Mesenchymal stem cell proliferation promoter, culture medium for mesenchymal stem cells, mesenchymal stem cells and culture supernatant

Abstract

The purpose of the present invention is to provide a culture medium for culturing having excellent proliferation of mesenchymal stem cells. The present invention is a mesenchymal stem cell proliferation promoter containing 16: 0 lysophosphatidic acid, a derivative of 16: 0 lysophosphatidic acid, and / or a salt of the 16: 0 lysophosphatidic acid and / or the derivative of 16: 0 lysophosphatidic acid. It is preferable that the mesenchymal stem cell proliferation promoter additionally contains 18: 1 lysophosphatidic acid, a derivative of 18: 1 lysophosphatidic acid, and / or a salt of the 18: 1 lysophosphatidic acid and / or the derivative of 18: 1 lysophosphatidic acid. The present invention also includes a culture medium for mesenchymal stem cells containing 16: 0 lysophosphatidic acid, a derivative of 16: 0 lysophosphatidic acid, and / or salts of the 16: 0 lysophosphatidic acid and the derivative of 16: 0 lysophosphatidic acid.

Description

Technical Field

[0001] This invention relates to mesenchymal stem cell proliferation promoters, culture media for mesenchymal stem cells, mesenchymal stem cells, and culture supernatants. Background Technology

[0002] Regenerative medicine utilizes cells and tissues to assist or regenerate dysfunctional tissues and organs. This type of regenerative medicine has the potential to become a new treatment method for diseases that are difficult to address with previous therapies, making its practical application an urgent issue. Among these, regenerative medicine utilizing stem cells derived from living organisms has attracted attention and is currently under research. Of these, ES cells and iPS cells are primarily being studied as tissue regeneration technologies. On the other hand, adult stem cells such as mesenchymal stem cells, in addition to their role in replenishing cells during tissue damage, are expected to have various effects such as anti-inflammatory and immunosuppressive effects, thus attracting attention as a more feasible cell therapy.

[0003] Mesenchymal stem cells (MSCs) are progenitor cells with multi-differentiation capabilities first isolated from bone marrow by Friedenstein (1982) (see Non-Patent Literature 1). These MSCs are known to exist in various tissues such as bone marrow, umbilical cord, and adipose tissue, and MSC transplantation is expected to be a novel treatment for various refractory diseases (see Patent Literature 1-2). Furthermore, MSCs have been reported to have immunosuppressive effects and to aggregate in tumors. Studies have also been conducted on using MSCs to prevent post-transplant rejection (Non-Patent Literature 2; Stem Cell Res Ther. 2021; 12: 192.) and for the delivery of cancer treatment drugs (Non-Patent Literature 3; Cancer Res (2013) 73(1): 364-372). Recently, it has been learned that there exist cells with functions equivalent to mesenchymal cells in adipose tissue, placenta, umbilical cord, and oviduct; MSCs are sometimes also referred to as mesenchymal cells.

[0004] Various culture media have been developed for culturing mesenchymal stem cells (see, for example, patent documents 3 and 4), but there is a desire to develop a culture medium that enables mesenchymal stem cells, which are more suitable for disease treatment, to proliferate in large quantities and efficiently while remaining undifferentiated.

[0005] Existing technical documents

[0006] Patent documents

[0007] Patent Document 1: Japanese Patent Application Publication No. 2012-157263

[0008] Patent Document 2: Japanese Patent Publication No. 2012-508733

[0009] Patent Document 3: US Patent Publication No. 2010 / 0015710

[0010] Patent Document 4: Japanese Patent Application Publication No. 2010-094062

[0011] Non-patent literature

[0012] Non-patent literature 1: Pittenger FM et al., Science 284, pp. 143-147, 1999

[0013] Non-patent literature 2: Stem Cell Res Ther., 12, pp. 192-, 2021

[0014] Non-patent literature 3: Cancer Res, 73(1), pp.364-372, 2013 Summary of the Invention

[0015] The problem the invention aims to solve

[0016] The object of the present invention is to provide a method for enabling mesenchymal stem cells to proliferate efficiently while maintaining their undifferentiated state under the conditions described above, that is, to provide a preparation with excellent proliferative promoting effect on mesenchymal stem cells and a culture medium for mesenchymal stem cells.

[0017] Solution for solving the problem

[0018] In order to solve the above-mentioned problems, in-depth research was conducted, and as a result, the inventors discovered that mesenchymal stem cells (MSCs) in a culture medium supplemented with a specific lysophosphatidic acid (LPA) exhibit excellent proliferative properties, thus completing the present invention. According to the present invention, it is possible to promote the proliferation of mesenchymal stem cells while maintaining their undifferentiated state. That is, the main points of the present invention are as follows.

[0019] [1] A mesenchymal stem cell proliferation promoter containing 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid and / or their salts.

[0020] [2] The mesenchymal stem cell proliferation promoter according to [1] also contains 18:1 lysophosphatidic acid, derivatives of 18:1 lysophosphatidic acid and / or their salts.

[0021] [3] According to the mesenchymal stem cell proliferation promoter described in [2], the content (μg / mL) of 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and / or their salts is 0.1 to 10 relative to the content (μg / mL) of 18:1 lysophosphatidic acid, derivatives of 18:1 lysophosphatidic acid and / or their salts.

[0022] [4] The mesenchymal stem cell proliferation promoter according to [1], wherein the mesenchymal stem cells are derived from adipose tissue.

[0023] [5] An additive for culturing mesenchymal stem cells, comprising 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and / or their salts.

[0024] [6] A culture medium for mesenchymal stem cells containing 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and / or their salts.

[0025] [7] The culture medium for mesenchymal stem cells according to [6] also contains 18:1 lysophosphatidylcholine, derivatives of 18:1 lysophosphatidylcholine and / or their salts.

[0026] [8] The culture medium for mesenchymal stem cells according to [7] wherein the content (μg / mL) of 16:0 lysophosphatidylcholine, derivatives of 16:0 lysophosphatidylcholine and / or their salts is in the ratio of 0.1 to 10 of 18:1 lysophosphatidylcholine, derivatives of 18:1 lysophosphatidylcholine and / or their salts.

[0027] [9] The culture medium for mesenchymal stem cells described in [6] to [8] is used to promote the proliferation of mesenchymal stem cells.

[0028]

[10] A mesenchymal stem cell obtained by culturing it using any one of the mesenchymal stem cell proliferation promoters in [1] to [4], the mesenchymal stem cell culture additive in [5], or the mesenchymal stem cell culture medium in any one of [6] to [9].

[0029]

[11] A culture supernatant containing 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid and / or their salts, said culture supernatant being obtained by culturing mesenchymal stem cells in a culture medium for mesenchymal stem cells as described in any one of [6] to [9].

[0030]

[12] A method for culturing mesenchymal stem cells, comprising the step of culturing mesenchymal stem cells in a culture medium containing 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid and / or salts thereof.

[0031]

[13] The culture method described in

[12] is a floating / stirred culture.

[0032]

[14] Use of 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and / or their salts for promoting the proliferation of mesenchymal stem cells.

[0033]

[15] Use of 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and / or their salts, and 18:1 lysophosphatidic acid, derivatives of 18:1 lysophosphatidic acid and / or their salts for promoting the proliferation of mesenchymal stem cells.

[0034]

[16] Use of 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and / or their salts for the manufacture of mesenchymal stem cell proliferation promoters.

[0035]

[17] Use of 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and / or their salts, and 18:1 lysophosphatidic acid, derivatives of 18:1 lysophosphatidic acid and / or their salts for the manufacture of mesenchymal stem cell proliferation promoters.

[0036]

[18] A method for promoting the proliferation of mesenchymal stem cells, characterized in that a culture medium containing 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid and / or their salts is used.

[0037]

[19] A method for promoting the proliferation of mesenchymal stem cells, characterized in that a culture medium containing 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and / or their salts, and 18:1 lysophosphatidic acid, derivatives of 18:1 lysophosphatidic acid and / or their salts is used.

[0038]

[20] A method for promoting the proliferation of mesenchymal stem cells, characterized in that 16:0 lysophosphatidylcholine, derivatives of 16:0 lysophosphatidylcholine and / or their salts are used.

[0039]

[21] A method for promoting the proliferation of mesenchymal stem cells, characterized in that 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and / or their salts are used, as well as 18:1 lysophosphatidic acid, derivatives of 18:1 lysophosphatidic acid and / or their salts.

