TW202444895A - Mesenchymal stem cell proliferation promoter, culture dish for mesenchymal stem cells, mesenchymal stem cells and culture supernatant - Google Patents

Abstract

The purpose of the present invention is to provide a medium for culture excellent in growth of mesenchymal stem cells. The present invention is a mesenchymal stem cell growth promoter containing 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof. The mesenchymal stem cell growth promoter of the present invention preferably also contains 18:1 lysophosphatidic acid, a derivative of 18:1 lysophosphatidic acid, and/or a salt thereof. The present invention also includes a medium for mesenchymal stem cells that contains16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof.

Description

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

The present invention relates to a mesenchymal stem cell proliferation promoter, a culture dish for mesenchymal stem cells, mesenchymal stem cells and a culture supernatant.

Regenerative medicine is a medical treatment that uses cells or tissues to assist and regenerate dysfunctional tissues or organs. Since this regenerative medicine can be a novel treatment for diseases that are difficult to respond to existing treatments, its practical application has become an urgent issue. Among them, regenerative medicine using biologically derived stem cells has attracted much attention and has been widely studied. Among the above-mentioned biologically derived stem cells, ES cells and iPS cells are mainly studied as tissue regeneration technologies. On the other hand, adult stem cells such as mesenchymal stem cells, in addition to their role as repair cells in tissue damage, are also expected to have various effects such as anti-inflammatory and immunosuppressive effects, and are attracting much attention as a more feasible cell therapy.

Mesenchymal stem cells are differentiated multipotent progenitor cells first isolated from the bone marrow by Friedenstein (1982) (see non-patent document 1). These mesenchymal stem cells are known to exist in various tissues such as the bone marrow, umbilical cord, and fat, and mesenchymal stem cell transplantation is highly anticipated as a new treatment method for various intractable diseases (see patent documents 1-2). In addition, some reports have shown that mesenchymal stem cells have immunosuppressive effects or are cumulative in tumors, and some studies have been conducted on the use of mesenchymal stem cells to prevent rejection after transplantation (non-patent document 2; Stem Cell Res Ther. 2021; 12: 192.), or to deliver cancer therapeutic drugs (non-patent document 3; Cancer Res (2013) 73 (1): 364-372). Recently, it has been known that there are cells with the same functions as mesenchymal cells such as adipose tissue, placenta, umbilical cord, and oocyte membranes, and some have called mesenchymal stem cells mesenchymal stromal cells.

Various culture dishes have been developed for culturing mesenchymal stem cells (see, for example, Patent Documents 3 and 4), but it is desirable to develop a culture dish that can allow mesenchymal stem cells that are more suitable for disease treatment to proliferate in large quantities and efficiently while maintaining their undifferentiated state. [Prior Art Document] [Patent Document]

[Patent Document 1] Japanese Patent Publication No. 2012-157263 [Patent Document 2] Japanese Patent Publication No. 2012-508733 [Patent Document 3] U.S. Patent Publication No. 2010/0015710 [Patent Document 4] Japanese Patent Publication No. 2010-094062 [Non-Patent Document]

[Non-patent document 1] Pittenger F. M. et al., Science 284, pp.143-147, 1999 [Non-patent document 2] Stem Cell Res Ther., 12, pp.192-, 2021 [Non-patent document 3] Cancer Res, 73(1), pp.364-372, 2013

[The problem that the invention wants to solve]

Under the above circumstances, the present invention aims to provide a method that can effectively proliferate mesenchymal stem cells while maintaining their undifferentiated state, that is, to provide an agent having an excellent effect of promoting the proliferation of mesenchymal stem cells and a culture dish for mesenchymal stem cells. [Means for solving the problem]

As a result of the research to solve the above problems, the inventors of this case found that the culture dish added with specific lysophosphatidic acid (LPA) has excellent proliferation of mesenchymal stem (stromal) cells (MSC), and completed the present invention. According to the present invention, the proliferation of mesenchymal stem cells can be promoted while maintaining the undifferentiated nature. That is, the gist of the present invention is as follows.

[1] A mesenchymal stem cell proliferation promoting agent comprising 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and/or salts thereof. [2] The mesenchymal stem cell proliferation promoting agent as described in [1], further comprising 18:1 lysophosphatidic acid, derivatives of 18:1 lysophosphatidic acid and/or salts thereof. [3] The mesenchymal stem cell proliferation promoting agent as described in [2], wherein the ratio of the content (μg/mL) of 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and/or salts thereof to the content (μg/mL) of 18:1 lysophosphatidic acid, derivatives of 18:1 lysophosphatidic acid and/or salts thereof is 0.1 to 10. [4] A mesenchymal stem cell proliferation promoter as described in [1], wherein the mesenchymal stem cells are derived from adipose tissue. [5] A mesenchymal stem cell culture additive, comprising 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid and/or a salt thereof. [6] A mesenchymal stem cell culture dish, comprising 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid and/or a salt thereof. [7] A mesenchymal stem cell culture dish as described in [6], further comprising 18:1 lysophosphatidic acid, a derivative of 18:1 lysophosphatidic acid and/or a salt thereof. [8] A culture dish for mesenchymal stem cells as described in [7], wherein the ratio of the content (μg/mL) of 16:0 lysophosphatidic acid, its derivatives and/or its salts to the content (μg/mL) of 18:1 lysophosphatidic acid, its derivatives and/or its salts is 0.1 to 10. [9] A culture dish for mesenchymal stem cells as described in [6] to [8], which is used to promote the proliferation of mesenchymal stem cells. [10] A mesenchymal stem cell obtained by culturing the mesenchymal stem cells using a mesenchymal stem cell proliferation promoter as described in any one of [1] to [4], a mesenchymal stem cell culture additive as described in [5], or a mesenchymal stem cell culture dish as described in any one of [6] to [9]. [11] A culture supernatant containing 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof, obtained by culturing the mesenchymal stem cells using a mesenchymal stem cell culture dish as described in any one of [6] to [9]. [12] A method for culturing mesenchymal stem cells, comprising the step of culturing mesenchymal stem cells in a culture dish containing 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and/or salts thereof. [13] The culturing method of [12], wherein the culturing is a planktonic or agitated culture. [14] A method for promoting the proliferation of mesenchymal stem cells using 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and/or salts thereof. [15] Use of 16:0 lysophosphatidic acid, its derivatives and/or its salts, and 18:1 lysophosphatidic acid, its derivatives and/or its salts for promoting the proliferation of mesenchymal stem cells. [16] Use of 16:0 lysophosphatidic acid, its derivatives and/or its salts for producing a mesenchymal stem cell proliferation promoter. [17] Use of 16:0 lysophosphatidic acid, its derivatives and/or its salts, and 18:1 lysophosphatidic acid, its derivatives and/or its salts for producing a mesenchymal stem cell proliferation promoter. [18] A method for promoting the proliferation of mesenchymal stem cells, characterized by using a culture dish containing 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and/or salts thereof. [19] A method for promoting the proliferation of mesenchymal stem cells, characterized by using a culture dish containing 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and/or salts thereof, and 18:1 lysophosphatidic acid, derivatives of 18:1 lysophosphatidic acid and/or salts thereof. [20] A method for promoting the proliferation of mesenchymal stem cells, characterized by using 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and/or salts thereof. [21] A method for promoting the proliferation of mesenchymal stem cells, characterized by using 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and/or salts thereof, and 18:1 lysophosphatidic acid, derivatives of 18:1 lysophosphatidic acid and/or salts thereof. [Effect of the invention]

According to the mesenchymal stem cell proliferation promoter and the mesenchymal stem cell culture dish of the present invention, mesenchymal stem cells can be effectively proliferated while maintaining undifferentiated state. Furthermore, the culture supernatant obtained by culturing mesenchymal stem cells using the mesenchymal stem cell culture dish of the present invention is rich in various humoral factors such as exosomes and HGF, and can be used for disease treatment, etc.

