JP2023071331A - Mesenchymal stem cell, culture supernatant of mesenchymal stem cell, and pharmaceutical composition - Google Patents

Abstract

To provide a mesenchymal stem cell with a high survival rate.SOLUTION: A mesenchymal stem cell has a high content of fat-soluble components. It is preferable that the content of the fat-soluble components in a cell membrane be high in the mesenchymal stem cell. Further, it is more preferable that the fat-soluble components be long chain fatty acids, ethanolamine/amide derivatives, sphingolipids, compounds classified as cholesterol and phosphocholine, and at least one selected from the group consisting of β-estradiol, 17α-estradiol, and α-tocopherol acetate.SELECTED DRAWING: None

Description

The present invention relates to mesenchymal stem cells, mesenchymal stem cell culture supernatants, and pharmaceutical compositions.

Mesenchymal stem cells are multipotent progenitor cells isolated from bone marrow for the first time by Friedenstein in 1982 (see Non-Patent Document 1). It has been clarified that mesenchymal stem cells exist in various tissues such as bone marrow, umbilical cord, and adipose tissue, and mesenchymal stem cell transplantation is expected as a new treatment method for various intractable diseases ( See Patent Documents 1 and 2). Recently, it is known that stromal cells such as adipose tissue, placenta, umbilical cord, and egg membrane contain cells having similar functions. Therefore, mesenchymal stem cells are sometimes called stromal cells (Mesenchymal Stromal Cells).

Mesenchymal stem cells have been reported to exhibit various effects such as anti-inflammatory effects and anti-fibrotic effects, and are being clinically developed for various target diseases. However, it has been suggested that when these mesenchymal stem cells are administered directly into the blood as a therapeutic agent, the cells are damaged by the intensity of blood flow and die immediately after administration (non-patented). Reference 1). Therefore, depending on the disease, local administration is recommended instead of direct administration into the blood, but there is an inconvenience that the application is limited (Non-Patent Document 1). In a more versatile intravascular administration method, development of cells that can maintain a high survival rate after administration has been desired.

JP 2012-157263 A Japanese Patent Publication No. 2012-508733

Pittenger F. M. et al. , Science 284, pp. 143-147, 1999

An object of the present invention is to provide mesenchymal stem cells with a high survival rate under the circumstances as described above.

As a result of intensive research to solve the above problems, the present inventors have found mesenchymal stem (stromal) cells characterized by a high content of fat-soluble components such as long-chain fatty acids and cholesterols. The inventors have found that cells (MSCs) have a higher survival rate than cells obtained by conventional general culture methods, and have completed the present invention. INDUSTRIAL APPLICABILITY According to the present invention, therapeutic agents effective for treating diseases can be provided. That is, the gist of the present invention is as follows.

[1] Mesenchymal stem cells characterized by having a high fat-soluble component content.
[2] The mesenchymal stem cell according to [1], characterized by having a high content of fat-soluble components in the cell membrane.
[3] The fat-soluble component is selected from the group consisting of long-chain fatty acids, ethanolamine/amide derivatives, sphingolipids, cholesterol and phosphocholine compounds, and β-estradiol, 17α-estradiol, and α-tocopherol acetate. The mesenchymal stem cell according to [1] or [2], which is at least one kind of mesenchymal stem cell according to [1] or [2].
[4] the long-chain fatty acid is at least one selected from the group consisting of dihomo-γ-linolenic acid, palmitoleic acid, oleic acid, myristic acid, palmitic acid, linoleic acid, and eicosadienoic acid;
at least the ethanolamine/amide derivative is selected from the group consisting of 1-palmitoyl-sn-glycero-3-phosphocholineethanolamine, palmitoylethanolamide, oleoylethanolamine, stearoylethanolamide, and dipalmitoylphosphatidylethanolamine; is one type,
The sphingolipid is at least one selected from the group consisting of sphingosine, sphinganine, and sphingomyelin,
the cholesterol is at least one selected from the group consisting of desmosterol-27-dehydrocholesterol-2, 22-hydroxycholesterol and 20α-hydroxycholesterol-2;
The phosphocholine is 1-myristoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-sn-glycero-3-phosphocholine, 1-oleoyl-sn-glycero-3-phosphocholine, 1-hexadecyl-2-acetyl-sn -glycero-3-phosphocholine-1,1-stearoyl-sn-glycero-3-phosphocholine, and at least one selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine, [ 3].
[5] The mesenchymal stem cell according to any one of [1] to [4], which is derived from umbilical cord tissue.
[6] A pharmaceutical composition comprising the mesenchymal stem cells of any one of [1] to [5].
[7] The culture supernatant of mesenchymal stem cells according to any one of [1] to [5].
[8] A pharmaceutical composition comprising the culture supernatant of [7].