[0040] The effects of the invention

[0041] The mesenchymal stem cell proliferation promoter and culture medium for mesenchymal stem cells according to the present invention enable mesenchymal stem cells to proliferate efficiently while maintaining their undifferentiated state. Furthermore, the culture supernatant obtained by culturing mesenchymal stem cells using the culture medium for mesenchymal stem cells of the present invention contains a large number of exosomes, HGF, and various humoral factors, and is therefore suitable for use in disease treatment, etc. Attached Figure Description

[0042] Figure 1 This graph illustrates the effect of LPA-containing medium on the proliferation-promoting effect of planar adhesion culture on mesenchymal stem cells. AD: Adipose-derived mesenchymal stem cells; UC: Umbilical cord-derived mesenchymal stem cells.

[0043] Figure 2 This figure shows the effect of LPA-containing medium on the proliferation-promoting effect of adipose-derived mesenchymal stem cells in planar adhesion culture.

[0044] Figure 3 This figure shows the effect of LPA-containing medium on the proliferation-promoting effect of umbilical cord-derived mesenchymal stem cells in planar adhesion culture.

[0045] Figure 4 This is a graph showing the results of a study on the concentration ratio of 16:0 LPA to 18:1 LPA in the culture of mesenchymal stem cells using LPA-containing medium.

[0046] Figure 5 This is a graph showing the results of a study on the concentration of LPA in the culture medium containing LPA for mesenchymal stem cells.

[0047] Figure 6 This figure shows the effect of LPA-containing medium in planktonic / stirred culture on the proliferation-promoting effect of adipose-derived mesenchymal stem cells.

[0048] Figure 7 This figure illustrates the effect of LPA-containing medium on exosome production in planar adhesion culture of mesenchymal stem cells.

[0049] Figure 8 This is a graph showing the results of evaluating the adhesion of mesenchymal stem cells cultured in a medium containing LPA to various culture flasks.

[0050] Figure 9 This figure shows the results of a study on the effect of LPA added to the culture medium on the number of cell divisions in planar adhesion culture of adipose-derived mesenchymal stem cells.

[0051] Figure 10 This figure shows the results of a study on the effect of LPA added to the culture medium on batch-to-batch deviation of cell division number in planar adhesion culture of adipose-derived mesenchymal stem cells.

[0052] Figure 11 This is a graph showing the results of counting cell divisions in over-passaged culture of adipose-derived mesenchymal stem cells in planar adhesion culture in LPA-containing medium.

[0053] Figure 12 This is a photograph showing the results of an angiogenesis assay using HUVECs.

[0054] Figure 13 This is a graph showing the effect of LPA-containing culture medium on the number of divisions of umbilical cord-derived mesenchymal stem cells.

[0055] Figure 14 This is a graph showing the effect of LPA-containing culture medium on the number of divisions of dental pulp-derived mesenchymal stem cells. Detailed Implementation

[0056] The following provides a detailed description of the mesenchymal stem cell proliferation promoter, the culture medium for mesenchymal stem cells, the culture method for mesenchymal stem cells, the mesenchymal stem cells, and the culture supernatant of the present invention.

[0057] [Mesenchymal stem cell proliferation promoter]

[0058] The mesenchymal stem cell proliferation promoter of the present invention is characterized by containing specific lysophosphatidic acid, derivatives of lysophosphatidic acid, and / or salts thereof. Using the mesenchymal stem cell proliferation promoter of the present invention enables mesenchymal stem cells to proliferate efficiently while maintaining their undifferentiated state. In addition to the aforementioned lysophosphatidic acid, derivatives of lysophosphatidic acid, and / or salts thereof, the mesenchymal stem cell proliferation promoter of the present invention may also contain other components without impairing the effects of the present invention. The mesenchymal stem cell proliferation promoter of the present invention will be described in detail below.

[0059] In this invention, for mesenchymal stem cells, the state of "maintaining undifferentiated state" refers to the state of maintaining the ability of mesenchymal stem cells to differentiate into osteocytes, chondrocytes, and adipocytes.

[0060] In this invention, mesenchymal stem cells refer to cells capable of differentiating into one or more types of mesenchymal cells (bone cells, cardiomyocytes, chondrocytes, tendon cells, adipocytes, etc.) and capable of proliferating while maintaining this ability. The term "mesenchymal stem cells" used in this invention refers to cells identical to mesenchymal cells, without special distinction between the two. Additionally, it is sometimes abbreviated as mesenchymal cells. Examples of tissues containing mesenchymal stem cells include adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amnion, chorion, decidua, dermis, skeletal muscle, periosteum, dental sac, periodontal ligament, dental pulp, and tooth germ. As the mesenchymal stem cells in this invention, examples include mesenchymal stem cells derived from adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amnion, chorion, decidua, dermis, skeletal muscle, periosteum, dental sac, periodontal ligament, dental pulp, and tooth germ. Among these, mesenchymal stem cells derived from adipose tissue, umbilical cord, bone marrow, and dental pulp are preferred, while those derived from adipose tissue, umbilical cord, and dental pulp are more preferred, and those derived from adipose tissue are even more preferred.

[0061] In this invention, adipose tissue refers to tissue containing interstitial cells such as adipocytes and microvascular cells, for example, tissue obtained by surgically removing or extracting subcutaneous fat from mammals.

[0062] In this invention, the umbilical cord refers to the white tubular tissue that connects the fetus and the placenta. It is composed of the umbilical vein, umbilical artery, colloid tissue (Wharton's Jelly), and the umbilical stroma itself, and contains a large number of mesenchymal stem cells.

[0063] In this invention, bone marrow refers to the parenchyma, the solid tissue filling the cavity of bone, and is a hematopoietic organ. Bone marrow contains bone marrow fluid, and the cells present within it are called bone marrow cells. In addition to erythrocytes, granulocytes, megakaryocytes, lymphocytes, and adipocytes, bone marrow cells also include mesenchymal stem cells, hematopoietic stem cells, and vascular endothelial progenitor cells. Bone marrow cells can be collected, for example, from the human iliac bone, long bones, or other bones.

[0064] In this invention, dental pulp refers to the connective tissue rich in blood vessels and nerves observed at the center of the tooth, and is the tissue that grows from the dental papilla in the tooth germ. It is the soft tissue that fills the pulp cavity inside the tooth.

[0065] As for the type of mesenchymal stem cells used in this invention, mammals are preferred. Examples of mammals include humans, horses, cattle, sheep, pigs, dogs, cats, rabbits, mice, rats, and monkeys. Among these, humans, horses, cattle, and cats are preferred.

[0066] Mesenchymal stem cells can be cells provided by companies such as PromoCell, Lonza, Biological Industries, Veritas, R&D Systems, and Corning, or cells prepared using methods known to those skilled in the art. Furthermore, mesenchymal stem cells can be primary cells isolated from donor tissue or cell lineages.

[0067] In this invention, lysophosphatidic acid (LPA) is a lysophosphatidylcholine having an unsubstituted phosphate group, and it exists in various combinations with different alkyl chain lengths and double bond numbers (carbon number to unsaturation). Examples of such carbon number to unsaturation combinations include 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:0, 20:1, 20:2, 20:3, 20:4, 20:5, 22:0, 22:1, 22:2, 22:3, 22:4, 22:5, and 22:6. From the viewpoint of excellent mesenchymal stem cell proliferation promotion effect, 16:0, 16:1, 18:0, 18:1, 18:2, and 18:3 are preferred, 16:0 and 18:1 are more preferred, and 16:0 is even more preferred. Furthermore, the mesenchymal stem cell proliferation promoter of the present invention may also contain various lysophosphatidic acids with different combinations of alkyl chain length and double bond number (carbon number - degree of unsaturation). Additionally, the lysophosphatidic acid contained in the mesenchymal stem cell proliferation promoter of the present invention may also be a derivative of lysophosphatidic acid. Examples of lysophosphatidic acid derivatives include lysophosphatidylcholine, lysophosphatidylserine, lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylglycerol. The lysophosphatidic acid contained in the mesenchymal stem cell proliferation promoter of the present invention may be in any form, such as a salt. When lysophosphatidic acid is in the form of a salt, examples of its salts include monosodium salts, disodium salts, monopotassium salts, dipotassium salts, magnesium salts, and calcium salts.

[0068] As a mesenchymal stem cell proliferation promoter of the present invention, from the viewpoint of more significantly exerting the effects of the present invention, it is preferable to contain 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and / or salts thereof, and more preferably, in addition to 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and / or salts thereof, it also contains 18:1 lysophosphatidic acid, derivatives of 18:1 lysophosphatidic acid and / or salts thereof.

