JP7152389B2 - Cell population containing mesenchymal stem cells, method for producing the same, mesenchymal stem cells, and pharmaceutical composition - Google Patents

Description

The present invention relates to a method for producing a cell population containing mesenchymal stem cells. Furthermore, the present invention relates to cell populations containing mesenchymal stem cells, mesenchymal stem cells, and pharmaceutical compositions.

Mesenchymal stem cells, also called mesenchymal stromal cells, are somatic stem cells that have been reported to exist in bone marrow, adipose tissue, dental pulp, and the like. It has also been found to be present in fetal appendages such as Mesenchymal stem cells are attracting attention as a promising cell source in regenerative medicine because they have the ability to differentiate into bone, cartilage, fat, and the like.

In addition, since mesenchymal stem cells have not only differentiation ability but also immunosuppressive ability, it has been reported that immune-related diseases, inflammatory diseases, and the like can be treated by intravenous administration.

Patent Document 1 describes a method for producing an amniotic mesenchymal cell composition, a method for cryopreservation, and a therapeutic agent. In particular, by cryopreserving a mixture containing amniotic mesenchymal cells in a solution containing 5 to 10% by mass of dimethyl sulfoxide and 5 to 10% by mass of hydroxylethyl starch or 1 to 5% by mass of dextran, It is described that cryopreserved amniotic mesenchymal cells can be produced as cell preparations optimized for transplantation.

Patent Document 2 discloses (D) collecting a cell population of mesenchymal cells from the amniotic membrane of a mammal, (E) seeding the collected cell population at a cell concentration of 400 to 35000/cm ² , (F) seeding at a cell concentration of 1/5000 or more and less than 1/10 of the initial culture, and subculturing twice a week for subculturing 3 to 4 times. (G) when a colony of cells having a spindle-shaped morphology is formed in the subculture, maintaining the culture in the same culture dish until the cells become confluent. Methods for preparing lineage stem cell populations are described.

JP 2015-61520 A International publication WO2013/077428

In recent years, it has been found that mesenchymal stem cells derived from fetal appendages are heterogeneous cell populations containing various cells with different differentiation, proliferation, and cytokine-producing abilities. In order to manufacture cell preparations of stable quality, it is necessary to prepare a purified and highly homogeneous cell population.

In Patent Document 1, a mixture containing amniotic mesenchymal cells is cryopreserved in a specific cryopreservation solution to suppress the decrease in survival rate of amniotic mesenchymal cells after thawing, and cryopreserved amniotic membrane It has been described that mesenchymal cells can be produced as cell preparations optimized for transplantation. However, selective preparation of mesenchymal stem cells having specific superior characteristics from among mesenchymal stem cells, specifically, a cell population rich in mesenchymal stem cells exhibiting a high immunosuppressive effect, There is no description whatsoever about selective preparation using the characteristics of mesenchymal stem cells as an index. Moreover, in Patent Document 2, a mesenchymal stem cell population having high proliferative ability and differentiation ability is prepared by seeding cells at low density, but mesenchymal stem cells contained in the mesenchymal stem cell population There is no description or suggestion about selecting a cell population containing a large amount of mesenchymal stem cells exhibiting a high immunosuppressive effect, using the characteristics of .

An object of the present invention is to provide a cell population containing mesenchymal stem cells exhibiting a high immunosuppressive effect, a method for producing the same, a mesenchymal stem cell exhibiting a high immunosuppressive effect, and a pharmaceutical composition containing the cell population. do.

As a result of intensive studies to solve the above problems, the present inventors found that a cell population containing cells collected from fetal appendages contains mesenchymal stem cells that are positive for CD97 and CD358. Furthermore, the ratio of mesenchymal stem cells positive for CD97 is 30% or more, and in the cell population including mesenchymal stem cells positive for CD358, genes encoding immunosuppression-related cytokines (TGFB2 gene, TNFAIP6 , IL1A gene, IL1B gene and CCL2 gene), it was found to exhibit a high immunosuppressive effect. The present invention has been completed based on these findings.

That is, according to this specification, the following inventions are provided.
[1] A method for producing a cell population containing mesenchymal stem cells, comprising obtaining a cell population having the following cell characteristics (a) and (b):
(a) the proportion of CD97-positive mesenchymal stem cells in the cell population is 30% or more; and (b) the cell population contains CD358-positive mesenchymal stem cells.
[2] A cell population of mesenchymal stem cells having the following cell characteristics (a) and (b):
(a) the proportion of CD97-positive mesenchymal stem cells in the cell population is 30% or more; and (b) the cell population contains CD358-positive mesenchymal stem cells.
[3] The cell population of [2], wherein the mesenchymal stem cells are derived from fetal appendages.
[4] Mesenchymal stem cells positive for CD97 and CD358.
[5] A pharmaceutical composition comprising the cell population of [2] or [3] or the mesenchymal stem cells of [4] and a pharmaceutically acceptable medium.
[6] The pharmaceutical composition of [5], wherein the dose of mesenchymal stem cells to humans is 10 ⁹ cells/kg body weight or less.
[7] The pharmaceutical composition of [5] or [6], wherein the pharmaceutical composition is an injection preparation.
[8] The pharmaceutical composition of [5] or [6], wherein the pharmaceutical composition is a preparation for transplantation having a cell aggregate or sheet-like structure.
[9] The pharmaceutical composition according to any one of [5] to [8], which is a therapeutic agent for immune-related diseases.

[10] The cell population has a relative expression level of the TGFB2 gene with respect to the SDHA gene expression level of 1.5 or more, a relative expression level of the TNFAIP6 gene with respect to the SDHA gene expression level of 0.2 or more, The relative expression level of the IL1A gene with respect to the SDHA gene expression level is 0.2 or more, the relative expression level of the IL1B gene with respect to the SDHA gene expression level is 1.3 or more, or CCL2 with respect to the SDHA gene expression level The cell population of [2] or [3], which satisfies any one or more of relative gene expression levels of 1.0 or more.
[11] A cell population obtained by the production method of [1].
[12] Use of the cell population of [2] or [3] or the mesenchymal stem cell of [4] for the production of a pharmaceutical composition.
[13] The use of [12], wherein the pharmaceutical composition is a mesenchymal stem cell dose of 10 ⁹ cells/kg body weight or less per human dose.
[14] The use of [12] or [13], wherein the pharmaceutical composition is an injectable preparation.
[15] The use according to [12] or [13], wherein the pharmaceutical composition is a preparation for transplantation of cell mass or sheet-like structure.
[16] The use according to any one of [12] to [15], wherein the pharmaceutical composition is a therapeutic agent for immune-related diseases.
[17] The cell population of [2] or [3] or the mesenchymal stem cell of [4] for use in treating a disease.
[18] The cell population or mesenchymal stem cells according to [17], wherein the dose of mesenchymal stem cells to a human is 10 ⁹ cells/kg body weight or less.
[19] The cell population or mesenchymal stem cells of [17] or [18], which is an injectable preparation.
[20] The cell population or mesenchymal stem cells of [17] or [18], which is a preparation for transplantation with a cell mass or sheet-like structure.
[21] The cell population or mesenchymal stem cells of any one of [17] to [20], wherein the disease is an immune-related disease.
[22] A method for treating a disease, which comprises administering the cell population of [2] or [3] or the mesenchymal stem cell of [4] to a patient in need of treatment.
[23] The method for treating the disease of [22], wherein the dose of mesenchymal stem cells to humans is 10 ⁹ cells/kg body weight or less.
[24] The method for treating the disease of [22] or [23], which is an injectable preparation.
[25] The method for treating the disease of [22] or [23], which is a preparation for transplantation with a cell mass or sheet-like structure.
[26] The method for treating the disease of any one of [22] to [25], wherein the disease is an immune-related disease.
[27] A composition comprising the cell population of [2] or [3] or the mesenchymal stem cells of [4], and a vehicle.

