Stem cell filtrate formulation and method for preparing the same

Abstract

To provide a stem cell filtrate preparation useful for hair regeneration, alleviation of pain caused by a disease of an interosseous joint, such as osteoarthrosis or rheumatoid arthritis, and treatment of the disease, treatment of adipose tissue or skin cells, and in addition, restoration of a damaged muscle, and prevention or treatment of decubitus.SOLUTION: A stem cell filtrate preparation according to one aspect of the present invention includes a filtrate of a stem cell from which a cell membrane is removed. The stem cell may be at least one selected from an adipose-tissue-derived mesenchymal stem cell, an epithelial stem cell, a pigmented stem cell, an iPS cell, and an ES cell. Furthermore, the stem cell filtrate preparation may include an adipocyte or an effective component extracted from cellular fat.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a stem cell filtrate preparation that is useful for hair regeneration, relieving pain caused by diseases of the interosseous junctions such as osteoarthritis and rheumatoid arthritis and treating such diseases, treating adipose tissue or skin cells, and further for repairing damaged muscles and preventing or treating bedsores. [Background technology]

[0002] In recent years, many people, regardless of gender, suffer from thinning hair and hair loss due to changes in the living environment, imbalances in hormone secretion caused by stress, etc. Therefore, expectations and social demands for the development of hair restorers that are effective in promoting hair growth, hair growth, and hair loss prevention are increasing. Conventionally, various hair restorers containing low molecular weight compounds, plant extracts, fermented plant materials, etc. have been reported (see, for example, Patent Document 1 (JP Patent Publication No. 2011-132812 A) and Patent Document 2 (JP Patent Publication No. 2010-265252 A)). In recent years, hair restorers containing minoxidil, capronium chloride, trans-3,4'-dimethyl-3-hydroxyflavanone, nicotinamide, piroctone olamine, adenosine, etc. have been put to practical use. However, conventional hair restorers only promote the proliferation and activation of hair papilla cells, and their effects are said to be limited and largely depend on individual differences.

[0003] Meanwhile, in recent years, with the advancement of regenerative medicine technology using pluripotent stem cells, research into hair regenerative medicine has also been actively conducted. For example, it has been reported that hair follicles, the tissue that produces hair, have been partially regenerated using induced pluripotent stem cells (iPS cells). It has also been reported that hair follicles can be regenerated by artificially creating hair follicle primordia from hair follicle stem cells and transplanting them into the skin. It has also been known that adipose tissue and preadipocytes were cultured, and the medium recovered from the culture solution containing these cells was tested for its growth-promoting activity on isolated human hair follicles, and hair growth was promoted (see Patent Document 3 (JP Patent Publication No. 2005-519591)).

[0004] When using a medium collected from a culture solution containing such adipose tissue and preadipocytes (adipose stem cells), it is necessary to culture tissue collected from the patient himself, so it takes a long time to obtain a specific cell preparation for hair growth. In response to this, the present inventor has disclosed an invention of a cell preparation for hair regeneration that does not require culture and contains adipose tissue-derived mesenchymal stem cells and adipocytes collected from adipose tissue (see Patent Document 4 (International Publication WO2014 / 178438)). This cell preparation for hair regeneration is a state in which adipose tissue-derived mesenchymal stem cells coexist with adipocytes, and when this is transplanted into a site where hair growth is desired, hair growth is observed at the site and hair can be regenerated.

[0005] Furthermore, hyaluronic acid injection, fat cell transplantation, etc. are known as means for increasing the volume of human soft tissues, and are widely used in the fields of cosmetic surgery and plastic surgery, such as facial wrinkle removal, depression treatment, and breast reconstruction. However, while hyaluronic acid injection treatment is easy, it has a problem that the injected hyaluronic acid is absorbed in about half a year, so the effect is not sustained. In addition, fat cell transplantation may cause necrosis or absorption in the body after transplantation. Therefore, there are disadvantages such as the need for reoperation and, in some cases, scarring.

[0006] In order to eliminate these drawbacks, transplantation of adipose stem cells has been proposed, and a method for efficiently isolating adipose stem cells has been developed (see, for example, Patent Document 5 (JP Patent Publication No. 2005-519883) and Patent Document 6 (JP Patent Publication No. 2007-509601)). By transplanting adipose stem cells, the rate of cell settlement after transplantation has been improved, and a certain effect on soft tissue regeneration has been observed. However, adipose stem cells obtained by such a method may contain impurities, which may cause necrosis of adipose cells or the formation of lumps (calcification) after transplantation. Adipose cells are generally obtained by liposuction, and the aspirated fat is a mixture containing various impurities in addition to adipose cells and adipose stem cells. Impurities to be removed from such a mixture include blood (especially red blood cells), dead cells, senescent cells, etc.

[0007] In response to this, a method has been proposed in which a mixture containing fat cells and adipose stem cells collected by liposuction is centrifuged using a syringe equipped with a weight filter as described in Patent Document 7 (JP Patent Publication 2007-533396) to remove impurities that cause calcification and fat necrosis, to prepare a cell preparation (Condensed Rich Fat: CRF (registered trademark)) containing healthy concentrated fat cells and adipose stem cells, and then this cell preparation is injected. This method improves the rate of cell attachment and is less likely to cause problems such as lumps, and is therefore becoming widely accepted.

[0008] Thus, various techniques for separating and concentrating adipocytes and adipose stem cells have been developed, and safe and highly fixed adipocytes and adipose stem cells are being provided. Furthermore, as shown in Patent Document 8 (Patent Publication No. 5572777), cell preparations containing these adipocytes and adipose stem cells are being used as cell preparations useful for relieving pain caused by diseases of bone junctions such as osteoarthritis and for treating said diseases, and as cell filtrate preparations useful for repairing damaged muscles. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] JP 2011-132812 A [Patent Document 2] JP 2010-265252 A [Patent Document 3] Special Publication No. 2005-519591 [Patent Document 4] International Publication No. WO2014 / 178438 [Patent Document 5] Special Publication No. 2005-519883 [Patent Document 6] Special Publication No. 2007-509601 [Patent Document 7] Special Publication No. 2007-533396 [Patent Document 8] Patent No. 5572777 Summary of the Invention [Problem to be solved by the invention]

[0010] According to the above-mentioned cell preparation for hair regeneration or cell preparation useful for relieving pain caused by diseases of bone joints such as osteoarthritis and treating said diseases, it is possible to obtain a desired therapeutic effect without performing a culture process by only collecting adipose tissue-derived mesenchymal stem cells and adipocytes from the collected crude adipose tissue and allowing them to coexist. However, since the stem cells in these cell preparations are living cells that can further differentiate, when these cell preparations are transplanted to a specific site, the cells may proliferate or differentiate on the spot, which may have a negative effect on the transplant site. In addition, there are various legal restrictions on the use of stem cells, which means that they cannot be used freely for treatment.

[0011] The inventors have conducted extensive research to solve the problems that arise when using cell preparations containing such stem cells, and have discovered that a method of using the contents of stem cells (hereinafter referred to as the "filtrate"; in this state, the stem cells are no longer stem cells) by removing the cell membranes of the stem cells (hereinafter referred to as "destroying the stem cells") contains components secreted from the stem cells that are effective for hair regeneration or the treatment of certain diseases as described above, and thus is effective for hair regeneration or the treatment of certain diseases, thereby completing the present invention.

[0012] In other words, another object of the present invention is to provide a stem cell filtrate preparation capable of regenerating hair, which uses a stem cell filtrate preparation from which the cell membrane of stem cells has been removed, a stem cell filtrate preparation that is useful for relieving pain caused by diseases of the interosseous junctions such as osteoarthritis and for treating such diseases, and further a stem cell filtrate preparation that is useful for repairing damaged muscles and preventing or treating bedsores. [Means for solving the problem]

[0013] The stem cell filtrate preparation according to the first aspect of the present invention is characterized in that it contains a filtrate of stem cells from which the cell membrane has been removed.

