Cell population containing mesenchymal cells, pharmaceutical composition containing the same, and method for producing the same

Abstract

The problem of the present invention is to provide a cell population containing mesenchymal cells capable of forming a cell sheet that can spontaneously peel off from a substrate. The present invention is a cell population containing mesenchymal cells, in which the ratio of cells positive for CD324 is 70% or more, and the ratio of mesenchymal cells positive for CD90 is 90% or more.

Description

Technical Field

[0001] The present invention relates to a cell population containing mesenchymal cells, a pharmaceutical composition containing the cell population, and a method for producing the cell population, which can be used in the fields of medicine, biochemistry, and the like. Background Art

[0002] Regenerative medicine is a medical treatment that restores the functions of an organism by applying stem cells artificially cultured in vitro to a patient's body to regenerate damaged organs and tissues. In recent years, the practical application of new treatment methods that involve applying cells such as mesenchymal stem cells, skeletal muscle myoblasts, and epithelial cells to patients to promote the regeneration and functional improvement of tissues and organs has been rapidly developing. For example, a bone marrow mesenchymal stem cell preparation for patients with acute graft-versus-host disease (GVHD) and spinal cord injury, and a skeletal muscle myoblast sheet for patients with severe heart failure have been sold in Japan as products for regenerative medicine and the like.

[0003] Most of the cells used in regenerative medicine and the like have the property of adhering to a culture substrate. When manufacturing the above cell preparations, in addition to the process of culturing the cells while adhering them to the culture substrate for proliferation, there is also a process of detaching the cells adhered to the culture substrate. The above-mentioned bone marrow mesenchymal stem cells also have the property of adhering to a culture substrate. After culturing the cells while adhering them to the culture substrate for proliferation, the cells are detached from the culture substrate, and a cell suspension is recovered. As the method for detaching the cells from the culture substrate, for example, a chemical method using a proteolytic enzyme such as trypsin or a chemical reagent, and a physical method using an instrument such as a cell scraper for physically detaching the cells can be cited. For example, Patent Document 1 discloses a method of adding trypsin to a culture substrate to detach mesenchymal stem cells.

[0004] In addition, Patent Document 2 discloses a method of detaching cells from a culture substrate by changing the temperature, using a culture substrate obtained by coating the surface with a temperature-responsive polymer whose cell adhesiveness changes with temperature.

[0005] Prior Art Documents

[0006] Patent Documents

[0007] Patent Document 1: Japanese Patent No. 5394932 Gazette

[0008] Patent Document 2: International Publication WO2001 / 068799 Summary of the Invention

[0009] Problems to be Solved by the Invention

[0010] As described above, various methods for detaching adherent cells such as mesenchymal stem cells from a culture substrate have been developed. However, in the method of Patent Document 1, cultured cells are damaged by chemicals such as trypsin. In addition, when cultured cells are detached from a culture substrate by trypsin treatment, the detached cells become single cells and cannot be detached in the form of a cell sheet.

[0011] In addition, in the method of Patent Document 2, as a culture substrate that serves as a scaffold for cell culture, a specific material needs to be used, so the manufacturing cost increases.

[0012] Therefore, the problem of the present invention is to provide a cell population that spontaneously detaches from a culture substrate without using chemical, physical, or special culture substrates, a method for producing the same, and a pharmaceutical composition containing the same.

[0013] Means for Solving the Problem

[0014] The present inventors conducted intensive studies to solve the above problems, and as a result, found that a cell population containing mesenchymal cells with a ratio of CD324-positive cells of 70% or more and a ratio of CD90-positive mesenchymal cells of 90% or more can spontaneously detach from a substrate without special means or instruments. The present invention was completed based on such an insight.

[0015] That is, according to the present specification, the following inventions can be provided.

[0016] (1) A cell population comprising a cell population containing mesenchymal cells,

[0017] In the above cell population, the ratio of CD324-positive cells is 70% or more, and the ratio of CD90-positive mesenchymal cells is 90% or more.

[0018] (2) The cell population according to (1), wherein, in the above cell population, the ratio of CD326-positive cells is 10% or less.

[0019] (3) The cell population according to (1) or (2), wherein, in the above cell population, the ratio of CD73-positive mesenchymal cells is 80% or more, the ratio of CD166-positive mesenchymal cells is 80% or more, the ratio of CD45-positive cells is 10% or less, and the ratio of CD105-positive mesenchymal cells is 70% or more.

[0020] (4) The cell population according to any one of (1) to (3), wherein the above mesenchymal cells are derived from fetal appendages.

[0021] (5) A method for producing a cell population containing mesenchymal cells, the method comprising:

[0022] A process for culturing a cell population containing mesenchymal cells, and

[0023] A process for screening a cell population having the following characteristics (a) and (b) from the above cell population containing mesenchymal cells,

[0024] (a) In the above cell population, the ratio of cells positive for CD324 is 70% or more,

[0025] (b) In the above cell population, the ratio of mesenchymal cells positive for CD90 is 90% or more.

[0026] (6) A pharmaceutical composition comprising the cell population according to any one of (1) to (4) and a pharmaceutically acceptable medium.

[0027] (7) The pharmaceutical composition according to (6), wherein the human is administered in such a manner that the single dose of mesenchymal cells is 10 12 or less.

[0028] (8) The pharmaceutical composition according to (6) or (7), wherein the above pharmaceutical composition is a preparation for injection.

[0029] (9) The pharmaceutical composition according to (6) or (7), wherein the above pharmaceutical composition is a preparation for transplantation of cell aggregates or sheet structures.

[0030] (10) The pharmaceutical composition according to any one of (6) to (9), which is a therapeutic agent for a disease selected from immune-related diseases, ischemic diseases, lower limb ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, nervous system diseases, graft-versus-host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, lupus erythematosus, collagen diseases, stroke, cerebral infarction, cerebral hematoma, cerebrovascular paralysis, brain tumor, liver cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes, mycosis fungoides, scleroderma, diseases caused by degeneration and / or inflammation of connective tissues such as cartilage, articular cartilage defect, meniscus injury, osteochondritis dissecans, aseptic osteonecrosis, knee osteoarthritis, inflammatory arthritis, rheumatoid arthritis, eye diseases, angiogenesis-related diseases, ischemic heart disease, coronary heart disease, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular heart disease, trauma, epithelial injury, fibrosis, lung diseases, muscular dystrophy, chronic pancreatitis, chronic nephritis, and cancer.

[0031] (11) Use of a cell population containing mesenchymal cells according to any one of (1) to (4) in the manufacture of a pharmaceutical composition.

[0032] (12) Use of a cell population containing mesenchymal cells according to any one of (1) to (4) in the manufacture of a cell therapeutic agent.

[0033] (13) Use of a cell population containing mesenchymal cells according to any one of (1) to (4) in the manufacture of a drug for myocardial regeneration, cardiomyocyte generation, angiogenesis, blood vessel repair, or suppression of immune response.

[0034] (14) A cell population containing mesenchymal cells according to any one of (1) to (4) for the treatment of a disease.

[0035] (15) The cell population according to (14), wherein the disease is the disease according to (10).

[0036] (16) A cell population containing mesenchymal cells according to any one of (1) to (4) for administration to a patient or subject and for myocardial regeneration, cardiomyocyte generation, angiogenesis, blood vessel repair, or suppression of immune response.

[0037] (17) A method for treating a disease of a patient or subject, the method comprising: administering a therapeutically effective amount of a cell population containing mesenchymal cells according to any one of (1) to (4) to the patient or subject.

[0038] (18) The method according to (17), wherein the disease is the disease according to (10) above, and the patient or subject is a patient or subject in need of treatment for the disease.

[0039] (19) The method according to (17) or (18), wherein the patient is a human, and the dose per administration is 10 12 cells or less.

[0040] (20) The method according to (17), (18) or (19), wherein the cell population has a cell mass or sheet-like structure.

[0041] (21) A method for myocardial regeneration, cardiomyocyte generation, angiogenesis, blood vessel repair, or suppression of immune response in a patient or subject, the method comprising: administering a therapeutically effective amount of a cell population containing mesenchymal cells according to any one of (1) to (4) to the patient or subject.

[0042] (22) The method according to (21), wherein the patient is a human, and the dose per administration is 10 12Less than cells.

[0043] (23) The method according to (21) or (22), wherein the cell population has a cell aggregate or sheet-like structure.

[0044] This specification incorporates the disclosure of Japanese Patent Application No. 2019-111470 which is the basis of priority for this application.

[0045] Effects of the Invention

[0046] The cell population according to one or more embodiments of the present invention can spontaneously detach from the culture substrate without using chemical and / or physical methods.

[0047] The method for producing a cell population according to one or more embodiments of the present invention can efficiently produce a cell population having the above-mentioned effects.

[0048] The pharmaceutical composition according to one or more embodiments of the present invention can be used for the treatment of immune-related diseases and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is an observation image of the cell morphology of the cell population of the fifth passage of Comparative Example 1 prepared from donor #1 in Evaluation 1 on the 21st day of culture (magnification 40 times).

[0050] Figure 2 This is an observation image (magnification 40 times) of the cell morphology of the cell population of the fifth passage of Example 1 prepared from donor #1 on the 10th day of culture in Evaluation 2. Partial cell detachment was observed.

[0051] Figure 3 This is an observation image (magnification 100 times) of the cell morphology of the second passage cell population of Example 2 prepared from donor #3 on the 10th day of culture in Evaluation 2. Partial cell detachment was observed.

[0052] Figure 4 This is an observation image (magnification 40 times) of the cell morphology of the second passage cell population of Example 2 prepared from donor #3 on the 11th day of culture in Evaluation 2. A mass structure (cell agglomerate) formed by aggregation of cell sheets peeled off from the substrate was observed. DETAILED DESCRIPTION

[0053] [1] Notes on terminology

[0054] "Mesenchymal cells" in this specification refer to cells that are positive for at least one of CD73, CD90, CD166, CD105, negative for CD45, and negative for CD326. Representatively, they refer to cells that satisfy the following characteristics i) and ii). "Mesenchymal stromal cells" and "Mesenchymal stem cells (MSC)" are also included in the mesenchymal cells of the present invention.

