previously contained Matrigel (the number of cells per well is 1 × 10⁶). 4The plates were placed at 37°C and 5% CO2 and cultured for 4-6 hours. Cells on the cultured plates were imaged at 40x magnification using a phase-contrast microscope (Leica). The degree of lumen formation was evaluated using ImageJ image analysis software, based on the length of the formed lumen, using five randomly taken microscope images.

[0105] <Result> The measurement results are shown in Figure 13. Compared to the control group, no significant changes were observed in the group treated with CM dilution, while the group treated with concentrated dilution showed an effect of promoting lumen formation.

[0106] Example 11: <Method> 8-week-old db / db mice (BKS.Cg-+Lepr db / +Lepr db / J), and its control mouse (BKS.Cg-Dock7 m + / Dock7 m + / J) was used in the experiment. Mice were housed individually in separate cages for individual identification. The backs of the mice were shaved with clippers under isoflurane inhalation, and the entire skin layer was dissected using a sterile biopsy punch (6.0 mm diameter, Kai Industries) to create ulcers. A donut-shaped silicone splint (donut hole diameter 9 mm, outer diameter 15 mm, thickness 1 mm, Kyowa Kogyo) was fixed with 6-0 nylon to prevent wound contraction. On days 0, 3, and 6, with the ulcer creation day being day 0, 0.1 ml of the concentrated CCM stock solution listed in Table 1 was injected evenly into the subcutaneous tissue around the ulcer in four directions using a syringe. The wounds were dressed with Moistkin pads to prevent silicone splint detachment and wound drying. CCM used was the purified and concentrated culture supernatant from ASC culture. The wound progression from day 0 to day 21 was photographed using a single-lens reflex camera, and the wound healing promotion effect was evaluated based on the wound area. The mice were euthanized on day 21 by inhaling 5% isoflurane.

[0107] [Table 3]

[0108] <Result> The evaluation results are shown in Figure 14. It was revealed that administering CCM significantly improved delayed wound healing in type 2 diabetic mice.

[0109] Example 12: Effects of platelet lysis solution on cell proliferation and HGF production in ASC <Reagents and Kits> Human platelet lysates (hPL) Fetal bovine serum (FBS) TrypLE Express(Gibco,#12604-021) Human HGF Quantikine ELISA Kit (R&D Systems, #DHG00B)

[0110] <Sample to be measured> (1) Collection of culture supernatant sample hASCs 2.5 × 10 4 Seeds were seeded in 100 mm dishes at cells / mL and cultured in a 37°C, 5% CO2 incubator. After 24 hours, the culture medium was replaced with D-MEM / Ham's F12 supplemented with 10% FBS and 5% hPL, respectively, and cultured for 7 days.

[0111] (2) Processing of culture supernatant samples After 7 days of incubation, the culture supernatant was collected from each dish, cell debris was removed by centrifugation, and the supernatant was stored at -80°C. After collecting the culture supernatant, the cells were detached using TrypLE Express, and the number of nucleated cells was counted using a cell counter (Logos Biosystems, #L30001).

[0112] <Result> The measurement results are shown in Figure 15. As shown in the left panel of Figure 15, when ASCs were cultured using a medium supplemented with 10% FBS, the cell number increased by approximately twice as much by Day 7 compared to Day 0 (D0) seeding. On the other hand, it was confirmed that the cell number increased by approximately eight times when cultured in a medium supplemented with 5% hPL. A comparison of HGF concentrations in the culture supernatant recovered on D7 confirmed that the amount of HGF released was significantly higher from hASCs cultured in 5% hPL than from hASCs cultured in 5% hPL (right panel in Figure 15).

[0113] <Consideration> Human platelet lysates (hPL) were found to enhance the cell proliferation capacity and HGF production capacity of hASCs compared to FBS.

[0114] Example 13: Effects of various growth factors on HGF production capacity in ASC <Reagents and Kits> Recombinant Human FGF basic / FGF2 (R&D Systems, #233-GMP) Recombinant Human LR3 IGF-1 / IGF-1 GMP (R&D Systems, #8335D-GMP) Recombinant Human PDGF-BB (R&D Systems, #AF220) Recombinant Human EGF (R&D Systems, #236-EG-200) Human HGF Quantikine ELISA Kit (R&D Systems, #DHG00B)

[0115] <Sample to be measured> (1) Collection of culture supernatant sample hASCs 5 × 10 4Cells were seeded in 48-well plates at cells / mL and cultured at 37°C, 5% CO2, and 21% O2. After 24 hours, the culture medium was replaced with D-MEM / Ham's F12 supplemented with bFGF (basic fibroblast growth factor), IGF (insulin-like growth factor), EGF (epidermal growth factor), and PDGF-BB (platelet-derived growth factor BB), respectively, and cultured for 72 hours. The final concentrations of various growth factors in hASCs were 0, 2.5, 5.0, 10.0, 20.0, 50.0, and 100.0 ng / mL, respectively.