[Form of implementing the invention]

The mesenchymal stem cell proliferation promoter, mesenchymal stem cell culture dish, mesenchymal stem cell culture method, mesenchymal stem cells and culture supernatant of the present invention are described in detail below.

[Mesenchymal stem cell proliferation promoter] The mesenchymal stem cell proliferation promoter of the present invention is characterized by containing specific lysophosphatidic acid, lysophosphatidic acid derivatives and/or salts thereof. If the mesenchymal stem cell proliferation promoter of the present invention is used, mesenchymal stem cells can be effectively proliferated while maintaining undifferentiated properties. The mesenchymal stem cell proliferation promoter of the present invention may contain other ingredients in addition to the above-mentioned lysophosphatidic acid, lysophosphatidic acid derivatives and/or salts thereof, as long as the effects of the present invention are not impaired. The mesenchymal stem cell proliferation promoter of the present invention is described in detail below.

In the present invention, the state of "maintaining undifferentiated" of mesenchymal stem cells refers to a state in which the differentiation potential of mesenchymal stem cells into bone cells, chondrocytes and fat cells is maintained.

In the present invention, mesenchymal stem cells refer to cells that have the ability to differentiate into one or more cells belonging to the mesenchymal system (bone cells, cardiac muscle cells, chondrocytes, tendon cells, fat cells, etc.) and can proliferate while maintaining this ability. The term "mesenchymal stem cells" used in the present invention refers to the same cells as mesenchymal cells, without specifically distinguishing the two. In addition, sometimes they are simply expressed as mesenchymal cells. Tissues containing mesenchymal stem cells include, for example, adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amnion, chorionic membrane, chorionic membrane, dermis, skeletal muscle, periosteum, dental follicle, periodontal membrane, dental pulp, tooth germ, etc. Mesenchymal stem cells in the present invention include those derived from adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amnion, chorionic villus, tunica, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, tooth germ, etc. Among them, mesenchymal stem cells derived from adipose tissue, mesenchymal stem cells derived from umbilical cord, mesenchymal stem cells derived from bone marrow, and mesenchymal stem cells derived from dental pulp are preferred. Mesenchymal stem cells derived from adipose tissue, mesenchymal stem cells derived from umbilical cord, and mesenchymal stem cells derived from dental pulp are more preferred. Mesenchymal stem cells derived from adipose tissue, mesenchymal stem cells derived from umbilical cord, and mesenchymal stem cells derived from dental pulp are even more preferred. Mesenchymal stem cells derived from adipose tissue are even more preferred.

In the present invention, adipose tissue refers to a tissue containing interstitial cells including fat cells and microvascular cells, and is, for example, a tissue obtained by surgically removing or aspirating subcutaneous fat of a mammal.

The umbilical cord in the present invention refers to the white tubular tissue connecting the fetus and the placenta, which is composed of the umbilical cord vein, umbilical cord artery, colloid tissue (Wharton’s Jelly), umbilical cord matrix itself, etc., and is rich in mesenchymal stem cells.

In the present invention, bone marrow refers to the soft tissue that fills the inner cavity of bones and is a hematopoietic organ. There is bone marrow fluid in the bone marrow, and the cells in it are called bone marrow cells. In addition to red blood cells, granulocytes, megakaryocytes, lymphocytes, adipocytes, etc., bone marrow cells also include mesenchymal stem cells, hematopoietic stem cells, endothelial progenitor cells, etc. Bone marrow cells can be taken from, for example, human iliac bones, long tubular bones or other bones.

In the present invention, dental pulp refers to the connective tissue rich in blood vessels and nerves that can be found in the core of the tooth, which means the growth of the dental papilla in the tooth germ. It is the soft tissue that fills the pulp cavity inside the tooth.

The species of the mesenchymal stem cells in the present invention are preferably mammals. Examples of mammals include humans, horses, cows, sheep, pigs, dogs, cats, rabbits, mice, rats, and monkeys. Among them, humans, horses, cows, and cats are preferred.

Mesenchymal stem cells may be cells provided by, for example, PromoCell, Lonza, Biological Industries, Veritas, R&D Systems, and Corning, or cells prepared by methods well known in the art. Furthermore, mesenchymal stem cells may be primary cells isolated from the tissue of the provider or cells of an established cell line.

In the present invention, lysophosphatidic acid (LPA) is a lysophospholipid having an unsubstituted phosphate group, and there are multiple types of lysophosphatidic acid having different combinations of alkyl chain length and double bond number (carbon number and unsaturation degree). The above-mentioned carbon number-unsaturation combination (carbon number: unsaturation) includes 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, 22:6, etc. Among these, based on the excellent effect of promoting the proliferation of mesenchymal stem cells, 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 further preferred. Furthermore, the mesenchymal stem cell proliferation promoting agent of the present invention may also contain a plurality of lysophosphatidic acids with different combinations of alkyl chain length-double bond number (carbon number-unsaturation degree). Furthermore, the lysophosphatidic acid contained in the mesenchymal stem cell proliferation promoting agent of the present invention may be a derivative of lysophosphatidic acid. Examples of the derivatives of lysophosphatidic acid include lysophosphatidylcholine, lysophosphatidylserine, lysophosphatidylethanolamine, lysophosphatidylinositol, lysophosphatidylglycerol, and the like. The lysophosphatidic acid contained in the mesenchymal stem cell proliferation promoting agent of the present invention may be in any form such as a salt. When lysophosphatidic acid is in the form of salt, it can be listed as monosodium salt, disodium salt, monopotassium salt, dipotassium salt, magnesium salt, calcium salt, etc.

In order to more significantly exert the effect of the present invention, the mesenchymal stem cell proliferation promoter of the present invention preferably contains 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and/or salts thereof. In addition to 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and/or salts thereof, it is also more preferable to further contain 18:1 lysophosphatidic acid, derivatives of 18:1 lysophosphatidic acid and/or salts thereof.

The concentration of each lysophosphatidic acid contained in the mesenchymal stem cell proliferation promoting agent of the present invention, measured as the concentration when applied to cells, is 0.0025 μg/mL to 1 mg/mL, can be 0.01 μg/mL to 1 mg/mL, preferably 0.1 μg/mL to 100 μg/mL, more preferably 1 μg/mL to 50 μg/mL, even more preferably 1.5 μg/mL to 25 μg/mL, particularly preferably 2 μg/mL to 20 μg/mL, even more preferably 2 μg/mL to 10 μg/mL, and most preferably 2.5 μg/mL to 5 μg/mL.

The mesenchymal stem cell proliferation promoter of the present invention may also contain a mixture of multiple lysophosphatidic acids, in which case the total concentration of all lysophosphatidic acids, measured as the concentration when applied to cells, is 0.05 μg/mL to 1.25 mg/mL, preferably 0.1 μg/mL to 100 μg/mL, more preferably 0.3 μg/mL to 50 μg/mL, even more preferably 1 μg/mL to 40 μg/mL, particularly preferably 2 μg/mL to 30 μg/mL, and even more preferably 5 μg/mL to 20 μg/mL. Furthermore, the mixing ratio of the plurality of lysophosphatidic acids is not particularly limited. When 16:0 lysophosphatidic acid and 18:1 lysophosphatidic acid are contained, based on the viewpoint of excellent effect of promoting the proliferation of mesenchymal stem cells, the ratio of the content (μg/mL) of 16:0 lysophosphatidic acid and/or its salt to the content (μg/mL) of 18:1 lysophosphatidic acid and/or its salt 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.

