Method for isolating single mesenchymal stem cell

Abstract

The present invention addresses the problem of providing a new method for efficiently isolating a mesenchymal stem cell as a single cell and efficiently proliferating the single cell.　Provided is a method for isolating a single mesenchymal stem cell from a cell population including mesenchymal stem cells, the method comprising: a first culture step for culturing the cell population in a first culture medium containing a human platelet lysate; a recovery step for recovering the cell population after the first culture step; and an isolation step for isolating a single mesenchymal stem cell from the cell population after the recovery step.

Description

Method for isolating single mesenchymal stem cells

[0001] The present invention relates to a method for isolating a single mesenchymal stem cell, a method for producing a single mesenchymal stem cell, and the like.

[0002] Pluripotent stem cells are cells that have the ability to differentiate into various somatic cells. Examples of pluripotent stem cells include iPS cells obtained by introducing reprogramming factors into somatic cells, ES cells derived from the inner cell mass, and mesenchymal stem cells, which are somatic stem cells.

[0003] Among these, mesenchymal stem cells are known to have the ability to differentiate into osteoblasts, chondrocytes, adipocytes, etc., and have been shown to exist in various tissues such as bone marrow, fat, dental pulp, and fetal appendages (placenta, umbilical cord, fetal membrane, etc.) (Patent Document 1). Based on their pluripotency, mesenchymal stem cells are being put to practical use as regenerative medicine materials. In fact, based on their immunosuppressive ability, clinical applications are being advanced for a wide range of diseases such as acute graft-versus-host disease (GVHD), Crohn's disease, spinal cord injury, and osteoarthritis of the knee. Furthermore, because mesenchymal stem cells have low immunogenicity and can be transplanted allogeneically, they are also useful as regenerative medicine materials for ex vivo gene therapy.

[0004] To isolate and utilize mesenchymal stem cells from mesenchymal tissues or fetal appendages, it is necessary to remove cells other than mesenchymal stem cells contained in the same tissue. For example, bone marrow fluid contains many hematopoietic stem cells in addition to mesenchymal stem cells, and fat contains many adipocytes in addition to mesenchymal stem cells, so it is desirable to remove these cells. Furthermore, the amniotic membrane, a type of fetal appendage, has attracted attention as a promising biological tissue rich in mesenchymal stem cells, but because it contains many epithelial cells in addition to mesenchymal stem cells, it is desirable to remove these epithelial cells.

[0005] Furthermore, from the viewpoint of cell quality control, it is more desirable to isolate mesenchymal stem cells as single cells and expand and culture the single cells to produce a highly homogeneous cell population. For example, when mesenchymal stem cells transformed with a plasmid vector or a viral vector are used for therapy, differences in the quality between cells may occur based on differences in the number of introduced copies of the vector, the position of genome insertion, etc. In order to obtain a highly homogeneous cell population, it is necessary to isolate single cells from mesenchymal stem cells after gene introduction and expand and culture them.

[0006] Therefore, a new method is needed for efficiently isolating mesenchymal stem cells as single cells and efficiently proliferating the single cells.

[0007] WO2015 / 025810

[0008] An object of the present invention is to provide a new method for efficiently isolating mesenchymal stem cells as single cells and efficiently proliferating the single cells.

[0009] In single-cell cloning, a single cell is isolated from a cell population containing multiple cells and grown. After gene transfer, if necessary, cells are isolated one by one using a cell sorter or the like, and then seeded one cell per well in a multi-well plate and cultured to obtain a cell population derived from the single cell (Figure 4).

[0010] To isolate mesenchymal stem cells as single cells, the inventors cultured mesenchymal stem cells collected from amniotic membranes in a commonly used medium containing fetal bovine serum (FBS). After culture, the amniotic mesenchymal stem cells were isolated and seeded individually into each well of a 96-well plate using a cell sorter, and then further cultured to attempt cell proliferation. However, after isolation as single cells, amniotic mesenchymal stem cells cultured in FBS-containing medium showed no cell proliferation in any of the 96-well plates.

[0011] Next, the inventors cultured amniotic mesenchymal stem cells in a medium containing human platelet lysate (hPL) instead of fetal bovine serum (FBS), and isolated one cell per well of a 96-well plate using a cell sorter. As a result, cell proliferation of amniotic mesenchymal stem cells was observed in nearly half of the wells.

[0012] Furthermore, the inventors conducted a similar experiment using bone marrow mesenchymal stem cells as the target cells. After culturing bone marrow mesenchymal stem cells in a medium containing FBS, they isolated single cells into each well of a 96-well plate using a cell sorter and cultured them. As a result, cell proliferation was observed in only 20 wells out of the 96 wells. On the other hand, when bone marrow mesenchymal stem cells were cultured in a medium containing hPL and then isolated and cultured individually, cell proliferation was observed in 45 wells out of the 96 wells.