According to the present invention, novel mesenchymal stem cells with high viability can be provided. The mesenchymal stem cells of the present invention have a high content of lipid-soluble components such as long-chain fatty acids and cholesterols, have reduced membrane fluidity, and have increased cell membrane strength. This strengthening of the cell membrane allows cells to maintain a high survival rate, so after administration to patients, it is expected that they will survive in the bloodstream with a higher probability, reach the affected area alive, and exhibit a higher therapeutic effect. can.

FIG. 1 is a diagram showing a comparison of fine viability of mesenchymal stem cells in flat culture (ADH) and in suspension culture (SUS).

The mesenchymal stem cells of the present invention are described in detail below.

[Mesenchymal stem cells]
The mesenchymal stem cells of the present invention are characterized by a high content of fat-soluble components such as long-chain fatty acids and cholesterols.

The mesenchymal stem cells of the present invention should have a higher content of fat-soluble components such as long-chain fatty acids and cholesterols than other cells. Specifically, long-chain fatty acids, ethanolamine/amide derivatives, High content of sphingolipids, compounds classified as cholesterol or phosphocholine, or β-Estradiol, 17α-Estradiol, α-Tocopherol acetate.

Long-chain fatty acids are fatty acids containing 11 or more carbon atoms in the molecule, and specifically include dihomo-γ-linolenic acid, palmitoleic acid, oleic acid, myristic acid, palmitic acid, linoleic acid, and eicosadiene. acids and the like.

As ethanolamine and ethanolamide derivatives, specifically, 1-palmitoyl-sn-glycero-3-phosphoethanolamine (1-Palmitoyl-glycero-3-phosphoethanolamine-1, 1-Palmitoyl-glycero-3-phosphoethanolamine-2 ), Palmitoylethanolamide-2, Oleoyl ethanolamine, Stearoyl ethanolamide, Dipalmitoylphosphatidylethanolamine (1,2-Dipalmitoyl-glycero-3-phosphoethanolamine).

As sphingolipids, specifically, sphingosine, sphinganine, sphingomyelin ((Sphingomyelin (d18:1/16:0)-1, Sphingomyelin (d18:1/16:0)-2, Sphingomyelin (d18:1/16:0)-3, Sphingomyelin(d18:1/16:0)-4, Sphingomyelin(d18:1/18:0)-2, Sphingomyelin(d18:1/18:0)-1 )).

Specific examples of cholesterol include Desmosterol-27-Dehydrocholesterol-2, 22-Hydroxycholesterol, and 20α-Hydroxycholesterol-2.

As phosphocholine, specifically, 1-myristoyl-sn-glycero-3-phosphocholine (1-Myristoyl-glycero-3-phosphocholine-1, 1-Myristoyl-glycero-3-phosphocholine-2), 1-palmitoyl-sn -glycero-3-phosphocholine (1-Palmitoyl-glycero-3-phosphocholine-1, 1-Palmitoyl-glycero-3-phosphocholine-2), 1-oleoyl-sn-glycero-3-phosphocholine (1-Oleoyl-glycero -3-phosphocholine-1, 1-Oleoyl-glycero-3-phosphocholine-2), 1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine-1 (1-Hexadecyl-2-acetyl-glycero-3- phosphocholine-1), 1-stearoyl-sn-glycero-3-phosphocholine (1-Staroyl-glycero-3-phosphocholine-1), 1,2-distearoyl-sn-glycero-3-phosphocholine (1,2-Distearoyl -glycero-3-phosphocholine-1, 1,2-Distearoyl-glycero-3-phosphocholine-2).