[0055] As "mesenchymal cells", among somatic cells (tissue cells) that can be collected from various tissues and organs, cells that are positive for at least one of CD73, CD90, CD166, CD105, negative for CD45, and negative for CD326 can be used. Preferably, cells that satisfy the following characteristics i) and ii) can be used. There is no particular limitation on the above-mentioned somatic cells, and examples can include: adipocytes, adipose stem cells, nerve cells, neural stem cells, cardiomyocytes, cardiac stem cells, hepatocytes, hepatic stem cells, epithelial cells, epithelial stem cells, skeletal muscle cells, skeletal muscle stem cells, hematopoietic cells, hematopoietic stem cells, mesenchymal cells, mesenchymal stem cells, amnion-derived mesenchymal cells, amnion-derived mesenchymal stem cells, amniotic epithelial-derived mesenchymal cells, amniotic epithelial-derived mesenchymal stem cells, amniotic extracellular matrix layer-derived mesenchymal cells, amniotic extracellular matrix layer-derived mesenchymal stem cells, gastrointestinal epithelial cells, gastrointestinal epithelial stem cells, osteoblasts, chondrocytes, synovial cells, synovial stem cells, etc.

[0056] Typical characteristics of mesenchymal cells in this specification

[0057] i) Show adhesion to plastic under the culture conditions of a standard medium. Here, the so-called standard medium refers to a medium in which serum, a serum replacement reagent, or a proliferation factor (for example, human platelet lysate as a serum replacement reagent) is added to a basal medium (for example, αMEM medium).

[0058] ii) Positive for surface antigens CD73 and CD90, and negative for CD45 and CD326.

[0059] The above-mentioned "mesenchymal cells" may be any cells that exhibit at least one of CD73 positivity, CD90 positivity, CD166 positivity, CD105 positivity, CD45 negativity, and CD326 negativity. For example, they may be cells that exhibit CD73 positivity, CD90 positivity, CD45 negativity, and CD326 negativity. There is no particular limitation on whether they have the differentiation ability to differentiate into bone, cartilage, fat, etc. The "mesenchymal cells" in this specification also include cells such as mesenchymal stem cells that have the differentiation ability to differentiate into bone, cartilage, and fat. In addition, the above-mentioned "mesenchymal cells" also include cells that exhibit at least one of CD73 positivity, CD90 positivity, CD166 positivity, CD105 positivity, CD45 negativity, and CD326 negativity but do not have the differentiation ability to differentiate into bone, cartilage, and fat. In addition, the above-mentioned "mesenchymal cells" also include cells that exhibit at least one of CD73 positivity, CD90 positivity, CD166 positivity, CD105 positivity, CD45 negativity, and CD326 negativity but only differentiate into one or two of bone, cartilage, and fat.

[0060] In this specification, "fetal appendages" refer to fetal membranes, placenta, umbilical cord, and amniotic fluid. In addition, the "fetal membranes" are the fetal sac containing the amniotic fluid of the fetus and are formed from the amnion, chorion, and decidua from the inside. Among them, the amnion and chorion originate from the fetus. The "amnion" refers to a transparent membrane lacking blood vessels located in the innermost layer of the fetal membranes, and the inner wall is covered by a layer of epithelial cells with secretory functions and secretes amniotic fluid. The inner layer of the amnion (also called the epithelial cell layer) is covered by a layer of epithelial cells with secretory functions and secretes amniotic fluid, and the outer layer of the amnion (also called the extracellular matrix layer, equivalent to the matrix) contains mesenchymal cells.

[0061] The morphology of the "cell population containing mesenchymal cells" in this specification is not particularly limited, and examples thereof may include: cell pellets, cell aggregates, cell sheets, cell floating liquids, cell suspensions, their cryopreserved products, etc.

[0062] In this specification, the "ratio of (mesenchymal) cells positive for a given surface antigen" is as described in the examples below, and represents the ratio of cells positive for the given surface antigen obtained by flow cytometry analysis in the cell population as the object. In this specification, the ratio of cells positive for a given surface antigen is sometimes referred to as the "positive rate", and the ratio of cells negative for a given surface antigen is sometimes referred to as the "negative rate".

[0063] In this specification, the ratio of a given cell in a cell population refers to the proportion of the number of the above-mentioned given cells to the total number of cells in the cell population as the object.

[0064] In this specification, "mesenchymal cells", "cell population containing mesenchymal cells", and "fetal appendages" are preferably of human origin.

[0065] [2] Cell population containing mesenchymal cells

[0066] The cell population of one or more embodiments of the present invention is a cell population containing mesenchymal cells. In the above cell population, the ratio of CD324-positive cells is 70% or more, and the ratio of CD90-positive mesenchymal cells is 90% or more. The CD324-positive cells may be mesenchymal cells.

[0067] When the cell population containing mesenchymal cells with such characteristics is cultured on a substrate, even without using conventional chemical methods and / or physical methods, the cell population containing mesenchymal cells will spontaneously detach from the substrate. In a more preferred mode, the above cell population containing mesenchymal cells spontaneously detaches from the substrate in the form of a cell sheet.

[0068] The "cell population containing mesenchymal cells" in this specification only needs to be a cell population containing at least mesenchymal cells, and there is no particular limitation. It may be a population containing other cells. The ratio of mesenchymal cells in the above cell population containing mesenchymal cells may be 70% or more, 75% or more, 80% or more, 85% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 97% or more, 99% or more, 100%, but there is no particular limitation.

[0069] In addition, the ratio of other cells in the above cell population containing mesenchymal cells may be 30% or less, 20% or less, 10% or less, 5% or less, 3% or less, 1% or less, 0%. It should be noted that the above other cells only need to be other than mesenchymal cells, and there is no particular limitation. Examples may include blood cell types such as lymphocytes, granulocytes, and red blood cells.

[0070] CD324 of the surface antigen refers to cluster of differentiation 324, which is a protein known as epithelial cadherin (E-cadherin).

[0071] CD90 of the surface antigen refers to cluster of differentiation 90, which is a protein known as Thy-1.

[0072] In the above cell population, the ratio of CD324-positive cells may be more preferably 75% or more, 80% or more, 85% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more.

[0073] In the above cell population, the ratio of mesenchymal cells positive for CD90 can be more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, and can be 100%.

[0074] In a cell population of one or more embodiments of the present invention, the ratio of cells positive for CD326 is preferably 10% or less.

[0075] CD326 refers to cluster of differentiation 326, which is a protein known as EpCAM.

[0076] In the above cell population, the ratio of cells positive for CD326 can be more preferably 5% or less (negative rate 95% or more), 4% or less (negative rate 96% or more), 3% or less (negative rate 97% or more), 2% or less (negative rate 98% or more), 1% or less (negative rate 99% or more), and can be 0% (negative rate 100%).

[0077] According to one aspect of the present invention, a cell population containing mesenchymal cells provided by the present invention preferably satisfies one or more of the following, and more preferably all satisfy: the ratio of mesenchymal cells positive for CD73 is 90% or more, the ratio of mesenchymal cells positive for CD166 is 80% or more, the ratio of mesenchymal cells positive for CD105 is 70% or more, and the ratio of cells positive for CD45 is 10% or less. In addition, according to one aspect of the present invention, a cell population containing mesenchymal cells provided by the present invention preferably has a ratio of cells positive for CD34 of 10% or less.

[0078] CD73 refers to cluster of differentiation 73, which is a protein known as 5'-nucleotidase or Ecto-5'-nucleotidase.

[0079] CD166 refers to cluster of differentiation 166, which is a protein known as Activated leukocyte cell adhesion molecule (ALCAM).

[0080] CD105 refers to cluster of differentiation 105, which is a protein known as Endoglin.

[0081] CD45 refers to cluster of differentiation 45, which is a protein known as PTPRC (Protein tyrosine phosphatase, receptor type, C) or LCA (Leukocyte common antigen).

[0082] CD34 refers to cluster of differentiation 34, a protein known as Hematopoietic progenitor cell antigen CD34.

[0083] Among the above cell populations, the ratio of mesenchymal cells positive for CD73 can be more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, and can be 100%.

[0084] Among the above cell populations, the ratio of mesenchymal cells positive for CD166 can be more preferably 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.

[0085] Among the above cell populations, the ratio of mesenchymal cells positive for CD105 can be more preferably 74% or more, 75% or more, 80% or more, 85% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more.

[0086] Among the above cell populations, the ratio of cells positive for CD45 can be more preferably 5% or less (negative rate 95% or more), 4% or less (negative rate 96% or more), 3% or less (negative rate 97% or more), 2% or less (negative rate 98% or more), 1% or less (negative rate 99% or more), and can be 0% (negative rate 100%).

[0087] Among the above cell populations, the ratio of cells positive for CD34 can be more preferably 5% or less (negative rate 95% or more), 4% or less (negative rate 96% or more), 3% or less (negative rate 97% or more), 2% or less (negative rate 98% or more), 1% or less (negative rate 99% or more), and can be 0% (negative rate 100%).

[0088] Here, cells or mesenchymal cells positive for CD324, CD90, CD326, CD73, CD166, CD105, CD45, and CD34 respectively refer to cells or mesenchymal cells with positive expression of CD324, CD90, CD326, CD73, CD166, CD105, CD45, and CD34.

[0089] In one or more embodiments of the present invention, expression markers (CD324, CD90, CD326, CD73, CD166, CD105, CD45, or CD34) used as indicators can be detected by any detection method known in the art. Examples of methods for detecting expression markers include, but are not limited to, flow cytometry or cell staining. In flow cytometry using a fluorescently labeled antibody, when cells that emit stronger fluorescence than the negative control (isotype control) are detected, those cells are determined to be "positive" for the marker. The fluorescently labeled antibody can be any antibody known in the art, and examples include antibodies labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), etc., but are not limited thereto. In cell staining, when cells that are colored or emit fluorescence are observed under a microscope, those cells are determined to be "positive" for the marker. Cell staining can be immunocytochemical staining using an antibody or non-immunocytochemical staining without using an antibody. There is no particular limitation on the antibody used in immunocytochemical staining, and to detect the target marker, a generally known antibody can be used, and preferably the antibody used in the examples described below is used. For example, there is no particular limitation on the antibody used to detect CD324 positivity, and antibodies prepared from clones of REA811, 67A4, or SPM381 can be used to detect CD324 positivity. In one or more embodiments of the present invention, it is more preferable to use an antibody prepared from a clone of REA811 to detect CD324 positivity. It should be noted that the expression marker has the same meaning as the surface antigen, and the two can be used interchangeably.