The form of the mesenchymal stem cell proliferation promoting agent of the present invention is not particularly limited, and may be the above-mentioned lysophosphatidic acid itself or a composition composed of the above-mentioned lysophosphatidic acid and other components. In addition, the form of the above-mentioned composition is not particularly limited. The above-mentioned composition may be, for example, a mesenchymal stem cell culture additive mixed in a liquid culture dish for culture of mesenchymal stem cells or during the preparation of the liquid culture dish.

A preferred embodiment of the mesenchymal stem cell proliferation promoting agent of the present invention is a liquid culture dish containing the above-mentioned lysophosphatidic acid at the above-mentioned concentration and/or ratio.

When the mesenchymal stem cell proliferation promoting agent of the present invention is implemented as a liquid culture dish, the mesenchymal stem cell proliferation promoting agent of the present invention is a basal culture dish for animal cell culture known in the art that contains the above-mentioned lysophosphatidic acid at the above-mentioned concentration and/or ratio.

The basal culture dish for animal cell culture in the present invention refers to a culture dish containing carbon sources, nitrogen sources, inorganic salts, etc. required for animal cell culture. The animal cells referred to herein refer to mammalian cells, especially human cells. The basal culture dish for animal cell culture in the present invention may also contain raw materials of biological origin, but considering the possibility of using the cultured cells or their culture supernatant for the treatment of diseases of animals (including humans), it is preferred to use a culture dish that does not contain raw materials of biological origin as much as possible from the perspective of infectious risk.

The above-mentioned base culture dish for animal cell culture can use any culture dish for animal cell culture well known to those skilled in the art. Specifically, there can be mentioned minimal essential medium (MEM) such as Eagle's dish, Dulbecco's modified Eagle's dish (DMEM), minimal essential medium α (MEM-α), mesenchymal cell basal dish (MSCBM), Ham's F-12 and F-10 dishes, DMEM/F12 dishes, Williams dish E, RPMI-1640 dish, MCDB dish, 199 dish, Fisher dish, Iscove modified Dulbecco's dish (IMDM), McCoy modified dish, and mixed dishes thereof.

Furthermore, the above-mentioned basic culture dish for animal cell culture can be a known culture dish adjusted for mesenchymal stem cells, for example, Mesenchymal Stem Cell Growth Medium 2 (Ready-to-use) manufactured by PromoCell, Mesenchymal Stem Cell Growth Medium XF (Ready-to-use), manufactured by PromoCell, MSCGM BulletKittm, MSCGMtm Mesenchymal Stem Cell Growth Medium BulletKittm (manufactured by Lonza), xeno-free culture dish for human mesenchymal stem cells (MSC NutriStem (registered trademark) XF, manufactured by Biological Industries), MesenCult-ACF Plus (manufactured by Veritas), StemXVivotm Serum-Free Human MSC Expansion Media (manufactured by R&D Systems, Corning), serum-free culture dish for adipose-derived stem cells (KBM ADSC-4, manufactured by KOHJIN BIO), serum-free culture dish for mesenchymal stem cells (R: STEM Medium for hMSC High Growth, manufactured by Rohto), etc.

When the mesenchymal stem cell proliferation promoting agent of the present invention is implemented as a liquid culture dish, the mesenchymal stem cell proliferation promoting agent of the present invention may further contain amino acids such as glutamine, sugars such as glucose, metal salts such as sodium chloride or magnesium sulfate, trace metals such as selenium, lipids, etc., in addition to the above-mentioned animal cell culture base culture dish and the above-mentioned lysophosphatidic acid, as required. substances (except the above-mentioned lysophosphatidic acid and its salts), pantothenic acid and other vitamins, albumin, insulin, transferrin, insulin, growth factors (such as epidermal growth factor, basic fibroblast growth factor), growth factors, interleukins and other proteins, polysaccharides, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts and other substances.

When the mesenchymal stem cell proliferation promoting agent of the present invention is implemented in a liquid culture dish, the mesenchymal stem cell proliferation promoting agent of the present invention may contain serum as required in addition to the above-mentioned animal cell culture base culture dish and the above-mentioned lysophosphatidic acid. The above-mentioned serum is not particularly limited as long as it does not inhibit the proliferation of mesenchymal stem cells, and is serum derived from animals, preferably serum derived from mammals (such as fetal bovine serum, human serum, etc.), and more preferably human serum. The concentration of the above-mentioned serum in the mesenchymal stem cell proliferation promoting agent of the present invention can be within the concentration range well known to those skilled in the art. When cultured mesenchymal stem cells are used for medical purposes, other animal-derived components may become sources of infection of blood-borne pathogens or foreign antigens. Therefore, the mesenchymal stem cell proliferation promoter of the present invention is preferably serum-free. When serum-free, serum substitutes (e.g., Knockout Serum Replacement (KSR) (Invitrogen), Chemically-defined Lipid concentrated (Gibco), etc.) may also be used.

When the mesenchymal stem cell proliferation promoting agent of the present invention is implemented in a liquid culture dish, the osmotic pressure is preferably 0.9 to 1.1. The pH is preferably 6.0 to 9.0, and more preferably 6.5 to 8.5. Furthermore, the liquid culture dish is sterile, and the endotoxin content is preferably 2.5 EU/mL or less.

The mesenchymal stem cell proliferation promoting agent of the present invention can be prepared by mixing the above components by a common method. In addition, the mesenchymal stem cell proliferation promoting agent of the present invention is provided in a concentrated state, and can also be in a form to be diluted when used.

As can be understood from the above description, in the present invention, "the use of 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and/or salts thereof is used to promote the proliferation of mesenchymal stem cells", "the use of 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and/or salts thereof, and 18:1 lysophosphatidic acid, derivatives of 18:1 lysophosphatidic acid and/or salts thereof is used to promote the proliferation of mesenchymal stem cells" , "the use of 16:0 lysophosphatidic acid, its derivatives and/or its salts for the manufacture of a mesenchymal stem cell proliferation promoting agent", "the use of 16:0 lysophosphatidic acid, its derivatives and/or its salts, and 18:1 lysophosphatidic acid, its derivatives and/or its salts for the manufacture of a mesenchymal stem cell proliferation promoting agent" are also included in its scope.

As can be understood from the above description, in the present invention, "a method for promoting the proliferation of mesenchymal stem cells, characterized by using a culture dish containing 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid and/or a salt thereof", "a method for promoting the proliferation of mesenchymal stem cells, characterized by using 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid and/or a salt thereof, and 18:1 lysophosphatidic acid, a derivative of 18:1 lysophosphatidic acid and/or a salt thereof" "A culture dish containing salts of 16:0 lysophosphatidic acid, etc.", "A method for promoting the proliferation of mesenchymal stem cells, characterized by using 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and/or salts thereof", "A method for promoting the proliferation of mesenchymal stem cells, characterized by using 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and/or salts thereof, and 18:1 lysophosphatidic acid, derivatives of 18:1 lysophosphatidic acid and/or salts thereof" are also included in its scope.

[Mesenchymal stem cell culture dish] The present invention is a mesenchymal stem cell culture dish containing a basal culture dish for animal cell culture and specific lysophosphatidic acid, lysophosphatidic acid derivatives and/or salts thereof. The mesenchymal stem cell culture dish of the present invention is excellent in promoting the proliferation of mesenchymal stem cells and can be used as a dish for promoting stem cell proliferation. In addition, the mesenchymal stem cell culture dish of the present invention is equivalent to one embodiment of the above-mentioned mesenchymal stem cell proliferation promoter of the present invention (when it is a liquid culture dish).