The mesenchymal stem cells of the present invention are cells that have a higher content of fat-soluble components such as long-chain fatty acids and cholesterols than other cells. For example, mesenchymal stem cells obtained by a conventional culture method (for example, a method of culturing by a flat culture method) can be mentioned. In addition, the flat culture method may be a method in which cells are cultured by static culture in which cells are adhered to a flat surface such as a flask or petri dish, and the container, medium, etc. are not particularly specified, but for example, Examples thereof include cells obtained by inoculating leaf stem cells in a cell culture flask and performing planar culture using a serum-free medium for mesenchymal stem cells (Rohto, etc.). The mesenchymal stem cells of the present invention have a higher content of fat-soluble components such as long-chain fatty acids and cholesterols than mesenchymal stem cells obtained by conventional culture methods (for example, a method of culturing by a planar culture method). It should be as high as possible, preferably 1.5 times or more, more preferably 3 times or more, still more preferably 5 times or more, particularly preferably 10 times, compared to mesenchymal stem cells obtained under conventional culture conditions when cultured for 8 days. It is sufficient if the content of fat-soluble components such as long-chain fatty acids and cholesterols is at least twice as high.

In the present invention, mesenchymal stem cells have the ability to differentiate into one or more types of cells belonging to the mesenchymal system (osteocytes, cardiomyocytes, chondrocytes, tendon cells, adipocytes, etc.), and maintain this ability. It means a cell that can proliferate. The term mesenchymal stem cells used in the present invention means the same cells as stromal cells, and there is no particular distinction between the two. In addition, they are sometimes simply referred to as mesenchymal cells. Tissues containing mesenchymal stem cells include, for example, adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amniotic membrane, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, Examples include dental pulp and tooth germ. For example, adipose tissue-derived mesenchymal stem cells mean mesenchymal stem cells contained in adipose tissue, and may also be referred to as adipose tissue-derived stromal cells. Among these, adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and placenta-derived mesenchymal stem cells are selected from the viewpoints of efficacy for treatment of neuropathic diseases, availability, etc. Stem cells and dental pulp-derived mesenchymal stem cells are preferred, adipose tissue-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells are more preferred, and umbilical cord-derived mesenchymal stem cells are most preferred.

The mesenchymal stem cells in the present invention may be derived from the same species as the subject (subject) to be treated, or may be derived from a different species. Species of mesenchymal stem cells in the present invention include humans, horses, cows, sheep, pigs, dogs, cats, rabbits, mice, and rats, preferably cells derived from the same species as the subject (subject) to be treated. . The mesenchymal stem cells in the present invention may be derived from the subject (subject) to be treated, that is, autologous cells, or may be derived from another subject of the same type, that is, allogeneic cells. Allogeneic cells are preferred.

Mesenchymal stem cells are less likely to cause rejection even in allogeneic subjects. Can be used as stem cells. Therefore, compared to the case of preparing and using autologous mesenchymal stem cells, the mesenchymal stem cells of the present invention are easier to commercialize and more likely to stably obtain a certain effect. Allogeneic is more preferred.

In the present invention, tissue-derived mesenchymal stem cells such as adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells refer to adipose tissue-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells, respectively. An arbitrary cell population including tissue-derived mesenchymal stem cells such as stem cells and bone marrow-derived mesenchymal stem cells. The cell population is at least 20%, preferably 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98% % or 99% are tissue-derived mesenchymal stem cells such as adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells.