[0090] For the cell population of one or more embodiments of the present invention, there is no particular limitation on the presence or absence of differentiation ability. It is preferably capable of differentiating into cartilage tissue and does not have the ability to differentiate into adipose tissue or has a low ability to differentiate into adipose tissue, and more preferably has a low ability to differentiate into bone tissue or does not have the ability to differentiate into bone tissue.

[0091] The cell population of one or more embodiments of the present invention can be stored in a frozen state until immediately before use. The above cell population can contain any components in addition to mesenchymal cells and other cells. Examples of such components include, but are not limited to, salts, polysaccharides (e.g., HES, dextran, etc.), proteins (e.g., albumin, etc.), DMSO, culture medium components (e.g., components contained in RPMI1640 medium, etc.).

[0092] [3] Method for producing a cell population containing mesenchymal cells

[0093] The method for manufacturing a cell population containing mesenchymal cells according to one or more embodiments of the present invention includes: a step of culturing a cell population containing mesenchymal cells, and a step of screening a cell population having the following characteristics from the cell population containing mesenchymal cells: (a) in the cell population, the ratio of cells positive for CD324 is 70% or more; (b) in the cell population, the ratio of mesenchymal cells positive for CD90 is 90% or more.

[0094] According to the method for manufacturing a cell population containing mesenchymal cells according to one or more embodiments of the present invention, a cell population containing mesenchymal cells having the characteristics of (a) and (b) above can be prepared. These characteristics are useful as indices when obtaining a cell population containing mesenchymal cells that can spontaneously detach from a substrate. In addition, these characteristics are useful as indices when obtaining a mesenchymal cell population in the form of a cell sheet without making the detached mesenchymal cell population into a single-cell form.

[0095] In the method for manufacturing the cell population, the step of culturing a cell population containing mesenchymal cells and the step of screening a cell population having the above characteristics from the cell population containing mesenchymal cells may be different steps or may be an integrated step. As an example of the integrated step of the above two steps, for example, as described later, a cell population containing mesenchymal cells is cultured under specific conditions capable of screening a cell population satisfying one or both of the above (a) and (b) characteristics.

[0096] In the method for manufacturing the cell population, the step of screening a cell population satisfying the above characteristics may be any step as long as it can screen a cell population satisfying the above characteristics, and there is no particular limitation. As such a step, for example, a cell population satisfying (a) can be selected using a cell sorter, and then cultured under conditions capable of screening a cell population satisfying (b) from the obtained cell population. In addition, a cell population satisfying (b) can be selected using a cell sorter, and then cultured under conditions capable of screening a cell population satisfying (a) from the obtained cell population. In addition, as another method for preparing a cell population satisfying the above characteristics, culturing a cell population under specific conditions capable of screening a cell population satisfying the above (a) and (b) can be cited. Details of the culture conditions and culture methods will be described below. In addition, the method described in the cell population screening method described later can also be used to screen a cell population satisfying the above characteristics.

[0097] The method for manufacturing a cell population containing mesenchymal cells according to one or more embodiments of the present invention may preferably further include: a cell population obtaining step of obtaining a cell population containing mesenchymal cells as a raw material by subjecting fetal appendages such as amniotic membranes to enzymatic treatment.

[0098] The amniotic membrane is formed by an epithelial cell layer and an extracellular matrix layer, the latter containing mesenchymal cells. The above-described cell population obtaining step may further include a step of obtaining the amniotic membrane by cesarean section.

[0099] The cell population containing cells collected from fetal appendages is more preferably a cell population obtained by treating a specimen collected from fetal appendages with at least collagenase, the specimen containing an epithelial cell layer and an extracellular matrix layer containing mesenchymal cells.

[0100] The enzymatic treatment of a specimen collected from fetal appendages (preferably a specimen containing an epithelial cell layer and an extracellular matrix layer containing mesenchymal cells) is preferably a treatment using an enzyme (or a combination thereof) capable of freeing the mesenchymal cells contained in the extracellular matrix layer of the fetal appendages and not decomposing the epithelial cell layer. As such an enzyme, there is no particular limitation, and examples thereof include collagenase and / or metalloprotease. As the metalloprotease, there is no particular limitation, and examples thereof include thermolysin and / or Dispase which are metalloproteases that cleave the N-terminal side of non-polar amino acids.

[0101] The activity concentration of collagenase is preferably 50 PU / ml or more, more preferably 100 PU / ml or more, and still more preferably 200 PU / ml or more. In addition, the activity concentration of collagenase is not particularly limited, and for example, it is 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, 500 PU / ml or less. Here, PU (Protease Unit) is defined as the amount of enzyme that decomposes 1 μg of FITC-collagen in 1 minute at pH 7.5 and 30°C.

[0102] The activity concentration of metalloproteinase (e.g., thermolysin and / or dispase) is preferably 50 PU / ml or more, more preferably 100 PU / ml or more, further preferably 150 PU / ml or more, and further preferably 190 PU / ml or more. In addition, the activity concentration of metalloproteinase is preferably 1000 PU / ml or less, more preferably 900 PU / ml or less, further preferably 800 PU / ml or less, further preferably 700 PU / ml or less, further preferably 600 PU / ml or less, further preferably 500 PU / ml or less, and further preferably 300 PU / ml or less. Here, in the case of using dispase as the metalloproteinase, PU (Protease Unit) is defined as the amount of enzyme that liberates amino acids equivalent to 1 μg tyrosine from casein lactate within 1 minute at pH 7.5 and 30°C. Within the above enzyme concentration range, the contamination of epithelial cells contained in the epithelial cell layer of fetal appendages can be prevented, and at the same time, the mesenchymal cells contained in the extracellular matrix layer can be efficiently liberated. The preferred concentration combination of collagenase and / or metalloproteinase can be determined by microscopic observation of fetal appendages after enzyme treatment and flow cytometry of the obtained cells.

[0103] From the viewpoint of efficiently recovering viable cells, it is preferable to treat fetal appendages with a combination of collagenase and metalloproteinase. It is more preferable to simultaneously treat fetal appendages by the above combination. As the metalloproteinase in this case, thermolysin and / or Dispase can be used, but it is not limited thereto. Mesenchymal cells can be easily obtained by treating fetal appendages only once with an enzyme solution containing collagenase and metalloproteinase. In addition, by simultaneously treating, the risk of contamination by bacteria, viruses, etc. can be reduced.

[0104] Enzymatic treatment of fetal appendages preferably involves immersing the amniotic membrane that has been washed with a cleaning solution such as physiological saline or Hank's balanced salt solution in an enzyme solution and performing the treatment while stirring with a stirring device. As such a stirring device, from the perspective of efficiently dissociating the mesenchymal cells contained in the extracellular matrix layer of the fetal appendages, a stirrer or a vibrator can be used, for example, but is not limited thereto. The stirring speed is not particularly limited. When a stirrer or a vibrator is used, for example, it is 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. Additionally, the stirring speed is not particularly limited. When a stirrer or a vibrator is used, for example, it is 100 rpm or less, 90 rpm or less, 80 rpm or less, 70 rpm or less, or 60 rpm or less. The enzymatic treatment time is not particularly limited. For example, it is 10 minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes or more, 50 minutes or more, 60 minutes or more, 70 minutes or more, 80 minutes or more, or 90 minutes or more. Additionally, the enzymatic treatment time is not particularly limited. For example, it is 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 enzymatic treatment temperature is not particularly limited. For example, it is 15°C or more, 16°C or more, 17°C or more, 18°C or more, 19°C or more, 20°C or more, 21°C or more, 22°C or more, 23°C or more, 24°C or more, 25°C or more, 26°C or more, 27°C or more, 28°C or more, 29°C or more, 30°C or more, 31°C or more, 32°C or more, 33°C or more, 34°C or more, 35°C or more, or 36°C or more. Additionally, the enzymatic treatment temperature is not particularly limited. For example, it is 40°C or less, 39°C or less, 38°C or less, or 37°C or less.

[0105] In the manufacturing method of one or more embodiments of the present invention, free mesenchymal cells can be separated and / or recovered from an enzyme solution containing free mesenchymal cells by using known methods such as filters, centrifugation, hollow fiber separation membranes, cell sorters, etc., according to the desire. It is preferable to filter the enzyme solution containing free mesenchymal cells by using a filter. In the method of filtering the above enzyme solution by using a filter, only free cells pass through the filter, and the undigested epithelial cell layer cannot pass through the filter and remains on the filter. Therefore, not only can free mesenchymal cells be easily separated and / or recovered, but also the risk of contamination by bacteria, viruses, etc. can be reduced. As the filter, there is no particular limitation, and for example, a mesh filter can be cited. The pore size (the size of the mesh holes) of the mesh filter is not particularly limited, for example, it is 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. In addition, the pore size of the mesh filter is not particularly limited, for example, it is 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. Regarding the filtration rate, there is no particular limitation. By setting the pore size of the mesh filter within the above range, the enzyme solution containing mesenchymal cells can be filtered by allowing it to drip naturally, thereby preventing a decrease in cell viability.

[0106] As the material of the mesh filter, nylon is preferably used. Tubes having nylon mesh filters of 40 μm, 70 μm, 95 μm or 100 μm, such as Falcon cell filters widely used for research purposes, can be used. In addition, medical mesh fabrics (nylon and polyester) used in hemodialysis, etc. can be used. In addition, arterial filters (polyester mesh filters, pore size: 40 μm or more and 120 μm or less) used during extracorporeal circulation can also be used. Other materials can also be used, for example, stainless steel mesh filters, etc.

[0107] When allowing mesenchymal cells to pass through the filter, it is preferably natural dripping (free fall). It can also be forced filtration through suction using a pump or the like. In order to avoid damaging the cells, it is preferably set to the weakest possible pressure.

[0108] The cell population containing mesenchymal cells that has passed through the filter can be recovered by centrifugation after diluting the filtrate with a double volume or more of a culture medium or a balanced salt buffer. As the balanced salt buffer, Dulbecco’s phosphate buffer (DPBS), Earle’s balanced salt solution (EBSS), Hank’s balanced salt solution (HBSS), phosphate buffer (PBS), etc. can be used, but it is not limited thereto.

[0109] The manufacturing method of one or more embodiments of the present invention includes: a step of culturing a cell population containing mesenchymal cells.