[0069] The concentration of each lysophosphatidic acid contained in the mesenchymal stem cell proliferation promoter of the present invention, calculated based on the concentration when applied to cells, is 0.0025 μg / mL or more and 1 mg / mL or less, preferably 0.01 μg / mL or more and 1 mg / mL or less, more preferably 0.1 μg / mL or more and 100 μg / mL or less, more preferably 1 μg / mL or more and 50 μg / mL or less, further preferably 1.5 μg / mL or more and 25 μg / mL or less, particularly preferably 2 μg / mL or more and 20 μg / mL or less, further more preferably 2 μg / mL or more and 10 μg / mL or less, and most preferably 2.5 μg / mL or more and 5 μg / mL or less.

[0070] The mesenchymal stem cell proliferation promoter of the present invention may also contain a variety of lysophosphatidic acids. In this case, the total concentration of all lysophosphatidic acids, calculated based on the concentration when applied to cells, is 0.05 μg / mL or more and 1.25 mg / mL or less, preferably 0.1 μg / mL or more and 100 μg / mL or less, more preferably 0.3 μg / mL or more and 50 μg / mL or less, further preferably 1 μg / mL or more and 40 μg / mL or less, particularly preferably 2 μg / mL or more and 30 μg / mL or less, and even more preferably 5 μg / mL or more and 20 μg / mL or less. Furthermore, the mixing ratio of various lysophosphatidic acids is not particularly limited. In the case of including both 16:0 and 18:1 lysophosphatidic acids, from the viewpoint of excellent mesenchymal stem cell proliferation promotion effect, the ratio of the content (μg / mL) of 16:0 lysophosphatidic acid and / or their salts to the content (μg / mL) of 18:1 lysophosphatidic acid and / or their salts is 0.001 to 1000, preferably 0.01 to 100, more preferably 0.1 to 10, further preferably 0.1 to 5, particularly preferably 0.3 to 3, and even more preferably 0.5 to 1.5.

[0071] The form of the mesenchymal stem cell proliferation promoter of the present invention is not particularly limited, and it may be the aforementioned lysophosphatidylcholine itself, or a composition composed of the aforementioned lysophosphatidylcholine and other components. Furthermore, the form of the aforementioned composition is not particularly limited. The aforementioned composition may, for example, be a liquid culture medium for culturing mesenchymal stem cells, or a mesenchymal stem cell culture additive prepared during the preparation of the liquid culture medium.

[0072] A preferred embodiment of the mesenchymal stem cell proliferation promoter of the present invention is a liquid culture medium containing the above-mentioned lysophosphatidic acid at the above-mentioned concentration and / or ratio.

[0073] In the case where the mesenchymal stem cell proliferation promoter of the present invention is a liquid culture medium, the mesenchymal stem cell proliferation promoter of the present invention is prepared by containing the above-mentioned lysophosphatidic acid in a conventionally known animal cell culture basal culture medium at the above-mentioned concentration and / or ratio.

[0074] The basal culture medium for animal cell culture in this invention refers to a culture medium containing carbon sources, nitrogen sources, and inorganic salts necessary for culturing animal cells. Here, animal cells refer to mammalian cells, particularly human cells. The basal culture medium for animal cell culture in this invention may contain biologically derived materials; however, considering the possibility of using the cells obtained through culture and their culture supernatant to treat diseases in animals (including humans), from the viewpoint of infectious risk, a culture medium that contains as few biologically derived materials as possible is preferred.

[0075] As the basal medium for the aforementioned animal cell culture, animal cell culture media known to those skilled in the art can be used. Specifically, examples include minimum essential medium (MEM) such as Igor medium, DMEM modified Igor medium (DMEM), minimum essential medium α (MEM-α), mesenchymal cell basal medium (MSCBM), Ham's F-12 and F-10 media, DMEM / F12 medium, Williams medium E, RPMI-1640 medium, MCDB medium, 199 medium, Fisher medium, Iscove modified DMEM medium (IMDM), McCoy modified medium, and mixtures thereof.

[0076] In addition, as the basal culture medium for the aforementioned animal cell cultures, known culture media adapted for mesenchymal stem cell use can be used. Examples include: Mesenchymal Stem Cell Growth Medium 2 (Ready-to-use), PromoCell; Mesenchymal Stem Cell Growth Medium XF (Ready-to-use), PromoCell; MSCGMM Bullet Kittm; MSCGMtm Mesenchymal Stem Cell Growth Medium Bullet Kittm (Lonza); Allogeneic-free culture medium for human mesenchymal stem cells (MSC NutriStem (registered trademark), Biological Industries); MesenCult-ACF Plus (Veritas); StemXVivotm serum-free human MSC proliferation medium (R&D Systems, Corning); serum-free culture medium for adipose-derived stem cells (KBM ADSC-4, Kohjin Bio); and serum-free culture medium for mesenchymal stem cells (R: STEM Medium for hMSC). HighGrowth (and Rohto Corporation), etc.

[0077] When the mesenchymal stem cell proliferation promoter of the present invention is implemented in a liquid culture medium, in addition to the above-mentioned basic culture medium for animal cell culture and the above-mentioned lysophosphatidic acid, the mesenchymal stem cell proliferation promoter of the present invention may also contain, as needed, amino acids such as glutamine, sugars such as glucose, metal salts such as sodium chloride and / or magnesium sulfate, trace metals such as selenium, lipids (except for the above-mentioned lysophosphatidic acid and their salts), vitamins such as pantothenic acid, albumin, insulin, transferrin, growth factors (e.g., epidermal growth factor, basic fibroblast growth factor), growth factors, cytokines and other proteins, polysaccharides, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts and other substances.

[0078] When the mesenchymal stem cell proliferation promoter of the present invention is implemented as a liquid culture medium, in addition to the above-mentioned basal culture medium for animal cell culture and the above-mentioned lysophosphatidic acid, the mesenchymal stem cell proliferation promoter of the present invention may also contain serum as needed. As for the serum, there are no particular limitations as long as it does not hinder the proliferation of mesenchymal stem cells; it can be serum derived from animals, preferably serum derived from mammals (e.g., fetal bovine serum, human serum, etc.), and more preferably human serum. The concentration of the serum in the mesenchymal stem cell proliferation promoter of the present invention is only required to be within a concentration range known to those skilled in the art. When using cultured mesenchymal stem cells for medical purposes, components derived from other animals may become sources of infection for blood-mediated pathogens or xenoantigens; therefore, the mesenchymal stem cell proliferation promoter of the present invention preferably does not contain serum. In the case of not containing serum, serum substitutes can be used (e.g., Knockout Serum Replacement (KSR) (Invitrogen), Chemically-defined Lipid Concentrated (Gibco), etc.).

[0079] When the mesenchymal stem cell proliferation promoter of the present invention is implemented as a liquid culture medium, the osmotic pressure ratio is preferably 0.9 to 1.1. The pH is preferably 6.0 to 9.0, more preferably 6.5 to 8.5. In addition, the above-mentioned liquid culture medium is preferably sterile and has an endotoxin content of 2.5 EU / mL or less.

[0080] The mesenchymal stem cell proliferation promoter of the present invention can be manufactured by mixing the above-mentioned components using conventional methods. Furthermore, the mesenchymal stem cell proliferation promoter of the present invention can be provided in a concentrated state and diluted before use.

[0081] As can be understood from the above description, the scope of the present invention also includes "the use of 16:0 lysophosphatidylcholine, derivatives of 16:0 lysophosphatidylcholine and / or their salts for promoting the proliferation of mesenchymal stem cells", "the use of 16:0 lysophosphatidylcholine, derivatives of 16:0 lysophosphatidylcholine and / or their salts, as well as 18:1 lysophosphatidylcholine, derivatives of 18:1 lysophosphatidylcholine and / or their salts for promoting the proliferation of mesenchymal stem cells", "the use of 16:0 lysophosphatidylcholine, derivatives of 16:0 lysophosphatidylcholine and / or their salts for manufacturing mesenchymal stem cell proliferation promoters", and "the use of 16:0 lysophosphatidylcholine, derivatives of 16:0 lysophosphatidylcholine and / or their salts, as well as 18:1 lysophosphatidylcholine, derivatives of 18:1 lysophosphatidylcholine and / or their salts for manufacturing mesenchymal stem cell proliferation promoters".