[0014] The filtrate obtained by removing the cell membrane of the stem cells, i.e., the stem cell filtrate, contains ingredients secreted from the stem cells that are effective for alleviating pain caused by diseases of the interosseous junction such as hair regeneration or osteoarthritis, treating the diseases, repairing damaged muscles, and preventing or treating bedsores. The stem cell filtrate preparation of the first aspect of the present invention contains such a stem cell filtrate, and therefore can be used to provide good effects when used for alleviating pain caused by diseases of the interosseous junction such as hair regeneration or osteoarthritis, treating the diseases, treating adipose tissue or skin cells, repairing damaged muscles, and preventing or treating bedsores.

[0015] In addition, the stem cell filtrate preparation of the first aspect of the present invention does not contain living stem cells, so that even if transplanted to a specific site, the stem cells will not proliferate or differentiate, and therefore will not have any adverse effect on the transplant site. In addition, although there are various legal restrictions on the use of stem cells, the stem cell filtrate preparation of the first aspect of the present invention does not contain living stem cells, so it is not subject to legal restrictions and can be freely used for various treatments.

[0016] In addition, in the stem cell filtrate preparation of this embodiment, conventionally known pharma- ceutically acceptable carriers, excipients, anti-inflammatory agents, analgesics, immunosuppressants, etc. may be added as necessary. Furthermore, collagen, fibroblasts, growth factors, cytokines, etc. may be added within a range that does not impair the effect of the stem cell filtrate preparation of this embodiment, and other cell preparations known to have tissue regeneration effects, such as PRP (Platelet Rich Plasma), may also be used in combination.

[0017] In the stem cell filtrate preparation of this embodiment, the stem cells can be at least one type selected from adipose tissue-derived mesenchymal stem cells, epithelial stem cells, pigmented stem cells, iPS cells (induced pluripotent stem cells), and ES cells (embryonic stem cells).

[0018] These various stem cells are known to have therapeutic effects on various diseases corresponding to each of them, and the stem cell filtrate preparation of this embodiment can be effective in treating various diseases corresponding to each of the stem cells.

[0019] Also, at least two types of mesenchymal stem cells, epithelial stem cells, pigmented stem cells, iPS cells, and ES cells derived from adipose tissue may be mixed. In the case of filtrates of iPS cells or ES cells, a sufficient effect can be obtained with one type, but an even better effect can be obtained by adding and mixing filtrates of mesenchymal stem cells, epithelial stem cells, and pigmented stem cells derived from adipose tissue. In addition, in the case of only filtrates of mesenchymal stem cells, epithelial stem cells, and pigmented stem cells derived from adipose tissue, a combination of two or more types of these is highly effective, and a combination of three types is even more effective.

[0020] In addition, the stem cell filtrate preparation of this embodiment may further contain active ingredients extracted from adipocytes or cellular fat.When the preparation contains not only stem cell filtrate but also active ingredients extracted from adipocytes or cellular fat, in addition to the various therapeutic effects of the stem cell filtrate itself, the effect based on the coexistence of active ingredients extracted from adipocytes or cellular fat is improved.In particular, it can be effective for hair regeneration, for relieving pain caused by bone junction diseases such as osteoarthritis and for treating said diseases, for repairing damaged muscles, and for preventing or treating bedsores.

[0021] Moreover, when the preparation contains fat cells or ECM (extracellular matrix) present around the fat cells, the slippage of the joints is improved, and joint pain can be alleviated early, and pain caused by diseases of the interosseous joint is alleviated, while the stem cell filtrate promotes the regeneration of bone tissue, cartilage tissue, soft tissue, and other tissues in the joints. In addition, by injecting the stem cell filtrate preparation of this type into the intervertebral disc, it not only acts as a cushion and improves (improves) the movement of the vertebral body, but also has a long-lasting effect of alleviating joint pain. In addition, when fat cells and stem cell filtrate are used in combination, the stem cell filtrate is retained in the affected area by the action of the fat cells, and the engraftment efficiency and tissue regeneration efficiency of the stem cell filtrate are further promoted, making it even more effective in treating diseases of the interosseous joint.

[0022] Furthermore, the combination of fat cells and stem cell filtrate has an excellent repair effect, especially on muscles damaged by muscle degeneration or surgical muscle resection, and can quickly relieve pain caused by muscle damage, thereby contributing to improving the patient's quality of life (QOL). In addition, the addition of active ingredients extracted from fat cells, such as the inflammatory cytokine IL-1β, to stem cell filtrate suppresses the expression of IL-1β-induced protease in chondrocytes and protects cartilage tissue.

[0023] Furthermore, the method for preparing a stem cell filtrate preparation according to the second aspect of the present invention is characterized by comprising the following steps (1) and (2): (1) a step of destroying the cell membrane of a stem cell and obtaining components consisting of the destroyed cell membrane, the cell nucleus, and the cytoplasm; (2) A process of removing cell membranes from the component obtained in the process (1) and collecting a stem cell filtrate.

[0024] In the method for preparing a stem cell filtrate preparation of this embodiment, at least one type of stem cell selected from adipose tissue-derived mesenchymal stem cells, epithelial stem cells, pigmented stem cells, iPS cells (induced pluripotent stem cells), and ES cells (embryonic stem cells) can be used as the stem cells. In addition, at least two types of adipose tissue-derived mesenchymal stem cells, epithelial stem cells, and pigmented stem cells may be mixed. In the case of adipose tissue-derived mesenchymal stem cells, epithelial stem cells, and pigmented stem cells, the combination of two or more types of these is highly effective, and the combination of three types is even more effective. In the case of iPS cell or ES cell filtrate, even one type can be sufficiently effective.

[0025] Furthermore, in the method for preparing a stem cell filtrate preparation of this embodiment, the step (1) may be a step of irradiating adipose tissue removed from the body or adipose tissue within the body with laser light or ultrasound to destroy the cell membrane of mesenchymal stem cells within the adipose tissue.

[0026] Furthermore, in the method for preparing a stem cell filtrate preparation of this embodiment, an active ingredient extracted from adipocytes or cellular fat may be further added to the stem cell filtrate obtained in step (2) above.

[0027] According to the method for preparing a stem cell filtrate preparation of the second aspect of the present invention, the stem cell filtrate preparation of the first aspect of the present invention can be easily prepared. Effect of the Invention

[0028] As described above, the stem cell filtrate preparation of the present invention contains stem cell contents, and therefore can be used to alleviate pain caused by diseases of bone junctions such as hair regeneration or osteoarthritis, and to treat the diseases, as well as to repair damaged muscles and prevent or treat bedsores. Moreover, since it does not contain living stem cells, the stem cells do not proliferate or differentiate even when transplanted to a specific site, so there is no adverse effect on the transplant site. In addition, although there are various legal restrictions on the use of stem cells, the stem cell filtrate preparation of the present invention does not contain living stem cells, so it is not subject to legal restrictions and can be freely used for various treatments. Furthermore, according to the preparation method of the stem cell filtrate preparation of the present invention, it is possible to easily prepare a stem cell filtrate preparation that has the effect of alleviating pain caused by diseases of bone junctions such as hair regeneration or osteoarthritis, and to treat the diseases, as well as to repair damaged muscles and prevent or treat bedsores. [Brief description of the drawings]

[0029] [Figure 1] This shows the results of observing the hair growth condition on the forehead of subject A before and after administration of a stem cell filtrate preparation of the present invention. [Diagram 2] This shows the results of observing the hair growth condition on the forehead of subject B before and after administration of the stem cell filtrate preparation of the present invention. [Diagram 3] Figure 3A is an X-ray photograph showing the right knee joint of subject C before administration of the stem cell filtrate preparation, and Figure 3B is an X-ray photograph showing the right knee joint of subject C approximately two months after administration of the stem cell filtrate preparation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The stem cell filtrate preparation and its preparation method according to the present invention will be described in detail below using various embodiments. However, the various embodiments shown below are examples for embodying the technical idea of ​​the present invention, and are not intended to limit the present invention to these embodiments. The present invention can be equally applied to other embodiments included in the scope of the claims.