(Method for preparing mesenchymal stem cells)
The method for preparing mesenchymal stem cells with a high content of fat-soluble components such as long-chain fatty acids and cholesterols is not particularly limited, but they can be prepared, for example, as follows. That is, after separating mesenchymal stem cells from tissues such as fat, umbilical cord, and bone marrow according to a method known to those skilled in the art, by performing a suspension culture production method (stirring culture) using a specific medium, etc. Mesenchymal stem cells having a high content of fat-soluble components such as long-chain fatty acids and cholesterols in mesenchymal stem cells can be obtained. In the cell population obtained by this method, 50% or more of the cell population is preferably cells with a high content of fat-soluble components such as long-chain fatty acids and cholesterols, and 70% or more is long-chain fatty acids, cholesterols, etc. It is more preferable that the cells have a high content of lipid-soluble components of 80% or more, and more preferably 80% or more are cells that have a high content of lipid-soluble components such as long-chain fatty acids and cholesterols. Cells with a high content of fat-soluble components such as chain fatty acids and cholesterols are particularly preferred, and are substantially uniform cell populations of cells with a high content of fat-soluble components such as long-chain fatty acids and cholesterols. is most preferred. A method for preparing mesenchymal stem cells having a high content of fat-soluble components such as long-chain fatty acids and cholesterols will be specifically described below.

Mesenchymal stem cells can be prepared by methods well known to those skilled in the art. As an example, a method for preparing umbilical cord tissue-derived mesenchymal stem cells and adipose tissue-derived mesenchymal stem cells will be described below.

The umbilical cord can be collected by removing the placenta as appropriate from postpartum tissues including the placenta and umbilical cord delivered by vaginal delivery and caesarean section. After removing the cord blood from the collected umbilical cord, aseptic or bacteriostatic treatment may be performed. Removal of cord blood is accomplished by rinsing with an anticoagulant solution such as a heparin-containing solution. Aseptic or bacteriostatic treatments include, but are not limited to, application of povidone-iodine, or media supplemented with one or more antibiotics and/or antimycotics such as penicillin, streptomycin, amphotericin B, gentamicin, and nystatin. Alternatively, it may be immersed in a buffer. Moreover, a step of selectively lysing erythrocytes may be included as necessary. Methods well known in the art can be used to selectively lyse red blood cells, such as incubation in hypertonic or hypotonic media with lysis with ammonium chloride.

The umbilical cord-derived cells of the present invention mean a cell population prepared using umbilical cord as a raw material, and may be obtained by a known production method, for example, by a method including the following steps (i) to (iii). can do:
(i) cutting the umbilical cord;
(ii) culturing the umbilical cord obtained in step (i); and (iii) passaging.

Further, as another method for preparing the cells, (i) instead of the step of cutting the umbilical cord, the step of (i') dissociating the tissue by enzymatic treatment of the umbilical cord may be included. Furthermore, in addition to the step of (i) cutting the umbilical cord, the step of (i′) subjecting the umbilical cord to enzyme treatment to dissociate tissue may be included.

In the step (i) of cutting the umbilical cord of the present invention, the umbilical cord obtained by the above-described method is cut by mechanical force (shredding force or shearing force) in a state containing amniotic membrane, blood vessels, perivascular tissue, and Walton's Jelly. can be done by Although not particularly limited, the umbilical cord segment obtained by cutting has a size of 1 to 10 mm ³ , 1 to 5 mm ³ , 1 to 4 mm ³ , 1 to 3 mm ³ or 1 to 2 mm ³ . In the step (i') of the present invention, in which the umbilical cord is enzymatically treated to dissociate the tissue, the umbilical cord obtained by the above-described method is enzymatically treated in a state containing the amniotic membrane, blood vessels, perivascular tissue, and Walton's jelly to dissociate the tissue. It can be carried out in a dissociation step. Enzymatic treatments include, but are not limited to, enzyme treatments using one or more enzymes such as collagenase, dispase and hyaluronidase.

In the step of culturing the umbilical cord obtained in step (ii) (i) of the present invention, the umbilical cord obtained in step (i) is cultured at an appropriate cell density and under culture conditions using an appropriate cell culture medium. culture.