[0082] As can be understood from the above description, the scope of the present invention also includes "a method for promoting the proliferation of mesenchymal stem cells, characterized by using a culture medium containing 16:0 lysophosphatidylcholine, a derivative of 16:0 lysophosphatidylcholine, and / or salts thereof", "a method for promoting the proliferation of mesenchymal stem cells, characterized by using a culture medium containing 16:0 lysophosphatidylcholine, a derivative of 16:0 lysophosphatidylcholine, and / or salts thereof, as well as 18:1 lysophosphatidylcholine, a derivative of 18:1 lysophosphatidylcholine, and / or salts thereof", "a method for promoting the proliferation of mesenchymal stem cells, characterized by using 16:0 lysophosphatidylcholine, a derivative of 16:0 lysophosphatidylcholine, and / or salts thereof", and "a method for promoting the proliferation of mesenchymal stem cells, characterized by using 16:0 lysophosphatidylcholine, a derivative of 16:0 lysophosphatidylcholine, and / or salts thereof, as well as 18:1 lysophosphatidylcholine, a derivative of 18:1 lysophosphatidylcholine, and / or salts thereof".

[0083] Culture medium for mesenchymal stem cells

[0084] This invention comprises a culture medium for mesenchymal stem cells, which contains a basal culture medium for animal cell culture and specific lysophosphatidic acid, lysophosphatidic acid derivatives, and / or their salts. The mesenchymal stem cell culture medium of this invention is suitable for use in promoting the proliferation of mesenchymal stem cells due to its excellent effect on promoting mesenchymal stem cell proliferation. It should be noted that the mesenchymal stem cell culture medium of this invention corresponds to one embodiment of the mesenchymal stem cell proliferation promoter of this invention described above (in the case of a liquid culture medium).

[0085] As the aforementioned lysophosphatidic acid, there are various combinations of alkyl chain length and double bond number (carbon number - degree of unsaturation). Specifically, examples of such carbon number-degree of unsaturation combinations (carbon number: degree of unsaturation) include 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:0, 20:1, 20:2, 20:3, 20:4, 20:5, 22:0, 22:1, 22:2, 22:3, 22:4, 22:5, and 22:6. From the viewpoint of excellent mesenchymal stem cell proliferation promotion effect, 16:0, 16:1, 18:0, 18:1, 18:2, and 18:3 are preferred, 16:0 and 18:1 are more preferred, and 16:0 is even more preferred. Furthermore, the mesenchymal stem cell culture medium of the present invention may also contain various lysophosphatidic acids with different combinations of alkyl chain length and double bond number (carbon number - degree of unsaturation). Additionally, the lysophosphatidic acid contained in the mesenchymal stem cell culture medium of the present invention may be a derivative of lysophosphatidic acid, or any form such as a salt. Specific examples of the above-mentioned derivatives and salts can be found in the section on [Mesenchymal Stem Cell Proliferation Promoters].

[0086] As the culture medium for mesenchymal stem cells of the present invention, it preferably contains 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and / or their salts, and more preferably, in addition to 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and / or their salts, it also contains 18:1 lysophosphatidic acid, derivatives of 18:1 lysophosphatidic acid and / or their salts.

[0087] The concentration of each lysophosphatidic acid contained in the culture medium for mesenchymal stem cells of the present invention is 0.0025 μg / mL or more and 1 mg / mL or less, preferably 0.01 μg / mL or more and 1 mg / mL or less, more preferably 0.1 μg / mL or more and 100 μg / mL or less, more preferably 1 μg / mL or more and 50 μg / mL or less, even more preferably 1.5 μg / mL or more and 25 μg / mL or less, particularly preferably 2 μg / mL or more and 20 μg / mL or less, even more preferably 2 μg / mL or more and 10 μg / mL or less, and most preferably 2.5 μg / mL or more and 5 μg / mL or less.

[0088] The culture medium for mesenchymal stem cells of the present invention may contain a variety of lysophosphatidic acids. The total concentration of all lysophosphatidic acids at this time is preferably 0.1 μg / mL or more and 100 μg / mL or less, more preferably 0.3 μg / mL or more and 50 μg / mL or less, even more preferably 1 μg / mL or more and 40 μg / mL or less, particularly preferably 2 μg / mL or more and 30 μg / mL or less, and even more preferably 5 μg / mL or more and 20 μg / mL or less. Furthermore, the mixing ratio of various lysophosphatidic acids is not particularly limited. In the case of including both 16:0 and 18:1 lysophosphatidic acids, from the viewpoint of excellent mesenchymal stem cell proliferation promotion effect, the ratio of the content (μg / mL) of 16:0 lysophosphatidic acid, its derivatives and / or their salts to the content (μg / mL) of 18:1 lysophosphatidic acid, its derivatives and / or their salts is 0.001 to 1000, preferably 0.01 to 100, more preferably 0.1 to 10, further preferably 0.1 to 5, particularly preferably 0.3 to 3, and even more preferably 0.5 to 1.5.

[0089] The mesenchymal stem cell culture medium of the present invention is equivalent to one embodiment of the mesenchymal stem cell proliferation promoter of the present invention described above (in the case of a liquid culture medium). Therefore, the description of the liquid culture medium of the mesenchymal stem cell proliferation promoter described above, as well as the basic culture medium for animal cell culture, other components, characteristics, etc., can be directly applied.

[0090] The mesenchymal stem cell culture medium of the present invention also includes a culture medium in which a gelling material is added to the above-mentioned liquid culture medium to form a gel. As the gelling material, any material capable of making the culture medium gel-like and suitable for cell culture can be used; examples include agar, agarose, alginate, collagen, gelatin, cellulose, etc. The concentration of these gelling materials added is sufficient to make the culture medium gel-like, and those skilled in the art can set it appropriately as needed. It should be gel-like under cell culture or preservation conditions; for example, it is preferably gel-like at any temperature within the range of -80°C to 100°C.

[0091] [Additives for Mesenchymal Stem Cell Culture]

[0092] This invention also includes a mesenchymal stem cell culture additive containing 16:0 lysophosphatidylcholine, a derivative of 16:0 lysophosphatidylcholine, and / or salts thereof. The mesenchymal stem cell culture additive of this invention, by containing the aforementioned specific lysophosphatidylcholine, a derivative of lysophosphatidylcholine, and / or salts thereof, exhibits excellent effects in promoting the proliferation of mesenchymal stem cells when added to the culture medium or the like during mesenchymal stem cell culture, and is therefore suitable for use in mesenchymal stem cell culture and / or for promoting mesenchymal stem cell proliferation. It should be noted that the mesenchymal stem cell culture additive of this invention corresponds to one embodiment of the mesenchymal stem cell proliferation promoter of this invention described above. Therefore, for a detailed description of the mesenchymal stem cell culture additive of this invention, the description of the mesenchymal stem cell proliferation promoter of this invention should be applied.

[0093] [Mesenchymal stem cell culture methods]

[0094] The present invention also includes a method for culturing mesenchymal stem cells, comprising the step of culturing mesenchymal stem cells in a culture medium containing specific lysophosphatidic acid, derivatives of lysophosphatidic acid, and / or salts thereof. According to the culture method of the present invention, mesenchymal stem cells can proliferate efficiently while maintaining their undifferentiated state.

[0095] As the aforementioned lysophosphatidic acid, there are various combinations of alkyl chain length and double bond number (carbon number - degree of unsaturation). Specifically, examples of such carbon number-unsaturation combinations (carbon number: degree of unsaturation) include 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:0, 20:1, 20:2, 20:3, 20:4, 20:5, 22:0, 22:1, 22:2, 22:3, 22:4, 22:5, and 22:6. From the viewpoint of excellent mesenchymal stem cell proliferation promotion, 16:0, 16:1, 18:0, 18:1, 18:2, and 18:3 are preferred, 16:0 and 18:1 are more preferred, and 16:0 is even more preferred. Furthermore, the aforementioned culture medium may also contain various lysophosphatidic acids with different combinations of alkyl chain length and double bond number (carbon number - degree of unsaturation). Furthermore, the lysophosphatidylcholine contained in the above-mentioned culture medium can be a derivative of lysophosphatidylcholine or any form of its salts. Specific examples of the above-mentioned derivatives and salts can be found in the section on [Mesenchymal Stem Cell Proliferation Promoters].