[0031] [Embodiment 1] In embodiment 1, a stem cell filtrate preparation consisting of a mixture of adipose tissue-derived stem cell filtrate and adipose cells is prepared. The stem cell filtrate preparation of embodiment 1 is suitable for hair regeneration. Note that adipose tissue-derived mesenchymal stem cells are stem cells that can mainly differentiate into adipose tissue, muscle cells, bone, cartilage, ligament, skin cells, and nerve cells.

[0032] The adipose tissue-derived mesenchymal stem cells used in the first embodiment may be those collected from adipose tissue collected from a living body, or those contained in the adipose tissue of a living body may be used as they are. In particular, adipose tissue-derived mesenchymal stem cells present in subcutaneous adipose tissue can be collected easily with relatively low impact, and can be collected in relatively large quantities. They can also be collected simultaneously with fat cells, so that the stem cell filtrate preparation of the first embodiment can be prepared by destroying the adipose tissue-derived mesenchymal stem cell membrane and filtering, and mixing the collected adipose tissue as it is. In addition, in the stem cell filtrate preparation of the first embodiment, the more filtrate components corresponding to adipose tissue-derived mesenchymal stem cells than fat cells, the better the therapeutic effect.

[0033] In addition, fat cells are cells that have the function of synthesizing, storing, and releasing fat, and form adipose tissue. In addition, fat cells do not undergo cell division because they are fully differentiated. There are white fat cells and brown fat cells, and either of these may be used, or a mixture of both may be used. As fat cells, fat cells in an isolated state may be used, or a mixture of fat cells and extracellular matrix (ECM) present around the fat cells may be used. Specifically, adipose tissue collected as fat cells may be used as it is, or adipose tissue from which the extracellular matrix has been removed may be used.

[0034] Furthermore, the concentration of the filtrate components corresponding to adipose tissue-derived mesenchymal stem cells in the stem cell filtrate preparation of embodiment 1 is not particularly limited, but from the viewpoint of efficiently promoting hair regeneration, for example, a filtrate component concentration corresponding to 10,000 or more adipose tissue-derived mesenchymal stem cells, preferably 100,000 or more, more preferably 150,000 or more, and particularly preferably 150,000 to 200,000, per 1 mL of stem cell filtrate preparation can be mentioned.

[0035] Furthermore, the number of adipocytes in the stem cell filtrate preparation of embodiment 1 is not particularly limited, but from the viewpoint of efficiently promoting hair regeneration, examples include 1,000 or more adipocytes, preferably 5,000 or more, more preferably 10,000 or more, and particularly preferably 10,000 to 20,000 adipocytes per mL of stem cell filtrate preparation, or a filtrate component concentration corresponding thereto.

[0036] In the stem cell filtrate preparation of embodiment 1, the ratio of the number of filtrate components corresponding to adipose tissue-derived mesenchymal stem cells and fat cells is not particularly limited, and it is considered that the more the content of the filtrate components corresponding to adipose tissue-derived mesenchymal stem cells, the more excellent the hair regeneration effect can be obtained. For example, the filtrate components corresponding to adipose tissue-derived mesenchymal stem cells are at least 1-fold, preferably 100-fold, more preferably about 500-1000-fold, per fat cell.

[0037] In the stem cell filtrate preparation of embodiment 1, by using adipose tissue-derived mesenchymal stem cells and adipocytes derived from the patient to be administered for the purpose of autologous transplantation, immune rejection can also be avoided. However, the stem cell filtrate preparation of embodiment 1 is not limited to being prepared using adipose tissue-derived mesenchymal stem cells and adipocytes derived from a person other than the patient to be administered and used for allogeneic transplantation. When using adipose tissue-derived mesenchymal stem cells and adipocytes derived from a person other than the patient to be administered, it is preferable to perform a treatment to remove antigenicity, such as gamma irradiation, on the adipocytes (including the extracellular matrix (ECM) present around the adipocytes) collected from another person before preparing the filtrate. In addition, although adipose tissue-derived mesenchymal stem cells are usually non-immunogenic cells, those subjected to the above treatment may be used as necessary. Furthermore, adipose tissue-derived mesenchymal stem cells can be cultured, and a stem cell filtrate preparation can be prepared from the cultured adipose tissue-derived mesenchymal stem cells.

[0038] Furthermore, since adipose tissue typically contains adipose tissue-derived mesenchymal stem cells and adipocytes, the stem cell filtrate preparation of embodiment 1 may be prepared using the harvested adipose tissue as is, or may be prepared using the harvested adipose tissue from which impurities (for example, the anesthetic liquid injected during fat harvesting (so-called tumescent fluid), aged adipocytes, blood, tissue fluid, etc.) have been removed, or the harvested adipose tissue from which impurities and some adipocytes have been removed, etc.

[0039] <Other ingredients> Furthermore, conventionally known pharma- ceutically acceptable carriers, excipients, anti-inflammatory agents, analgesics, immunosuppressants, etc. may be added to the stem cell filtrate preparation of embodiment 1 as necessary. Furthermore, collagen, fibroblasts, growth factors, proliferation factors, cytokines, etc. may be added within a range that does not impair the effects of the stem cell filtrate preparation of embodiment 1.

[0040] In addition, other cell preparations known to have tissue regeneration effects, such as PRP (Platelet Rich Plasma), may be used in combination with the stem cell filtrate preparation of embodiment 1, so long as the effect of the stem cell filtrate preparation of embodiment 1 is not impaired. However, from the viewpoint of further increasing the efficiency of hair regeneration, it is preferable to use the preparation after removing transforming growth factor β1 (TGFβ1).

[0041] <Method of preparing stem cell filtrate preparation of Experimental Example 1> The stem cell filtrate preparation of embodiment 1 contains a liquid fraction obtained by filtering and removing solid components from a liquid component obtained from adipose tissue-derived mesenchymal stem cells having a disrupted cell membrane or iPS cells having a disrupted cell membrane. Therefore, the following methods (a) to (c) can be used to prepare the stem cell filtrate of embodiment 1.

[0042] <Method (a)> (1a) removing a liquid fraction from adipose tissue collected from a body and obtaining a cellular fraction; (2a) separating adipose tissue-derived mesenchymal stem cells and adipocytes from at least a portion of the cell fraction obtained in the step (1a); (3a) a step of irradiating the separated adipose tissue-derived mesenchymal stem cells with laser light or ultrasound to destroy the cell membrane of the adipose tissue-derived mesenchymal stem cells to obtain a liquid component; (4a) filtering the liquid component obtained in the step (3a) to remove solid components and obtain a filtrate of adipose tissue-derived mesenchymal stem cells from which cell membranes have been removed; and (5a) mixing a portion of the adipocytes obtained in the step (2a) with the filtrate of the adipose tissue-derived mesenchymal stem cell filtrate obtained in the step (4a).

[0043] <Method (b)> (1b) a step of obtaining adipose tissue-derived mesenchymal stem cells in a state in which the cell membrane is destroyed together with a cell fraction containing adipocytes while irradiating adipose tissue in vivo with laser light using a Lipolaser (registered trademark name) or the like; (2b) a step of filtering solid components from the adipose tissue-derived mesenchymal stem cells having their cell membranes destroyed obtained in (1b) above to obtain a filtrate of adipose tissue-derived mesenchymal stem cells from which the cell membranes have been removed; (3b) obtaining adipocytes by separating the adipocytes from the cell fraction obtained in (1b) above; and (4b) mixing the filtrate of the adipose tissue-derived mesenchymal stem cells obtained in the step (2b) with a portion of the adipocytes obtained in the step (3b).