[0013] From the above results, it was revealed that single cells of mesenchymal stem cells having the ability to proliferate can be efficiently obtained by culturing cells before and / or after cell sorting in a medium containing hPL. The present invention is based on the above findings and provides the following.

[0014] (1) A method for isolating a single mesenchymal stem cell from a cell population containing mesenchymal stem cells, comprising: a first culturing step of culturing the cell population in a first culture medium containing human platelet lysate; a recovering step of recovering the cell population after the first culturing step; and an isolating step of isolating a single mesenchymal stem cell from the cell population after the recovering step. (2) The method according to (1), wherein the cell population is derived from amniotic membrane or bone marrow. (3) The method according to (1) or (2), wherein the first culture medium contains 3 v / v% or more of the human platelet lysate. (4) The method according to (3), wherein the first culture medium contains 3 to 20 v / v% of the human platelet lysate. (5) The method according to any one of (1) to (4), wherein the first culture medium further contains heparin or a heparin substitute. (6) The method according to any one of (1) to (5), wherein the first culture medium is a basal medium or a serum-free medium. (7) The method of any of (1) to (6), wherein the recovery step involves recovering the cell population from the first culture medium. (8) The method of any of (1) to (7), wherein dead or live cells are labeled after the recovery step, and only live cells are isolated in the isolation step. (9) The method of any of (1) to (8), further comprising an introduction step of introducing a gene expression vector into the cell population before the isolation step. (10) The method of (9), wherein the gene expression vector contains a marker gene. (11) The method of (9) or (10), wherein the introduction step is performed before the first culture step. (12) The method of (10) or (11), wherein the isolation step involves isolating the single mesenchymal stem cell expressing the marker protein using the expression of a marker protein encoded by the marker gene as an indicator. (13) The method of any of (10) to (12), wherein the marker gene is an antibiotic resistance gene that confers resistance to an antibiotic. (14) The method according to any one of (1) to (13), further comprising a second culturing step of culturing the single mesenchymal stem cell after the isolation step in a second culture medium that does not contain other mesenchymal stem cells. (15) The method according to (14), wherein the second culture medium contains human platelet lysate. (16) The method according to (14) citing (13), or (15) citing (13) and (14), wherein the second culture medium contains the antibiotic.(17) A method for producing a single mesenchymal stem cell, the method comprising producing the single mesenchymal stem cell using the method described in any one of (1) to (16). (18) A method for producing a mesenchymal stem cell population derived from a single mesenchymal stem cell, the method comprising producing the mesenchymal stem cell population by proliferating the single mesenchymal stem cell in the second culture step using the method described in any one of (14) to (16). (19) A method for culturing a single mesenchymal stem cell, the method comprising: a first culture step of culturing a cell population containing a mesenchymal stem cell in a first medium; a collection step of recovering the cell population after the first culture step; an isolation step of isolating a single mesenchymal stem cell from the cell population after the collection step; and a second culture step of culturing the single mesenchymal stem cell after the isolation step in a second medium not containing other mesenchymal stem cells, wherein the second medium comprises human platelet lysate. This specification incorporates the disclosure of Japanese Patent Application No. 2024-003123, from which the present application claims priority.

[0015] According to the present invention, a new method is provided for efficiently isolating mesenchymal stem cells as single cells and efficiently growing the single cells.

[0016] 1 is an image diagram showing an outline of an experiment in which mesenchymal stem cells after FBS culture or hPL culture are isolated as single cells, and then wells containing proliferating cells are identified. 2 is a diagram showing the results of counting the number of wells containing proliferating cells in a 96-well plate after isolating bone marrow mesenchymal stem cells (bone marrow MSCs in the diagram) or amniotic mesenchymal stem cells (amniotic MSCs in the diagram) after FBS culture or hPL culture as single cells. 3 is a diagram showing the number of doublings of bone marrow mesenchymal stem cells and amniotic mesenchymal stem cells during long-term FBS culture. 4 is an image diagram showing a method for isolating single cells into which a marker gene has been introduced in one embodiment of the present invention.