[0096] The culture medium containing specific lysophosphatidic acid, lysophosphatidic acid derivatives, and / or their salts in the culture method of the present invention corresponds to one embodiment of the mesenchymal stem cell proliferation promoter of the present invention (in the case of a liquid culture medium). Therefore, for details regarding the aforementioned culture medium, the description of the liquid culture medium in the mesenchymal stem cell proliferation promoter section can be directly applied. Furthermore, for the description of mesenchymal stem cells, the description in the mesenchymal stem cell proliferation promoter section can also be directly applied.

[0097] The culture method of the present invention is not particularly limited as long as it includes the step of culturing mesenchymal stem cells in a culture medium containing specific lysophosphatidylcholine, derivatives of lysophosphatidylcholine, and / or their salts; otherwise, conventional methods can be used. Typically, the culture temperature is 30°C to 40°C, preferably 30°C to 37°C. The CO2 concentration during culture is 2% to 10%, preferably 2% to 7%, more preferably 5%. The O2 concentration during culture is 0% to 100%, preferably 0.5% to 22%, more preferably 5% to 21%. Furthermore, during culture, mesenchymal stem cell passage and culture medium replacement can be performed as needed, and the timing and method are not particularly limited as long as they are suitable for each type of mesenchymal stem cell. The morphology of the mesenchymal stem cells can be observed while performing these procedures as conventionally done. The passage number is preferably 0 to 8, more preferably 1 to 6. The culture period is preferably 3 to 50 days, more preferably 3 to 40 days, and even more preferably 3 to 30 days. The seeding density can be determined appropriately based on the purpose of culture. Low-density seeding with long-term culture or high-density seeding with short-term culture can be used, with 2500 cells / cm² being the preferred density. 2~10,000 cells / cm 2 More preferably, 5000 cells / cm² 2 ~7500 cells / cm 2 .

[0098] In the mesenchymal stem cell culture method of the present invention, mesenchymal stem cells can be cultured by adhering them to a general planar adhesion culture using cell culture flasks, culture dishes, plates, or other culture vessels. Alternatively, they can be cultured by adhering them to microbeads, microcarriers, etc., and allowing the microbeads, microcarriers, etc., to float in the culture medium, which is called float culture (suspension culture) or float / stirred culture. They can also be cultured by adhering them to fibrous scaffolds such as microfibers, or they can be cultured using spherical bodies.

[0099] The culture vessel used in the culture of mesenchymal stem cells of the present invention is not particularly limited as long as it can culture mesenchymal stem cells. Examples include: flasks, tissue culture flasks, culture dishes, Piper culture dishes, tissue culture dishes, multiple culture dishes, microplates, microwell plates, multiple plates, multiwell plates, microslides, chamber slides, culture trays, tubes, trays, culture bags, roller bottles, etc.

[0100] [Mesenchymal stem cells]

[0101] This invention also includes mesenchymal stem cells obtained by culturing them using the mesenchymal stem cell proliferation promoter, mesenchymal stem cell culture additive, or mesenchymal stem cell culture medium of this invention. The mesenchymal stem cells of this invention, when cultured in a medium containing specific lysophosphatidic acid, lysophosphatidic acid derivatives, and / or their salts, not only exhibit promoted proliferation, high viability, and good condition, but also, compared to conventional mesenchymal stem cells, are able to release large amounts of exosomes, HGF, and other humoral factors, thus making them suitable for the treatment of various diseases.

[0102] The mesenchymal stem cells of the present invention can be obtained by culturing conventional mesenchymal stem cells using the mesenchymal stem cell proliferation promoter of the present invention or the mesenchymal stem cell culture medium of the present invention. Typically, the culture temperature is 30°C to 40°C, preferably 30°C to 37°C. The CO2 concentration during culture is 2% to 10%, preferably 2% to 7%, more preferably 5%. The O2 concentration during culture is 0% to 100%, preferably 0.5% to 22%, more preferably 5% to 21%. Furthermore, during culture, mesenchymal stem cell passages and culture medium changes can be performed as needed. The timing and method are not particularly limited as long as they are suitable for each type of mesenchymal stem cell, and the morphology of the mesenchymal stem cells can be observed while proceeding in the same manner as before. The passage number is preferably 0 to 8, more preferably 1 to 6. The culture period is preferably 3 to 50 days, more preferably 3 to 40 days, and even more preferably 3 to 30 days. The seeding density is typically preferably 2500 cells / cm². 2 ~10,000 cells / cm 2 More preferably, 5000 cells / cm² 2 ~7500 cells / cm 2 .

[0103] The mesenchymal stem cells of the present invention can increase the amount of exosomes and various humoral factors such as HGF released into the culture supernatant by increasing the seeding density and cell density. A preferred seeding density is 5000 cells / cm³. 2 ~40,000 cells / cm 2 More preferably 7500 cells / cm³ 2 ~30,000 cells / cm 2 Further optimized to 15,000 cells / cm 2 ~20,000 cells / cm 2 In addition, by reducing the inoculation density and conducting long-term culture, it is also possible to increase the concentration of various humoral factors such as exosomes and HGF in the culture supernatant.

[0104] The mesenchymal stem cells of this invention are CD73, CD90, and CD105 positive and maintain undifferentiated characteristics. Furthermore, compared to conventional mesenchymal stem cells, the mesenchymal stem cells of this invention release more CD9 and CD63 positive exosomes. Moreover, compared to conventional mesenchymal stem cells, the mesenchymal stem cells of this invention release more various humoral factors, including hepatocyte growth factor (HGF), insulin-like growth factors (IGF-related factors), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF).

[0105] Examples of diseases for which the mesenchymal stem cells of the present invention can be used as medicines include: chondrolysis, rheumatoid arthritis, psoriatic arthritis, spondyloarthritis, osteoarthritis, gout, psoriasis, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, congestive heart failure, stroke, aortic stenosis, renal failure, lupus erythematosus, pancreatitis, allergy, fibrosis, anemia, atherosclerosis, restenosis, chemotherapy / radiotherapy-related complications, type I diabetes, type II diabetes, autoimmune hepatitis, hepatitis C, primary biliary cirrhosis, primary sclerosing cholangitis, fulminant hepatitis, celiac disease, and nonspecific colitis. Allergic conjunctivitis, diabetic retinopathy, Sjögren's syndrome, uveitis, allergic rhinitis, asthma, asbestosis, silicosis, chronic obstructive pulmonary disease, chronic granulomatous inflammation, cystic fibrosis, sarcoidosis, glomerulonephritis, vasculitis, dermatitis, HIV-related cachexia, cerebral malaria, ankylosing spondylitis, leprosy, pulmonary fibrosis, esophageal cancer, gastroesophageal reflux, Barrett's esophagus, gastric cancer, duodenal cancer, small bowel cancer, appendix cancer, large colorectal cancer, colon cancer, rectal cancer, anal cancer, pancreatic cancer, liver cancer, gallbladder cancer, spleen cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, ovarian cancer, breast cancer, lung cancer, thyroid cancer, fibromyalgia, etc.

[0106] There are no particular limitations on the method of administration when using the mesenchymal stem cells of the present invention as a medicine. Preferred methods include intravascular administration (preferably intravenous administration), intraperitoneal administration, enteral administration, and subcutaneous administration, with intravascular administration being more preferred.

[0107] The dosage for using the mesenchymal stem cells of the present invention as a pharmaceutical product can vary depending on the type of disease, the severity of its symptoms, the dosage form, the weight of the recipient, etc. In the case of human administration, the dosage can be 1 × 10⁻⁶ per day. 5 1×109 Mesenchymal stem cells can be administered within a range of [number] doses. When administered to small animals such as mice, the dosage can be 1 × 10 [units] per day. 4 1×10 9 Dosage within a range of 1×10, preferably 1×10 5 1×10 9 The range is specified. It should be noted that when the mesenchymal stem cells of the present invention are used as a medicine, administration can be performed once or multiple times a day. Furthermore, the above administration can be a single dose or continuous administration. In the case of continuous administration, for example, it can be administered more than twice at a frequency of once every three days.

[0108] There are no particular limitations on the mammals that can be used as drug subjects when the mesenchymal stem cells of the present invention are used as pharmaceuticals, but humans, monkeys, mice, rats, hamsters, guinea pigs, cattle, pigs, horses, rabbits, sheep, goats, cats, dogs, etc., are preferred, with humans being more preferred. Furthermore, when using the mesenchymal stem cells of the present invention as pharmaceuticals, from the viewpoint of obtaining more stable and superior preventive and / or therapeutic effects against diseases, it is preferable that the species is consistent with the mammalic species used as drug subjects.