The umbilical cord tissue-derived mesenchymal stem cells of the present invention can be produced, for example, using the following suspension culture production method. As a suspension culture production method, a method of stirring and culturing spherical cell masses formed by any method in a culture vessel, and a method of adhering cells on microcarriers and stirring the microcarriers to perform agitation culture in a culture vessel. There are ways to do so. From the viewpoint of obtaining mesenchymal stem cells with a higher content of lipid-soluble components such as long-chain fatty acids and cholesterols, cells are adhered to microcarriers and the microcarriers are agitated in a culture tank. A method of culturing is preferred. Specifically, a cell suspension containing mesenchymal stem cells and microcarriers are mixed, added to a culture tank, stirred and cultured using a serum-free medium for mesenchymal stem cells, etc., and suspended for a certain period of time. By culturing, it is possible to obtain cells with a high content of fat-soluble components such as long-chain fatty acids and cholesterols, compared to conventional planar culture in a flask or the like. The culture period can be appropriately selected according to the state of the cells, the number of cells, the culture scale, etc., and is, for example, 24 hours to 2 weeks, preferably 2 days to 10 days. More preferably, the total number of culture days is 3 to 21 days. During this period, the cell/microcarrier suspension may be fractionated and transferred to new microcarriers and culture medium once or twice a week, if necessary. For stirring, there are a method of rotating a stirring blade in a container with a stirrer, and a method of suspending the culture solution by placing the bag containing the culture solution and cells on a shaker and shaking the bag together. In addition, the medium used in the suspension culture production method is not particularly limited as long as it is a medium capable of culturing mesenchymal stem cells, and examples include serum-free medium for mesenchymal stem cells (manufactured by Rohto). The microcarrier is not particularly limited as long as it can be used in suspension culture, and examples include polyester, polystyrene, glass, dextran, gelatin, and collagen. Specific examples of Micro Career that can be obtained by GE Healthcare's CYTODEX1, CYTODEX3, CYTODEX3, CYTOPORE1, CULTISPORE2, CULTISPHERE, CYTOLINE 1, CYTOLINE 2, CORNING CELLBI. ND, low concentration Synthemax II, high concentration Synthemax II, Positive Charge Examples include microcarriers, collagen-coated microcarriers, soluble microcarriers, and SoloHill microcarriers from Pall. The material of the microcarrier preferably has properties such as solubility and biodegradability, and more preferably has a chemically defined composition, and is xeno-free and animal-free. . When cells are adhered to a new microcarrier, it may be preliminarily soaked overnight in the medium to be used for equilibration.

In order to confirm that the obtained cells are the mesenchymal stem cells of the present invention, fat-soluble components extracted from the cells are quantified using a liquid chromatograph time-of-flight mass spectrometer (LC-TOFMS, etc.) or the like. , to a control cell as described above. Alternatively, the analysis may be performed by a conventional method using flow cytometry or the like. Additionally, the ability to differentiate into each cell lineage may be confirmed, and such differentiation can be performed by conventional methods.

The mesenchymal stem cells of the present invention can be prepared as described above, and are mesenchymal stem cells with a high content of fat-soluble components such as long-chain fatty acids and cholesterols. may be defined as being cells with the properties of
(1) shows adhesion to plastic under culture conditions in a standard medium;
(2) positive for surface antigens CD44, CD73, CD90, and CD105, negative for CD31, CD34, and CD45; and (3) capable of differentiating into osteocytes, adipocytes, and chondrocytes under specific culture conditions.

Cells with a higher content of fat-soluble components such as long-chain fatty acids and cholesterols are selectively separated from the mesenchymal stem cells obtained by the above-mentioned steps by an immunological technique using a cell sorter, magnetic beads, or the like. Thus, mesenchymal stem cells with a higher content of fat-soluble components such as long-chain fatty acids and cholesterols can be obtained. In addition, by performing a specific culture method that can increase the content of fat-soluble components such as long-chain fatty acids and cholesterols, or by culturing in a specific medium, etc., long-chain fatty acids, cholesterols, etc. in mesenchymal stem cells It is also possible to increase the content of fat-soluble components and efficiently obtain mesenchymal stem cells with a higher content of fat-soluble components such as long-chain fatty acids and cholesterols. As an example, a specific method for selective isolation by an immunological technique using a cell sorter will be described below.