The above-mentioned lysophosphatidic acid exists in a plurality of types having different combinations of alkyl chain length-double bond number (carbon number-unsaturation degree). Specifically, the above-mentioned carbon number-unsaturation degree combinations (carbon number: unsaturation degree) 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, 22:6, etc. Among these, from the viewpoint of excellent effect of promoting the proliferation of mesenchymal stem cells, 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 culture dish for mesenchymal stem cells of the present invention may also contain a plurality of lysophosphatidic acids with different combinations of alkyl chain length-double bond number (carbon number-unsaturation degree). Furthermore, the lysophosphatidic acid contained in the culture dish for mesenchymal stem cells of the present invention may be in any form such as a derivative or salt of lysophosphatidic acid. As for specific examples of the above-mentioned derivatives and salts, the description in the item [Mesenchymal stem cell proliferation promoting agent] can be applied.

The culture dish for mesenchymal stem cells of the present invention preferably contains 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and/or salts thereof, and more preferably further contains 18:1 lysophosphatidic acid, derivatives of 18:1 lysophosphatidic acid and/or salts thereof in addition to 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and/or salts thereof.

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

The culture dish for mesenchymal stem cells of the present invention may also contain a mixture of multiple lysophosphatidic acids, in which case the total concentration of all lysophosphatidic acids is preferably 0.1 μg/mL to 100 μg/mL, more preferably 0.3 μg/mL to 50 μg/mL, even more preferably 1 μg/mL to 40 μg/mL, particularly preferably 2 μg/mL to 30 μg/mL, and even more preferably 5 μg/mL to 20 μg/mL. Furthermore, the mixing ratio of the plurality of lysophosphatidic acids is not particularly limited. When 16:0 lysophosphatidic acid and 18:1 lysophosphatidic acid are contained, based on the viewpoint of excellent effect of promoting the proliferation of mesenchymal stem cells, the ratio of the content (μg/mL) of 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and/or salts thereof to the content (μg/mL) of 18:1 lysophosphatidic acid, derivatives of 18:1 lysophosphatidic acid and/or salts thereof is 0.001-1000, preferably 0.01-100, more preferably 0.1-10, still more preferably 0.1-5, particularly preferably 0.3-3, and still more preferably 0.5-1.5.

Since the culture dish for mesenchymal stem cells of the present invention is equivalent to one embodiment of the above-mentioned mesenchymal stem cell proliferation promoting agent of the present invention (in the case of a liquid culture dish), the description of the liquid culture dish in the mesenchymal stem cell proliferation promoting agent can be directly applied to the above-mentioned basic culture dish for animal cell culture, other components, characteristics, etc.

The culture dish for mesenchymal stem cells of the present invention also includes one in which a gelling material is added to the above-mentioned liquid culture dish to form a gel. The above-mentioned gelling material can be any material that can make the culture dish gel-like and can be used for cell culture, and examples thereof include agar, agarose, alginic acid, collagen, gelatin, cellulose, etc. The added concentration of such gelling material can be any concentration that can make the culture dish gel-like, and the industry can set it appropriately according to needs. It can be in a gel-like state under the cell culture or storage conditions, and preferably in a gel-like state at any temperature within the temperature range of, for example, -80°C to 100°C.

[Supplement for mesenchymal stem cell culture] The present invention also includes a supplement for mesenchymal stem cell culture, which contains 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and/or salts thereof. The supplement for mesenchymal stem cell culture of the present invention, by containing the above-mentioned specific lysophosphatidic acid, derivatives of lysophosphatidic acid and/or salts thereof, can be used for mesenchymal stem cell culture and/or mesenchymal stem cell proliferation promotion because it can exert an excellent effect of promoting the proliferation of mesenchymal stem cells when added to a culture dish or the like when culturing mesenchymal stem cells. In addition, the additive for culturing mesenchymal stem cells of the present invention is equivalent to one embodiment of the above-mentioned mesenchymal stem cell proliferation promoting agent of the present invention. Therefore, the detailed description of the additive for culturing mesenchymal stem cells of the present invention can be applied to the description of the mesenchymal stem cell proliferation promoting agent of the present invention.

[Method for culturing mesenchymal stem cells] The present invention also includes a method for culturing mesenchymal stem cells, which includes the step of culturing mesenchymal stem cells in a culture dish containing specific lysophosphatidic acid, lysophosphatidic acid derivatives and/or salts thereof. According to the culturing method of the present invention, mesenchymal stem cells can be effectively proliferated while maintaining undifferentiated properties.

The above-mentioned lysophosphatidic acid exists in a plurality of types having different combinations of alkyl chain length-double bond number (carbon number-unsaturation degree). Specifically, the above-mentioned carbon number-unsaturation degree combinations (carbon number: unsaturation degree) 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, 22:6, etc. Among these, from the viewpoint of excellent effect of promoting the proliferation of mesenchymal stem cells, 16:0, 16:1, 18:0, 18:1, 18:2, 18:3 are preferred, 16:0, 18:1 are more preferred, and 16:0 is even more preferred. Furthermore, the above-mentioned culture dish may also contain a plurality of lysophosphatidic acids with different combinations of alkyl chain length-double bond number (carbon number-unsaturation). Furthermore, the lysophosphatidic acid contained in the above-mentioned culture dish may be any form of a derivative of lysophosphatidic acid or such salts. As for the specific examples of the above-mentioned derivatives and salts, the description in the item [Mesenchymal stem cell proliferation promoter] can be applied.

Since the culture dish containing specific lysophosphatidic acid, lysophosphatidic acid derivatives and/or salts thereof in the culture method of the present invention is equivalent to one embodiment of the mesenchymal stem cell proliferation promoting agent of the present invention (a liquid culture dish), the details of the culture dish can be directly applied to the description of the liquid culture dish in the mesenchymal stem cell proliferation promoting agent. Moreover, the description of mesenchymal stem cells can also be directly applied to the description of the mesenchymal stem cell proliferation promoting agent.

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 dish containing specific lysophosphatidic acid, derivatives of lysophosphatidic acid and/or salts thereof, and the same method as usual can be used otherwise. The culture temperature is usually 30°C to 40°C, preferably 30 to 37°C. The _CO2 concentration during culture is 2% to 10%, preferably 2% to 7%, and more preferably 5%. The _O2 concentration during culture is 0% to 100%, preferably 0.5% to 22%, and more preferably 5 to 21%. Furthermore, mesenchymal stem cells may be subcultured or culture dishes may be replaced as needed during the culture. The timing and method are not particularly limited as long as they are suitable for each mesenchymal stem cell, and the process may be performed in the same manner as usual while observing the morphology of the mesenchymal stem cells. The number of subcultures is preferably 0 to 8, more preferably 1 to 6. The culture time 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 appropriately determined according to the purpose of cultivation, and can be low density seeding and long time cultivation, or high density seeding and short time cultivation. Generally, the preferred density is 2500 cells/cm ² to 10000 cells/cm ² , and more preferably 5000 cells/cm ² to 7500 cells/cm ² .

The culturing method of mesenchymal stem cells of the present invention can be carried out by attaching the mesenchymal stem cells to a general flat surface of a culture vessel such as a flask, a culture dish, or a culture plate for cell culture, or by attaching the mesenchymal stem cells to microbeads, microcarriers, etc. and culturing the microbeads, microcarriers, etc. by floating culture (suspension culture), floating and stirring culture, or by attaching the mesenchymal stem cells to a fibrous scaffold such as microfibers, or by using spheroid culture.

The culture device used for culturing mesenchymal stem cells of the present invention is not particularly limited as long as it can culture mesenchymal stem cells, and examples thereof include flasks, tissue culture flasks, culture dishes, petri dishes, tissue culture dishes, multi-dishes, microtiter plates, microplates, multi-plates, multi-well plates, microscope slides, chamber slides, dishes, tubes, trays, culture bags, rollers, and the like.