[0109] [Culture supernatant]

[0110] This invention also includes a culture supernatant containing 16:0 lysophosphatidylcholine, 16:0 lysophosphatidylcholine derivatives, and / or their salts, obtained by culturing mesenchymal stem cells in the aforementioned mesenchymal stem cell culture medium of this invention. Compared to culture supernatants obtained by culturing mesenchymal stem cells in conventional culture media, the culture supernatant of this invention contains more CD9 and CD63-positive exosomes. Furthermore, it also contains more hepatocyte growth factor (HGF), insulin-like growth factors (IGF-related factors), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and other humoral factors. Therefore, the culture supernatant of this invention is suitable for the treatment of various diseases. Additionally, it can also be applied to cosmetics and food.

[0111] (Preparation of culture supernatant)

[0112] The culture supernatant of mesenchymal stem cells obtained by the following method is used as the culture supernatant in this invention. Alternatively, substances obtained by removing unwanted components from the supernatant by means of dialysis / ultrafiltration, fractions obtained by fractionating the supernatant by column fractionation, fractions selected by using antibodies against specific molecules, fractions obtained by centrifugation, etc., may also be used as the culture supernatant in this invention.

[0113] The culture medium used to obtain the culture supernatant is the mesenchymal stem cell culture medium of the present invention described above. The method for obtaining the culture supernatant is not particularly limited as long as it is suitable for the culture of each mesenchymal stem cell. Generally, the culture temperature is 30°C to 40°C, preferably 30°C to 37°C. The CO2 concentration during culture is 2% to 10%, preferably 2% to 7%, more preferably 5%. The O2 concentration during culture is 0% to 100%, preferably 0.5% to 22%, more preferably 5% to 21%. Furthermore, during culture, mesenchymal stem cell passages and culture medium changes can be performed as needed. The timing and method are not particularly limited as long as they are suitable for each mesenchymal stem cell. The morphology of the mesenchymal stem cells can be observed while performing these procedures in the same manner as before. The passage number of mesenchymal stem cells is preferably 0 to 8, more preferably 1 to 6. The culture period is preferably 3 to 50 days, more preferably 3 to 40 days, and even more preferably 3 to 30 days. The seeding density is generally preferably 2500 cells / cm². 2 ~10,000 cells / cm 2 More preferably, 5000 cells / cm² 2 ~7500 cells / cm 2 .

[0114] When obtaining the culture supernatant, increasing the seeding density and cell density of mesenchymal stem cells can increase the concentration of various humoral factors such as exosomes and HGF in the culture supernatant. A preferred seeding density is 5000 cells / cm³. 2 ~40,000 cells / cm 2 More preferably 7500 cells / cm³ 2 ~30,000 cells / cm 2 Further optimized to 15,000 cells / cm 2 ~20,000 cells / cm 2 In addition, by reducing the seeding density of mesenchymal stem cells and culturing them for a longer period of time, it is also possible to increase the concentration of various humoral factors such as exosomes and HGF in the culture supernatant.

[0115] After obtaining the culture supernatant, the cells are passaged and cultured in the culture medium for mesenchymal stem cells of the present invention is repeated multiple times, and the culture supernatant can be obtained multiple times.

[0116] The culture used to obtain the culture supernatant of the present invention can be a planar adhesion culture attached to a flask, culture dish, plate or the like used for cell culture, or a floating / stirred culture attached to a microcarrier, microbead or the like.

[0117] When the culture supernatant of the present invention is provided, it is preferably sterilized by aseptic treatment, and the endotoxin content is preferably below 2.5 EU / mL.

[0118] The culture supernatant of the present invention can be used for the same diseases as those for which the mesenchymal stem cells of the present invention can be used as pharmaceuticals. Furthermore, the species to which the drug is intended can also be used. The culture supernatant of the present invention can also be used as a quasi-pharmaceutical, cosmetic, or food product. When the culture supernatant of the present invention is used as a pharmaceutical product, regarding the method of administration, intravenous administration (preferably intravenous), intraperitoneal administration, enteral administration, subcutaneous administration, etc., are preferred, with intravenous administration being more preferred.

[0119] The dosage of the culture supernatant of the present invention when used as a medicine can vary depending on the type of disease, the severity of its symptoms, the dosage form, and the weight of the recipient. It should be noted that the culture supernatant of the present invention can be administered once or multiple times a day. Furthermore, the above administration can be a single dose or continuous administration. In the case of continuous administration, for example, it can be administered more than twice at a frequency of once every three days.

[0120] Example

[0121] The present invention will be described in detail below with examples and test cases, but the present invention is not limited to these examples.

[0122] [Example 1: Study on the Proliferation-Promoting Effect of Planar Adhesion Culture / LPA-Containing Medium on Mesenchymal Stem Cells - 1]

[0123] Human adipose-derived mesenchymal stem cells (AD-MSC, Promo Cell preparation) and human umbilical cord-derived mesenchymal stem cells (UC-MSC, Promo Cell preparation) were used at a concentration of 5,000 cells / cm³. 2Cells were seeded in T25 flasks (Corning) for adhesion cells and cultured in R:STEM Medium for hMSC High Growth (Rohto Pharmaceutical Co., Ltd., serum-free medium, hereinafter referred to as RS medium, RS or RSTEM), or in RS medium supplemented with 18:1 LPA and / or 16:0 LPA. When only 18:1 LPA or only 16:0 LPA was added, the final concentration was 5.0 μg / mL; when both 18:1 LPA and 16:0 LPA were added, the final concentration was 2.5 μg / mL. After culturing at 37°C and 5% CO2 for 3 days in each medium, cells were peeled with trypsin, and the cell count in each flask was determined. Cells cultured only in RS medium without either 18:1 LPA or 16:0 LPA were used as a control (RS). The number of recovered cells in the control was set as 100%, and the relative percentage of recovered cells under each medium condition was calculated. The results are shown below. Figure 1 and Figure 2 . Figure 1 The effects of adding 16:0 LPA are shown (AD-MSC, UC-MSC). Figure 2 The addition of either 18:1 LPA or 16:0 LPA and the effect of both are shown (AD-MSC).

[0124] When cultured in medium supplemented with 16:0 LPA, the proliferation of both AD-MSCs and UC-MSCs was significantly promoted. Figure 1 Furthermore, culture media supplemented with both 18:1 LPA and 16:0 LPA showed a synergistic proliferation-promoting effect on AD-MSCs compared to culture media supplemented with only 18:1 LPA or 16:0 LPA. Figure 2 ).

[0125] [Example 2: Study on the Proliferation-Promoting Effect of Planar Adhesion Culture / LPA-Containing Medium on Mesenchymal Stem Cells - 2]

[0126] Human umbilical cord-derived mesenchymal stem cells (UC-MSC, Promo Cell) were used at a rate of 5,000 cells / cm². 2Cells were seeded in T25 flasks (Corning) for adhesion cells and cultured in basal medium (MEM, bFGF, albumin, insulin, transferrin, 3% FBS) supplemented with 18:1 LPA and / or 16:0 LPA. In cases with only 18:1 LPA or only 16:0 LPA, the final concentration was added at 2.5 μg / mL; in cases with both 18:1 LPA and 16:0 LPA, the final concentration was added at 2.5 μg / mL for each. After culturing for 3 days at 37°C and 5% CO2 in each medium, cells were peeled with trypsin, and the cell count in each flask was determined. Cells cultured in basal medium without either 18:1 LPA or 16:0 LPA served as a control. The cell recovery count in the control was set as 100%, and the relative percentage of cell recovery under each medium condition was calculated. The results are presented as follows. Figure 3 .

[0127] like Figure 3 As shown, for UC-MSCs, both the medium supplemented with 18:1 LPA and the medium supplemented with 16:0 LPA showed excellent proliferation-promoting effects compared to the basal medium. Furthermore, in the mediums supplemented with both 18:1 LPA and 16:0 LPA, a significant proliferation-promoting effect was obtained compared to the control. It should be noted that although data are not shown, for AD-MSCs, the same situation as in Example 1 confirmed the proliferation-promoting effect of adding 18:1 LPA and / or 16:0 LPA.