The cell suspension obtained by treating the above-prepared mesenchymal stem cells attached to a microcarrier or the like with a trypsin/EDTA solution or the like is centrifuged (room temperature, 400 G, 5 minutes) to remove the supernatant. . Add Staining Buffer (1% BSA-PBS) to the cells to prepare 1×10 ⁶ cells/500 μL, homogenize the cell suspension concentration by pipetting, and transfer 50 μL to a new 1.5 mL microtube. dispense one by one. A fluorescence-labeled detection reagent is added to the dispensed cell suspension, suspended, and then reacted for 30 minutes to 1 hour under light shielding and refrigeration. Any fluorescence-labeled detection reagent can be used as long as it can specifically bind to long-chain fatty acids and cholesterols on the cell membrane. After washing three times with 1 mL of Staining Buffer, add Staining Buffer to make 50 μL, add secondary antibody at a concentration of 1 to 10 μg/mL, suspend, and react for 30 minutes to 1 hour under light shielding and refrigeration. . After washing three times with 1 mL of staining buffer, 300 μL of PI buffer (prepared by adding 28.8 μL of propidium iodide solution (manufactured by SIGMA, P4864) to 14.4 mL of staining buffer) was added and well suspended. Separation can be performed by passing through a tube with fluorescence activated cell sorting (FACS).

The mesenchymal stem cells of the present invention can be used as medicines for treatment of diseases, improvement of various symptoms, and the like. It can also be used as a material for obtaining a culture supernatant, which will be described later.

[Culture supernatant of mesenchymal stem cells]
The present invention also includes the culture supernatant of the mesenchymal stem cells of the present invention described above. The culture supernatant of the present invention is a supernatant obtained by culturing the mesenchymal stem cells described above by the following method, a supernatant obtained by removing unnecessary components from the supernatant by means of dialysis, ultrafiltration, or the like. It includes a fraction obtained by fractionating the supernatant with a column or the like, a fraction selected using an antibody against a specific molecule or the like, a fraction obtained by centrifugation, and the like.

As the medium used for obtaining the culture supernatant of the present invention, the same medium as the medium described in the preparation of mesenchymal stem cells can be used. The method for culturing mesenchymal stem cells is not particularly limited as long as it is suitable for each mesenchymal stem cell, but it is preferable to use the suspension culture method described above for the culture for obtaining the culture supernatant. . Usually, it is carried out at a temperature of 30° C.-37° C., under 2%-7% CO ₂ environment, under 5%-21% O ₂ environment, preferably at 37° C., 5% CO ₂ environment. In addition, the culture period for obtaining the culture supernatant is not particularly limited, but can be appropriately set while observing the state of the cells.

The cell culture supernatant of the mesenchymal stem cells cultured in the above medium may be collected only once, or may be collected multiple times over multiple days. The collected culture supernatant can be subjected to filter sterilization, dialysis, concentration, column fractionation, dilution, etc. as necessary to obtain the culture supernatant of the present invention.

The mesenchymal stem cell culture supernatant of the present invention can be used for pharmaceuticals, quasi-drugs, cosmetics, and the like, depending on the purpose.

[Pharmaceutical composition]
The present invention also includes pharmaceutical compositions containing the above-described mesenchymal stem cells of the present invention and pharmaceutical compositions containing the culture supernatant of the present invention.

Subjects to which the pharmaceutical composition of the present invention is administered are mammals, preferably humans, horses, cows, dogs, cats and the like, more preferably humans.

EXAMPLES The present invention will be described in detail below with reference to examples and test examples, but the present invention is not limited to these examples.