[0110] The seeding density of cells in the step of culturing the cell population containing mesenchymal cells is not particularly limited. For example, it can be seeded at a density of, for example, 500 to 10,000 cells / cm 2 . As the lower limit of the above seeding density, for example, it is preferably 500 cells / cm 2 or more, 1,000 cells / cm 2 or more, 2,000 cells / cm 2 or more, 3,000 cells / cm 2 or more, 4,000 cells / cm 2 or more, 5,000 cells / cm 2 or more. In addition, as the upper limit of the above seeding density, for example, it is preferably 10,000 cells / cm 2 or less, 9,000 cells / cm 2 or less, 8,000 cells / cm 2 or less, 7,000 cells / cm 2 or less.

[0111] It should be noted that the above step of culturing may include a subculture step or a step of repeating the culture multiple times under different culture conditions.

[0112] As the culture period of the above one-time culture, for example, it can be 4 to 10 days, and more specifically, it can be 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days.

[0113] The culture medium used for the above culture can be prepared by using any liquid culture medium for animal cells as the basal medium and appropriately adding other components (serum, serum replacement reagents, growth factors, etc.) as needed. It should be noted that in the method of adding a growth factor to the above basal medium, it can be prepared by further adding a reagent (such as heparin) for stabilizing the growth factor in the growth factor, or the growth factor can be stabilized in advance with a gel, polysaccharide, etc., and then the stabilized growth factor is added to the above basal medium to prepare. Thus, in order to culture a cell population containing mesenchymal cells, the culture medium obtained by adding serum, serum replacement reagent or growth factor to the basal medium is defined as the standard culture medium.

[0114] As the basal medium, 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 of 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 their mixed media (e.g., DMEM / F12 medium (Dulbecco’s Modified Eagle’s Medium / Nutrient Mixture F-12 Ham)) etc. can be used, without particular limitation. As a preferred basal medium, αMEM medium can be exemplified.

[0115] As other components that can be added to the basal medium, for example, albumin, bovine serum, serum replacement reagent, or growth factor etc. can be listed. A serum replacement reagent is preferred, and human platelet lysate is particularly preferred. Among them, it is preferably cultured in a basal medium containing a serum replacement reagent and not containing albumin, bovine serum, and growth factor. As the lower limit of the concentration of human platelet lysate in the medium, for example, it can be listed as 1 wt% or more, 2 wt% or more, 3 wt% or more in terms of the final concentration. In addition, as the upper limit of the concentration of human platelet lysate in the medium, for example, it is preferably 20 wt% or less, 10 wt% or less, 7 wt% or less.

[0116] In addition, the medium used for the above culture can be a commercially available serum-free medium. For example, STK1, STK2 (DS PHARMA BIOMEDICAL), EXPREP MSC Medium (BioMimetics Sympathies), Corning stemgro human mesenchymal stem cell medium (Corning) etc. can be listed, without particular limitation.

[0117] The cultivation of a cell population containing mesenchymal cells can be carried out, for example, in the following steps. First, the cell suspension is centrifuged, the supernatant is removed, and the obtained cell pellet is suspended in a culture medium. Next, the cells are inoculated into a culture vessel (e.g., a plastic culture vessel) and cultured using the culture medium in an environment with a CO2 concentration of 3% or more and 5% or less and at 37°C. The cells obtained through the above cultivation are the cells after the first cultivation.

[0118] The cells after the first cultivation described above can be further passaged and cultured as follows. First, the cells cultured to a confluence of 95% or less in the first cultivation are treated with ethylenediaminetetraacetic acid (EDTA) and then treated with trypsin to detach them from the culture vessel (e.g., a plastic culture vessel). The cell population produced in one or more embodiments of the present invention is a cell population capable of forming a cell culture that can be detached from a substrate under stable conditions and can also be easily detached when detached from the culture vessel using trypsin treatment, so that the damage to the cells caused by trypsin treatment can be reduced. In addition, when a cell sheet is formed on the substrate by the cell population, the cell sheet can be easily detached from the substrate, and the cell sheet can be easily dispersed by enzyme treatment to obtain a cell suspension, so that the damage to the cells can be reduced. Next, the obtained cell suspension is centrifuged, the supernatant is removed, and the obtained cell pellet is suspended in a culture medium. Finally, the cells are inoculated into a culture vessel (e.g., a plastic culture vessel) and cultured using the culture medium in an environment with a CO2 concentration of 3% or more and 5% or less and at 37°C. The cells obtained through the above passage and cultivation are the cells after the first passage. By performing the same passage and cultivation, cells after the nth passage (n represents an integer of 1 or more) can be obtained. In the case of performing passage cultivation, the cells can be cultured to a confluence of 95% or less, and the cells can be detached and recovered according to the above steps for the next generation of cultivation. From the viewpoint of mass-producing cells, the lower limit of the passage number n is, for example, 1 or more, preferably 2 or more, more preferably 3 or more, further preferably 4 or more, and further preferably 5 or more. In addition, from the viewpoint of suppressing cell aging, the upper limit of the passage number n is, for example, preferably 50 or less, 45 or less, 40 or less, 35 or less, and 30 or less. In the case of recovering the cultured cells without further passage cultivation, the cells can be cultured to a confluence of 100% or more, allowed to form a cell sheet on the substrate, and further cultured to cause the cell sheet to spontaneously detach from the substrate.

[0119] The manufacturing method of one or more embodiments of the present invention may include a step of screening a cell population having the characteristics shown in (a) and (b) below.

[0120] (a) The ratio of CD324-positive cells is 70% or more,

[0121] (b) The ratio of mesenchymal cells positive for CD90 is 90% or more.

[0122] As a method for screening a cell population using the above characteristics as indicators, the following method can be used. The method includes the step of screening a cell population that satisfies the characteristics of (a) and / or (b) above with a cell sorter as described above, and further includes, if necessary, a step of culturing under conditions capable of screening a cell population that satisfies the characteristics not screened in the above step. In addition, as a screening method other than the cell sorter, physical methods such as FACS and magnetic bead-based sorting can be used.

[0123] In addition, as another screening step, a step of culturing a cell population under specific conditions capable of screening a cell population that satisfies the characteristics of (a) and (b) above can be cited. For example, in the above cell population culturing step, by appropriately combining preferred culturing conditions, a cell population that satisfies the characteristics of (a) and (b) above can also be obtained. In this case, part or all of the above cell population culturing step is also a step of screening a cell population that satisfies the characteristics of (a) and (b) above. As an example, a cell population containing mesenchymal cells can be cultured in a medium supplemented with a serum replacement reagent such as human platelet lysate. Specifically, it can be cited: a chemical method of purifying a cell population containing mesenchymal cells to meet the above indicators by eliminating cells that do not meet the indicators using appropriate culturing conditions such as a basal medium supplemented with human platelet lysate; or a method of culturing a cell population to change it into a cell population that meets the above indicators by selecting an appropriate standard medium such as a basal medium supplemented with human platelet lysate. However, the method is not particularly limited and can be appropriately selected according to the culturing method, etc.

[0124] It should be noted that before the above screening, a step of identifying a cell population containing pluripotent stem cells using the above characteristics as indicators can be included.

[0125] The timing for screening a cell population having the characteristics of (a) and (b) above is not particularly limited, and examples include: before culturing, during culturing, after culturing, before cell population recovery, after cell population recovery, before preparing a frozen stock solution of the cells, after thawing the frozen stock solution of the cells, etc.

[0126] The method for screening a cell population having the characteristics of (a) and (b) above will be described in more detail.

[0127] As a method for screening a cell population having the above-mentioned characteristics (a) and (b) by culturing a cell population, for example, a cell population containing mesenchymal cells is inoculated on a culture substrate and proliferatively cultured on the culture substrate, whereby positive selection is performed on mesenchymal cells that are CD324-positive and CD90-positive, and a cell population in which the ratio of CD324-positive cells is 70% or more and the ratio of CD90-positive mesenchymal cells is 90% or more is detached / recovered. At this time, a culture substrate coated with an anti-CD324 antibody and an anti-CD90 antibody can be used for culturing. The timing for screening a cell population containing mesenchymal cells by culturing mesenchymal cells that are CD324-positive and CD90-positive is not particularly limited, and screening can be performed in any passage culture. It is preferable to screen mesenchymal cells that are CD324-positive and CD90-positive from a cell population containing mesenchymal cells in the primary culture.

[0128] In addition, mesenchymal cells that are CD324-positive and CD90-positive can be screened from a cell population containing mesenchymal cells by flow cytometry or a cell separation method using magnetic beads.

[0129] In the screening of a cell population, it is preferable to further screen a cell population in which the ratio of CD326-positive cells is 10% or less; it is preferable to further screen a cell population that satisfies one or more of the following conditions: the ratio of CD73-positive mesenchymal cells is 80% or more, the ratio of CD166-positive mesenchymal cells is 80% or more, the ratio of CD105-positive mesenchymal cells is 70% or more, and the ratio of CD45-positive cells is 10% or less. More preferably, a cell population that satisfies all of the above conditions is screened; it is preferable to further screen a cell population in which the ratio of CD34-positive cells is 10% or less. The screened cell population can be further cultured by the above method. In addition, the step of screening a cell population having these additional characteristics can be a step integrated with the step of culturing the cell population, or can be a separate step. The step of screening a cell population having these additional characteristics can be a step integrated with the step of screening a cell population having the above-mentioned characteristics (a) and (b), or can be a separate step.

[0130] In addition, the manufacturing method of one or more embodiments of the present invention may include a step of cryopreserving a cell population containing the above-mentioned mesenchymal cells. In a mode including the step of cryopreserving the cell population, after thawing the cell population, the cell population can be separated, recovered, and / or cultured as needed. In addition, it can also be used directly after thawing the cell population.