According to the present invention, a cell population containing mesenchymal stem cells exhibiting a high immunosuppressive effect can be prepared. In addition, according to the present invention, surface antigen positivity and relative expression levels of various genes with respect to housekeeping genes can be used as indicators of the formation of cell populations containing mesenchymal stem cells that exhibit a high immunosuppressive effect. Thereby, a cell preparation (pharmaceutical composition) can be produced.

FIG. 1 shows the results of measurement of the ratio of CD97-positive cells using a flow cytometer with respect to amniotic MSCs cultured in Example 1. FIG. 4 shows the results of measuring the ratio of CD97-positive cells using a flow cytometer with respect to amniotic MSCs cultured in Comparative Example 1. FIG. 4 shows the results of measuring the ratio of CD358-positive cells using a flow cytometer with respect to amniotic MSCs cultured in Example 1. FIG. 4 shows the results of measuring the ratio of CD358-positive cells using a flow cytometer with respect to amniotic MSCs cultured in Comparative Example 1. FIG.

Embodiments of the present invention will be specifically described below, but the following description is for facilitating understanding of the present invention, and the scope of the present invention is not limited to the following embodiments. Other embodiments in which a person skilled in the art appropriately replaces the configurations of the following embodiments are also included in the scope of the present invention.

[1] Explanation of Terms "Fetal appendage" as used herein refers to egg membrane, placenta, umbilical cord and amniotic fluid. Furthermore, the "egg membrane" is the gestational sac containing the amniotic fluid of the fetus, which consists of the amniotic membrane, the chorion and the decidua from the inside. Of these, the amnion and chorion originate from the fetus. "Amniotic membrane" refers to the innermost layer of the egg membrane, the thin, transparent thin film lacking blood vessels. The inner layer of the amniotic membrane (also called the epithelial cell layer) is covered with a single layer of epithelial cells that have a secretory function and secretes amniotic fluid, while the outer layer of the amniotic membrane (also called the extracellular matrix layer, which corresponds to the stroma) stores mesenchymal stem cells. include.

As used herein, "mesenchymal stem cells (MSCs)" refer to stem cells that meet the definition below, and are used interchangeably with "mesenchymal stromal cells". As used herein, "mesenchymal stem cells" are sometimes referred to as "MSCs".

Definition of mesenchymal stem cells
i) Shows adhesiveness to plastic under culture conditions in standard medium.
ii) Positive for surface antigens CD105, CD73, CD90 and negative for CD45, CD34, CD11b, CD79alpha, CD19, HLA-DR.

The term "mesenchymal stem cell population" as used herein means a cell population containing mesenchymal stem cells, and its form is not particularly limited. etc.

The term "amniotic mesenchymal stem cells" as used herein refers to mesenchymal stem cells derived from the amniotic membrane, and is used interchangeably with "amniotic mesenchymal stromal cells." As used herein, "amniotic mesenchymal stem cells" may be referred to as "amniotic MSCs".

As used herein, the term “immunosuppressive action” refers to the action of suppressing the response of the immune system in vivo. The immunosuppressive effect can be evaluated by the expression level of one or more genes selected from TGFB2 gene, TNFAIP6 gene, IL1A gene, IL1B gene and CCL2 gene, and the evaluation method is described in Examples below. can be performed by microarray analysis.

As used herein, the term "proportion of CD97-positive mesenchymal stem cells" refers to the proportion of cells positive for the surface antigen analyzed by flow cytometry, as described in Examples below. In the present specification, the "proportion of CD97-positive mesenchymal stem cells" may be referred to as "positive rate".

[2] Cell population containing mesenchymal stem cells The cell population containing mesenchymal stem cells provided by the present invention is
(a) in the cell population, the ratio of CD97-positive mesenchymal stem cells is 30% or more, and (b) the cell population contains CD358-positive mesenchymal stem cells,
It is characterized by

CD97 stands for Cluster of Differentiation 97 and is a protein also known as BL-Ac[F2] encoded by the ADGRE5 (Adhesion G protein-coupled receptor E5) gene.
CD358 means differentiation cluster 358, and is a protein encoded by the TNFRSF21 (tumor necrosis factor receptor superfamily, member 21) gene.

In the cell population containing mesenchymal stem cells provided by the present invention, (a) the ratio of CD97-positive mesenchymal stem cells in the cell population is 30% or more, and (b) the cell population is , contain CD358-positive mesenchymal stem cells, a cell population containing mesenchymal stem cells exhibiting a high immunosuppressive effect is formed. Therefore, in the present invention, the above conditions can be used as an index for the formation of cell populations exhibiting a high immunosuppressive effect.

The ratio of CD97-positive mesenchymal stem cells in the cell population is 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, 50% or more, 51% or more, It may be 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, or 60% or more.

"Comprising CD358-positive mesenchymal stem cells" is not particularly limited as long as CD358-positive mesenchymal stem cells are present in the cell population, and the ratio is 5% or more. , for example, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, or 10% or more.

Surface antigen markers (CD97, CD358) can be detected by any detection method known in the art. Methods of detecting surface antigen markers include, but are not limited to, flow cytometry or cell staining. In flow cytometry using a fluorescently labeled antibody, if a cell that emits more fluorescence than the negative control (isotype control) is detected, the cell is determined to be "positive" for that marker. Any antibody known in the art can be used as the fluorescence-labeled antibody, and examples thereof include antibodies labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), and the like. include but are not limited to: In cell staining, when colored or fluorescent cells are observed under a microscope, the cells are determined to be "positive" for the marker. Cell staining may be immune cell staining using antibodies or non-immune cell staining without antibodies.

Specifically, the ratio (positive rate) of cells that are positive for surface antigen markers (CD97, CD358) is determined using dot plot development analysis of flow cytometry using the following procedures (1) to (8). can be measured at
(1) When the target cells are adherent cells, trypsin-EDTA (Thermo Fisher Scientific) is used to detach the adherent cells from the plastic culture vessel, and the cells are recovered by centrifugation. If the target cells are non-adherent cells, the cells are collected by centrifugation.
(2) After fixing the cells with 4% paraformaldehyde, the cells were washed with phosphate buffer (PBS) and adjusted to 1.0×10 ⁶ cells/mL with 2% BSA/PBS. Prepare a suspension. 100 μL of the cell suspension is dispensed.
(3) Centrifuge the dispensed cell suspension, and add 100 μL of 0.5% BSA/PBS to the resulting cell pellet. Antibodies corresponding to each surface antigen marker or their isotype control antibodies are then added. When analyzing CD358, which is a surface antigen marker, Mouse IgG (H+L) is then added as a secondary antibody. After mixing each reaction solution with a Voltex, the mixture is allowed to stand at 4° C. for 20 minutes.
(4) After adding 0.5% BSA/PBS and washing the cells by centrifugation, the cells were suspended in 0.5% BSA/PBS, and a cell strainer (35 μm nylon mesh filter) (Corning Inc./part number) was used. : 352235).
(5) The cell suspension obtained by filter filtration is analyzed with ALL Event 10000 using BD Accuri ^™ C6 Flow Cytometer (Becton Dickinson).
(6) For the measurement results, the vertical axis is SSC (side scattered light) (numerical range: 0 or more and 16777215 or less), and the horizontal axis is the fluorescence intensity of the antibody-labeled dye (numerical range: 10 ¹ or more 10 ^7.2 below).
(7) In the dot plot development, select all regions (gates) where the cell population with the higher fluorescence intensity is 1.0% or less of the total cells measured with the isotype control antibody.
(8) Calculate the percentage of cells included in the gate selected in (7) among the total cells measured with the antibody corresponding to the surface antigen marker.

The timing for detecting the surface antigen marker described above is not particularly limited. integer), during maintenance culture, before cryopreservation, after thawing, or before formulating as a pharmaceutical composition.