[0128] [Example 3: Planar Adhesion Culture / Study on the Concentration Ratio of 16:0 LPA to 18:1 LPA]

[0129] Human umbilical cord-derived mesenchymal stem cells (UC-MSC, Promo Cell) were used at a rate of 5,000 cells / cm². 2 Cells were seeded in T25 flasks (Corning) for adhesion cellularity and cultured in R: STEM Medium for hMSC High Growth (Rohto Pharmaceutical, serum-free medium, hereinafter referred to as RS medium), or in RS medium with a total addition of 5.0 μg / ml and various concentrations of 18:1 LPA and 16:0 LPA added. It should be noted that after culturing for 3 days at 37°C and 5% CO2 in each medium containing LPA, cells were peeled with trypsin, and the cell count in each flask was determined. Cells cultured only in RS medium without either 18:1 LPA or 16:0 LPA were used as a control. The number of recovered cells in the control was set as 100%, and the relative percentage of recovered cells under each medium condition was calculated. The results are presented below. Figure 4 .

[0130] like Figure 4 As shown, compared to UC-MSCs, a more significant proliferation-promoting effect was obtained when the ratio of 16:0 LPA to 18:1 LPA was 10:90 to 75:25. It should be noted that although data are not shown, the same proliferation-promoting effect of adding various concentrations of 18:1 LPA and 16:0 LPA was confirmed for AD-MSCs, similar to the case in Example 3.

[0131] [Example 4: Planar Adhesion Culture / Study on LPA Concentration]

[0132] Human umbilical cord-derived mesenchymal stem cells (UC-MSC, Promo Cell) were used at a rate of 5,000 cells / cm². 2 Cells were seeded in T25 flasks (Corning Chemicals) and cultured in R:STEM Medium for hMSC High Growth (Rohto Pharmaceutical Co., Ltd., serum-free medium, hereinafter referred to as RS medium) or in RS medium supplemented with equal amounts of 18:1 LPA and 16:0 LPA. After culturing for 3 days at 37°C and 5% CO2 in each medium, cells were peeled off with trypsin, and the cell count in each flask was determined. Cells cultured only in RS medium without either 18:1 LPA or 16:0 LPA were used as a control. The number of recovered cells in the control was set as 100%, and the relative percentage of recovered cells under each medium condition was calculated. The results are presented as follows: Figure 5 .

[0133] like Figure 5 As shown, the combined concentration of 18:1 LPA and 16:0 LPA was above 0.3125 μg / mL, demonstrating excellent proliferation-promoting effects, especially at 5 μg / mL to 20 μg / mL, where significant proliferation-promoting effects were obtained.

[0134] [Example 5: Planktonic Culture]

[0135] Human adipose-derived mesenchymal stem cells (AD-MSCs, produced by Promo Cell) were used at a concentration of 7,500 cells / cm². 2Low-concentration Corning Synthemax II microcarriers (Corning Incorporated) were inoculated into a disposable bioreactor (Able) and cultured in RS medium, or in RS medium supplemented with 2.5 μg / mL each of 18:1 LPA and 16:0 LPA. After culturing for 3 days at 37°C and 5% CO2, cells were peeled off with trypsin, and the cell count in each flask was determined. The cells cultured only in RS medium without either 18:1 LPA or 16:0 LPA were used as the control (RS). The number of recovered cells in the control was set as 100%, and the relative percentage of recovered cells under each medium condition was calculated. The results are presented as follows: Figure 6 .

[0136] like Figure 6 As shown, in the culture medium supplemented with both 18:1 LPA and 16:0 LPA, a significant proliferation-promoting effect was observed in AD-MSCs compared to the control. This effect obtained through planar culture was significantly greater than that obtained through planar adhesion culture (Example 1).

[0137] [Example 6: Evaluation of exosome production from adipose-derived mesenchymal stem cells]

[0138] Human adipose-derived mesenchymal stem cells (AD-MSC, Promo Cell) were prepared at a concentration of 5,000 cells / cm³. 2 Adhesion cells were seeded in T25 flasks (Corning) and cultured in RS medium (RSTEM) and in RS medium supplemented with 2.5 μg / mL of 18:1 LPA and 2.5 μg / mL of 16:0 LPA (with added LPA). After culturing in each medium at 37°C and 5% CO2 for 4 days, the culture supernatant was collected, and the exosome volume was quantified using a CD9 and CD63 ELISA kit (Cosmo Bio Inc.). The results are shown below. Figure 7 .

[0139] like Figure 7 As shown, in the culture medium supplemented with 18:1 LPA and 16:0 LPA, the amount of exosomes in the culture supernatant obtained from culturing AD-MSCs was about 4 times that in the serum culture medium and RS medium (RSTEM) mentioned above. The addition of 18:1 LPA and 16:0 LPA significantly promoted the production of exosomes derived from AD-MSCs.

[0140] [Example 7: Evaluation of humoral factor production in adipose-derived mesenchymal stem cells]

[0141] Human adipose-derived mesenchymal stem cells (AD-MSC, Promo Cell) were prepared at a concentration of 5,000 cells / cm³. 2Adhesion cells were seeded in T25 flasks (Corning) and cultured in RS medium, as well as in RS medium supplemented with 2.5 μg / mL of 18:1 LPA and 2.5 μg / mL of 16:0 LPA. After 4 days of culture at 37°C and 5% CO2, the culture supernatant was collected, and humoral factors in the supernatant were analyzed using a RayBio-tagged antibody array.

[0142] The results of the above analysis show that, compared with RS medium, RS medium supplemented with LPA (a mixture of 16:0 and 18:1) showed that hepatocyte growth factor (HGF) was more than 5 times higher, insulin-like growth factors (IGF-related factors) were more than 2 times higher, platelet-derived growth factor (PDGF) was more than 3 times higher, and vascular endothelial growth factor (VEGF) was more than 2 times higher.

[0143] [Example 8: Evaluation of the Adhesion of Mesenchymal Stem Cells]

[0144] Human adipose-derived mesenchymal stem cells (AD-MSC, Promo Cell) were prepared at a concentration of 5,000 cells / cm³. 2 Cells were seeded into commercially available adhesion cell flasks and cultured for 3 days at 37°C and 5% CO2 in RS medium or RS medium supplemented with 2.5 μg / mL each of 18:1 LPA and 16:0 LPA (RS+LPA). After 3 days of culture, cells were peeled off with trypsin, and the cell count in each flask was determined. The results are presented as follows: Figure 8 It should be noted that the details of the flask used are as follows.

[0145] Corning CellBind (registered trademark) CellBIND surface

[0146] By introducing oxygen-containing functional groups onto the polystyrene surface of the culture container, the surface becomes negatively charged.

[0147] ·Nunc / Nunc EasYFlask cell culture flask

[0148] The culture surface underwent a unique surface treatment.

[0149] • Falcon / Falcon (registered trademark) cell culture flasks

[0150] The culture surface was treated with vacuum gas plasma.

[0151] • Sumitomo Bakelite Adhesive Cell Culture Flask with Filter Cap (Green Cap)

[0152] The culture surface underwent a physical hydrophilic treatment.

[0153] like Figure 8 As shown, in cultures using RS medium supplemented with 18:1 LPA and 16:0 LPA, adhesion-based culture performance was demonstrated in both flasks compared to RS.

[0154] [Example 9: Evaluation of the proliferation-promoting effect and culture deviation of mesenchymal stem cells]

[0155] Three different batches of human adipose-derived mesenchymal stem cells (AD-MSC, Promo Cell) were used, at a concentration of 5,000 cells / cm³. 2 Cells were inoculated into RS medium or RS medium supplemented with 2.5 μg / mL each of 18:1 LPA and 16:0 LPA (RS+LPA) and cultured at 37°C and 5% CO2 for a total of 9 days. The number of cell divisions was measured after 9 days and shown in the figure. Figure 9 Additionally, the batch-to-batch deviation (variance factor) of the number of splits was compared, and the results are presented in... Figure 10 .

[0156] like Figure 9 As shown, when cultured using RS+LPA, the number of cell divisions after 9 days was approximately twice that in RS culture, demonstrating excellent culture performance. Furthermore, the batch-to-batch variation in cell division numbers was also smaller with RS+LPA culture compared to RS culture. This suggests that the improved adhesion properties of mesenchymal stem cells in LPA-added media contribute to increased proliferation and reduced batch-to-batch (donor) variability.

[0157] [Example 10: Overpassing of Mesenchymal Stem Cells]

[0158] Human adipose-derived mesenchymal stem cells (AD-MSC, Promo Cell) were prepared at a concentration of 5,000 cells / cm³. 2 Overpass culture experiments were conducted at 37°C and 5% CO2, with repeated passages every 3–4 days, in RS medium or RS medium supplemented with 2.5 μg / mL each of 18:1 LPA and 16:0 LPA (RS+LPA). The results are presented in [Table data would be inserted here]. Figure 11 .