[0131] The method for cryopreserving a cell population containing the above-mentioned mesenchymal cells is not particularly limited, and examples thereof include: a programmable freezer, a cryogenic freezer, immersion in liquid nitrogen, etc. The temperature during freezing is preferably -30°C or lower, -40°C or lower, -50°C or lower, -60°C or lower, -70°C or lower, -80°C or lower, -90°C or lower, -100°C or lower, -110°C or lower, -120°C or lower, -130°C or lower, -140°C or lower, -150°C or lower, -160°C or lower, -170°C or lower, -180°C or lower, -190°C or lower, or -196°C (liquid nitrogen temperature) or lower. The preferred freezing rate during freezing is, for example, -1°C / min, -2°C / min, -3°C / min, -4°C / min, -5°C / min, -6°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, or -15°C / min. When a programmable freezer is used as the above-mentioned freezing method, for example, the temperature can be lowered to a temperature between -50°C or higher and -30°C or lower (for example, -40°C) at a freezing rate of -2°C / min or higher and -1°C / min or lower, and further the temperature can be lowered to a temperature between -100°C or higher and -80°C or lower (for example, -90°C) at a freezing rate of -11°C / min or higher and -9°C / min or lower (for example, -10°C / min).

[0132] When freezing is performed by the above-mentioned freezing method, the above-mentioned cell population can be frozen in a state of being loaded into any storage container. Examples of such a storage container include, but are not limited to: cryotubes, cryovials, cryovials, infusion bags, etc.

[0133] From the viewpoint of improving the survival rate of mesenchymal cells with relatively high proliferative ability, the cryopreservation solution for freezing preferably contains albumin at a given concentration of more than 0% by mass. The preferred concentration of albumin is, for example, 0.5% by mass or higher, 1% by mass or higher, 2% by mass or higher, 3% by mass or higher, 4% by mass or higher, 5% by mass or higher, 6% by mass or higher, 7% by mass or higher, or 8% by mass or higher. In addition, the preferred concentration of albumin is, for example, 40% by mass or lower, 35% by mass or lower, 30% by mass or lower, 25% by mass or lower, 20% by mass or lower, 15% by mass or lower, 10% by mass or lower, or 9% by mass or lower. Examples of albumin include, but are not limited to: bovine serum albumin, mouse albumin, human albumin, etc.

[0134] [4] Pharmaceutical composition

[0135] A cell population containing mesenchymal cells according to one or more embodiments of the present invention can be used as a pharmaceutical composition. That is, according to one or more embodiments of the present invention, a pharmaceutical composition can be provided, which contains the above-mentioned cell population and a pharmaceutically acceptable medium.

[0136] The pharmaceutical composition of one or more embodiments of the present invention can be used as a cell therapy agent, such as a therapeutic agent for refractory diseases.

[0137] The pharmaceutical composition of one or more embodiments of the present invention can be used as a therapeutic agent for diseases selected from immune-related diseases, ischemic diseases, lower limb ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, nervous system diseases, graft-versus-host disease (GVHD), inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, lupus erythematosus, collagen diseases, stroke, cerebral infarction, cerebral hematoma, cerebrovascular paralysis, brain tumor, liver cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes, mycosis fungoides (Alibert-Bazin syndrome), scleroderma, diseases caused by degeneration and / or inflammation of connective tissues such as cartilage, articular cartilage defect, meniscus injury, osteochondritis dissecans, aseptic osteonecrosis, knee osteoarthritis, inflammatory arthritis, rheumatoid arthritis, eye diseases, angiogenesis-related diseases, ischemic heart disease, coronary heart disease, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular heart disease, trauma, epithelial injury, fibrosis, lung diseases, muscular dystrophy, chronic pancreatitis, chronic nephritis, and cancer. The pharmaceutical composition of one or more embodiments of the present invention, in an amount capable of measuring the effect by administering to the treatment site, can treat the above diseases.

[0138] According to one or more embodiments of the present invention, there can be provided a cell population containing mesenchymal cells of one or more embodiments of the present invention for a pharmaceutical composition.

[0139] According to one or more embodiments of the present invention, there can be provided a cell population containing mesenchymal cells of one or more embodiments of the present invention for a cell therapy agent.

[0140] According to one or more embodiments of the present invention, there can be provided a cell population containing mesenchymal cells of one or more embodiments of the present invention for the treatment of the above diseases.

[0141] According to one or more embodiments of the present invention, there can be provided a cell population containing mesenchymal cells of one or more embodiments of the present invention for administering to a patient or subject to regenerate myocardium, generate cardiomyocytes, promote angiogenesis, repair blood vessels, or inhibit immune response.

[0142] According to one or more embodiments of the present invention, there can be provided a method for transplanting cells to a patient or subject, and a method for treating a disease of a patient or subject, the method comprising: a step of administering to the patient or subject a therapeutically effective amount of a cell population containing mesenchymal cells of one or more embodiments of the present invention.

[0143] According to one or more embodiments of the present invention, there can be provided the use of a cell population containing mesenchymal cells according to one or more embodiments of the present invention in the manufacture of a pharmaceutical composition.

[0144] According to one or more embodiments of the present invention, there can be provided the use of a cell population containing mesenchymal cells according to one or more embodiments of the present invention in the manufacture of a cell therapeutic agent.

[0145] According to one or more embodiments of the present invention, there can be provided the use of a cell population containing mesenchymal cells according to one or more embodiments of the present invention in the manufacture of a therapeutic agent for the above-mentioned disease.

[0146] According to one or more embodiments of the present invention, there can be provided the use of a cell population containing mesenchymal cells according to one or more embodiments of the present invention in the manufacture of a therapeutic agent required for myocardial regeneration, cardiomyocyte generation, angiogenesis, blood vessel repair, or suppression of immune response by administering to a patient or subject.

[0147] As the dosage of the pharmaceutical composition according to one or more embodiments of the present invention, in the case of administering to a patient or subject, it is the amount of cells that can obtain a therapeutic effect for the disease compared to a patient or subject who has not been administered. The specific dosage can be appropriately determined according to the administration route, administration method, purpose of use, and age, weight, and symptoms of the patient or subject, etc. The dosage is not particularly limited. For example, in terms of the number of mesenchymal cells, it is 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. In addition, the dosage is not particularly limited. For example, in terms of the number of mesenchymal cells, it is 10 9 cells / kg body weight or less, 10 9 cells / kg body weight or less, or 10 9 cells / kg body weight or less. In addition, as the dosage for one administration, in terms of the number of mesenchymal cells, it is preferably 10 12 cells or less, more preferably 10 11 cells or less, and even more preferably 10 10 cells or less.

[0148] The administration method of the pharmaceutical composition according to one or more embodiments of the present invention is not particularly limited, and examples thereof include: subcutaneous injection, intranodal injection, intravenous injection, intraperitoneal injection, intrathoracic injection, or direct injection to a local area, or direct transplantation to a local area, etc.

[0149] The pharmaceutical composition of one or more embodiments of the present invention can also be used in the form of an injectable preparation for treating other diseases, or a preparation for transplanting cell masses or sheet structures, or a gel preparation mixed with any gel. As described above, in a preferred embodiment of the present invention, since the obtained cell population can be peeled off from the substrate in the form of a cell sheet, the obtained cell population in sheet form can also be directly (or with minimal processing) used as a sheet-like transplant preparation. That is, a sheet-like cell population containing mesenchymal cells is also one embodiment of the present invention.

[0150] The patient or subject targeted by the pharmaceutical composition of one or more embodiments of the present invention is typically a human, and can also be other animals. Examples of other animals include: mammals such as dogs, cats, cows, horses, pigs, goats, monkeys, ferrets, etc., and birds such as chickens.

[0151] The pharmaceutical composition of one or more embodiments of the present invention can be stored in a frozen state until immediately before use. The pharmaceutical composition of one or more embodiments of the present invention can contain any components used in human treatment. Examples of such components include, but are not limited to: salts, polysaccharides (e.g., HES, dextran, etc.), proteins (e.g., albumin, etc.), DMSO, culture medium components (e.g., components contained in RPMI1640 medium, etc.).

[0152] In addition, the pharmaceutical composition of one or more embodiments of the present invention can be a pharmaceutical composition obtained by diluting a cell population containing mesenchymal cells with an infusion preparation used as a pharmaceutically acceptable medium. As the "infusion preparation (pharmaceutically acceptable medium)" in this specification, any solution used in human treatment can be used, and there is no particular limitation. Examples include: normal saline, 5% glucose solution, Ringer's solution, lactated Ringer's solution, acetate Ringer's solution, starting solution (solution No. 1), dehydration replenishment solution (solution No. 2), maintenance infusion (solution No. 3), postoperative recovery solution (solution No. 4), etc.

[0153] Other examples of diseases and the like that can be treated by using a cell population containing mesenchymal cells for a patient or subject, other specific examples of the above diseases and the like, and specific steps of the treatment can be referred to the matters described in Hare et al., J. Am. Coll. Cardiol., December 8, 2009; 54(24): 2277-2286, Honmou et al., Brain 2011: 134; 1790-1807, Makhoul et al., Ann. Thorac. Surg. 2013; 95: 1827-1833, Japanese Patent No. 590577, Japanese Patent Application Laid-Open No. 2010-518096, Japanese Patent Application Laid-Open No. 2012-509087, Japanese Patent Application Laid-Open No. 2014-501249, Japanese Patent Application Laid-Open No. 2013-256515, Japanese Patent Application Laid-Open No. 2014-185173, Japanese Patent Application Laid-Open No. 2010-535715, Japanese Patent Application Laid-Open No. 2015-038059, Japanese Patent Application Laid-Open No. 2015-110659, Japanese Patent Application Laid-Open No. 2006-521121, Japanese Patent Application Laid-Open No. 2009-542727, Japanese Patent Application Laid-Open No. 2014-224117, Japanese Patent Application Laid-Open No. 2015-061862, Japanese Patent Application Laid-Open No. 2002-511094, Japanese Patent Application Laid-Open No. 2004-507454, Japanese Patent Application Laid-Open No. 2010-505764, Japanese Patent Application Laid-Open No. 2011-514901, Japanese Patent Application Laid-Open No. 2013-064003, Japanese Patent Application Laid-Open No. 2015-131795, etc.

[0154] The present invention will be specifically described by the following examples, but the present invention is not limited to the examples.

[0155] Examples

[0156] <Comparative Example 1>

[0157] (Step 1-1: Collection of amniotic membrane)

[0158] The fetal membranes and placenta as fetal appendages were aseptically collected from a pregnant woman (donor #1) in an elective cesarean section case who had given informed consent. The obtained fetal membranes and placenta were placed in a sterilized basin filled with physiological saline, and the amniotic membrane was manually peeled off from the cut end of the fetal membranes. The amniotic membrane was washed with Hank's balanced salt solution (without Ca / Mg) to remove the attached blood and blood clots.