The immunosuppressive effect can be evaluated by the expression level of one or more genes selected from TGFB2 gene, TNFAIP6 gene, IL1A gene, IL1B gene and CCL2 gene. The immunosuppressive effect may be evaluated as the relative expression level of each of the above genes with respect to the expression level of the SDHA gene by microarray analysis described in Examples below.
The sequence of the SDHA (Succinate dehydrogenase complex, subunit A) gene is registered as ID: 6389 in the gene database of the National Center for Biotechnology Information.
The sequence of the TGFB2 (Transforming growth factor, beta 2) gene is registered as ID: 7042 in the gene database of the National Center for Biotechnology Information.
The sequence of the TNFAIP6 (tumor necrosis factor, alpha-induced protein 6) gene is registered as ID: 7130 in the gene database of the National Center for Biotechnology Information.

The IL1A (Interleukin 1, alpha) gene sequence is registered as ID: 3552 in the gene database of the National Center for Biotechnology Information.
The IL1B (Interleukin 1, beta) gene sequence is registered as ID: 3553 in the gene database of the National Center for Biotechnology Information.
The sequence of CCL2 (chemokine (CC motif) ligand 2) gene is registered as ID: 6347 in the gene database of the National Center for Biotechnology Information.

The relative expression level of the TGFB2 gene with respect to the expression level of the SDHA gene is preferably 1.5 or more, 1.6 or more, 1.7 or more, 1.8 or more, 1.9 or more, 2.0 or more. It may be 1 or more, 2.2 or more, 2.3 or more, 2.4 or more, 2.5 or more, 2.6 or more, or 2.7 or more.
The relative expression level of the TNFAIP6 gene with respect to the SDHA gene expression level is preferably 0.2 or more, and may be 0.3 or more.
The relative expression level of the IL1A gene with respect to the expression level of the SDHA gene is preferably 0.2 or more, and may be 0.3 or more.

The relative expression level of the IL1B gene with respect to the expression level of the SDHA gene is preferably 1.3 or more, and may be 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, or 1.8 or more. .
The relative expression level of the CCL gene with respect to the expression level of the SDHA gene is preferably 1.0 or more, 1.1 or more, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.5 or more. It may be 6 or more, or 1.7 or more.

The timing of measuring the gene expression level described above is not particularly limited. integer), during maintenance culture, before cryopreservation, after thawing, or before formulating as a pharmaceutical composition.

The origin of mesenchymal stem cells is not particularly limited, but for example, mesenchymal stem cells derived from fetal appendages, bone marrow, fat, or dental pulp can be used. The mesenchymal stem cells are preferably fetal appendage-derived mesenchymal stem cells, more preferably amniotic membrane-derived mesenchymal stem cells.

The mesenchymal stem cell population of the present invention can be stored in a frozen state until immediately before use. The mesenchymal stem cell population described above may contain any component other than mesenchymal stem cells. Examples of such components include salts, polysaccharides (e.g., HES, dextran, etc.), proteins (e.g., albumin, etc.), DMSO, amino acids, medium components (e.g., components contained in RPMI1640 medium, etc.), and the like. It can be, but is not limited to.

A cell population of the invention may be provided as a composition in combination with a vehicle. As a medium, a liquid medium (eg, a medium or a pharmaceutically acceptable medium described later) can be preferably used.

The cell populations of the invention can contain any number of mesenchymal stem cells. The cell population of the present invention is, for example, 1 x 10 ¹ , 2 x 10 ¹ , 5 x 10 ¹ , 1 x 10 ² , 2 x 10 ² , 5 x 10 ² , 1 x 10 ³ , 2 x 10 ³ , 5 x 10 ³ , 1 x 10 ⁴ , 2 x 10 ⁴ , 5 x 10 ⁴ , 1 x 10 ⁵ , 2 x 10 ⁵ , 5 x 10 ⁵ , 1 x10 ⁶ , 2 x 10 ⁶ , 5 x 10 ⁶ , 1 x 10 ⁷ , 2 x 10 ⁷ , 5 x 10 ⁷ , 1 x 10 ⁸ , 2 x 10 ⁸ , 5 x 10 ⁸ , 1 x 10 ⁹ , 2 x 10 ⁹ , 5 x 10 ⁹ , 1 x 10 ¹⁰ , 2 x 10 ¹⁰ , 5 x 10 ¹⁰ , 1 x 10 ¹¹ , 2 x 10 ¹¹ , 5×10 ¹¹ , 1×10 ¹² , 2×10 ¹² , 5×10 ¹² or more or less mesenchymal stem cells.

[3] Method for producing a cell population containing mesenchymal stem cells According to the method for producing a cell population containing mesenchymal stem cells according to the present invention, a cell population having the following cell characteristics (a) and (b) is obtained. It is a method that includes
(a) the proportion of CD97-positive mesenchymal stem cells in the cell population is 30% or more; and (b) the cell population contains CD358-positive mesenchymal stem cells.

That is, in the method for producing a cell population containing mesenchymal stem cells according to the present invention, the cell population containing mesenchymal stem cells has a ratio of CD97-positive mesenchymal stem cells of 30% or more and is CD358-positive. The method includes the step of preparing under conditions that maintain the presence of mesenchymal stem cells. The above condition that "the ratio of CD97-positive mesenchymal stem cells is 30% or more and CD358-positive mesenchymal stem cells are included" is a cell containing mesenchymal stem cells that exhibits a high immunosuppressive effect. It is an index of cluster formation, and the production method of the present invention is not particularly limited as long as it satisfies the above index.

The production method of the present invention includes a cell population obtaining step of obtaining a cell population containing mesenchymal stem cells by enzymatically treating a sample containing mesenchymal stem cells (for example, fetal appendages such as amniotic membrane). It's okay.

The amniotic membrane consists of an epithelial cell layer and an extracellular matrix layer, the latter containing amniotic MSCs. Amniotic epithelial cells, like other epithelial cells, characteristically express epithelial cadherin (E-cadherin: CD324) and epithelial adhesion factor (EpCAM: CD326), whereas amniotic MSCs express these epithelial-specific surface antigen markers. and are readily distinguishable by flow cytometry. The above cell population acquisition step may include a step of obtaining an amniotic membrane by caesarean section.

The cell population containing mesenchymal stem cells in the present invention is preferably a cell population obtained by treating a sample containing an epithelial cell layer and an extracellular matrix layer collected from fetal appendages with at least collagenase.

Enzymatic treatment of a sample collected from the fetal appendage (preferably a sample containing an epithelial cell layer and an extracellular matrix layer) preferably liberates the mesenchymal stem cells contained in the extracellular matrix layer of the fetal appendage. It is a treatment with an enzyme (or a combination thereof) that can and does not degrade the epithelial cell layer. Examples of such enzymes include, but are not limited to, collagenase and/or metalloproteinase. Metalloproteinases include, but are not limited to, thermolysin and/or dispase, which are metalloproteinases that cleave the N-terminal side of nonpolar amino acids.

The activity concentration of collagenase is preferably 50 PU/ml or more, more preferably 100 PU/ml or more, still more preferably 200 PU/ml or more, still more preferably 300 PU/ml or more, still more preferably 400 PU/ml or more. The activity concentration of collagenase is not particularly limited, but is, for example, 1000 PU/ml or less, 900 PU/ml or less, 800 PU/ml or less, 700 PU/ml or less, 600 PU/ml or less, or 500 PU/ml or less. Here, PU (Protease Unit) is defined as the amount of enzyme that decomposes 1 ug of FITC-collagen in 1 minute at pH 7.5 and 30°C.

The activity concentration of the metalloproteinase (for example, thermolysin and/or dispase) is preferably 50 PU/ml or more, more preferably 100 PU/ml or more, still more preferably 200 PU/ml or more, still more preferably 300 PU/ml or more, still more preferably 300 PU/ml or more. 400 PU/ml or more. In addition, the activity concentration of the metalloproteinase is preferably 1000 PU/ml or less, more preferably 900 PU/ml or less, still more preferably 800 PU/ml or less, still more preferably 700 PU/ml or less, still more preferably 600 PU/ml or less, still more preferably 600 PU/ml or less. is less than or equal to 500 PU/ml. Here, in the embodiment using dispase as the metalloproteinase, PU (Protease Unit) is defined as the amount of enzyme that releases an amino acid equivalent to 1 μg of tyrosine per minute from lactic casein at pH 7.5 and 30°C. be. Within the above enzyme concentration range, it is possible to efficiently liberate mesenchymal stem cells contained in the extracellular matrix layer while preventing epithelial cells contained in the epithelial cell layer of fetal appendages from being mixed. Preferred concentration combinations of collagenase and/or metalloproteinases can be determined by microscopic observation of fetal appendages after enzymatic treatment and by flow cytometry of the obtained cells.