[0116] (2) Processing of culture supernatant samples After 72 hours of incubation, the culture supernatant was collected from each well, cell debris was removed by centrifugation, and the supernatant was stored at -80°C.

[0117] <Result> The measurement results for HGF production capacity are shown in Figure 16. In hASCs, bFGF and PDGF-BB showed a promoting effect on HGF production. The optimal concentration for bFGF was 20 ng / mL, and for PDGF-BB it was 100 ng / mL. On the other hand, IGF and EGF did not show any effect on HGF production.

[0118] <Consideration> Both bFGF and PDGF-BB factors promoted HGF production, but bFGF showed its effect at relatively low concentrations, and this effect continued up to a final bFGF concentration of 20 ng / mL. On the other hand, PDGF-BB promoted HGF production in a concentration-dependent manner. PDGF-BB is a platelet-derived growth factor and is present in large quantities in platelet lysates (PL). In ASC culture, PL may also be useful as a supplement in addition to FBS.

[0119] Example 14: Effects of adjusting stem cell culture method and purification concentration process on the quality of purified concentrate <Reagents and Kits> Human platelet lysates (hPL) Human HGF Quantikine ELISA Kit(R&D Systems,#DHG00B) Fuji Drychem NX10N (Fujifilm Medical Co., Ltd.) Fuji Drychem Slide NH3-PII (for plasma) (Fujifilm Medical Co., Ltd.)

[0120] <Sample to be measured> (1) Collection of culture supernatant sample hASCs 2.5 × 10 5 Cells were seeded in 150 mm dishes at a concentration of cells / mL and cultured at 37°C in a 5% CO2 incubator. The culture medium used was D-MEM / Ham's F12 supplemented with 5% hPL and specific supplements. The culture supernatant was collected and the medium was replaced every 7 days. A lot number was assigned on each collection day.

[0121] (2) Ultrafiltration of the culture supernatant The recovered culture supernatant was centrifuged to remove cell debris and other impurities, and the supernatant was subjected to primary filtration using a stirred ultrafiltration apparatus to concentrate it approximately 30 times. Subsequently, the "primary concentrate" obtained after primary filtration was diluted 30 times with phosphate-buffered saline (PBS) and subjected to ultrafiltration again. Thus, approximately 10 mL of purified concentrate was obtained from each 340 mL of culture supernatant from each lot recovered in (1) after two ultrafiltrations. The HGF concentration and ammonia concentration were measured in the purified concentrate after secondary filtration.

[0122] <Measurement method> HGF concentration was measured using the Human HGF Quantikine ELISA Kit. Ammonia concentration was measured using the Fuji Drychem NX10N and Fuji Drychem Slide NH3-PII (for plasma).

[0123] <Measurement results> The measurement results are shown in Table 4.

[0124] [Table 4]

[0125] <Consideration> By adjusting the culture method (supplements added to the basal culture medium) and the number of ultrafiltration cycles, we were able to obtain a purified concentrate with a higher HGF concentration and a lower ammonia concentration.

[0126] Example 15: Effects of culture supernatant and purified concentrate on inflammatory cytokine production <Reagents and Kits> Human IL-6 Quantikine ELISA Kit (R&D Systems, #D6050) Lipopolysaccharides from Escherichia coliO55:B5 (Sigma-Aldrich, #L6529) The culture supernatant and purified concentrate were prepared as shown in the table below.