[0159] (Step 1-2: Enzymatic treatment of amniotic membrane and recovery of mesenchymal cells)

[0160] The amniotic membrane containing an epithelial cell layer and an extracellular matrix layer containing mesenchymal cells was immersed in Hank's balanced salt solution (containing Ca / Mg) containing 240 PU / mL collagenase and 200 PU / mL Dispase I, and subjected to shaking and stirring at 37 °C for 90 minutes at 50 rpm, thereby subjecting the amniotic membrane to enzymatic treatment. The solution after enzymatic treatment was filtered through a nylon sieve with a mesh size of 95 μm to remove undigested matter of the amniotic membrane, and a cell suspension containing mesenchymal cells was recovered.

[0161] (Step 1-3: Cultivation of mesenchymal cells)

[0162] The cell population containing mesenchymal cells obtained in the above-mentioned "Step 1-2: Enzymatic treatment of amniotic membrane and recovery of mesenchymal cells" was inoculated into a culture vessel, CellSTACK (registered trademark) (manufactured by Corning). The inoculation density was 6,000 cells / cm 2 at this density. After cell inoculation, it was adherently cultured in αMEM (Alpha Modification of Minimum Essential Medium Eagle) containing fetal bovine serum (FBS) at a final concentration of 10% and basic fibroblast growth factor (bFGF) at 10 ng / mL until near confluence. After cultivation, 15 mL of TrypLE Select was added to each layer of CellSTACK (registered trademark), and incubated at 37 °C for 3 minutes. As a result, about 30% of the cell population remained adhered to CellSTACK (registered trademark) without detachment and remained in the culture vessel. Therefore, the incubation was extended for 5 minutes (total 8 minutes) to completely detach the above cell population, and the remaining cell population was also recovered. The cell population obtained here was the cell population of the 0th passage. Then, 1 / 5 of the above cell population was inoculated into a CellSTACK (registered trademark) of the same scale as the previous culture, and thus subculture was carried out in αMEM containing FBS at a final concentration of 10% and bFGF at 10 ng / mL. The medium was changed at a frequency of once every 2 to 4 days. At the moment of reaching near confluence, 15 mL of TrypLE Select was added to each layer of CellSTACK (registered trademark), and incubated at 37 °C for 3 minutes. As a result, about 30% of the cell population remained adhered to CellSTACK (registered trademark) without detachment and remained in the culture vessel. Therefore, the incubation was extended for 5 minutes (total 8 minutes) to completely detach the above cell population, and the remaining cell population was also recovered. The cell population obtained here was the cell population of the 1st passage. Then, RPMI 1640 was added to adjust the cell concentration to 2×10 7cells / mL. An equal volume of CP-1 (registered trademark) solution (a solution mixed at a ratio of CP-1 (registered trademark) : 25% human serum albumin = 34 : 16) was added thereto, and transferred to cryovials at 1 mL per portion, slowly frozen to -80°C, and then cryopreserved in liquid nitrogen for 1 day. Then, the cryopreserved cell population was thawed, and the cell population of the first passage was inoculated onto CellSTACK (registered trademark) at a density of about 15,000 - 18,000 cells / cm 2 and adherently cultured to near confluence in αMEM (Alpha Modification of Minimum Essential Medium Eagle) containing fetal bovine serum (FBS) at a final concentration of 10% and basic fibroblast growth factor (bFGF) at 10 ng / mL. After the culture, 15 mL of TrypLE Select was added to each layer of CellSTACK (registered trademark), and incubated at 37°C for 3 minutes. As a result, about 30% of the cell population did not detach and remained adhered to CellSTACK (registered trademark) in the culture vessel. Therefore, the incubation was extended for 5 minutes (total 8 minutes) to completely detach the above cell population, and the remaining cell population was also recovered. The cell population obtained here was the cell population of the second passage. Then, 1 / 5 volume of the above cell population was inoculated onto CellSTACK (registered trademark) of the same scale as the previous culture, and thus subcultured in αMEM containing FBS at a final concentration of 10% and bFGF at 10 ng / mL. The medium was changed at a frequency of once every 2 - 4 days. At the time of near confluence, 15 mL of TrypLE Select was added to each layer of CellSTACK (registered trademark), and incubated at 37°C for 3 minutes. As a result, about 30% of the cell population did not detach and remained adhered to CellSTACK (registered trademark) in the culture vessel. Therefore, the incubation was extended for 5 minutes (total 8 minutes) to completely detach the above cell population, and the remaining cell population was also recovered. The cell population obtained here was the cell population of the third passage. For the above cell population, RPMI 1640 was added to make the cell concentration reach 4×10 6 cells / mL. An equal volume of CP-1 (registered trademark) solution (a solution mixed at a ratio of CP-1 (registered trademark) : 25% human serum albumin = 34 : 16) was added thereto, transferred to cryovials at 1 mL per portion, slowly frozen to -80°C, and then cryopreserved in liquid nitrogen for 1 day. Then, the cryopreserved cell population was thawed, and at a density of about 6,000 cells / cm 2The density of the above-mentioned cell population at the 3rd passage was inoculated into CellSTACK (registered trademark) and adherently cultured in αMEM (Alpha Modification of Minimum Essential Medium Eagle) containing 10% fetal bovine serum (FBS) and 10 ng / mL basic fibroblast growth factor (bFGF) at a final concentration until nearly confluent. After culturing, 15 mL of TrypLE Select was added to each layer of CellSTACK (registered trademark) and incubated at 37 °C for 3 minutes. As a result, about 30% of the cell population did not detach and remained adhered to CellSTACK (registered trademark) in the culture vessel. Therefore, incubation was extended for 5 minutes (total 8 minutes) to completely detach the above cell population, and the remaining cell population was also recovered. The cell population obtained here was the cell population at the 4th passage. Then, 1 / 5 of the above cell population was inoculated into CellSTACK (registered trademark) of the same scale as the previous culture, and thus subculture was carried out in αMEM containing 10% FBS and 10 ng / mL bFGF at a final concentration. The medium was changed at a frequency of once every 2 - 4 days. At the moment of reaching nearly confluent, 15 mL of TrypLE Select was added to each layer of CellSTACK (registered trademark) and incubated at 37 °C for 3 minutes. As a result, about 30% of the cell population did not detach and remained adhered to CellSTACK (registered trademark) in the culture vessel. Therefore, incubation was extended for 5 minutes (total 8 minutes) to completely detach the above cell population, and the remaining cell population was also recovered. The cell population obtained here was the cell population at the 5th passage. Then, RPMI 1640 was added to make the cell concentration reach 4×10 6 cells / mL. An equal volume of CP-1 (registered trademark) solution (a solution mixed in the ratio of CP-1 (registered trademark)∶25% human serum albumin = 34∶16) was added thereto, transferred to cryovials at 1 mL per portion, slowly frozen to -80 °C, and then cryopreserved in liquid nitrogen.

[0163] (Step 1-4: Surface antigen analysis of mesenchymal cells)

[0164] Regarding the cell population at the 5th passage cultured by the above-mentioned culture method, various surface antigens (positive rate of CD324, positive rate of CD73, positive rate of CD90, positive rate of CD105, positive rate of CD166, negative rate of CD45, negative rate of CD326) were analyzed using a flow cytometer. As a result, the positive rate of CD324 was less than 70% (specifically 33%), the positive rate of CD105 was 70% or more (specifically 93%), and the positive rates of CD73, CD90, and CD166 were all 90% or more (specifically CD73: 99%, CD90: 93%, CD166: 97%). The negative rates of CD45 and CD326 were both 95% or more (specifically CD45: 100%, CD326: 100%). From the above results, it can be known that the cell population cultured by the above-mentioned culture method is a cell population containing mesenchymal cells. Additionally, it can be known that the cell population at the 5th passage in Comparative Example 1 is a cell population that, although meeting the condition that the ratio of mesenchymal cells positive for CD90 is 90% or more, does not meet the condition that the ratio of cells positive for CD324 is 70% or more.

[0165] Note that in this assay, as the isotype control antibody, REAControl(S) APC (Miltenyi Biotec, Clone: REA293, Catalog No.: 130-113-434) was used. As the antibody against CD324 antigen, CD324-APC, Human (Miltenyi Biotec, Clone: REA811, Catalog No.: 130-111-840) was used. As the antibody against CD73 antigen, CD73-APC, Human (Miltenyi Biotec, Clone: REA804, Catalog No.: 130-111-909) was used. As the antibody against CD90 antigen, CD90-APC, Human (Miltenyi Biotec, Clone: REA897, Catalog No.: 130-114-861) was used. As the antibody against CD105 antigen, CD105-APC, Human (Miltenyi Biotec, Clone: REA794, Catalog No.: 130-112-166) was used. As the antibody against CD166 antigen, CD166-APC, Human (Miltenyi Biotec, Clone: REA442, Catalog No.: 130-106-576) was used. As the antibody against CD45 antigen, CD45-APC, Human (Miltenyi Biotec, Clone: REA747, Catalog No.: 130-110-633) was used. As the antibody against CD326 antigen, CD326-APC, Human (Miltenyi Biotec, Clone: REA764, Catalog No.: 130-111-000) was used. Surface antigen analysis was performed using Guava easyCyte from Merck, and the measurement conditions were set as 30,000 cells for analysis and a flow rate of 35.4 μL / min. In addition, the ratio (positive rate) of positive cells for each antigen was calculated according to the following steps.

[0166] (1) Plot the measurement results of the isotype control in the form of a dot plot with SSC on the vertical axis and FSC on the horizontal axis.

[0167] (2) Set the gate for the cell population that conforms to mesenchymal cells, and plot it in the form of a histogram with the count on the vertical axis and the fluorescence intensity of APC on the horizontal axis for this cell population.

[0168] (3) In the histogram of (2), select all regions (gates) where the cell population with stronger fluorescence intensity is 0.5% or less among all the cells measured with the isotype control antibody.

[0169] (4) Calculate the proportion of the cells contained in the selected gate in (3) among all the cells assayed using the antibody corresponding to the surface antigen marker.

[0170] <Example 1>

[0171] (Step 2-1: Collection of amniotic membrane)

[0172] Amniotic membrane was obtained from the same donor (Donor #1) as in Comparative Example 1 by the same method as in Comparative Example 1.