From the viewpoint of efficiently collecting living cells, it is preferable to simultaneously treat the fetal appendages with a combination of collagenase and metalloproteinase. As the metalloproteinase in this case, thermolysin and/or dispase can be used, but not limited thereto. Mesenchymal stem cells can be conveniently obtained by treating fetal appendages only once with an enzyme solution containing collagenase and metalloproteinase. In addition, the simultaneous batch processing can reduce the risk of contamination with bacteria, viruses, and the like.

The enzymatic treatment of fetal appendages is preferably carried out by immersing the amniotic membrane, which has been washed with a washing solution such as physiological saline or Hank's balanced salt solution, in the enzyme solution and stirring the enzyme solution with a stirring means. From the viewpoint of efficiently liberating the mesenchymal stem cells contained in the extracellular matrix layer of the fetal appendage, such stirring means can be, for example, a stirrer or shaker, but is not limited thereto. The stirring speed is not particularly limited, but when a stirrer or shaker is used, it is, for example, 5 rpm or more, 10 rpm or more, 20 rpm or more, 30 rpm or more, 40 rpm or more, or 50 rpm or more. The stirring speed is not particularly limited, but when using a stirrer or shaker, it is, for example, 100 rpm or less, 90 rpm or less, 80 rpm or less, 70 rpm or less, or 60 rpm or less. The enzyme treatment time is not particularly limited, but is, for example, 10 minutes or longer, 20 minutes or longer, 30 minutes or longer, 40 minutes or longer, 50 minutes or longer, 60 minutes or longer, 70 minutes or longer, 80 minutes or longer, or 90 minutes or longer. The enzyme treatment time is not particularly limited, but is, for example, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, 110 minutes or less, or 100 minutes or less. The enzyme treatment temperature is not particularly limited, but is, for example, 15°C or higher, 16°C or higher, 17°C or higher, 18°C or higher, 19°C or higher, 20°C or higher, 21°C or higher, 22°C or higher, 23°C or higher, 24°C. 25°C or higher, 26°C or higher, 27°C or higher, 28°C or higher, 29°C or higher, 30°C or higher, 31°C or higher, 32°C or higher, 33°C or higher, 34°C or higher, 35°C or higher, or 36°C or higher be. Moreover, although the enzyme treatment temperature is not particularly limited, it is, for example, 40° C. or lower, 39° C. or lower, 38° C. or lower, or 37° C. or lower.

In the production method of the present invention, if desired, separated and/or collected separated mesenchymal stem cells from the enzyme solution containing the separated mesenchymal stem cells by a known method such as filter, centrifugation, hollow fiber separation membrane, cell sorter, or the like. can do. Preferably, the enzyme solution containing mesenchymal stem cells released by a filter is filtered. In the embodiment in which the enzyme solution is filtered through a filter, only the released cells pass through the filter, and the undegraded epithelial cell layer remains on the filter without passing through the filter. Not only can it be separated and/or collected in an efficient manner, but also the risk of contamination with bacteria, viruses, etc. can be reduced. Examples of filters include, but are not limited to, mesh filters. The pore size (mesh size) of the mesh filter is not particularly limited, but is, for example, 40 μm or more, 50 μm or more, 60 μm or more, 70 μm or more, 80 μm or more, or 90 μm or more. The pore size of the mesh filter is not particularly limited, but is, for example, 200 μm or less, 190 μm or less, 180 μm or less, 170 μm or less, 160 μm or less, 150 μm or less, 140 μm or less, 130 μm or less, 120 μm or less, 110 μm or less, or 100 μm or less. . The filtration rate is not particularly limited, but by setting the pore size of the mesh filter within the above range, the enzyme solution containing mesenchymal stem cells can be filtered by gravity, thereby preventing a decrease in cell viability. be able to.

Nylon is preferably used as the material of the mesh filter. Tubes containing 40 μm, 70 μm, 95 μm or 100 μm nylon mesh filters are available, such as the Falcon cell strainer commonly used for research. In addition, medical mesh cloths (nylon and polyester) used in hemodialysis and the like can be used. Furthermore, an arterial filter (polyester mesh filter, pore size: 40 μm or more and 120 μm or less) used for extracorporeal circulation can also be used. Other materials such as stainless steel mesh filters can also be used.

When mesenchymal stem cells are allowed to pass through a filter, natural fall (free fall) is preferred. Forced passage through the filter, such as suction using a pump or the like, is also possible, but it is desirable to use as weak a pressure as possible in order to avoid damaging the cells.

The mesenchymal stem cells that have passed through the filter can be recovered by centrifugation after diluting the filtrate with a double volume or more of medium or balanced salt buffer. Examples of balanced salt buffers that can be used include, but are not limited to, Dulbecco's phosphate buffer (DPBS), Earle's balanced salt solution (EBSS), Hank's balanced salt solution (HBSS), phosphate buffer (PBS), and the like.

The cell population obtained in the cell population acquisition step is prepared under the conditions that the ratio of CD97-positive mesenchymal stem cells is 30% or more and CD358-positive mesenchymal stem cells are included. . The above conditions are useful as indicators for obtaining cell populations containing mesenchymal stem cells exhibiting a high immunosuppressive effect. The preparation method is not particularly limited as long as it satisfies the above criteria. Examples of such a method include sorting a cell population that satisfies the above conditions using a cell sorter. In addition, as another preparation method that satisfies the above criteria, the cell population has a ratio of CD97-positive mesenchymal stem cells of 30% or more and contains CD358-positive mesenchymal stem cells. Culturing in.

A culture method that satisfies the above criteria includes, for example, a step of seeding a cell population in an uncoated plastic culture vessel at a density of 400 to 5,000 cells/cm ² and culturing it multiple times. can be done. The density at which the cell population is seeded is more preferably 500 cells/cm ² or more, more preferably 600 cells/cm ² or more, still more preferably 700 cells/cm ² or more, still more preferably 800 cells. /cm ² or more, more preferably 900 cells/cm ² or more, still more preferably 1000 cells/cm ² or more, still more preferably 1100 cells/cm ² or more, still more preferably 1200 cells/cm ² or more, more preferably 1,300 cells/cm ² or more, still more preferably 1,400 cells/cm ² or more, still more preferably 1,500 cells/cm ² or more, still more preferably 1,600 cells/cm ² or more , more preferably 1700 cells/cm ² or more, more preferably 1800 cells/cm ² or more, still more preferably 1900 cells/cm ² or more, still more preferably 2000 cells/cm ² or more . The density when seeding the cell population is more preferably 4800 cells/cm ² or less, more preferably 4600 cells/cm ² or less, still more preferably 4400 cells/cm ² or less, still more preferably 4200 cells. /cm ² or less, more preferably 4000 cells/cm ² or less, still more preferably 3800 cells/cm ² or less, still more preferably 3600 cells/cm ² or less, still more preferably 3400 cells/cm ² or less, more preferably 3200 cells/cm ² or less, more preferably 3000 cells/cm ² or less, still more preferably 2800 cells/cm ² or less, still more preferably 2600 cells/cm ² or less , more preferably 2400 cells/cm ² or less, more preferably 2200 cells/cm ² or less.

Another culture method that satisfies the above criteria includes, for example, a step of seeding a cell population in a plastic culture vessel coated with a coating agent at a density of 400 to 5,000 cells/cm ² and culturing the cells multiple times. can be mentioned. Preferred conditions for density when seeding the cell population are the same as those described above.