[0044] <Method (c)> (1c) culturing the iPS cells in a culture medium; (2c) removing a liquid fraction from the culture medium of step (1c) to obtain a cell fraction; (3c) isolating iPS cells from at least a portion of the cell fraction obtained in the step (2c); (4c) destroying the cell membrane of the isolated iPS cells by irradiating the cells with laser light or ultrasound to obtain a liquid component; (5c) filtering the liquid component obtained in the step (4c) to remove solid components and obtain a filtrate of adipose tissue-derived mesenchymal stem cells from which cell membranes have been removed; and (6c) adding fat cells to the filtrate obtained in step (5c).

[0045] <Details of method (a)> The method (a) for preparing a stem cell filtrate formulation of embodiment 1 includes: (1a) removing a liquid fraction from adipose tissue collected from a body and obtaining a cellular fraction; (2a) separating adipose tissue-derived mesenchymal stem cells and adipocytes from at least a portion of the cell fraction obtained in the step (1a); (3a) a step of irradiating the separated adipose tissue-derived mesenchymal stem cells with laser light or ultrasound to destroy the cell membrane of the adipose tissue-derived mesenchymal stem cells to obtain a liquid component; (4a) filtering the liquid component obtained in the step (3a) to remove solid components and obtain a filtrate of adipose tissue-derived mesenchymal stem cells from which cell membranes have been removed; and (5a) mixing a portion of the adipocytes obtained in the step (2a) with the filtrate of the adipose tissue-derived mesenchymal stem cell filtrate obtained in the step (4a).

[0046] Here, the collected adipose tissue refers to a mixture of cellular and liquid fractions derived from adipose tissue collected from the adipose tissue by liposuction, lipectomy, delipidation, etc. The cellular fraction includes fat cells, adipose stem cells, and impurities (blood cells, dead cells, senescent cells, etc.). The liquid fraction includes tumescent fluid, cell-tissue fluid, etc.

[0047] The liquid fraction and the cell fraction can be separated simply by leaving the collected fat to settle and separate the cell fraction. The method for removing the liquid fraction is not particularly limited, but examples thereof include decantation, suction, etc. In addition, centrifugation, filtration, etc. may be performed as necessary.

[0048] The conditions for centrifugation and filtration are not particularly limited as long as they do not damage the adipose tissue-derived mesenchymal stem cells and adipocytes and can remove impurities, but examples of such conditions include 700 to 2500 × g for 1 to 15 minutes, and preferably 2000 to 2200 × g for 5 to 10 minutes.

[0049] In addition, when filtration is performed, the collected adipose tissue can be passed through a mesh filter having a pore size of, for example, 10 to 300 μm, preferably 10 to 150 μm, more preferably 10 to 100 μm, and even more preferably 15 to 20 μm, to remove blood, tissue fluid, anesthetics (tumescent fluid) administered to the site where the fat is collected, and the like, to obtain a mixture of adipose tissue-derived mesenchymal stem cells and adipocytes on the filter. In addition, the collected adipose tissue containing impurities can be filled into a container equipped with such a mesh filter and subjected to centrifugation.

[0050] In this way, a cell fraction containing adipose tissue-derived mesenchymal stem cells and adipocytes (including extracellular matrix) from which the liquid fraction has been removed can be obtained. In step (2a), adipose tissue-derived mesenchymal stem cells are separated from at least a portion, preferably half, of the cell fraction obtained in step (1a). Then, in step (3a), the cell membrane of the adipose tissue-derived mesenchymal stem cells obtained in step (2a) is destroyed by irradiating the adipose tissue-derived mesenchymal stem cells with laser light or ultrasound to obtain a liquid component. Next, in step (4a), the liquid component obtained in step (3a) is filtered to remove solid components, and a filtrate of adipose tissue-derived mesenchymal stem cells from which the cell membrane has been removed is obtained. Furthermore, in step (5a), the filtrate of adipose tissue-derived mesenchymal stem cells from which the cell membrane has been removed obtained in step (4a) is added to a portion of the adipocytes obtained in step (2a), and can be used as the stem cell filtrate preparation of embodiment 1.

[0051] After the step (2a), a step of culturing the separated adipose tissue-derived mesenchymal stem cells may be provided.

[0052] In addition, after the step (2a), a step of micronizing the separated fat cells may be included. The micronization of fat cells can be performed by a method commonly employed in the art, and is not particularly limited. For example, the micronization of fat cells can be performed by cutting the separated fat cells with a drill or the like as necessary. Here, the micronized fat cells refer to fat cells having a size that can pass through a 26-30 gauge, preferably 18-30 gauge, injection needle. Such a treatment for micronizing fat cells can be performed at least once, preferably 1 to several times, more preferably 1 to 2 times. By micronizing fat cells, the concentration of fat stem cells per unit volume of the stem cell filtrate preparation can be increased, and not only the hair regeneration effect but also the effect of alleviating pain caused by diseases of the interosseous joints and muscle damage, the effect of tissue regeneration in the interosseous joints, and the effect of muscle repair are more significantly exhibited.

[0053] Here, an example of a method for separating adipose tissue-derived mesenchymal stem cells is the method described in Patent Document 5 (JP Patent Publication No. 2005-519883). More specifically, a protease is added to the cell fraction in order to facilitate separation of adipose tissue-derived mesenchymal stem cells and adipocytes by decomposing the bonds between cells. Examples of protease include collagenase, trypsin, lipase, etc., and one selected from these may be used alone, or two or more may be used in combination.

[0054] The method for separating adipose tissue-derived mesenchymal stem cells and adipocytes from the enzyme-treated cell fraction is not particularly limited and may be appropriately selected from conventionally known methods, for example, centrifugation, filtration, etc. The conditions for centrifugation are not particularly limited as long as they can separate adipose tissue-derived mesenchymal stem cells and adipocytes without damaging them, for example, 100 to 500×g for 1 to 20 minutes, preferably 100 to 300×g for 5 to 10 minutes. By subjecting the cells to such centrifugation, adipose tissue-derived mesenchymal stem cells are obtained as a precipitate, and adipocytes are concentrated in the supernatant.

[0055] The adipose tissue-derived mesenchymal stem cells in the precipitate can then be passed through a mesh filter having a hole size of 10 to 100 μm, preferably 10 to 50 μm, and more preferably 15 to 20 μm, to obtain adipose tissue-derived mesenchymal stem cells on the filter, and fat cells can be obtained in the filtrate.

[0056] The enzyme-treated adipose tissue-derived mesenchymal stem cells and adipocytes are preferably subjected to a washing treatment after separation, and washing can be performed with phosphate buffered saline (PBS), physiological saline, Ringer's solution, dextran, etc. The washing treatment may be repeated multiple times as necessary, and when recovering the cells after washing, they may be subjected to centrifugation as necessary. The centrifugation conditions may be the same as those used when separating the adipose tissue-derived mesenchymal stem cells and adipocytes from the enzyme-treated cell fraction.

[0057] Then, the filtrate containing the adipocytes after separation of the adipose tissue-derived mesenchymal stem cells can be filtered using a finer filter to separate the adipocytes on the filter.

[0058] The cell membrane of the adipose tissue-derived mesenchymal stem cells thus separated is destroyed, for example, by laser or ultrasonic method, and then the solid components such as the destroyed cell membrane are filtered out by a filter to obtain a filtrate of adipose tissue-derived mesenchymal stem cells from which the cell membrane has been removed.Then, the filtrate of adipose tissue-derived mesenchymal stem cells from which the cell membrane has been removed is mixed with fat cells in a predetermined ratio to obtain the stem cell filtrate preparation of embodiment 1.