[0173] (Step 2-2: Enzymatic treatment of amniotic membrane and recovery of mesenchymal cells)

[0174] A cell population containing mesenchymal cells was obtained by the same method as in Comparative Example 1.

[0175] (Step 2-3: Culture of mesenchymal cells)

[0176] The cell population containing mesenchymal cells obtained by the above-mentioned "Step 2-2: Enzymatic treatment of amniotic membrane and recovery of mesenchymal cells" was inoculated into a culture vessel, CellSTACK (registered trademark) (manufactured by Corning). The seeding density was 6,000 cells / cm 2 of density. After cell inoculation, the cells were adherently cultured in αMEM containing human platelet lysate (hPL) at a final concentration of 5% until near confluence. After culturing, 15 mL of TrypLE Select was added to each layer of CellSTACK (registered trademark), and the cells were incubated at 37 °C for 3 minutes to completely detach the above cell population. The cell population obtained here was the cell population of the 0th passage. Then, 1 / 5 of the above cell population was inoculated into CellSTACK (registered trademark) of the same scale as the previous culture, and thus passage culture was carried out in αMEM containing hPL at a final concentration of 5%. The medium was changed at a frequency of once every 2 - 4 days. At the moment of reaching near confluence, 15 mL of TrypLE Select was added to each layer of CellSTACK (registered trademark), and the cells were incubated at 37 °C for 3 minutes to completely detach the above cell population. The cell population obtained here was the cell population of the 1st passage. Then, RPMI1640 was added to make the cell concentration reach 2 × 10 7 cells / mL. An equal volume of CP-1 (registered trademark) solution (a solution mixed at a ratio of CP-1 (registered trademark) : 25% human serum albumin = 34 : 16) was added thereto, and transferred to cryovials at 1 mL per portion, and then slowly frozen to -80 °C, and then cryopreserved in liquid nitrogen for 1 day. Then, the above cryopreserved cell population was thawed, and seeded at about 15,000 - 18,000 cells / cm 2The density of the cell population of the first passage was inoculated into CellSTACK (registered trademark) and adherently cultured in αMEM containing 5% human platelet lysate (hPL) at a final concentration until near confluence. Then, 15 mL of TrypLE Select was added to each layer of CellSTACK (registered trademark) and incubated at 37 °C for 3 minutes to completely detach the above cell population. The cell population obtained here is the cell population of the second passage. Next, 1 / 5 of the above cell population was inoculated into CellSTACK (registered trademark) of the same scale as the previous culture, and thus subculture was carried out in αMEM containing 5% hPL at a final concentration. The medium was changed at a frequency of once every 2 - 4 days. At the moment of near confluence, 15 mL of TrypLE Select was added to each layer of CellSTACK (registered trademark) and incubated at 37 °C for 3 minutes to completely detach the above cell population. The cell population obtained here is the cell population of the third passage. Then, RPMI 1640 was added to make the cell concentration reach 4×10 6 cells / mL. An equal volume of CP-1 solution (registered trademark) (a solution mixed in the ratio of CP-1 (registered trademark)∶25% human serum albumin = 34∶16) was added thereto, transferred to cryovials at 1 mL per portion, slowly frozen to -80 °C, and then cryopreserved in liquid nitrogen for 1 day. Then, the above cryopreserved cell population was thawed, and the cell population of the third passage was inoculated into CellSTACK (registered trademark) at a density of about 6,000 cells / cm 2 The density of the cell population of the third passage was inoculated into CellSTACK (registered trademark) and adherently cultured in αMEM containing 5% human platelet lysate (hPL) at a final concentration until near confluence. Then, 15 mL of TrypLE Select was added to each layer of CellSTACK (registered trademark) and incubated at 37 °C for 3 minutes to completely detach the above cell population. The cell population obtained here is the cell population of the fourth passage. Next, 1 / 5 of the above cell population was inoculated into CellSTACK (registered trademark) of the same scale as the previous culture, and thus subculture was carried out in αMEM containing 5% hPL at a final concentration. The medium was changed at a frequency of once every 2 - 4 days. At the moment of near confluence, 15 mL of TrypLE Select was added to each layer of CellSTACK (registered trademark) and incubated at 37 °C for 3 minutes to completely detach the above cell population. The cell population obtained here is the cell population of the fifth passage. For the above cell population, RPMI 1640 was added to make the cell concentration reach 4×10 6cells / mL. An equal volume of CP-1 (registered trademark) solution (a solution mixed at a ratio of CP-1 (registered trademark) ∶ 25% human serum albumin = 34 ∶ 16) was added thereto. After transferring 1 mL per portion to cryovials, it was slowly frozen to -80°C and then cryopreserved in liquid nitrogen.

[0177] (Step 2-4: Analysis of surface antigens of mesenchymal cells)

[0178] Regarding the fifth-passage mesenchymal cell population cultured by the above-described culture method, various surface antigens (positive rate of CD324, positive rate of CD73 known as an MSC marker, positive rate of CD90, positive rate of CD105, positive rate of CD166, negative rate of CD45, negative rate of CD326) were analyzed using a flow cytometer. As a result, the positive rates of CD324 and CD105 were 70% or more (specifically, CD324: 91%, CD105: 95%), and the positive rates of CD73, CD90, and CD166 were all 90% or more (specifically, CD73: 100%, CD90: 100%, CD166: 99%). The negative rates of CD45 and CD326 were both 95% or more (specifically, CD45: 100%, CD326: 99%). From the above results, it can be seen that the cells cultured by the above-described culture method are a cell population containing mesenchymal cells. In addition, it was confirmed that the cell population described in Example 1 is a cell population in which the ratio of CD324-positive cells is 70% or more and the ratio of CD90-positive mesenchymal cells is 90% or more.

[0179] It should be noted that the method and reagents for this measurement are the same as those in Comparative Example 1.

[0180] <Example 2>

[0181] Compared with Comparative Example 1 and Example 1, a mesenchymal cell population with different donors, enzyme treatment conditions, and culture conditions was obtained in Example 2 shown below.

[0182] Differing from Comparative Example 1 and Example 1, fetal membranes and placentas as fetal appendages were aseptically collected from 3 pregnant women (donors #2 to #4) with elective cesarean section cases who had given informed consent.

[0183] (Step 3-1: Collection of amniotic membrane)

[0184] Amniotic membrane was obtained by the same method as in Comparative Example 1 and Example 1.

[0185] (Step 3-2: Enzyme treatment of amniotic membrane and recovery of mesenchymal cells)

[0186] The amniotic membrane containing an epithelial cell layer and an extracellular matrix layer containing mesenchymal cells was immersed in Hank's balanced salt solution (containing Ca / Mg) containing 480 PU / mL of collagenase and 400 PU / mL of dispase I, and subjected to shaking and stirring at 37 °C for 90 minutes at 50 rpm, thereby performing enzymatic treatment on the amniotic membrane. By filtering the solution after enzymatic treatment through a nylon sieve with a mesh size of 95 μm, undigested matter of the amniotic membrane was removed, and a cell suspension containing mesenchymal cells was recovered.

[0187] (Step 3-3: Cultivation of mesenchymal cells)

[0188] The cell population containing mesenchymal cells obtained through the above "Step 3-2: Enzymatic treatment of amniotic membrane and recovery of mesenchymal cells" was inoculated into a culture container, CellSTACK (registered trademark). The inoculation density was set to 1,000 cells / cm 2 . After cell inoculation, it was adherently cultured in αMEM containing 5% (final concentration) of human platelet lysate (hPL) until near confluence. The medium was changed at a frequency of once every 3 to 5 days. After cultivation, 15 mL of TrypLE Select was added to each layer of CellSTACK (registered trademark), and incubated at 37 °C for 3 minutes to completely detach the above cell population. The cell population obtained here was the cell population of the 0th passage. Then, physiological saline was added to make the cell concentration reach 2×10 7 cells / mL. An equal volume of CP-1 (registered trademark) solution (a solution mixed at a ratio of CP-1 (registered trademark): 25% human serum albumin = 34:16) was added thereto, transferred to cryovials at 1 mL per portion, slowly frozen to -80 °C, and then cryopreserved in liquid nitrogen for 1 day. Then, the cryopreserved cell population was thawed, and the cell population of the 1st passage was inoculated into CellSTACK (registered trademark) at a density of approximately 1,000 cells / cm 2 and adherently cultured in αMEM containing 5% (final concentration) of human platelet lysate (hPL) for 5 days until near confluence. Then, 15 mL of TrypLE Select was added to each layer of CellSTACK (registered trademark), and incubated at 37 °C for 3 minutes to completely detach the above cell population. The cell population obtained here was the cell population of the 1st passage. Next, physiological saline was added to make the cell concentration reach 2×10 7 cells / mL. An equal volume of CP-1 (registered trademark) solution (a solution mixed at a ratio of CP-1 (registered trademark): 25% human serum albumin = 34:16) was added thereto, transferred to cryovials at 1 mL per portion, slowly frozen to -80 °C, and then cryopreserved in liquid nitrogen for 1 day. Then, the cryopreserved cell population was thawed, and at a density of approximately 1,000 cells / cm2 The density of the cell population at the second passage was inoculated into CellSTACK (registered trademark), and adherently cultured in αMEM containing 5% human platelet lysate (hPL) at a final concentration for 5 days until near confluence. Then, 15 mL of TrypLE Select was added to each layer of CellSTACK (registered trademark), and incubated at 37 °C for 3 minutes to completely detach the above cell population. The cell population obtained here was the cell population at the second passage. For the above cell population, physiological saline was added to make the cell concentration reach 4×10 6 cells / mL. An equal volume of CP-1 solution (a solution mixed in a ratio of CP-1 (registered trademark): 25% human serum albumin = 34:16) was added thereto, transferred to cryovials at 1 mL per portion, slowly frozen to -80 °C, and then cryopreserved in liquid nitrogen.