[0127] [Table 5]

[0128] <Sample to be measured> (1) Collection of normal human dermal fibroblast culture medium samples 3 × 10⁶ normal human dermal fibroblasts (NHDF) 5Seed cells / mL were seeded into 6-well plates (two plates) and cultured at 37°C, 5% CO2, and 21% O2. After 24 hours, the medium was changed to D-MEM / Ham's F12 and kept serum-free for 6 hours. Starved NHDF cells were then cultured in the medium prepared in Table 5. After 24 hours, the medium was removed using an aspirator, and the NHDF cells were washed twice with HBSS (Hanks' Balanced Salt Solution). Subsequently, the NHDF cells were cultured for 6 hours in D-MEM / Ham's F12 medium with or without Lipopolysaccharides (LPS) added, or with a final LPS concentration of 100 ng / mL added. After 6 hours, the NHDF cell conditioned cultures under each condition were collected.

[0129] (2) Processing of NHDF cell-conditioned culture medium samples The NHDF cell-conditioned culture medium collected in (1) above was centrifuged to remove cell debris and other impurities, and the supernatant was stored at -80°C.

[0130] <Measurement method> The concentration of the inflammatory cytokine IL-6 in NHDF cell-conditioned culture medium was measured using the ELISA method.

[0131] <Result> The measurement results are shown in Figure 17. In NHDF cells, the addition of 100 ng / mL of LPS promoted the secretion of the inflammatory cytokine IL-6. Furthermore, despite the presence of LPS, the group treated with the culture supernatant (CM) suppressed the IL-6 secretion ability of NHDF cells compared to the control group. The groups treated with a 100-fold dilution of the concentrated solution (CCM_1 / 100) and a 30-fold dilution of the concentrated solution (CCM_1 / 30) significantly suppressed IL-6 production.

[0132] <Consideration> IL-6 is a cytokine that plays a crucial role in the regulation of immune and inflammatory responses, and it is known that excessive production of IL-6 can cause various pathological conditions. While stem cell culture supernatant showed a mild anti-inflammatory effect, the culture supernatant concentrate showed a clear anti-inflammatory effect even at small amounts. The culture supernatant concentrate also suggests potential applications as an anti-inflammatory agent.

Claims

1. It contains at least one of the following secreted by mesenchymal stem cells or their progenitor cells: IGFBP, HGF, VEGF, PDGF, EGF, KGF (FGF-7), PDGFR, TGFα, and TGFβ. The lactic acid concentration is less than 0.3 mM, the ammonia concentration is 20 μg / dL or less, and the HGF concentration is 50,000 pg / mL or more. The aforementioned progenitor cells are adipocytes, osteoblasts, chondrocytes, myofibroblasts, nerve sheath progenitor cells, muscle progenitor cells, bone progenitor cells, cutaneous fibroblasts, or vascular endothelial progenitor cells. A purified concentrate of the culture supernatant of mesenchymal stem cells or their derived progenitor cells.

2. The purified concentrate according to claim 1, for use as a regenerative medicine therapeutic agent, immunosuppressant, anti-inflammatory agent, or anti-fibrotic agent.

3. A purified concentrate according to claim 1 or 2, comprising all of the following secreted by mesenchymal stem cells or precursor cells derived therefrom: IGFBP, HGF, VEGF, PDGF, EGF, KGF (FGF-7), PDGFR, TGFα, and TGFβ.

4. The purified concentrate according to any one of claims 1 to 3, wherein the mesenchymal stem cells or progenitor cells derived therefrom are adipose-derived mesenchymal stem cells or adipose-derived vascular endothelial progenitor cells.

5. A process for obtaining a culture supernatant by culturing mesenchymal stem cells or precursor cells derived therefrom in a culture medium; A concentration step for concentrating useful components from the culture supernatant: and A purification step to remove metabolic waste products from the concentrated culture supernatant; A method for producing a purified concentrate according to any one of claims 1 to 4, including

6. The method according to claim 5, wherein in the step of obtaining the culture supernatant, the culture supernatant is obtained from a culture of mesenchymal stem cells or precursor cells derived therefrom that are in the proliferative or confluent phase.

7. The method according to claim 5 or 6, wherein in the concentration step, the concentration of at least one of IGFBP, HGF, VEGF, PDGF, EGF, KGF (FGF-7), PDGFR, TGFα, and TGFβ is concentrated to 1.2 times or more compared to the culture supernatant before concentration.

8. The method according to any one of claims 5 to 7, wherein the metabolic waste products removed in the purification process are lactic acid and ammonia.

9. The method according to any one of claims 5 to 8, wherein the purification process is carried out by ultrafiltration.

10. The method according to claim 9, wherein ultrafiltration is performed using a filtration membrane having a molecular weight cutoff of 2 kDa to 30 kDa.

Images