[0159] like Figure 11As shown, when RS+LPA is used, AD-MSCs can be cultured until passage number 13 (P13) while maintaining the proliferation rate.

[0160] [Example 11: Angiogenesis Assay]

[0161] Human adipose-derived mesenchymal stem cells (AD-MSC, Promo Cell) were prepared at a concentration of 5,000 cells / cm³. 2 Cells were seeded into adhesion cell culture flasks (Corning) in various media (negative control, positive control, RSTEM, RSTEM+LPA (2.5 μg / mL each of 16:0 LPA and 18:1 LPA)) and cultured for 3 days at 37°C and 5% CO2. Then, the media were removed, cells were peeled with trypsin, and seeded into fresh media of the same type, and cultured for 3 days under the same conditions. Cells were then peeled with trypsin and cultured at 120,000 cells / cm³. 2 The culture medium was inoculated into 2% FBS-HuMedia and further cultured at 37°C and 5% CO2 for 3 days, after which the supernatant was recovered. It should be noted that 2% FBS-HuMedia was used as a negative control. Additionally, a medium containing 2% FBS-HuMedia supplemented with hEGF (final concentration 10 ng / mL) and hFGF-b (final concentration 5 ng / mL) was used as a positive control.

[0162] 40 μL / well of matrix gel basement membrane matrix (Corning) was added to each well of a black 96-well plate on ice and incubated at 37°C and 5% CO2 for at least 1 hour. Human umbilical vein endothelial cells (HUVECs) dispersed in the supernatant were seeded at 7,500 cells / well in the matrix gel-coated 96-well plates. Incubation was performed at 37°C and 5% CO2 for 5 hours. After culture, HUVECs were stained with calcein. The percentage of HUVEC staining area in the overall field of view was calculated using ImageJ to evaluate the proliferation rate of HUVECs (angiogenic capacity of each supernatant). Photographs taken during staining and comparisons of staining areas are shown below. Figure 12 Table 1.

[0163] [Table 1]

[0164] Table 1

[0165] Percentage of HUVEC staining area (%) SD negative control 9.455 0.186 Positive control 10.948 0.884 RSTEM 9.815 1.442 RSTEM+LPA 13.523 1.085

[0166] like Figure 12As shown in Table 1, the angiogenesis area was significantly larger in the culture supernatant of RSTEM+LPA, confirming a significantly superior angiogenesis capacity. Furthermore, proteomic analysis of the supernatants revealed that the culture supernatant of RSTEM+LPA contained higher levels of known angiogenesis-related factors (data not recorded) compared to the culture supernatant of RSTEM.

[0167] [Example 12: Evaluation of the proliferation-promoting effect of umbilical cord-derived mesenchymal stem cells]

[0168] One batch of human umbilical cord-derived mesenchymal stem cells (UC-MSC, manufactured by LIFELINE) was used, at a concentration of 4000 cells / cm³. 2 Cells were inoculated and cultured in RS medium (RSTEM) and mediums supplemented with 2.5 μg / mL each of 18:1 LPA and 16:0 LPA (containing LPA) at 37°C and 5% CO2 for a total of 10 days. Cell division count (PDL) was measured after 10 days and is shown in the figure. Figure 13 .

[0169] like Figure 13 As shown, when cultured in LPA-containing medium, the number of cell divisions increased after 10 days compared to culture in RS medium, demonstrating excellent culture performance. This suggests that cultured in LPA-added medium, even when using umbilical cord-derived mesenchymal stem cells, contributes to improved proliferative capacity.

[0170] [Example 13: Evaluation of cell surface antigen markers]

[0171] UC-MSCs cultured for 10 days under the conditions of Example 12 were thawed using a thermostat set to 37°C. 3.4 × 10⁶ cells were aliquoted from the thawed cell suspension and resuspended in culture medium. After centrifugation, the supernatant was removed, and the cells were resuspended in 1.7 mL of 1% BSA·PBS. 100 μL of the cell suspension and each antibody reagent were vortexed and reacted for 30 minutes under light and ice-cooled conditions. After the reaction, the cells were washed twice with 1% BSA·PBS, the supernatant was removed, and 500 μL of 1% BSA·PBS was added to homogenize the cell suspension. The cell suspension was added to tubes through a cell filter and reacted with 7-AAD at room temperature under light for 10 minutes. Flow cytometry was used to determine the cell surface markers. The results are shown in Table 2.

[0172] [Table 2]

[0173] CD11b 0.06％ CD19 0.03％ CD34 0.12％ CD45 0.12％ CD73 99.97％ CD90 99.39％ CD105 99.88％ IgG2a,k 0.06％ HLA-DR 0.06％

[0174] As shown in Table 2, it can be confirmed that UC-MSCs cultured using LPA-containing medium meet the definition of mesenchymal stem cells as defined by the International Society for Cellular Therapy (ISCT).

[0175] [Example 14: Evaluation of the proliferation-promoting effect of dental pulp-derived mesenchymal stem cells]

[0176] Using human dental pulp-derived mesenchymal stem cells (manufactured by Lonza), at a concentration of 3000 cells / cm³ 2 Cells were inoculated and cultured in RS medium (RSTEM) and medium supplemented with 2.5 μg / mL each of 18:1 LPA and 16:0 LPA (containing LPA) at 37°C and 5% CO2 for a total of 10 days. Cell division count (PDL) was measured after 4 days, and the results are presented as follows: Figure 14 .

[0177] like Figure 14 As shown, when cultured in LPA-containing medium, the number of cell divisions increased after 4 days compared to culture in existing medium, demonstrating excellent culture performance. Furthermore, samples taken at 7 and 10 days in LPA-containing medium also showed a better number of cell divisions compared to culture in existing medium. These results suggest that LPA-added medium contributes to improved proliferation capacity, even when used for dental pulp-derived mesenchymal stem cells.

[0178] Industrial availability

[0179] The mesenchymal stem cell proliferation promoter and culture medium for mesenchymal stem cells according to the present invention enable mesenchymal stem cells to proliferate efficiently while maintaining their undifferentiated state. Furthermore, the culture supernatant obtained by culturing mesenchymal stem cells using the culture medium for mesenchymal stem cells of the present invention contains a large number of exosomes, HGF, and various humoral factors, and is therefore suitable for use in disease treatment, etc.

Claims

1. A mesenchymal stem cell proliferation promoter containing 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and / or salts thereof.

2. The mesenchymal stem cell proliferation promoter according to claim 1, further comprising 18:1 lysophosphatidic acid, derivatives of 18:1 lysophosphatidic acid, and / or their salts.

3. The mesenchymal stem cell proliferation promoter according to claim 2, wherein, The ratio of the content (μg / mL) of 16:0 lysophosphatidic acid, its derivatives and / or their salts to the content (μg / mL) of 18:1 lysophosphatidic acid, its derivatives and / or their salts is 0.1 to 10.

4. The mesenchymal stem cell proliferation promoter according to claim 1, wherein, Mesenchymal stem cells originate from adipose tissue.

5. An additive for mesenchymal stem cell culture, comprising 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and / or salts thereof.

6. A culture medium for mesenchymal stem cells, comprising 16:0 lysophosphatidylcholine, derivatives of 16:0 lysophosphatidylcholine, and / or salts thereof.

7. The culture medium for mesenchymal stem cells according to claim 6, further comprising 18:1 lysophosphatidylcholine, derivatives of 18:1 lysophosphatidylcholine, and / or salts thereof.

8. The culture medium for mesenchymal stem cells according to claim 7, wherein, The ratio of the content (μg / mL) of 16:0 lysophosphatidic acid, its derivatives and / or their salts to the content (μg / mL) of 18:1 lysophosphatidic acid, its derivatives and / or their salts is 0.1 to 10.

9. The culture medium for mesenchymal stem cells according to claim 6, which is used to promote the proliferation of mesenchymal stem cells.

10. A mesenchymal stem cell obtained by culturing it using the mesenchymal stem cell proliferation promoter of any one of claims 1 to 4, the mesenchymal stem cell culture additive of claim 5, or the mesenchymal stem cell culture medium of any one of claims 6 to 9.

11. A culture supernatant containing 16:0 lysophosphatidylcholine, a derivative of 16:0 lysophosphatidylcholine, and / or salts thereof, said culture supernatant being obtained by culturing mesenchymal stem cells in the culture medium for mesenchymal stem cells according to any one of claims 6 to 9.

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