[0189] (Step 3-4: Surface antigen analysis of mesenchymal cells)

[0190] Regarding the cell populations at the second passage (#2 to #4) cultured by the culture method described in Step 3-3, various surface antigens (positive rate of CD324, positive rate of CD73, positive rate of CD90, positive rate of CD105, positive rate of CD166, negative rate of CD45, negative rate of CD326) were analyzed using a flow cytometer. As a result, the positive rates of CD324 and CD105 were 70% or more (specifically, in the order of #2, #3, and #4, CD324: 90%, 87%, 87%, CD105: 89%, 91%, 74%), and the positive rates of CD73, CD90, and CD166 were all 90% or more (specifically, in the order of #2, #3, and #4, CD73: 100%, 99%, 100%, CD90: 100%, 99%, 100%, CD166: 99%, 97%, 98%). The negative rates of CD45 and CD326 were both 95% or more (specifically, in the order of #2, #3, and #4, CD45: 99%, 100%, 100%, CD326: 99%, 100%, 100%). From the above results, it can be seen that the cell populations of #2, #3, and #4 cultured by the above culture method are all cell populations containing cells positive for CD324. In addition, it was confirmed that the cell population described in Example 2 is a cell population in which the ratio of cells positive for CD324 is 70% or more and the ratio of mesenchymal cells positive for CD90 is 90% or more. It should be noted that the method and reagents for this measurement are the same as those in Comparative Example 1.

[0191] <Evaluation experiment: Evaluation of the detachment ability of mesenchymal cells from the culture substrate>

[0192] (Evaluation 1: Evaluation of the cell population obtained in Comparative Example 1)

[0193] The cell population (#1) at the 5th passage cultured by the culture method described in Steps 1-3 of Comparative Example 1 was thawed, and the cell population at the 5th passage was inoculated into a 6-well plate at a density of approximately 10,000 cells / cm 2 and adherent culture was started in αMEM (Alpha Modification of Minimum Essential Medium Eagle) containing fetal bovine serum (FBS) at a final concentration of 10% and basic fibroblast growth factor (bFGF) at 10 ng / mL. The medium was changed at a frequency of once every 3 to 5 days. Although the confluence reached 100% on the 5th day of culture, no detachment treatment was performed and the culture was continued. The cell morphology on the 4th, 5th, 6th, 7th, 10th, 11th, 12th, 13th, 14th, 17th, and 21st days of culture was observed using a phase-contrast microscope manufactured by Olympus Corporation. Overconfluency was reached on the 7th day of culture, and the cells were in a state of being closely packed without gaps. However, subsequently, there was no obvious change in the cell morphology, and the cells did not spontaneously detach from the 6-well plate even after 21 days of starting the culture ( Figure 1 : Cell morphology on the 21st day of culture).

[0194] (Evaluation 2: Evaluation of the cell populations obtained in Examples 1 and 2)

[0195] The cell population (#1) at the 5th passage cultured by the culture method described in Steps 2-3 of Example 1 and the cell population (#3) at the 2nd passage cultured by the culture method described in Steps 3-3 of Example 2 were thawed, and inoculated into a culture vessel (6-well plate) at a density of approximately 10,000 cells / cm 2 and adherent culture was started in αMEM containing human platelet lysate (hPL) at a final concentration of 5%. The medium was changed at a frequency of once every 3 to 5 days. Although the confluence reached 100% on the 4th day of culture, no detachment treatment was performed and the culture was continued. The cell morphology on the 4th, 5th, 6th, 7th, 10th, 11th, 12th, 13th, 14th, 17th, and 21st days of culture was observed using a phase-contrast microscope manufactured by Olympus Corporation. Overconfluency was reached on the 5th day of culture, and the cells were in a state of being closely packed without gaps with some cells overlapping locally. On the 10th day of culture, a part of the cell population spontaneously detached from the culture vessel ( Figure 2 、 Figure 3 : Detachment of some cells observed on the 10th day of culture). A part of the detached cell population had a sheet-like morphology. On the 11th day of culture, the entire cell population spontaneously detached from the culture vessel and became a single cell sheet ( Figure 4: A mass structure formed by aggregating the cell sheet after detachment observed on the 11th day of culture of the cell population of the second passage of Example 2 prepared from donor #3).

[0196] The above results indicate that a cell population with a ratio of CD324-positive cells of 70% or more and a ratio of CD90-positive mesenchymal cells of 90% or more spontaneously detaches from the culture vessel. That is, a cell population satisfying the above conditions can be easily recovered after culture without using chemical methods such as adding a detachment agent or physical methods of physically recovering the cell population using a cell scraper. Thus, the cell population of the present invention does not require the detachment process necessary in the existing methods and can reduce or inhibit damage to the cells.

[0197] In addition, a cell population satisfying the conditions of a ratio of CD324-positive cells of 70% or more and a ratio of CD90-positive mesenchymal cells of 90% or more can obtain a cell population after detachment in the form of a cell sheet. That is, according to the method described in the present invention, a cell sheet can be prepared without using the above chemical method or physical method and without special treatment of the culture vessel. From the viewpoint of operability, the cell sheet is a preparation form suitable for clinical use and is useful.

[0198] <Preparation of Pharmaceutical Composition>

[0199] A part of the mesenchymal cell populations (#2 to 4) obtained in Example 2 above was used for the preparation of a pharmaceutical composition. A pharmaceutical composition (cell preparation) containing 2.0×10 8 cells, 6.8 mL of CP-1 solution (registered trademark), 3.2 mL of 25% human serum albumin solution, and 10 mL of physiological saline was prepared. This pharmaceutical composition was sealed in a freezing bag and stored in a frozen state. It should be noted that the pharmaceutical composition can be thawed at the time of use and administered to patients.

[0200] <Summary of Negative and Positive Rates of Surface Antigens>

[0201] Table 1 summarizes the results of surface antigen analysis, enzyme treatment time during passage, and cell sheet formation ability of Comparative Example 1, Example 1, and Example 2.

[0202] Table 1

[0203]

[0204] <Reference Example>

[0205] (Step 4-1: Culture of Bone Marrow-Derived Mesenchymal Stem Cells)

[0206] Purchased human bone marrow-derived mesenchymal stem cells (hMSC mesenchymal stem cells, manufactured by Lonza), and after thawing, they were seeded at a density of 6,000 cells / cm 2 in a φ15 cm culture dish and adherently cultured in a dedicated medium manufactured by Lonza until near confluence. The medium was changed at a frequency of once every 3 to 5 days. Then, the cell population was detached using TrypLE Select, and physiological saline was added to make the cell concentration reach 2×10 6 cells / mL. An equal volume of CP-1 (registered trademark) solution (a solution mixed at a ratio of CP-1 (registered trademark)∶25% human serum albumin = 34∶16) was added thereto, and then transferred to cryovials at 1 mL per portion, slowly frozen to -80°C, and then cryopreserved in liquid nitrogen.

[0207] (Step 4-2: Surface antigen analysis of bone marrow-derived mesenchymal stem cells)

[0208] Regarding the bone marrow-derived mesenchymal stem cell population cultured by the culture method described in Step 4-1, various surface antigens (positive rate of CD324, positive rate of CD73 known as an MSC marker, positive rate of CD90, positive rate of CD105, positive rate of CD166, negative rate of CD45, negative rate of CD326) were analyzed using a flow cytometer. As a result, the positive rate of CD324 was 70% or less (specifically 1%), and the positive rates of CD73, CD90, CD105, and CD166 were all 90% or more (specifically CD73: 97%, CD90: 98%, CD105: 97%, CD166: 95%). The negative rates of CD45 and CD326 were both 95% or more (specifically CD45: 100%, CD326: 100%).

[0209] All publications, patents, and patent applications cited in this specification are incorporated herein by direct reference.

Claims

1. A cell population comprising mesenchymal cells, which is derived from amniotic membrane, when the cell population is cultured adherently, the ratio of cells positive for CD324 is 70% or more, the ratio of mesenchymal cells positive for CD90 is 90% or more, the ratio of cells positive for CD326 is 10% or less, the ratio of mesenchymal cells positive for CD73 is 80% or more, the ratio of mesenchymal cells positive for CD166 is 80% or more, the ratio of cells positive for CD45 is 10% or less, and the ratio of mesenchymal cells positive for CD105 is 70% or more, when the adherent culture is carried out, human platelet lysate is added to the culture medium, and the final concentration of human platelet lysate in the culture medium is 1% by weight or more and 10% by weight or less.

2. A method for producing a cell population comprising mesenchymal cells, the method comprising: a step of culturing a cell population derived from amniotic membrane and containing mesenchymal cells, and a step of screening from the cell population containing mesenchymal cells a cell population having the characteristics of (a) and (g) shown below during adherent culture, (a) In the cell population, the ratio of cells positive for CD324 is 70% or more; (b) In the cell population, the ratio of mesenchymal cells positive for CD90 is 90% or more; (c) The ratio of cells positive for CD326 is 10% or less; (d) The ratio of mesenchymal cells positive for CD73 is 80% or more; (e) The ratio of mesenchymal cells positive for CD166 is 80% or more; (f) The ratio of cells positive for CD45 is 10% or less; and (g) The ratio of mesenchymal cells positive for CD105 is 70% or more, when the adherent culture is carried out, human platelet lysate is added to the culture medium, and the final concentration of human platelet lysate in the culture medium is 1% by weight or more and 10% by weight or less.

3. A pharmaceutical composition, which comprises: the cell population according to claim 1, and a pharmaceutically acceptable medium.

4. The pharmaceutical composition according to claim 3, wherein, Administer to a human in such a manner that the dosage of mesenchymal cells per administration is 10 or less. 12 cells.

5. The pharmaceutical composition according to claim 3 or 4, wherein, the pharmaceutical composition is a preparation for injection.

6. The pharmaceutical composition according to claim 3 or 4, wherein, the pharmaceutical composition is a preparation for transplantation in the form of cell aggregates or sheet structures.

7. The pharmaceutical composition according to claim 3 or 4, which is a therapeutic agent selected from the following diseases: Immune-related diseases, ischemic diseases, lower limb ischemia, cerebral ischemia, renal ischemia, pulmonary ischemia, nervous system diseases, graft-versus-host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, lupus erythematosus, collagen diseases, stroke, cerebral infarction, cerebral hematoma, cerebral vascular paralysis, brain tumor, liver cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes, mycosis fungoides, scleroderma, diseases caused by degeneration and / or inflammation of connective tissue, articular cartilage defect, meniscus injury, osteochondritis dissecans, aseptic osteonecrosis, knee osteoarthritis, inflammatory arthritis, rheumatoid arthritis, eye diseases, angiogenesis-related diseases, ischemic heart disease, coronary heart disease, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular heart disease, trauma, epithelial injury, fibrosis, lung diseases, muscular dystrophy, chronic pancreatitis, chronic nephritis, and cancer.

Images