[Mesenchymal stem cells] The present invention also includes a mesenchymal stem cell obtained by culturing using the above-mentioned mesenchymal stem cell proliferation promoter, mesenchymal stem cell culture additive or mesenchymal stem cell culture dish of the present invention. The mesenchymal stem cells of the present invention are cultured in a culture dish containing specific lysophosphatidic acid, lysophosphatidic acid derivatives and/or salts thereof, and not only can promote proliferation, have a high survival rate and good condition, but also can release more exosomes or various humoral factors such as HGF compared to conventional mesenchymal stem cells, and can be used for the treatment of various diseases.

The mesenchymal stem cells of the present invention are obtained by culturing the known mesenchymal stem cells using the above-mentioned mesenchymal stem cell proliferation promoter of the present invention or the culture dish for mesenchymal stem cells of the present invention. The culture temperature is usually 30°C to 40°C, preferably 30 to 37°C. The _CO2 concentration during the culture is 2% to 10%, preferably 2% to 7%, and more preferably 5%. The _O2 concentration during the culture is 0% to 100%, preferably 0.5% to 22%, and more preferably 5% to 21%. Furthermore, mesenchymal stem cells can be subcultured or culture dishes can be replaced as needed during the culture. The time and method are not particularly limited as long as they are suitable for each mesenchymal stem cell, and the process can be performed in the same manner as usual while observing the morphology of the mesenchymal stem cells. The number of subcultures is preferably 0 to 8, more preferably 1 to 6. The culture time 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/cm ² to 10000 cells/cm ² , more preferably 5000 cells/cm ² to 7500 cells/cm ² .

The mesenchymal stem cells of the present invention can increase the amount of extracellular bodies or various humoral factors such as HGF released into the culture supernatant by increasing the seeding density and cell density. Such seeding density is preferably 5000 cells/cm ² to 40000 cells/cm ² , more preferably 7500 cells/cm ² to 30000 cells/cm ² , and even more preferably 15000 cells/cm ² to 20000 cells/cm ² . In addition, by reducing the seeding density and culturing for a long time, the concentration of various humoral factors such as extracellular bodies or HGF in the culture supernatant can also be increased.

The mesenchymal stem cells of the present invention are positive for CD73, CD90 and CD105 and remain undifferentiated. Moreover, the mesenchymal stem cells of the present invention release more CD9 and CD63 positive exosomes than known mesenchymal stem cells. Moreover, the mesenchymal stem cells of the present invention release more various humoral factors such as hepatocyte growth factor (HGF), insulin-like growth factors (IGF-related factors), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and the like than known mesenchymal stem cells.

Diseases for which the mesenchymal stem cells of the present invention can be used as a medicine include, for example, cartilage degeneration, rheumatoid arthritis, psoriasis arthritis, spondylitis, osteoarthritis, gout, psoriasis, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, congestive heart failure, stroke, aortic valvular stenosis. disease, renal failure, lupus, pancreatitis, allergy, fibrosis, anemia, atherosclerosis, restenosis, chemotherapy/radiation-related complications, type I diabetes, type II diabetes, autoimmune hepatitis, hepatitis C, primary biliary cirrhosis, primary sclerosing cholangitis, fulminant hepatitis, chylous diarrhea, nonspecific colitis, allergy Conjunctivitis, diabetic retinopathy, sicca syndrome, uveitis, allergic rhinitis, asthma, asbestosis, silicosis, chronic obstructive pulmonary disease, chronic granulomatous inflammation, cystic fibrosis, polyarteritis nodosa, glomerulonephritis, vasculitis, dermatitis, HIV-related cachexia, cerebral malaria, ankylosing spondylitis, leprosy disease, pulmonary fibrosis, esophageal cancer, gastroesophageal reflux disease, Barrett's esophagus, gastric cancer, duodenal cancer, small intestine cancer, coccyx 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.

The administration method when the mesenchymal stem cells of the present invention are used as a pharmaceutical is not particularly limited, and is preferably intravascular administration (preferably intravenous administration), intraperitoneal administration, intraintestinal administration, subcutaneous administration, etc., and more preferably intravascular administration.

The dosage of the mesenchymal stem cells of the present invention when used as a medicine may vary depending on the type of disease or the severity of its symptoms, dosage form, weight of the subject, etc. When administered to humans, the mesenchymal stem cells can be administered in the range of 1×10 ⁵ to 1×10 ⁹ per day. When administered to small animals such as mice, the dosage can be in the range of 1×10 ⁴ to 1×10 ⁹ per day, preferably in the range of 1×10 ⁵ to 1×10 ^9. In addition, the administration of the mesenchymal stem cells of the present invention when used as a medicine can be performed in one or more times per day. Moreover, the above-mentioned administration can be performed as a single administration or continuously. When the administration is continued, the drug may be administered continuously for 2 or more times, for example, at a frequency of once or more every 3 days.

When the mesenchymal stem cells of the present invention are used as a drug, the mammals to which they are administered are not particularly limited, and preferably humans, monkeys, mice, rats, hamsters, guinea pigs, cattle, pigs, horses, rabbits, sheep, goats, cats, dogs, etc., and more preferably humans. Furthermore, when the mesenchymal stem cells of the present invention are used as a drug, it is preferred that the species of the mammal to which they are administered be consistent from the viewpoint of obtaining a more stable and superior preventive and/or therapeutic effect on the disease.

[Culture supernatant] The present invention also includes a culture supernatant containing 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and/or salts thereof, which is obtained by culturing mesenchymal stem cells in the culture dish for mesenchymal stem cells of the present invention. The culture supernatant of the present invention contains more CD9 and CD63 positive exosomes than the culture supernatant obtained by culturing mesenchymal stem cells in a conventional culture dish. 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 the present invention can be used for the treatment of various diseases. Moreover, it can also be used in cosmetics and food.

(Preparation of culture supernatant) The culture supernatant of mesenchymal stem cells obtained by the following method can be prepared as the culture supernatant in the present invention. In addition, the culture supernatant in the present invention can be prepared by removing excess components from the supernatant by means of dialysis, superfiltration, etc., by fractionating the supernatant with a column, etc., by using antibodies against specific molecules, etc., by centrifugation, etc.

The culture dish used to obtain the culture supernatant is the culture dish for mesenchymal stem cells of the present invention. The method for obtaining the culture supernatant is not particularly limited as long as it is a method suitable for the culture of each mesenchymal stem cell. The culture temperature is usually 30°C to 40°C, preferably 30°C to 37°C. The _CO2 concentration during culture is 2% to 10%, preferably 2% to 7%, and more preferably 5%. The _O2 concentration during culture is 0% to 100%, preferably 0.5% to 22%, and more preferably 5% to 21%. Furthermore, mesenchymal stem cells may be subcultured or culture dishes may be replaced as needed during the culture. The timing and method are not particularly limited as long as they are suitable for each mesenchymal stem cell, and the culture may be performed in the same manner as usual while observing the morphology of the mesenchymal stem cells. The number of subcultures of mesenchymal stem cells is preferably 0 to 8, more preferably 1 to 6. The culture time 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/cm ² to 10000 cells/cm ² , more preferably 5000 cells/cm ² to 7500 cells/cm ² .

When obtaining the culture supernatant, the concentration of various humoral factors such as extracellular bodies or HGF in the culture supernatant can be increased by increasing the seeding density and cell density of mesenchymal stem cells. Such seeding density is preferably 5000 cells/cm ² to 40000 cells/cm ² , more preferably 7500 cells/cm ² to 30000 cells/cm ² , and even more preferably 15000 cells/cm ² to 20000 cells/cm ² . In addition, the concentration of various humoral factors such as extracellular bodies or HGF in the culture supernatant can also be increased by reducing the seeding density of mesenchymal stem cells and culturing for a long time.

After obtaining the culture supernatant, the cells can be subcultured, and the interleukin-1-type stem cells of the present invention can be cultured in the culture dish for multiple times to obtain the culture supernatant multiple times.