[Preparation and culture of umbilical cord-derived mesenchymal stem cells (UCMSC)]
Umbilical cord-derived mesenchymal stem cells were collected by the method described in Cytotherapy, 18, 229-241, 2016. Briefly, the umbilical cord collected with the donor's consent was cut into pieces of 1 to 2 mm ³ , seeded on a culture dish, cultured in a serum-free medium for mesenchymal stem cells (Rohto), Umbilical cord mesenchymal stem cells (hereinafter also referred to as “UCMSC”) were obtained. The obtained UCMSCs were detached using a cell detachment solution (TrypLE Select (1X), manufactured by Thermo Fisher), transferred to a centrifuge tube, and centrifuged at 400 xg for 5 minutes to obtain cell pellets. After removing the supernatant, an appropriate amount of cell cryopreservation medium (STEM-CELLBANKER (Xenoac)) was added and suspended. The cell suspension solution was dispensed into cryotubes and stored at -80°C in a freezer. It was then transferred to the vapor phase over liquid nitrogen and continued to be stored.

[Example 1: Measurement of fat-soluble components of suspension cultured cells (SUS) and planar cultured cells (ADH) 1]
UCMSCs stored at -80 ° C. in a freezer or stored in a liquid nitrogen tank under the gas phase are awakened, and a cell suspension containing cells and microcarriers (CellBIND microcarriers, manufactured by Corning) are mixed, The cells were added to the culture tank, stirred and cultured using a serum-free medium for mesenchymal stem cells (Rohto), and cultured for 4 days to obtain suspension cultured cells (hereinafter also referred to as "SUS"). CellBIND microcarriers were added 12 cm ² min per mL of medium and UCMSCs were seeded at 3000-5000 Cells/cm ² . The rotation speed in the culture vessel was set at 15-50 rpm (preferably, the tip speed is 5055 mm/min or less) depending on the degree of cell growth. Similarly, the above-mentioned UCMSCs were awakened, the cells were seeded in a cell culture flask, plate culture was performed using a serum-free medium for mesenchymal stem cells (Rohto), cultured for 4 and 8 days, and the plate culture cells ( Hereinafter, also referred to as "ADH") was obtained. The cultured cells were washed with a 5% (w/w) mannitol (Wako Fujifilm) aqueous solution, and ethanol (Wako Fujifilm) was added in an amount that spreads throughout the cells to lyse the cells. The cell lysis ethanol solution and cells were transferred to a 15 mL centrifuge tube and stored in a freezer at -80°C. Add 1,000 μL of Milli-Q water to the obtained sample, stir with ultrasonic waves for 5 minutes while cooling with ice, perform centrifugation (4,400×g, 4° C., 5 minutes), and remove the supernatant. Recovered. This was dried, dissolved again in 200 μL of 50% isopropanol aqueous solution (v/v), and subjected to measurement. Capillary electrophoresis-time of flight mass spectrometer (CE-TOFMS CAPILLARY ELECTROPHORESIS TIME OF FLIGHT MASS SPECTROMETER), Agilent CE-TOFMS system (manufactured by Agilent Technologies)) cation mode, anion mode and liquid chromatography-time-of-flight mass Lipid-soluble components extracted from cells were quantified in the positive mode and negative mode of an analyzer (LC-TOFMS (Liquid Chromatograph / Time-of-flight Mass Spectrometer), Agilent 1200 series RRLC system SL (manufactured by Agilent Technologies)). (Table 1). Table 1 shows the relative expression level in SUS when the expression level in ADH of each fat-soluble component is used as the denominator.

SUS was shown to contain more long-chain fatty acids and cholesterols in the cell membrane than ADH. In addition, although not shown in Table 1, more sphingomyelin and 1,2-distearoyl-glycero-3-phosphocholine-2 in the cell membrane were contained in SUS than in ADH.