Examples of coating agents include extracellular matrix, fibronectin, vitronectin, osteopontin, laminin, entactin, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, gelatin, poly-L-ornithine, poly-D. - Lysine, Matrigel® matrix, including but not limited to.

Still another culture method that satisfies the above criteria includes, for example, culturing by adding basic fibroblast growth factor (bFGF) to the basal medium used for culture. The concentration of basic fibroblast growth factor is preferably 2 ng/mL or more, more preferably 4 ng/mL or more, still more preferably 6 ng/mL or more, still more preferably 8 ng/mL or more, More preferably, it is 10 ng/mL or more. The concentration of basic fibroblast growth factor is preferably 20 ng/mL or less, more preferably 18 ng/mL or less, 16 ng/mL or less, even more preferably 14 ng/mL or less, still more preferably 12 ng/mL or less. The timing of adding the basic fibroblast growth factor is not particularly limited. shown), during maintenance culture, before cryopreservation, or after thawing.

The culture period of the above single culture can be, for example, 4 to 10 days, more specifically, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days. can be mentioned.

The medium used for the above culture can be prepared by using any liquid medium for animal cell culture as a basal medium and optionally adding other components (serum, serum replacement reagent, growth factor, etc.). .

The basal medium includes BME medium, BGJb medium, CMRL1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium (Iscove's Modified Dulbecco's Medium), Medium 199 medium, Eagle MEM medium, αMEM (Alpha Modification Minimum Essential Medium Eagle) medium, DMEM medium (Dulbecco's Modified Eagle's Medium), Ham's F10 medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof (e.g., DMEM/F12 medium ( A medium such as Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham)) can be used, but is not particularly limited.

In addition, the medium used for the above culture may be a commercially available serum-free medium. Examples include STK1 and STK2 (DS Pharma Biomedical), EXPREP MSC Medium (Biomimetics Sympathies), Corning stemgro human mesenchymal stem cell medium (Corning), but are not particularly limited.

Other components added to the basal medium include, for example, albumin, serum, serum replacement reagents, growth factors, and the like. In the embodiment in which albumin is added to the basal medium, the concentration of albumin is preferably more than 0.05% and 5% or less. In addition, in the embodiment in which serum is added to the basal medium, the concentration of serum is preferably 5% or more. In the embodiment in which a growth factor is added, a reagent (such as heparin) for stabilizing the growth factor in the medium may be added in addition to the growth factor, or the growth factor may be added in advance with a gel, polysaccharide, or the like. After stabilization, stabilized growth factors may be added to the basal medium.

Cultivation of mesenchymal stem cells can be performed, for example, by the following steps. First, the cell suspension is centrifuged, the supernatant is removed, and the resulting cell pellet is suspended in a medium. Next, the cells are seeded in a plastic culture vessel and cultured in a 37° C. environment with a CO ₂ concentration of 3% or more and 5% or less using a medium so that the confluency rate is 95% or less. Examples of the medium include, but are not limited to, αMEM, M199, or media based on these. The cells obtained by culturing as described above are cells that have been cultured once.

For example, the cells that have been cultured once can be further subcultured and cultured as follows. First, the cells cultured once are treated with ethylenediaminetetraacetic acid (EDTA) and then treated with trypsin to detach from the plastic culture vessel. Next, the resulting cell suspension is centrifuged, the supernatant is removed, and the resulting cell pellet is suspended in medium. Finally, the cells are seeded in a plastic culture vessel and cultured in a 37° C. environment with a CO ₂ concentration of 3% or more and 5% or less using a medium so that the confluency rate is 95% or less. Examples of the medium include, but are not limited to, αMEM, M199, or media based on these. Cells obtained by passage and culture as described above are cells that have been passaged once. By performing similar passages and cultures, cells that have been passaged N times can be obtained (N represents an integer of 1 or more). From the viewpoint of mass production of cells, the lower limit of the number of passages N is, for example, 1 or more, preferably 2 or more, more preferably 3 or more, still more preferably 4 or more, still more preferably 5 or more, More preferably 6 times or more, more preferably 7 times or more, still more preferably 8 times or more, still more preferably 9 times or more, still more preferably 10 times or more, still more preferably 11 times or more, still more preferably 12 times or more, and more preferably preferably 13 times or more, more preferably 14 times or more, more preferably 15 times or more, still more preferably 16 times or more, still more preferably 17 times or more, still more preferably 18 times or more, still more preferably 19 times or more, still more preferably is 20 times or more, more preferably 25 times or more. Moreover, the upper limit of the passage number N is preferably, for example, 50 times or less, 45 times or less, 40 times or less, 35 times or less, or 30 times or less, from the viewpoint of suppressing cell aging.

According to the production method of the present invention, a cell population containing mesenchymal stem cells exhibiting a high immunosuppressive effect can be obtained. The lower limit of the number of cells obtained per culture batch (the number of cells obtained per unit surface area and per unit culture days) varies depending on the number of seeded cells, seeding density, etc., but for example, 1.0 × 10 ⁵ (cells/cm ² /day) or more, 2.0×10 ⁵ (pieces/cm ² /day) or more, 3.0×10 ⁵ (pieces/cm ² /day) or more, 4.0×10 ⁵ (pieces/cm ² /day) or more day) or more, 5.0×10 ⁵ (pieces/cm ² /day) or more, 6.0×10 ⁵ (pieces/cm ² /day) or more, 7.0×10 ⁵ (pieces/cm ² /day) or more 8.0×10 ⁵ (pieces/cm ² /day) or more, 9.0×10 ⁵ (pieces/cm ² /day) or more, or 10.0×10 ⁵ (pieces/cm ² /day) or more be. In addition, the upper limit of the number of obtained cells per culture batch is not particularly limited, but for example, 10.0 × 10 ⁸ (cells/cm ² /day) or less, 9.0 × 10 ⁸ (cells/cm ² /day) ) or less, 8.0×10 ⁸ (pieces/cm ² /day) or less, 7.0×10 ⁸ (pieces/cm ² /day) or less, 6.0×10 ⁸ (pieces/cm ² /day) or less , 5.0×10 ⁸ (pieces/cm ² /day) or less, 4.0×10 ⁸ (pieces/cm ² /day) or less, 3.0×10 ⁸ (pieces/cm ² /day) or less, 2 0×10 ⁸ (pieces/cm ² /day) or less or 1.0×10 ⁸ (pieces/cm ² /day) or less.

According to the production method of the present invention, a cell population containing mesenchymal stem cells exhibiting a high immunosuppressive effect can be obtained. Thereby, the mesenchymal stem cells obtained by the production method of the present invention are preferably 40 days or more, more preferably 45 days or more, 50 days or more, or 55 days or more after the start of in vitro culture. 60 days or more, 65 days or more, 70 days or more, 75 days or more, 80 days or more, 85 days or more, 90 days or more, 95 days or more, 100 days or more, 105 days or more, Alternatively, it is possible to culture up to 110 days or more.

In addition, the mesenchymal stem cells obtained by the production method of the present invention have a doubling frequency of preferably 10 times or more, more preferably 15 times or more, 20 times or more, 25 times or more, or 30 times after the start of in vitro culture. It is possible to culture until 35 times or more, 40 times or more, 45 times or more, or 50 times or more.

The production method of the present invention is
(a) the proportion of CD97-positive mesenchymal stem cells in the cell population is 30% or more, and (b) the cell population contains CD358-positive mesenchymal stem cells. The index may include an identification step of identifying a cell population containing mesenchymal stem cells exhibiting a high immunosuppressive effect.

The means for identifying cell populations containing said mesenchymal stem cells is preferably flow cytometry.

The proportion of CD97- and CD358-positive mesenchymal stem cells (positive rate) in the cell population can be measured using flow cytometry dot plot development analysis according to the procedure described in paragraph 0029.

The timing of performing the above identification is not particularly limited, but for example, immediately after separating the cells from the biological sample, during the culture process, after purification in the culture process, immediately after passage N times (N is an integer of 1 or more ), during maintenance culture, before cryopreservation, after thawing, or before formulation as a pharmaceutical composition.