The culture for obtaining the culture supernatant of the present invention may be a flat-surface attachment culture in which the cells are attached to a flask, a culture dish, a culture plate, etc. for cell culture, or a floating or agitated culture in which the cells are attached to a microcarrier, microbeads, etc.

The culture supernatant of the present invention is preferably sterile after aseptic treatment when provided, and the endotoxin content is preferably below 2.5 EU/mL.

The culture supernatant of the present invention can be used for the same diseases as the diseases for which the mesenchymal stem cells of the present invention can be used as a medicine. And the same applies to the species used as the object of administration. The culture supernatant of the present invention can also be used in quasi-drugs, cosmetics, and foods. When the culture supernatant of the present invention is used as a medicine, the preferred method of administration is intravascular administration (preferably intravenous administration), intraperitoneal administration, intraintestinal administration, subcutaneous administration, etc., among which intravascular administration is more preferred.

The dosage of the culture supernatant of the present invention when used as a medicine may vary depending on the type of disease or the severity of its symptoms, dosage form, weight of the subject, etc. In addition, the culture supernatant of the present invention when used as a medicine can be administered once or more times a day. Moreover, the above administration can be a single administration or continuous administration. When it is performed continuously, it can be administered continuously for more than 2 times at a frequency of once or more than 3 days. [Example]

The present invention is described in detail with reference to the following embodiments and test examples, but the present invention is not limited to these embodiments and the like.

[Example 1: Study on the effect of promoting the proliferation of mesenchymal stem cells in flat-surface adhesion culture/culture dishes containing LPA-1] Human adipose-derived mesenchymal stem cells (AD-MSC, manufactured by Promo Cell) and human umbilical cord-derived mesenchymal stem cells (UC-MSC, manufactured by Promo Cell) were seeded at 5,000 cells/ ^cm2 in T25 flasks for adherent cells (manufactured by Corning) and cultured in R:STEM Medium for hMSC High Growth culture dishes (manufactured by Rohto Pharmaceutical Co., Ltd., serum-free culture dishes, hereinafter referred to as RS culture dishes, RS or RSTEM) or RS culture dishes supplemented with 18:1 LPA and/or 16:0 LPA. When 18:1 LPA and 16:0 LPA were 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 each culture dish at 37°C and 5% CO ₂ for 3 days, cells were detached with trypsin and the number of cells in each flask was measured. The RS culture dish without either 18:1 LPA or 16:0 LPA was used as the control group (RS). The number of recovered cells in the control group was set as 100%, and the relative value (%) of the number of recovered cells under the conditions of each culture dish was calculated. The results are shown in Figures 1 and 2. FIG1 shows the effect of adding 16:0 LPA (AD-MSC, UC-MSC), and FIG2 shows the effect of adding either 18:1 LPA or 16:0 LPA or both (AD-MSC).

When cultured in a dish supplemented with 16:0 LPA, AD-MSC and UC-MSC can significantly promote their proliferation (Figure 1). Moreover, when cultured in dishes supplemented with 18:1 LPA and 16:0 LPA, AD-MSC can achieve a synergistic effect in promoting their proliferation compared to dishes supplemented with either 18:1 LPA or 16:0 LPA alone (Figure 2).

[Example 2: Study on the effect of promoting the proliferation of mesenchymal stem cells in flat-surface adhesion culture/culture dishes containing LPA-2] Mesenchymal stem cells derived from human umbilical cord (UC-MSC, manufactured by Promo Cell) were seeded at 5,000 cells/ ^cm2 in T25 flasks for adherent cells (Corning) and cultured in a basic culture dish (MEM, bFGF, albumin, insulin, transferrin, 3% FBS) supplemented with 18:1 LPA and/or 16:0 LPA. When 18:1 LPA or 16:0 LPA was added, the final concentration was 2.5 μg/mL. When both 18:1 LPA and 16:0 LPA were added, the final concentration was 2.5 μg/mL. After culturing each culture dish at 37°C and 5% CO ₂ for 3 days, cells were detached with trypsin and the number of cells in each flask was measured. RS culture dishes without either 18:1 LPA or 16:0 LPA were used as the control group. The number of recovered cells in the control group was set as 100%, and the relative value (%) of the number of recovered cells under the conditions of each culture dish was calculated. The results are shown in Figure 3.

As shown in FIG3 , for UC-MSC, the culture dish with 18:1 LPA added alone and the culture dish with 16:0 LPA added alone showed a better proliferation promoting effect compared with the basal culture dish. Furthermore, the culture dish with both 18:1 LPA and 16:0 LPA added showed a more significant proliferation promoting effect compared with the control group. In addition, although the data are not shown, for AD-MSC, the proliferation promoting effect produced by adding 18:1 LPA and/or 16:0 LPA can be confirmed as in Example 1.

[Example 3: Planar adhesion culture/Study of the concentration ratio of 16:0 LPA and 18:1 LPA] Mesenchymal stem cells derived from human umbilical cord (UC-MSC, manufactured by Promo Cell) were seeded at 5,000 cells/ ^cm2 in a T25 flask for adherent cells (Corning). 18:1 LPA and 16:0 LPA were fixed in R:STEM Medium for hMSC High Growth culture dishes (manufactured by Rohto Pharmaceutical, serum-free culture dishes, hereinafter referred to as RS culture dishes) or RS culture dishes at a total addition amount of 5.0μg/ml, and culture was performed using culture dishes added with various concentration ratios. In addition, after culturing each LPA dish at 37°C and 5% CO ₂ for 3 days, cells were detached with trypsin and the number of cells in each flask was measured. RS dishes without either 18:1 LPA or 16:0 LPA were used as the control group, and the number of recovered cells in the control group was set as 100%. The relative value (%) of the number of recovered cells under the conditions of each dish was calculated. The results are shown in Figure 4.

As shown in Figure 4, for UC-MSC, a more significant proliferation promoting effect can be obtained when the ratio of 16:0 LPA to 18:1 LPA is 10:90 to 75:25. In addition, for AD-MSC, the proliferation promoting effect produced by adding various concentrations of 18:1 LPA and 16:0 LPA can be confirmed as in Example 3.

[Example 4: Planar Adhesion Culture/Study of LPA Concentration] Mesenchymal stem cells derived from human umbilical cord (UC-MSC, Promo Cell) were seeded at 5,000 cells/ ^cm2 in T25 flasks for adherent cells (Corning), and cultured in R: STEM Medium for hMSC High Growth culture dishes (Rohto Pharmaceutical, serum-free culture dishes, hereinafter RS culture dishes) or RS culture dishes supplemented with 18:1 LPA and 16:0 LPA in equal amounts at various concentrations. After culturing each culture dish at 37°C and 5% _CO2 for 3 days, the cells were detached with trypsin, and the number of cells in each flask was measured. RS culture dishes without either 18:1 LPA or 16:0 LPA were used as control groups, and the number of recovered cells in the control group was set as 100%, and the relative value (%) of the number of recovered cells under each culture dish condition was calculated. The results are shown in FIG5 .

As shown in FIG5 , when the total concentration of 18:1 LPA and 16:0 LPA was 0.3125 μg/mL or more, an excellent proliferation promoting effect was exhibited, and a significant proliferation promoting effect was obtained in particular when the total concentration was 5 μg/mL to 20 μg/mL.

[Example 5: Planktonic culture] Human adipose-derived mesenchymal stem cells (AD-MSC, manufactured by Promo Cell) were seeded at 7,500 cells/ ^cm2 in Corning (registered trademark) low-concentration Synthemax (registered trademark) II microcarriers (Corning) in a single-use bioreactor (manufactured by Able) and cultured in planktonic culture in RS culture dishes or RS culture dishes supplemented with 2.5 μg/mL of 18:1 LPA and 16:0 LPA, respectively. After culturing for 3 days at 37°C and 5% _CO2 , the cells were detached with trypsin and the number of cells in each flask was measured. The cells cultured in RS dishes without either 18:1 LPA or 16:0 LPA were used as the control group (RS). The number of recovered cells in the control group was set as 100%, and the relative value (%) of the number of recovered cells under each dish condition was calculated. The results are shown in FIG6 .