[0059] <Details of method (b)> The method (b) for preparing the stem cell filtrate formulation of embodiment 1 includes: (1b) a step of obtaining adipose tissue-derived mesenchymal stem cells in a state in which the cell membrane is destroyed together with a cell fraction containing adipocytes while irradiating adipose tissue in vivo with laser light using a lipolase laser or the like; (2b) a step of filtering solid components from the adipose tissue-derived mesenchymal stem cells having their cell membranes destroyed obtained in (1b) above to obtain a filtrate of adipose tissue-derived mesenchymal stem cells from which the cell membranes have been removed; (3b) obtaining adipocytes by separating the adipocytes from the cell fraction obtained in (1b) above; and (4b) mixing the filtrate of the adipose tissue-derived mesenchymal stem cells obtained in the step (2b) with a portion of the adipocytes obtained in the step (3b).

[0060] When a cell fraction is extracted while irradiating laser light from within the body using a lipolaser, adipose tissue-derived mesenchymal stem cells with destroyed cell membranes are obtained together with the cell fraction.

[0061] In step (2b), the solid components are filtered from the adipose tissue-derived mesenchymal stem cells in a state in which the cell membrane is destroyed obtained in step (1b) to obtain a filtrate of adipose tissue-derived mesenchymal stem cells from which the cell membrane has been removed. Then, the filtrate of adipose tissue-derived mesenchymal stem cells obtained in step (2b) is added to a portion of the adipocytes obtained in step (1b), and the filtrate can be used as the stem cell filtrate preparation of embodiment 1.

[0062] The conditions for centrifugation are not particularly limited as long as the filtrate can be separated, but examples of the conditions include 700 to 2500×g for 1 to 15 minutes, and preferably 2000 to 2200×g for 5 to 10 minutes.

[0063] In addition, after step (1b), a step of micronizing the adipocytes separated from the cell fraction may be included. The micronization of the adipocytes can be performed by a method commonly employed in the art, and is not particularly limited. For example, the micronization of the adipocytes can be performed by cutting the separated adipocytes with a drill or the like as necessary. Here, the micronized adipocytes refer to adipocytes having a size that can pass through a 26-30 gauge, preferably 18-30 gauge, injection needle. Such a treatment for micronizing the adipocytes can be performed at least once, preferably 1 to several times, more preferably 1 to 2 times. By micronizing the adipocytes, the concentration of adipose stem cells per unit volume of the stem cell filtrate preparation can be increased, and not only the hair regeneration effect but also the effect of alleviating pain caused by diseases of the interosseous joints and muscle damage, the effect of tissue regeneration in the interosseous joints, and the effect of muscle repair are more significantly exhibited.

[0064] In step (3b), adipocytes are obtained by separation from the cell fraction obtained in step (1b), which is similar to step (2a).

[0065] In step (4b), the stem cell filtrate preparation of embodiment 1 can be obtained by mixing the filtrate of adipose tissue-derived mesenchymal stem cells obtained in step (2b) with a portion of the adipocytes obtained in step (3b).

[0066] <Details of method (c)> Method (c) is (1c) culturing the iPS cells in a culture medium; (2c) removing a liquid fraction from the culture medium of step (1c) to obtain a cell fraction; (3c) isolating iPS cells from at least a portion of the cell fraction obtained in the step (2c); (4c) destroying the cell membrane of the isolated iPS cells by irradiating the cells with laser light or ultrasound to obtain a liquid component; (5c) filtering the liquid component obtained in the step (4c) to remove solid components and obtain a filtrate of adipose tissue-derived mesenchymal stem cells from which cell membranes have been removed; and (6c) adding fat cells to the filtrate obtained in the step (5c).

[0067] In step (1c), iPS cells are cultured in a culture medium, and in step (2c), a liquid fraction is removed from the culture medium of step (1c) to obtain a cell fraction. In step (3c), iPS cells are separated from at least a part of the cell fraction obtained in step (2c). In step (4c), the iPS cells obtained in step (3c) are irradiated with laser light or ultrasound to destroy the cell membrane of the iPS cells to obtain a liquid component. Next, in step (5c), the liquid component obtained in step (4c) is filtered to remove solid components, and a filtrate of adipose tissue-derived mesenchymal stem cells from which the cell membrane has been removed is obtained. Furthermore, in step (6c), fat cells are added to the filtrate obtained in step (5c), which can be used as the stem cell filtrate preparation of embodiment 1.

[0068] Here, the adipocytes added in step (6c) may be the adipocytes separated in step (2a) or step (3b). Alternatively, step (6c) may be omitted, and only the iPS cell filtrate in step (5c) may be used as the stem cell filtrate preparation of embodiment 1.

[0069] In the methods (a) to (c), examples of adding fat cells to the stem cell filtrate have been described, but instead of fat cells, it is also possible to add active ingredients extracted from fat cells. For example, anti-IL-1β antibodies, which are anti-inflammatory cytokines, are active ingredients extracted from fat cells and added to the stem cell filtrate, and they have the effect of suppressing the expression of IL-1β-induced protease in chondrocytes and protecting cartilage tissue.

[0070] <Application and administration method> The stem cell filtrate preparation of embodiment 1 can be administered to the dermis or subcutaneous tissue of the site where hair regeneration is required, thereby promoting hair growth at the site and regenerating hair.The stem cell filtrate preparation of embodiment 1 can improve thinning hair caused by diffuse alopecia, postpartum alopecia, androgenetic alopecia, seborrheic alopecia, senile alopecia, alopecia areata, cicatricial alopecia, trichotillomania caused by mental illness, etc.

[0071] The dosage of the stem cell filtrate preparation of embodiment 1 is appropriately set according to the amount of hair at the administration site, the sex and physique of the patient, and the filtrate concentration of the adipose tissue-derived mesenchymal stem cells or iPS cells is not particularly limited, but from the viewpoint of efficiently promoting hair regeneration, for example, in terms of the number of adipose tissue-derived mesenchymal stem cells or iPS cells contained per 1 mL of the stem cell filtrate preparation of embodiment 1, 10,000 or more, preferably 100,000 or more, more preferably 150,000 or more, and particularly preferably 150,000 to 200,000 filtrates can be mentioned. Similarly, in terms of the number of fat cells contained per 1 mL of the stem cell filtrate preparation of embodiment 1, the number of fat cells can be about 1,000 to 100,000, preferably about 1,000 to 50,000, and more preferably about 5,000 to 10,000. The dosage of the stem cell filtrate preparation can be, for example, 0.1 to 1.0 mL, preferably 0.1 to 0.5 mL, and more preferably 0.2 to 0.5 mL per 1 cm2 of the administration site.

[0072] The method of administering the stem cell filtrate preparation of embodiment 1 to the site where hair regeneration is required is not particularly limited, and any conventionally known administration method can be adopted, for example, a method of injecting with a syringe, a cannula, etc. Although the hair regeneration effect of the stem cell filtrate preparation of embodiment 1 varies from person to person, hair growth is usually observed about 2 to 6 weeks after administration, and hair is regenerated at the site.

[0073] <Destruction of the cell membrane of adipose tissue-derived mesenchymal stem cells> In preparing the stem cell filtrate preparation of embodiment 1, it is preferable to use laser light or ultrasound to destroy the cell membrane of the adipose tissue-derived mesenchymal stem cells. The laser light is usually irradiated to a specific adipose tissue by an optical fiber, and the cell membrane of the adipose tissue-derived mesenchymal stem cells is destroyed. In this case, if a laser light irradiation device known as a lipo laser is used, a wide range of adipose tissue can be destroyed at once. A normal laser light irradiation device irradiates laser light in a linear direction from the irradiation end of an optical fiber, but a lipo laser light irradiation device irradiates laser light from the irradiation end of an optical fiber in a diffuse direction of a solid angle of 360 degrees.