[Example 2: Measurement of fat-soluble components of suspension cultured cells (SUS) and planar cultured cells (ADH) 2]
UCMSCs were awakened in the same manner as in Example 1, and subjected to agitation culture and planar culture for 8 days using a serum-free medium for mesenchymal stem cells (Rohto), and the obtained cells were subjected to liquid chromatography time-of-flight mass spectrometry. The fat-soluble components extracted from the cells were quantified using an instrument (LC-TOFMS). SUS was shown to contain more long-chain fatty acids and cholesterols in the cell membrane than ADH. Although not shown in Table 2, myristic acid, palmitic acid, linoleic acid, eicosadienoic acid, palmitoylethanolamide, stearoylethanolamide, dipalmitoylphosphatidylethanolamine, sphingomyelin (d18:1/18:0) in cell membranes )-1, cholesterol, β-estradiol, 17α-estradiol, and α-tocopherol acetate were more abundant in SUS than in ADH.

[Measurement of viability of planar cultured cells (ADH) and suspension cultured cells (SUS)]
UCMSCs stored at -80 ° C in a freezer or stored in a liquid nitrogen tank under the gas phase are awakened, a cell suspension containing cells and a microcarrier are mixed, added to a culture tank, and mesenchymal stem cells are obtained. Agitation culture was performed using a serum-free medium (Rohto) for 4, 8, 11 and 15 days to obtain suspension cultured cells (hereinafter referred to as "SUS"). Similarly, the above-mentioned UCMSCs were awakened, the cells were seeded in a cell culture flask, subjected to planar culture using a serum-free medium for mesenchymal stem cells (Rohto), and cultured for 4, 8, 11 and 15 days, Planar cultured cells (hereinafter referred to as "ADH") were obtained. Cell viability was determined during the recovery of SUS and ADH. The results are shown in FIG. It was shown that the survival rate of SUS is higher than that of ADH in any number of culture days.

According to the present invention, mesenchymal stem cells with high viability can be provided. The mesenchymal stem cells of the present invention are considered to be cells capable of maintaining a high survival rate even after administration in a more versatile intravascular administration method, and can be suitably used for disease treatment.

Claims (8)

A mesenchymal stem cell characterized by having a high fat-soluble component content. The mesenchymal stem cell according to claim 1, characterized by having a high content of fat-soluble components in the cell membrane. The fat-soluble component is at least selected from the group consisting of long-chain fatty acids, ethanolamine/amide derivatives, sphingolipids, cholesterol and phosphocholine compounds, and β-estradiol, 17α-estradiol, and α-tocopherol acetate. The mesenchymal stem cell according to claim 1 or 2, which is one type. the long-chain fatty acid is at least one selected from the group consisting of dihomo-γ-linolenic acid, palmitoleic acid, oleic acid, myristic acid, palmitic acid, linoleic acid, and eicosadienoic acid;
at least the ethanolamine/amide derivative is selected from the group consisting of 1-palmitoyl-sn-glycero-3-phosphocholineethanolamine, palmitoylethanolamide, oleoylethanolamine, stearoylethanolamide, and dipalmitoylphosphatidylethanolamine; is one type,
The sphingolipid is at least one selected from the group consisting of sphingosine, sphinganine, and sphingomyelin,
the cholesterol is at least one selected from the group consisting of desmosterol-27-dehydrocholesterol-2, 22-hydroxycholesterol and 20α-hydroxycholesterol-2;
The phosphocholine is 1-myristoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-sn-glycero-3-phosphocholine, 1-oleoyl-sn-glycero-3-phosphocholine, 1-hexadecyl-2-acetyl-sn -glycero-3-phosphocholine-1,1-stearoyl-sn-glycero-3-phosphocholine, and at least one selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine Item 4. The mesenchymal stem cell according to item 3. 5. The mesenchymal stem cell according to any one of claims 1 to 4, which is derived from umbilical cord tissue. A pharmaceutical composition comprising the mesenchymal stem cells according to any one of claims 1 to 5. A culture supernatant of the mesenchymal stem cells according to any one of claims 1 to 5. A pharmaceutical composition comprising the culture supernatant of claim 7.

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