In addition, the production method of the present invention can include a step of selectively separating the identified cell population after identifying the cell population containing the mesenchymal stem cells using the above (a) and (b) as indicators. . The means for selectively isolating the identified cell population is not particularly limited, but examples thereof include sorting of the cell population by a cell sorter, purification of the cell population by culture, and the like.

Moreover, the production method of the present invention can include a step of cryopreserving the cell population containing the mesenchymal stem cells. In the aspect including the step of cryopreserving the cell population, after thawing the cell population, the cell population may be identified, separated, collected and/or cultured as necessary. Alternatively, the cell population may be used as it is after being thawed.

Means for cryopreserving the cell population containing the mesenchymal stem cells are not particularly limited, but examples thereof include program freezers, deep freezers, immersion in liquid nitrogen, and the like. When using a program freezer, the freezing temperature is preferably −30° C. or less, −40° C. or less, −50° C. or less, −60° C. or less, −70° C. or less, −80° C. or less, or −90° C. or less. , -100°C or less, -110°C or less, -120°C or less, -130°C or less, -140°C or less, -150°C or less, -160°C or less, -170°C or less, -180°C or less, -190°C or less , or −196° C. (liquid nitrogen temperature) or less. When using a program freezer, preferred freezing rates are, for example, -1°C/min, -2°C/min, -3°C/min, -4°C/min, -5°C/min, -6°C/min. °C/min, -7°C/min, -8°C/min, -9°C/min, -10°C/min, -11°C/min, -12°C/min, -13°C/min, -14°C/min minutes or −15° C./minute. When a program freezer is used as such freezing means, for example, the temperature is reduced to a temperature between -50°C and -30°C (e.g., -40°C) at a freezing rate of -2°C/min or more and -1°C/min or less. and further lower the temperature to -100°C or higher and -80°C or lower (e.g., -90°C) at a freezing rate of -11°C/min or higher and -9°C/min or lower (e.g., -10°C/min). be able to. Further, when immersion in liquid nitrogen is used as such a freezing means, for example, after freezing by rapidly lowering the temperature to -196°C, cryopreservation can be performed in liquid nitrogen (gas phase).

When freezing by the above freezing means, the above cell population may be frozen in any storage container. Examples of such storage containers include, but are not limited to, cryotubes, cryovials, freezing bags, transfusion bags, and the like.

When freezing by the above freezing means, the above cell population may be frozen in any cryopreservation medium. As such a cryopreservation solution, a commercially available cryopreservation solution may be used, for example, CryoNovo (Akron Biotechnology), MSC Freezing Solution (Biological Industries), CryoStor (HemaCare) and the like. Not limited.

The cryopreservation solution described above can contain a predetermined concentration of polysaccharide. Preferred concentrations of polysaccharides are, for example, 1% by mass or more, 2% by mass or more, 3% by mass or more, 4% by mass or more, 5% by mass or more, 6% by mass or more, 7% by mass or more, 8% by mass or more, 9 % by mass or more, 10% by mass or more, 11% by mass or more, or 12% by mass or more. The preferred concentration of the polysaccharide is, for example, 40% by mass or less, 35% by mass or less, 30% by mass or less, 25% by mass or less, 20% by mass or less, 19% by mass or less, 18% by mass or less, 17% by mass or less. , 16% by mass or less, 15% by mass or less, 14% by mass or less, or 13% by mass or less. Examples of polysaccharides include, but are not limited to, hydroxylethyl starch (HES) or dextran (such as Dextran 40).

The above cryopreservation solution can contain a predetermined concentration of dimethylsulfoxide (DMSO). A preferable concentration of DMSO is, for example, 1% by mass or more, 2% by mass or more, 3% by mass or more, 4% by mass or more, 5% by mass or more, 6% by mass or more, 7% by mass or more, 8% by mass or more, or 9% by mass. % or more. Further, the preferred concentration of DMSO is, for example, 20% by mass or less, 19% by mass or less, 18% by mass or less, 17% by mass or less, 16% by mass or less, 15% by mass or less, 14% by mass or less, 13% by mass or less, It is 12% by mass or less, 11% by mass or less, or 10% by mass or less.

The above cryopreservation solution may contain albumin at a predetermined concentration higher than 0% by mass. A preferable albumin concentration is, for example, 0.5% by mass or more, 1% by mass or more, 2% by mass or more, 3% by mass or more, 4% by mass or more, 5% by mass or more, 6% by mass or more, 7% by mass or more, or It is 8% by mass or more. Further, the preferred concentration of albumin is, for example, 40% by mass or less, 35% by mass or less, 30% by mass or less, 25% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, or 9% by mass or less. be. Examples of albumin include, but are not limited to, bovine serum albumin, mouse albumin, and human albumin.

[4] Pharmaceutical composition The cell population containing mesenchymal stem cells according to the present invention can be used as a pharmaceutical composition. Thus, according to the invention there is provided a pharmaceutical composition comprising a cell population according to the invention and a pharmaceutically acceptable vehicle.

The pharmaceutical composition of the present invention is preferably a solution, more preferably an injectable solution.
The pharmaceutical composition of the present invention can be used as a cell therapeutic agent, for example, a therapeutic agent for intractable diseases.
The pharmaceutical composition of the present invention can be used as a therapeutic agent for immune-related diseases. The above diseases can be treated by administering the pharmaceutical composition of the present invention to the treatment site in such an amount that the effect can be measured.

According to the present invention there is provided a cell population comprising mesenchymal stem cells according to the invention for use in pharmaceutical compositions.
According to the present invention, there is provided a cell population comprising mesenchymal stem cells according to the present invention, which is used for cell therapeutic agents.

According to the present invention, there is provided a cell population comprising mesenchymal stem cells according to the invention, which is used for the treatment of immune-related diseases.

According to the present invention, there is provided a cell population comprising mesenchymal stem cells according to the present invention, which is administered to a patient or subject and used for suppressing an immune response.

According to the present invention, a method of transplanting cells into a patient or subject, comprising the step of administering to the patient or subject a therapeutically effective amount of a cell population comprising mesenchymal stem cells according to the present invention, and treating a disease of the patient or subject. A method of treatment is provided.

According to the invention there is provided the use of a cell population comprising mesenchymal stem cells according to the invention for the manufacture of a pharmaceutical composition.
According to the present invention there is provided use of a cell population comprising mesenchymal stem cells according to the present invention for the production of a cell therapeutic agent.

According to the present invention, there is provided use of a cell population comprising mesenchymal stem cells according to the present invention for producing therapeutic agents for immune-related diseases.

According to the present invention, there is provided the use of a cell population comprising mesenchymal stem cells according to the present invention for the production of therapeutic agents necessary for suppressing immune responses for administration to a patient or subject.

The dosage of the pharmaceutical composition of the present invention is the amount of cells that, when administered to a patient or subject, can achieve a therapeutic effect on the disease compared to a patient or subject who has not been administered. . A specific dosage can be appropriately determined depending on the dosage form, administration method, purpose of use, age, body weight and symptoms of the patient or test subject. A single dose of mesenchymal stem cells to humans is not particularly limited, but is, for example, 10 ⁴ cells/kg body weight or more, 10 ⁵ cells/kg body weight or more, or 10 ⁶ cells/kg body weight or more. The dose of mesenchymal stem cells to humans is not particularly limited, but is, for example, 10 ⁹ cells/kg body weight or less, 10 ⁸ cells/kg body weight or less, or 10 ⁷ cells/kg body weight or less.

The method of administration of the pharmaceutical composition of the present invention is not particularly limited. For example, it can be directly transplanted to Methods for administering pharmaceutical compositions are described, for example, in JP-A-2015-61520, Onken JE, t al. American College of Gastroenterology Conference 2006 Las Vegas, NV, Abstract 121. , Garcia-Olmo D, et al. Dis Colon Rectum 2005;48:1416-23. Intravenous injection, drip intravenous injection, local direct injection, local direct transplantation, and the like are known. The pharmaceutical composition of the present invention can also be administered by various methods described in the above references.