[0074] In addition, the destruction of the cell membrane of adipose tissue-derived mesenchymal stem cells by ultrasonic waves can be performed using a well-known ultrasonic homogenizer. The destruction of the cell membrane by ultrasonic waves is performed by generating and bursting extremely small bubbles using mechanical energy from a probe, which repeatedly shocks the cells and breaks them down. Since heat is easily generated during the process, it is preferable to irradiate in pulse mode.

[0075] Regardless of which laser light irradiation device or ultrasonic homogenizer is used, the cell membrane of the adipose tissue-derived mesenchymal stem cell can be destroyed by directly irradiating the adipose tissue or adipose tissue-derived mesenchymal stem cell extracted from a living body, or the adipose tissue in a living body, with laser light or ultrasonic wave. In particular, the method of directly irradiating the adipose tissue in a living body with laser light is widely adopted in liposuction, and in this case, a mixture of a cell fraction and a liquid fraction containing adipose tissue-derived mesenchymal stem cells whose cell membranes have been destroyed can be obtained directly from the living body.

[0076] In addition, the well-known ultrasonic aspiration method can be used to directly collect adipose tissue-derived mesenchymal stem cells and adipocytes from living tissue, but since the cell membranes of the collected adipose tissue-derived mesenchymal stem cells and adipocytes are not destroyed, it is necessary to separately adopt the above-mentioned cell membrane destruction method using laser light or ultrasound. Note that, as a method for destroying the cell membrane, an example using a laser light irradiation device or an ultrasonic homogenizer has been described, but the method is not particularly limited as long as it is a device that irradiates laser light or ultrasound. In addition, the method for destroying the cell membrane is not limited to the use of a device that irradiates laser light or ultrasound, and the present invention also includes a method for destroying the cell membrane by repeating freezing and thawing one or more times, and other methods for destroying the cell membrane.

[0077] [Test Example 1] The action and effect of the stem cell filtrate preparation of embodiment 1 will be specifically explained below using Test Example 1, but the stem cell filtrate preparation of embodiment 1 should not be interpreted as being limited to these experimental examples.

[0078] (1) Preparation of stem cell filtrate preparation The stem cell filtrate preparation was prepared using the LIPOMAX-SC kit (Medikan Corp., Seoul, Korea). The preparation process for this stem cell filtrate preparation could be carried out without exposing the cells to the outside air. The specific method is as follows.

[0079] Liposuction was performed on each subject using a syringe (a syringe manufactured for LIPOMAX-SC: manufactured by Medikan Corp., Seoul, Korea). The syringe containing the collected fat was left to stand at room temperature (about 25°C) for about 10 minutes to separate it into a cell fraction and a liquid fraction, and the liquid fraction containing tumescent fluid was discarded. The syringe was then centrifuged at 2200 x g for 8 minutes. The centrifugation separated the fat into three layers: an upper layer (free oil), a middle layer (adipose tissue-derived mesenchymal stem cells and adipocytes), and a lower layer (liquid fraction such as cell tissue fluid). Then, only the middle layer was left in the syringe, and the upper and lower layers were discarded, thereby obtaining a concentrate of adipose tissue-derived mesenchymal stem cells and adipocytes. Next, the concentrate was separated into adipose tissue-derived mesenchymal stem cells and adipocytes.

[0080] Next, the fat cells were subjected to gelling treatment to break down relatively large fat cells. The gelling treatment was performed 1 to 3 times. For gelling, a drill (Filler Geller cutter: manufactured by MEDIKAN) was used to cut and break down the fat cells into small pieces, which were then centrifuged at 2200 x g for 5 minutes to obtain small fat cells.

[0081] The adipose tissue-derived mesenchymal stem cells were sucked into the same syringe as described above, and the cell membrane of the adipose tissue-derived mesenchymal stem cells was destroyed by irradiating the lipo-laser light in the syringe. The syringe was then centrifuged at 2200×g for 8 minutes. The centrifugation separated the cells into three layers: an upper layer (free oil), a middle layer (solid components such as destroyed cell membranes), and a lower layer (adipose tissue-derived mesenchymal stem cell filtrate). The filtrate was then collected, and the filtrate was added with micronized adipocytes, and administered to the affected area as the stem cell filtrate preparation of Test Example 1.

[0082] Since the fat cells and fat tissue-derived mesenchymal stem cells were derived from each patient, the above-mentioned procedure was performed for each patient. The number of cells corresponding to the filtrate components corresponding to fat tissue-derived mesenchymal stem cells and fat cells in the obtained stem cell filtrate preparation varied depending on the patient, but 1 mL of stem cell filtrate preparation contained filtrate components corresponding to approximately 150,000 to 200,000 fat tissue-derived mesenchymal stem cells and filtrate components corresponding to approximately 10,000 to 20,000 fat cells.

[0083] (2) Confirmation of hair regeneration effect Two subjects with thinning hair on the forehead due to alopecia, etc. were given 2.5 mL of the stem cell filtrate preparation obtained above, which was injected subcutaneously using a syringe into the right forehead area (approximately 12.5 cm2) where the hair was thinning.

[0084] The results of observing hair growth at the administration site 2 to 3 months after administration of the stem cell filtrate preparation for subjects A and B are shown in Figures 1 and 2, respectively. These results confirmed that the stem cell filtrate preparation containing adipose tissue-derived mesenchymal stem cells and adipocytes has the effect of promoting hair growth and regenerating hair in areas of thinning hair, as well as the therapeutic effect of adipose tissue or skin cells.

[0085] [Embodiment 2] In embodiment 2, the stem cell filtrate preparation containing adipose tissue-derived mesenchymal stem cell filtrate and adipocytes prepared as described above in (c) was used as a stem cell filtrate preparation for treating diseases of bone junctions. In addition, the stem cell filtrate preparation used in embodiment 2 may also be used in combination with other cell filtrate preparations known to have tissue regeneration effects, such as PRP (Platelet Rich Plasma), and further, the regeneration effect of cartilage tissue can be further enhanced by adding an angiotensin II receptor antagonist such as losartan to PRP. However, from the viewpoint of improving tissue regeneration efficiency, it is preferable to remove VEGF (vascular endothelial growth factor) from PRP when cartilage tissue regeneration is expected, and to remove BMPs (bone morphogenetic proteins) when bone regeneration is expected.

[0086] The stem cell filtrate preparation of embodiment 2 also has an excellent therapeutic effect on diseases of interosseous joints. Interosseous joints refer to the joint parts of bones, including cartilaginous joints connected by cartilage or a combination of cartilage and fibrous connective tissue, synovial joints connected by cartilage and the synovial membrane surrounding the joint capsule, and fibrous joints connected by fibrous connective tissue, but here, it particularly refers to the part connected by cartilaginous joints and synovial joints that have mobility. Specific examples of interosseous joints include spinal joints, knee joints, ankle joints, toe joints, hip joints, wrist joints, elbow joints, shoulder joints, finger joints, acromioclavicular joints, and sacroiliac joints. In addition, for example, in the case of knee joints, soft tissues such as ligaments and meniscus, and in the case of spinal joints, disks (intervertebral disks), etc. are also included in the interosseous joints.

[0087] Furthermore, examples of diseases of interosseous junctions include herniation (prolapse), deformation, degeneration, inflammation, etc., and specific examples include spinal diseases such as herniated disc and osteoarthritis of the spine; joint diseases such as osteoarthritis (including osteoarthritis of the hip joint and osteoarthritis of the knee) and chronic arthritis; autoimmune diseases such as rheumatoid arthritis; and osteonecrosis such as idiopathic osteonecrosis of the knee and necrosis of the femoral head, and preferably include herniated disc, osteoarthritis, chronic arthropathy, idiopathic osteonecrosis of the knee, and necrosis of the femoral head.