As shown in Figure 6, the culture dishes supplemented with 18:1 LPA and 16:0 LPA can achieve a more significant proliferation promoting effect on AD-MSCs compared to the control group. This effect obtained by planktonic culture is more significant than that obtained by planar attachment culture (Example 1).

[Example 6: Evaluation of exosome production by adipose-derived mesenchymal stem cells] Human adipose-derived mesenchymal stem cells (AD-MSC, manufactured by Promo Cell) were seeded at 5,000 cells/ ^cm2 in a T25 flask for adherent cells (manufactured by Corning) and cultured in RS culture dishes (RSTEM) and RS culture dishes supplemented with 2.5 μg/mL of 18:1 LPA and 16:0 LPA (LPA added). After culturing each culture dish at 37°C and 5% _CO2 for 4 days, the culture supernatant was recovered and the amount of exosomes was quantified using a CD9, CD63 ELISA kit (manufactured by COSMO BIO Co., Ltd.). The results are shown in FIG7 .

As shown in FIG7 , the amount of exosomes in the culture supernatant obtained by culturing AD-MSCs in the culture dishes supplemented with 18:1 LPA and 16:0 LPA was approximately 4 times greater than that in the above-mentioned serum culture dishes and RS culture dishes (RSTEM). It can be seen that the addition of 18:1 LPA and 16:0 LPA can significantly promote the production of exosomes from AD-MSCs.

[Example 7: Evaluation of Humoral Factors Production by Adipose-Derived Mesenchymal Stem Cells] Human adipose-derived mesenchymal stem cells (AD-MSC, Promo Cell) were seeded at 5,000 cells/ ^cm2 in a T25 flask for adherent cells (Corning), and cultured in RS culture dishes and RS culture dishes supplemented with 2.5 μg/ _mL of 18:1 LPA and 16:0 LPA, respectively. After culturing each culture dish at 37°C and 5% CO2 for 4 days, the culture supernatant was recovered and analyzed for each humoral factor in the culture supernatant using RayBio Label-Based Antibody Array.

The results of the above analysis showed that compared with RS culture dishes, RS culture dishes supplemented with LPA (a mixture of 16:0 and 18:1) had 5 times more hepatocyte growth factor (HGF), 2 times more insulin-like growth factors (IGF-related factors), 3 times more platelet-derived growth factor (PDGF), and 2 times more vascular endothelial growth factor (VEGF).

[Example 8: Evaluation of the adhesion of mesenchymal stem cells] Mesenchymal stem cells derived from human adipose (AD-MSC, Promo Cell) were seeded at 5,000 cells/ ^cm2 in commercially available adherent cell flasks and cultured for 3 days at 37°C and 5% CO2 using RS culture dishes or RS culture dishes supplemented with 18:1 LPA and 16:0 LPA at 2.5 μg/ _mL (RS+LPA). After 3 days of culture, the cells were detached with trypsin and the number of cells in each flask was measured. The results are shown in FIG8 . In addition, the details of the flasks used are as follows: ・Corning CellBind/Corning (registered trademark) CellBIND surface introduces oxygen-containing functional groups to the polystyrene surface of the culture container, making the surface charge negative. ・Nunc/Nunc EasYFlask Cell Culture Flasks The culture surface has a unique surface treatment. ・Falcon/Falcon (registered trademark) Cell Culture Flask The culture surface has been treated with vacuum gas plasma. ・Sumitomo Bakelite/Attached Cell Culture Flask with Filter Seal (Green Seal) The culture surface has been treated with physical hydrophilicity.

As shown in FIG8 , when the culture dishes with 18:1 LPA and 16:0 LPA were used for culturing, both flasks showed good culturing performance due to adhesion compared to RS.

[Example 9: Evaluation of proliferation-promoting effect and culture heterogeneity of mesenchymal stem cells] Three different batches of mesenchymal stem cells derived from human adipose (AD-MSC, Promo Cell) were used, seeded at 5,000 cells/ ^cm2 , and cultured for a total of 9 days at 37°C and 5% CO2 using RS culture dishes or RS culture dishes supplemented with 18:1 LPA and 16:0 LPA at 2.5μg/ _mL (RS+LPA). The number of cell divisions on day 9 was measured and is shown in Figure 9. In addition, the heterogeneity (coefficient of variation) of the number of divisions between batches was compared, and the results are shown in Figure 10.

As shown in Figure 9, when cultured with RS+LPA, the number of cell divisions on day 9 was about twice that of cells cultured with RS, indicating good culture performance. Moreover, regarding the unevenness of cell divisions between batches, cells cultured with RS+LPA also had a smaller unevenness than cells cultured with RS. This indicates that when cultured with LPA-added culture dishes, the improvement in the adhesion performance of mesenchymal stem cells helps to enhance proliferation and reduce differences between batches (providers).

[Example 10: Subculture of Mesenchymal Stem Cells] Mesenchymal stem cells derived from human adipose (AD-MSC, Promo Cell) were seeded at 5,000 cells/ ^cm2 in an adherent cell flask, and the cells were subcultured repeatedly every 3 to 4 days in RS culture dishes or RS culture dishes supplemented with 18:1 LPA and 16:0 LPA at 2.5 μg/ _mL (RS+LPA) at 37°C and 5% CO2. The results are shown in FIG11 .

As shown in FIG. 11 , when RS+LPA was used, AD-MSCs could be cultured to passage 13 (P13) while maintaining their proliferation rate.

[Example 11: Angiogenesis Assay] Mesenchymal stem cells derived from human adipose (AD-MSC, manufactured by Promo Cell) were seeded at 5,000 cells/ ^cm2 in each culture dish (negative control group, positive control group, RSTEM, RSTEM+LPA (16:0 LPA and 18:1 LPA, 2.5ug/mL each)) in an adherent cell flask (manufactured by Corning) and cultured at 37°C and 5% _CO2 for 3 days. Afterwards, each culture dish was removed and the cells were detached with trypsin, then seeded in a new culture dish of the same type and cultured under the same conditions for 3 days. Afterwards, cells were detached with trypsin, seeded at 120,000 cells/ ^cm2 in 2% FBS-HuMedia, and cultured for 3 days at 37°C and 5% _CO2 , and the supernatant was collected. In addition, the negative control group used 2% FBS-HuMedia. And the positive control group used a culture dish supplemented with hEGF (final concentration 10 ng/mL) and hFGF-b (final concentration 5 ng/mL) in 2% FBS-HuMedia.

Add 40ul/well of Matrigel basement membrane matrix (Corning) to each black 96-well plate on ice and culture at 37°C, 5% CO ₂ for more than 1 hour. In the 96-well plate coated with Matrigel, 7,500 cells/well of human umbilical vein endothelial cells (HUVEC) were seeded and dispersed in each of the supernatants prepared above. Culture was carried out at 37°C, 5% CO ₂ for 5 hours. After culture, HUVEC was stained with calcitonin. Furthermore, the proportion (%) of the HUVEC staining area to the entire visual field was calculated using ImageJ to evaluate the proliferation rate of HUVEC (angiogenesis ability of each supernatant). The photos of the staining and the comparison of the staining area are shown in Figure 12 and Table 1, respectively.

As shown in Figure 12 and Table 1, the angiogenesis area was larger in the culture supernatant using RSTEM+LPA, which significantly confirmed the excellent angiogenesis ability. In addition, the results of proteomic analysis of each supernatant showed that a large number of factors known to be related to angiogenesis were detected in the culture supernatant using RSTEM+LPA compared with the culture supernatant using RSTEM (data not recorded).