The pharmaceutical composition of the present invention can also be used for the purpose of treating other diseases as a formulation for injection, a formulation for transplantation of cell clusters or sheet-like structures, or a gel formulation mixed with any gel.

Patients or subjects of the present invention are typically humans, but may be other animals. Other animals include, for example, dogs, cats, cows, horses, pigs, goats, sheep, monkeys (cynomolgus monkeys, rhesus monkeys, common marmosets, Japanese monkeys), ferrets, rabbits, rodents (mouse, rats, gerbils, guinea pigs, mammals such as hamsters), and birds such as chickens and quails, but are not limited thereto.

The pharmaceutical composition of the present invention can be stored in a frozen state until immediately before use. When administering the pharmaceutical composition of the present invention to a patient or subject, it can be rapidly thawed at 37°C before use.

The pharmaceutical compositions of the invention may contain any ingredient used in the treatment of humans. Examples of such components include salts, polysaccharides (e.g., HES, dextran, etc.), proteins (e.g., albumin, etc.), DMSO, amino acids, medium components (e.g., components contained in RPMI1640 medium, etc.), and the like. It can be, but is not limited to.

In addition, the pharmaceutical composition of the present invention may be obtained by diluting a cell population containing mesenchymal stem cells with an infusion preparation used as a pharmaceutically acceptable medium. The "infusion preparation (pharmaceutically acceptable medium)" in the present specification is not particularly limited as long as it is a solution used in the treatment of humans. Examples include Ringer's solution, Ringer's acetate solution, initiation solution (No. 1 solution), dehydration replenishment solution (No. 2 solution), maintenance infusion solution (No. 3 solution), and postoperative recovery solution (No. 4 solution).

The pharmaceutical composition of the present invention may contain various additives such as emulsifiers, dispersants, buffers, preservatives, wetting agents to increase storage stability, sterility, isotonicity, absorption and/or viscosity. , antibacterial agents, antioxidants, chelating agents, thickeners, gelling agents, pH adjusters, and the like. Examples of the thickening agent include, but are not limited to, HES, dextran, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, and the like. The concentration of the thickening agent depends on the selected thickening agent, but can be arbitrarily set within a concentration range that achieves the desired viscosity while being safe when administered to a patient or subject.

The pH of the pharmaceutical composition of the present invention can be around neutral pH, for example, pH 6.5 or higher or pH 7.0 or higher, or pH 8.5 or lower or pH 8.0 or lower, It is not limited to these.

The present invention will be specifically described in the following examples, but the present invention is not limited by the examples.

<Comparative Example 1>
(Step 1: Collection of amniotic membrane)
Fetal appendages, the egg membrane and placenta, were aseptically harvested from pregnant women (donors) in cases of elective cesarean section with informed consent. The resulting egg membrane and placenta were placed in a sterile vat containing physiological saline, and the amniotic membrane was manually stripped from the egg membrane stump. The amniotic membrane was washed with Hank's balanced salt solution (Ca-Mg free) to remove adhering blood and clots.

(Step 2: enzymatic treatment of amniotic membrane and collection of amniotic membrane MSCs)
The amniotic membrane containing the epithelial cell layer and the extracellular matrix layer was immersed in a Hank's balanced salt solution (containing Ca and Mg) containing 300 PU/mL collagenase and 200 PU/mL dispase I, and heated at 37°C for 90 minutes at 50 rpm. The amniotic membrane was enzymatically treated by shaking and stirring under conditions. The enzyme-treated solution was filtered through a nylon mesh with an opening of 95 μm to remove undigested amniotic membrane, and a cell suspension containing amniotic membrane MSCs was recovered. Using a flow cytometer, the obtained cell suspension was analyzed for the ratio of cells positive for the expression of CD90, one of the surface antigens known as a representative positive marker for MSCs. It was confirmed that the amniotic MSCs could be separated from the amniotic membrane with high purity.

A BD Accuri ^™ C6 Flow Cytometer manufactured by Becton Dickinson (BD) was used for surface antigen analysis, and the measurement conditions were the number of cells analyzed: 10,000 cells, and the flow rate setting: Slow (14 μL/min). FITC Mouse Anti-Human CD90 (BD/model number: 561969) was used as an antibody corresponding to the CD90 antigen, and FITC Mouse IgG1, κ Isotype Control (BD/model number: 349041) was used as an isotype control antibody.

(Step 3: Cryopreservation of amniotic MSCs)
The cell population obtained in the above “Step 2: enzymatic treatment of amniotic membrane and collection of amniotic MSCs” was suspended in Bambanker (Lymphotech) to a concentration of 1.0×10 ⁷ cells/mL, and cryo-treated. Dispensed into tubes. The cryotube was placed in a bicell (freezing container) (Nippon Freezer Co.), stored at −80° C. for 12 hours, and then stored frozen at liquid nitrogen temperature.

(Step 4: Culture of amniotic MSCs)
The cell population obtained in the above "Step 3: Cryopreservation of amniotic membrane MSC" was seeded in an uncoated plastic culture vessel, and 10% fetal bovine serum (FBS) (inactivated) and 1 × Antibiotic- Adherent culture was performed in αMEM (Alpha Modification of Minimum Essential Medium Eagle) containing Antimycotic (manufactured by Thermo Fisher Scientific) until subconfluent. Cells were then detached using TrypLE Select (1x) (Thermo Fisher Scientific) and 1/4 of the cells were seeded in uncoated plastic culture vessels of the same scale as the previous culture. , was subcultured. Medium exchange was performed twice a week. Subculture was continued in this manner.

<Example 1>
(Step 1: Collection of amniotic membrane)
An amniotic membrane was obtained in the same manner as in step 1 of Comparative Example 1, except that the egg membrane and placenta, which are fetal appendages, were aseptically collected from a donor different from the donor in Comparative Example 1.

(Step 2: enzymatic treatment of amniotic membrane and collection of amniotic membrane MSCs)
A cell suspension containing amniotic membrane MSCs was recovered in the same manner as in step 2 of Comparative Example 1. The resulting cell suspension was analyzed using a flow cytometer in the same manner as in Comparative Example 1. Cells positive for the expression of CD90, one of the surface antigens known as a representative positive marker for MSCs. was analyzed, and it was confirmed that amniotic MSCs could be separated from the amniotic membrane with high purity.

(Step 3: Cryopreservation of amniotic MSCs)
The cell population obtained in the above “Step 2: enzymatic treatment of amniotic membrane and collection of amniotic membrane MSCs” was cryopreserved in the same manner as in Step 3 of Comparative Example 1.

(Step 4: Culture of amniotic MSCs)
The cell population obtained in the above "Step 3: cryopreservation of amniotic MSCs" was adherently cultured in the same manner as in Step 4 of Comparative Example 1 until subconfluency was achieved. Subculture was performed in the same manner as in step 4 of Comparative Example 1.

<Analysis of CD97 expression>
With respect to the 6th passage cell populations cultured in Comparative Example 1 and Example 1, the ratio of cells positive for CD97 was measured using a flow cytometer.

In this measurement, Anti-CD97-Mouse Mono IgG1 (Miltenyi Biotec/model number: 130-097-102) was used as an antibody corresponding to the CD97 antigen, and Mouse IgG1 (Miltenyi Biotec/model number: 130-098-) was used as an isotype control antibody. 845) was used.

<Analysis of CD358 expression>
With respect to the 6th passage cell populations cultured in Comparative Example 1 and Example 1, the ratio of cells positive for CD358 was measured using a flow cytometer.

In this measurement, Mouse Anti Human CD358 (Bio-Rad Inc./model number: MCA2333) was used as a primary antibody corresponding to the CD358 antigen, and Mouse Mono IgG1 (Bio-Rad Inc./model number: MCA928) was used as an isotype control antibody corresponding to the CD358 antigen. Mouse F(ab)2 IgG (H+L) APC-conjugated Antibody (R&D Systems/model number: F0101B) was used as a primary antibody and a secondary antibody corresponding to the isotype control antibody.