[0088] The content of the filtrate corresponding to adipose tissue-derived mesenchymal stem cells in the stem cell filtrate preparation of embodiment 2 is not particularly limited as long as it is within a range in which a therapeutic effect for diseases of the bone junction can be obtained, and it is considered that the higher the content of the filtrate corresponding to adipose tissue-derived mesenchymal stem cells, the better the therapeutic effect can be obtained. For example, the content of the filtrate corresponding to adipose tissue-derived mesenchymal stem cells per fat cell is at least 1, preferably 5 or more, more preferably 1 to 10, and even more preferably 5 to 10. In addition, it is preferable that the number of adipose tissue-derived mesenchymal stem cells contained per mL of the stem cell filtrate preparation of embodiment 2 is usually 100,000 or more, preferably 150,000 or more, and even more preferably 150,000 to 200,000 filtrate.

[0089] The amount of the stem cell filtrate preparation of the second embodiment administered is not particularly limited as long as it is within a range that provides a therapeutic effect against diseases of the interosseous junction, and can be appropriately set depending on the size of the administration site, the site of the disease, the severity of the disease, the sex and physical build of the patient, and can be 0.5 to 20 mL, preferably 1 to 10 mL, and more preferably 1 to 5 mL. For example, 1 to 20 mL, preferably 1 to 10 mL, and more preferably 5 to 10 mL of the stem cell filtrate preparation of the second embodiment can be administered to the knee joint. For example, 0.5 to 20 mL, preferably 1 to 15 mL, and more preferably 1 to 2 mL of the stem cell filtrate preparation of the second embodiment can be administered to the intervertebral disc.

[0090] Methods of administration include, but are not limited to, injection into diseased areas of interosseous junctions (e.g., joint cavities, intervertebral discs, etc.) using a syringe, cannula, etc. Furthermore, by stimulating the injection site with the tip of the injection needle or cannula during injection, even better therapeutic effects can be obtained. Methods of stimulation are not particularly limited, but include, for example, a method in which a cannula with an obliquely cut tip is rotated back and forth and side to side when injecting the stem cell filtrate to stimulate the injection site.

[0091] When the stem cell filtrate preparation of embodiment 2 is administered to a patient with these diseases, it may be administered after treatment by a known method as necessary. For example, in the case of herniated disc, the herniated disc can be removed by a conventionally known method such as PLDD (Percutaneous Laser Disc Decompression), and then the stem cell filtrate preparation of embodiment 2 can be administered (injected). The action of the fat cells contained in the stem cell filtrate preparation of embodiment 2 relieves pain (joint pain) caused by diseases of the interosseous junction, and further, when the preparation contains mesodermal stem cell filtrate, the action of the cells promotes regeneration of bone tissue, cartilage tissue, soft tissue, etc. at the interosseous junction.

[0092] In addition, in the stem cell filtrate preparation of embodiment 2, the adipocytes obtained by separating the adipose stem cells in step (2c) may be washed and then mixed with the stem cell filtrate in step (5c). The washing can be performed by the same method as the washing treatment performed on the adipose stem cells described above. The adipocytes obtained here are selected and concentrated from healthy micronized adipocytes, so that they have a more remarkable and excellent effect on alleviating pain caused by diseases of the interosseous junction. Here, the use of the stem cell filtrate preparation prepared by the above method (c) is described, but the use of the stem cell filtrate preparation prepared by the above methods (a), (b), (d) and (e) also has the same excellent effect as the above method (c).

[0093] [Test Example 2. Effect of relieving pain caused by osteoarthritis] The stem cell filtrate preparation of the above embodiment 2 is administered to subjects with osteoarthritis of the knee, and the change in symptoms is observed.As a result, the subjects who are injected with the stem cell filtrate preparation of the embodiment 2 have their knee joint pain reduced from the day of injection, and this effect continues for about one month.In addition, compared with the subjects who are injected with hyaluronic acid, the knee joint pain relief effect is clearly manifested early, and the pain relief effect is also significant.

[0094] [Test Example 3. Tissue regeneration effect on osteoarthritis] Subject C Age at time of treatment: 66 years old, male Past medical history: Femoral medial condyle necrosis (right knee) For subject C, the adipose tissue-derived mesenchymal stem cell filtrate from which the cell membrane had been removed and the stem cell filtrate preparation containing adipocytes (5 mL, containing approximately 750,000 to 2,000,000 adipose stem cells) prepared in the above step (c) were injected into the right knee joint using a cannula with an obliquely cut tip. X-ray photographs of the lateral joint space of the left knee joint of subject C taken before and approximately 2 months after the injection of the stem cell filtrate preparation are shown in Figures 3A and 3B, respectively.

[0095] (result) Subject C's knee joint pain improved significantly within 5 days of treatment, and the alleviated pain continued thereafter, and his walking also improved significantly.

[0096] X-ray findings of subject C showed improvement of osteoarthritis and expansion of the joint space. That is, as shown in Figure 3, the joint space of subject C's right knee joint expanded from the state shown in Figure 3A before treatment to the state shown in Figure 3B about one month after treatment, and the symptoms improved.

[0097] The results of Test Example 3 showed that tissue regeneration within the knee joint was promoted by injecting a stem cell filtrate preparation containing a filtrate of stem cells with their cell membranes removed and a filtrate of fat cells into the knee joint of subject C, who had osteoarthritis of the knee.

[0098] [Test Example 4. Tissue regeneration effect on bone necrosis] For subject C in Test Example 3, who had osteoarthritis of the knee as well as osteonecrosis, the stem cell filtrate from which the cell membrane had been removed, prepared in step (c) above, and a stem cell filtrate preparation containing adipocytes (containing 5-10 mL of filtrate equivalent to approximately 750,000-2,000,000 adipocytes) were injected into the site of osteonecrosis.

[0099] In subject C, symptoms such as difficulty walking and pain improved significantly within 1-2 days after the stem cell filtrate preparation was injected into the joint. The joint space improved significantly about 2 months after treatment. Furthermore, a significant improvement in the appearance of bone necrosis was observed 2 months after the stem cell filtrate preparation was injected into the joint of subject C.

[0100] [Test Example 5. Effect of adding anti-IL-1β, an anti-inflammatory cytokine, to stem cell filtrate] In Test Example 5, a stem cell filtrate preparation is used in which anti-IL-1β, an anti-inflammatory cytokine that is one of the active ingredients extracted from fat cells, is added to the stem cell filtrate in place of the adipose tissue-derived mesenchymal stem cell filtrate. When the stem cell filtrate preparation with the anti-IL-1β antibody added is injected into the joint of a subject, it suppresses the expression of IL-1β-induced protease in chondrocytes and protects the cartilage tissue.

[0101] [Embodiment 3. Muscle repair method] In embodiment 3, the same stem cell filtrate preparation as that used in embodiment 2 is administered to the muscle damaged site of a patient with muscle damage. In addition, this muscle repair stem cell filtrate preparation can also be added with conventionally known pharma- ceutically acceptable carriers, excipients, anti-inflammatory agents, analgesics, immunosuppressants, etc., as necessary. In addition, collagen, fibroblasts, growth factors, growth factors, cytokines, etc. may be added within a range that does not impair the effect of the stem cell filtrate preparation of embodiment 3.

[0102] Similarly, as long as the effect of the stem cell filtrate preparation of embodiment 3 is not impaired, other cell filtrate preparations known to have tissue regeneration effects, such as PRP (Platelet Rich Plasma), may be used in combination; however, from the viewpoint of further increasing the efficiency of muscle tissue regeneration, it is preferable to use the preparation after removing transforming growth factor β1 (TGFβ1).