[Example 12: Evaluation of the proliferation-promoting effect of mesenchymal stem cells derived from the umbilical cord] A batch of mesenchymal stem cells derived from the human umbilical cord (UC-MSC, manufactured by LIFELINE) was used, seeded at 4000 cells/ ^cm2 , and cultured in RS culture dishes (RSTEM) and culture dishes supplemented with 2.5 μg/mL of 18:1 LPA and 16:0 LPA (culture dishes containing LPA) at 37°C and 5% _CO2 for a total of 10 days. The number of cell divisions (PDL) on day 10 was measured and is shown in Figure 13.

As shown in Figure 13, when cultured in a culture dish containing LPA, the number of cell divisions on day 10 increased compared to when cultured in RS, indicating good culture performance. This indicates that when cultured in a culture dish supplemented with LPA, the proliferation ability of mesenchymal stem cells derived from the umbilical cord is also improved.

[Example 13: Evaluation of cell surface antigen markers] UC-MSC cultured for 10 days under the conditions of Example 12 were frozen and thawed in a thermostat set at 37°C. 3.4×10 ^{^} 6 cells were taken from the thawed cell suspension and suspended in a culture dish. After centrifugation, the supernatant was removed and resuspended in 1.7 mL of 1% BSA・PBS. 100 μL of the cell suspension was mixed with each antibody reagent using a vortex mixer and allowed to react for 30 minutes in a light-shielded and ice-cooled environment. After the reaction, the cells were washed twice with 1% BSA・PBS, and after removing the supernatant, 500 μL of 1% BSA・PBS was added to make the concentration of the cell suspension uniform. The cell suspension was added to the tube through a cell filter, and reacted with 7-AAD for 10 minutes at room temperature in the dark, and each cell surface marker was measured by flow cytometry. The results are shown in Table 2.

As shown in Table 2, it was confirmed that UC-MSCs cultured in a culture dish containing LPA met the definition of mesenchymal stem cells as defined by the International Society for Cellular Therapy (ISCT).

[Example 14: Evaluation of the proliferation-promoting effect of mesenchymal stem cells derived from dental pulp] Mesenchymal stem cells derived from human dental pulp (manufactured by Lonza) were seeded at 3000 cells/ ^cm2 and cultured in RS culture dishes (RSTEM) and culture dishes supplemented with 2.5 μg/mL of 18:1 LPA and 16:0 LPA (LPA-containing culture dishes) at 37°C and 5% CO2 for _a total of 10 days. The results of measuring the number of cell divisions (PDL) on day 4 are shown in FIG14 .

As shown in Figure 14, when using a culture dish containing LPA for culturing, the number of cell divisions on day 4 increased compared to when using an existing culture dish for culturing, showing good culturing performance. Moreover, when the culture dish containing LPA was taken after 7 days and 10 days, it showed a better number of divisions than when using an existing culture dish for culturing. These results indicate that the culture dish with the addition of LPA also helps improve the proliferation ability of mesenchymal stem cells derived from dental pulp. [Industrial Applicability]

According to the mesenchymal stem cell proliferation promoter and the mesenchymal stem cell culture dish of the present invention, mesenchymal stem cells can be effectively proliferated while maintaining undifferentiated state. Furthermore, the culture supernatant obtained by culturing mesenchymal stem cells using the mesenchymal stem cell culture dish of the present invention is rich in various humoral factors such as exosomes and HGF, and can be used for disease treatment, etc.

[Figure 1] is a graph showing the effect of LPA-containing culture dishes on the proliferation of mesenchymal stem cells in flat adhesion culture. AD: adipose-derived mesenchymal stem cells, UC: umbilical cord-derived mesenchymal stem cells [Figure 2] is a graph showing the effect of LPA-containing culture dishes on the proliferation of adipose-derived mesenchymal stem cells in flat adhesion culture. [Figure 3] is a graph showing the effect of LPA-containing culture dishes on the proliferation of umbilical cord-derived mesenchymal stem cells in flat adhesion culture. [Figure 4] is a graph showing the results of studying the concentration ratio of 16:0 LPA to 18:1 LPA in the culture of mesenchymal stem cells in LPA-containing culture dishes. [Figure 5] is a graph showing the results of studying the LPA concentration in the culture of mesenchymal stem cells in LPA-containing culture dishes. [Figure 6] is a graph showing the effect of LPA-containing culture dishes on the proliferation of adipose-derived mesenchymal stem cells in floating and stirring culture. [Figure 7] is a graph showing the effect of LPA-containing culture dishes on the promotion of exosome production in planar adhesion culture of mesenchymal stem cells. [Figure 8] is a graph showing the results of evaluating the adhesion of mesenchymal stem cells cultured in LPA-containing culture dishes to various culture flasks. [Figure 9] is a graph showing the results of investigating the effect of LPA added to the culture dish on the number of cell divisions in the flat-surface adhesion culture of adipose-derived mesenchymal stem cells. [Figure 10] is a graph showing the results of investigating the effect of LPA added to the culture dish on the cell batch-to-batch heterogeneity of the number of cell divisions in the flat-surface adhesion culture of adipose-derived mesenchymal stem cells. [Figure 11] is a graph showing the results of counting the number of cell divisions in the subculture of adipose-derived mesenchymal stem cells in the flat-surface adhesion culture of the culture dish containing LPA. [Figure 12] is a graph showing a photograph of the results of angiogenesis test using HUVEC. [Figure 13] is a graph showing the effect of a culture dish containing LPA on the number of divisions of mesenchymal stem cells derived from the umbilical cord. [Figure 14] is a graph showing the effect of a culture dish containing LPA on the number of divisions of mesenchymal stem cells derived from the dental pulp.

Claims (11)

A mesenchymal stem cell proliferation promoter contains 16:0 lysophosphatidic acid, its derivatives and/or its salts. The mesenchymal stem cell proliferation promoter of claim 1 further comprises 18:1 lysophosphatidic acid, a derivative of 18:1 lysophosphatidic acid and/or a salt thereof. The mesenchymal stem cell proliferation promoter of claim 2, wherein the ratio of the content (μg/mL) of 16:0 lysophosphatidic acid, its derivatives and/or its salts to the content (μg/mL) of 18:1 lysophosphatidic acid, its derivatives and/or its salts is 0.1-10. The mesenchymal stem cell proliferation promoter of claim 1, wherein the mesenchymal stem cells are derived from adipose tissue. A supplement for culturing mesenchymal stem cells contains 16:0 lysophosphatidic acid, its derivatives and/or its salts. A culture dish for mesenchymal stem cells, comprising 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid and/or salts thereof. The culture dish for mesenchymal stem cells according to claim 6 further contains 18:1 lysophosphatidic acid, a derivative of 18:1 lysophosphatidic acid and/or a salt thereof. The culture dish for mesenchymal stem cells of claim 7, wherein the ratio of the content (μg/mL) of 16:0 lysophosphatidic acid, its derivatives and/or its salts to the content (μg/mL) of 18:1 lysophosphatidic acid, its derivatives and/or its salts is 0.1-10. The culture dish for mesenchymal stem cells according to claim 6 is used for promoting the proliferation of mesenchymal stem cells. A mesenchymal stem cell obtained by culturing using the mesenchymal stem cell proliferation promoter according to any one of claims 1 to 4, the additive for culturing mesenchymal stem cells according to claim 5, or the culture dish for mesenchymal stem cells according to any one of claims 6 to 9. A culture supernatant containing 16:0 lysophosphatidic acid, its derivatives and/or salts thereof, which is obtained by culturing mesenchymal stem cells in a culture dish for mesenchymal stem cells as claimed in any one of claims 6 to 9.