The results of the analysis are shown in Table 1 and Figures 1-4.

As shown in Table 1, compared with the cell population of Comparative Example 1, the cell population of Example 1 improved the positive rate of CD97 and CD358. In the cell population of Comparative Example 1, the ratio of CD97-positive mesenchymal stem cells is less than 30% (specifically 20.6%), whereas in the cell population of Example 1, CD97 is positive. The ratio of mesenchymal stem cells exhibiting was 30% or more (specifically 60.6%). Further, the cell population of Comparative Example 1 does not contain CD358-positive mesenchymal stem cells (specifically 0.0%), whereas the cell population of Example 1 is CD358-positive. It was clarified that it contains mesenchymal stem cells exhibiting the following characteristics (specifically, 7.1%).

<Evaluation of immunosuppressive effect>
Regarding the 6th passage cell population cultured in Comparative Example 1 and Example 1, the expression of TGFB2 gene, TNFAIP6 gene, IL1A gene, IL1B gene, CCL2 gene and SDHA gene was analyzed by microarray analysis.

Microarray analysis was performed according to the following procedures (1) to (5). The following procedures (3) to (5) were entrusted to Riken Genesis Co., Ltd. and performed.
(1) Sixth-passage cell populations cultured in Comparative Example 1 and Example 1 were separated from the plastic culture vessel using a cell scraper (manufactured by Corning) and collected by centrifugation.
(2) RNAlater (manufactured by Thermo Fisher Scientific) was added to the obtained cell pellet to stably store RNA, and then total RNA was extracted and purified using RNeasy Plus Mini kit (manufactured by QIAGEN).
(3) cDNA was synthesized from 100 ng of total RNA by reverse transcription reaction, and the cDNA was transcribed into cRNA by in vitro transcription and labeled with biotin (using 3'IVT PLUS Reagent Kit).
(4) 10.0 µg of labeled cRNA was added to the hybridization buffer, and hybridization was performed for 16 hours on Human GeneGenome U133A 2.0 Array (manufactured by Affymetrix). After washing with GeneChip Fluidics Station 450 (manufactured by Affymetrix) and staining with phycoerythrin, scanning was performed with GeneChip Scanner 3000 7G (manufactured by Affymetrix) and AGCC (manufactured by Affymetrix GeneChip Command Image Console Software) (Software) It was analyzed and quantified using Affymetrix Expression Console (manufactured by Affymetrix).
(5) The numerical data file was analyzed using analysis software GeneSpring GX (manufactured by Agilent Technologies).

The expression level of each gene was obtained as a relative expression level with respect to the expression level of the SDHA gene. The results are shown in the table below.

Table 2 shows that the cell population of Example 1 has higher relative expression levels of TGFB2 gene, TNFAIP6 gene, IL1A gene, IL1B gene and CCL2 gene than the cell population of Comparative Example 1.

Mesenchymal stem cell-based therapy is a promising approach for immune-related diseases such as graft-versus-host disease and Crohn's disease. The anti-inflammatory and/or immunosuppressive ability of mesenchymal stem cells is believed to involve various cytokines produced by mesenchymal stem cells, including TGFB2 and TNFAIP6. Among them, TGFB2 is a major player that not only induces and maintains immune tolerance by affecting leukocyte proliferation, differentiation, activation and survival, but also plays an essential role in coordinating the initiation and resolution of inflammatory responses. It functions as a mediator (Kyurkchiev D, et al. World J Stem Cells. 2014;6(5):552-70.). The regulatory activities of TGFB2 on immune cell function are diverse, including inhibition of effector T cell proliferation and function, generation of regulatory T cells from naive T lymphocytes, attenuation of cytokine production and cytolytic activity by NK cells. , suppression of B cells, dendritic cells and macrophages (Kyurkchiev D, et al. World J Stem Cells. 2014;6(5):552-70.). In addition, TNFAIP6 produced by mesenchymal stem cells has been suggested to be involved in the reduction of macrophage activation in an in vitro co-culture system and the alleviation of inflammation in a zymosan-induced peritonitis model (Bartosh TJ, et al. Proc. Natl Acad Sci U S A. 2010;107(31):13724-9.). In addition, mesenchymal stem cells must be stimulated with inflammatory cytokines to acquire anti-inflammatory properties. (Cesarz Z, Tamama K. Stem Cells Int. 2016; 2016:9176357.). The mesenchymal stem cells of the present invention can highly produce TGFB2, TNFAIP6, IL1A, IL1B, and CCL2, and can exhibit high anti-inflammatory and/or immunosuppressive ability by means of these cytokines. Therefore, the mesenchymal stem cells of the present invention can improve the patient's immune status by being administered to patients with immune-related diseases (eg, graft-versus-host disease, Crohn's disease).
From the above, it was revealed that the cell population of Example 1 exhibits a higher immunosuppressive effect than the cell population of Comparative Example 1.

Therefore, it was found that a cell population having the following cell characteristics (a) and (b) exhibits a high immunosuppressive effect.
(a) the proportion of CD97-positive mesenchymal stem cells in the cell population is 30% or more; and (b) the cell population contains CD358-positive mesenchymal stem cells.

It was also suggested that the above conditions (a) and (b) are effective as indicators for obtaining cell populations exhibiting a high immunosuppressive effect.

<Example 2>
Pregnant women (donors different from Comparative Example 1 and Example 1) in cases of elective cesarean section who obtained informed consent, in the same manner as in Example 1, "Step 1: Collection of amniotic membrane" Enzyme treatment and collection of amniotic MSCs", "Step 3: Cryopreservation of amniotic MSCs" and "Step 4: Culture of amniotic MSCs" were carried out. In the amniotic MSC culture, a part of the cell population was sampled at each passage, and the following conditions (a) and (b) were evaluated for each sampled cell population. The conditions were evaluated using the same method as in the above "Analysis of CD97 expression" and "Analysis of CD358 expression".
(a) the proportion of CD97-positive mesenchymal stem cells in the cell population is 30% or more; and (b) the cell population contains CD358-positive mesenchymal stem cells.

In the collected cell populations, there were cell populations satisfying the conditions (a) and (b) and cell populations not satisfying the conditions, so the two types of cell populations were evaluated as described in paragraph 0116. Immunosuppressive action was evaluated by the same method.

As a result, compared with the cell population that did not satisfy the above conditions (a) and (b), the cell population that satisfied the above conditions (a) and (b) had TGFB2 gene, TNFAIP6 gene, IL1A gene, IL1B gene and It was found that the relative expression level of the CCL2 gene was high. Cell populations satisfying the above conditions (a) and (b) exhibit higher immunosuppressive effects than cell populations not satisfying the above conditions (a) and (b). Using the condition b) as an indicator, it is possible to selectively obtain a cell population exhibiting a high immunosuppressive effect.

<Example 3: Production of pharmaceutical composition>
A portion of the cell population obtained in Example 1 above is subjected to the preparation of a pharmaceutical composition. A pharmaceutical composition (cell preparation) is prepared consisting of 20 mL RPMI 1640 medium containing 4.0×10 ⁸ amniotic MSCs, 800 mg HES, 0.7 mL DMSO and 800 mg human serum albumin. The pharmaceutical composition is sealed in a freezer bag and stored in a frozen state. In addition, the pharmaceutical composition can be thawed and provided to the patient at the time of use.

Claims (3)

A method for producing a cell population containing mesenchymal stem cells, wherein a cell population obtained by enzymatically treating a sample containing mesenchymal stem cells is used, and the ratio of mesenchymal stem cells positive for CD97 is 30%. A production method comprising the above and an identification step of identifying a cell population containing mesenchymal stem cells positive for CD358 . 2. The production method according to claim 1, further comprising a separation step of selectively separating said identified cell population. The production method according to claim 1 or 2, wherein the mesenchymal stem cells are derived from fetal appendages.

Images