[0103] The stem cell filtrate preparation of embodiment 3 has an excellent muscle repair effect when applied to a muscle damaged site. Muscle damage includes muscle degeneration, muscle fibrosis, destruction or necrosis of muscle fibers, muscle atrophy, muscle paralysis, muscle weakness, etc., as well as muscle resection due to surgery, trauma, etc. The site and type of muscle to which the stem cell filtrate preparation of embodiment 3 is applied are not particularly limited, and examples include skeletal muscles such as trunk muscles (including upper limb or lower limb muscles, pectoral muscles); smooth muscles such as bladder sphincter; and cardiac muscles. For example, by administering the stem cell filtrate preparation of embodiment 3 to a damaged site of the upper limb muscle, pain caused by muscle damage is alleviated early, and the muscle is repaired, thereby expanding the ROM (Range of Motion) of the upper limb and improving motor function.

[0104] In addition, by administering the stem cell filtrate preparation of embodiment 3 to the bladder sphincter of a patient who has urinary incontinence, frequent urination, and other dysuria due to muscle weakness or damage to the bladder sphincter, the muscle strength of the bladder sphincter can be increased to safely and easily resolve problems such as urinary incontinence and urinary leakage. It can also be applied to muscle damaged sites in patients with muscle diseases such as myocardial infarction, ischemia-reperfusion injury, muscular dystrophy (e.g., Duchenne muscular dystrophy (DMD)), and myositis (myopathy).

[0105] In addition, muscle damage is often accompanied by pain, but the administration of the stem cell filtrate preparation of embodiment 3 to the muscle damage site can alleviate pain early. Although it is not intended to limit the interpretation of the present invention, such pain relieving effect is believed to be due to the effect of the stem cell filtrate contained in the stem cell filtrate preparation of embodiment 3 being synergistically exerted by fat cells.

[0106] When using the stem cell filtrate preparation of embodiment 3 for the purpose of muscle repair, the dosage is not particularly limited as long as it is within the range in which the muscle repair effect can be obtained, and can be appropriately set depending on the size of the administration site, the site of the disease, the severity of the disease, the sex and physical constitution of the patient, etc. For example, the dosage per unit volume of 10 mL of damaged muscle can be 100 to 4 million, preferably 200 to 2 million, and more preferably 200 to 1 million filtrates converted into the number of stem cells. In addition, when using a stem cell filtrate preparation obtained by a method including step (b), the dosage can be, for example, 0.5 to 20 mL, preferably 1 to 10 mL, and more preferably 1 to 5 mL per unit volume of 10 mL of damaged muscle.

[0107] When using the stem cell filtrate preparation of the present invention for the purpose of muscle repair, the administration method is not particularly limited, and any conventionally known administration method can be adopted, such as injection into the muscle damage site with a syringe, cannula, etc. In addition, when using a stem cell filtrate preparation prepared by any of the above methods (a) to (e), an excellent muscle repair effect is achieved.

[0108] In the above embodiments 1 to 3, examples using a mixture containing stem cell filtrate and fat cells are shown, but the present invention is not limited to this, and any of the following can be implemented: using only stem cell filtrate, using a mixture containing stem cell filtrate and active ingredients extracted from fat cells, or using multiple types of stem cell filtrate, and a significant therapeutic effect can be obtained similar to that described in embodiments 1 to 3. For example, a significant therapeutic effect can be obtained when only adipose tissue-derived mesenchymal stem cell filtrate is used, or when only iPS cell filtrate is used.

[0109] Here, the stem cell filtrate preparation of the present invention is used for muscle repair, but the stem cell filtrate preparation of the present invention can also be used to prevent or treat bedsores with a remarkable therapeutic effect.For example, to treat bedsores, the stem cell filtrate preparation of the present invention can be applied alone to the site to be treated, or can be used together with a known bedsore treatment agent to protect the affected area.In addition, to prevent bedsores, the stem cell filtrate preparation of the present invention can be applied alone or together with a known moisturizer to the site where bedsores are likely to occur.

[0110] In addition, in the first to third embodiments, an example containing a stem cell filtrate and adipocytes is shown, but the present invention is not limited thereto. Even if only a filtrate from which at least one cell membrane selected from adipose tissue-derived mesenchymal stem cells, epithelial stem cells, pigmented stem cells, iPS cells, and ES cells has been removed, or if the filtrate is a mixture of adipose tissue-derived mesenchymal stem cells, epithelial stem cells, pigmented stem cells, iPS cells, and ES cells, or if the filtrate is a mixture of adipose tissue-derived mesenchymal stem cells, epithelial stem cells, pigmented stem cells, iPS cells, and ES cells, a predetermined therapeutic effect according to the type of stem cells can be obtained. For example, a remarkable therapeutic effect can be obtained even when iPS cells are used. In addition, the mesenchymal stem cells, epithelial stem cells, pigmented stem cells, iPS cells, and ES cells derived from adipose tissue may be cultured. In addition, at least two or more types of mesenchymal stem cells, epithelial stem cells, and pigmented stem cells derived from adipose tissue may be mixed. In the case of adipose tissue-derived mesenchymal stem cells, epithelial stem cells, and pigmented stem cells, a combination of two or more of these is highly effective, and a combination of three of these is even more effective. In the case of the filtrate of iPS cells or ES cells, a sufficient effect can be obtained with just one type of cell, but it is also possible to add active ingredients extracted from adipocytes or cellular fat to this.

Claims

1. A stem cell filtrate preparation, comprising a filtrate of stem cells from which the cell membrane has been removed.

2. The stem cell filtrate preparation according to claim 1, wherein the stem cells are at least one type selected from adipose tissue-derived mesenchymal stem cells, epithelial stem cells, pigmented stem cells, iPS cells, and ES cells.

3. The stem cell filtrate preparation of claim 2, characterized in that it contains at least two types selected from the adipose tissue-derived mesenchymal stem cells, the epithelial stem cells, the pigmented stem cells, the iPS cells, and the ES cells.

4. The stem cell filtrate preparation according to any one of claims 1 to 3, further comprising an active ingredient extracted from adipocytes or cellular fat.

5. The stem cell filtrate preparation according to any one of claims 1 to 4, characterized in that it is used for at least one of hair regeneration, relief of pain caused by diseases of the interosseous junctions such as osteoarthritis and treatment of said diseases, treatment of adipose tissue or skin cells, repair of damaged muscles, and prevention or treatment of bedsores.

6. A method for preparing a stem cell filtrate preparation, comprising the following steps (1) and (2): (1) a step of disrupting the cell membrane of stem cells and obtaining components consisting of the disrupted cell membrane, cell nucleus, and cytoplasm; (2) A process of removing cell membranes from the components obtained in the process (1) and collecting the stem cell filtrate.

7. The method for preparing a stem cell filtrate preparation according to claim 6, characterized in that the stem cells used are at least one type selected from adipose tissue-derived mesenchymal stem cells, epithelial stem cells, pigmented stem cells, iPS cells and ES cells.

8. A method for preparing the stem cell filtrate preparation described in claim 7, characterized in that at least two or more types of the adipose tissue-derived mesenchymal stem cells, the epithelial stem cells, and the pigmented stem cells are mixed.

9. The method for preparing a stem cell filtrate preparation according to any one of claims 6 to 8, characterized in that the step (1) is a step of destroying the cell membrane of mesenchymal stem cells in the adipose tissue by at least one method selected from the group consisting of a method of irradiating the adipose tissue removed from the body or the adipose tissue inside the body with laser light or ultrasound, and a method of repeatedly freezing and thawing the adipose tissue removed from the body at least once.

10. A method for preparing a stem cell filtrate preparation described in any one of claims 6 to 9, characterized in that an active ingredient extracted from adipocytes or cellular fat is further added to the stem cell filtrate obtained in step (2).

Images