[0017] 1. Method for Isolating Single Mesenchymal Stem Cells 1-1. Overview A first aspect of the present invention is a method for isolating single mesenchymal stem cells from a cell population containing mesenchymal stem cells (hereinafter sometimes abbreviated as "isolation method"). The isolation method of this aspect essentially comprises a first culture step of culturing a cell population containing mesenchymal stem cells in a first medium containing human platelet lysate, a recovery step of recovering the cell population, and an isolation step of isolating single mesenchymal stem cells. Mesenchymal stem cells isolated by the isolation method of this aspect grow efficiently under single-cell culture conditions. Therefore, a highly homogeneous mesenchymal stem cell population can be efficiently produced.

[0018] 1-2. Definition of Terms The following terms frequently used in this specification are defined below. As used herein, "fetal appendages" refers to tissues or organs that support fetal development outside the fetal body within the uterus, specifically the amniotic membrane, placenta, umbilical cord, and amniotic fluid. "Melon" refers to the gestational sac that contains fetal amniotic fluid, and is composed of the amnion, chorion, and decidua from the inside out. "Amniotic membrane" refers to the thin, transparent membrane with few blood vessels that is the innermost layer of the amniotic membrane. The inner layer of the amniotic membrane (also called the epithelial cell layer) is covered with a single layer of epithelial cells with secretory function and secretes amniotic fluid, and the outer layer of the amniotic membrane (also called the extracellular matrix layer, equivalent to the interstitium) contains mesenchymal stem cells.

[0019] As used herein, "mesenchymal stem cells" refers to stem cells that (i) exhibit adhesion to the surface of a plastic culture vessel under culture conditions in a standard medium (a medium containing a basal medium supplemented with serum, a serum replacement reagent, or growth factors), and (ii) are positive for the surface antigens CD105, CD73, and CD90, and negative for CD45. As used herein, "mesenchymal stem cells" are also referred to as "MSCs," and are synonymous with "mesenchymal stromal cells." The biological species from which mesenchymal stem cells are derived is not particularly limited as long as they possess mesenchymal stem cells, and may be, for example, fish, birds, or mammals. Mesenchymal stem cells may be, for example, mouse cells, chimpanzee cells, or human cells. In addition, sources of mesenchymal stem cells include, but are not limited to, bone marrow, hematopoietic stem cells, umbilical cord blood, umbilical cord, amniotic membrane, amniotic fluid, placental villi, nerves, adipose tissue, pancreas, synovial membrane, dental pulp, deciduous teeth, sperm, testes, cornea, etc. In addition, mesenchymal stem cells may be immortalized mesenchymal stem cell lines.

[0020] As used herein, a "cell population containing mesenchymal stem cells" refers to a cell population containing mesenchymal stem cells derived from, for example, any of the tissues described above (e.g., amniotic membrane or bone marrow). The form of the cell population containing mesenchymal stem cells is not particularly limited, and may be, for example, a cell pellet, a cell aggregate, a cell suspension, or the like.

[0021] As used herein, "amniotic mesenchymal stem cells" refer to mesenchymal stem cells derived from the amniotic membrane. As used herein, amniotic mesenchymal stem cells are also referred to as "amniotic MSCs."

[0022] In addition, as used herein, "bone marrow mesenchymal stem cells" refer to mesenchymal stem cells derived from bone marrow. As used herein, bone marrow mesenchymal stem cells are also referred to as "bone marrow MSCs."

[0023] As used herein, the term "medium" is not limited as long as it is capable of maintaining cells, such as mesenchymal stem cells, as living cells. For example, the medium may be a medium commonly used in cell culture and known in the art. The medium may be any of basal medium, serum-free medium, low-serum medium, and serum-added medium, but typically may be a basal medium (e.g., standard cell culture medium) or serum-free medium, which can be prepared by adding other components as needed. The term "standard cell culture medium" as used herein refers to a highly versatile basal medium that is primarily used for culturing various types of cells derived from mammals. Specifically, for example, 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, MEM-α (Minimum Essential Medium α) medium, DMEM medium (Dulbecco's Modified Eagle's Medium), Ham's F10 medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof (for example, DMEM / F12 medium (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 Ham)) can be used, but are not particularly limited to these. Various commercially available serum-free media can also be used. Examples of other components added to the basal medium include albumin, blood-derived components, and growth factors.

[0024] As used herein, the term "single cell" refers to a single cell that does not contain any other cells. As used herein, a single cell is also referred to as a "single cell."

[0025] As used herein, "single cell cloning" refers to isolating a single cell (i.e., a single cell) that has the ability to proliferate from a cell population containing two or more cells, and the isolated single cell is then cultured. For example, when single cell cloning is performed on mesenchymal stem cells, the isolated single mesenchymal stem cell is cultured in a medium that does not contain other cells, such as other mesenchymal stem cells.

[0026] As used herein, "proliferative potential" refers to the ability of a cell to increase its number by cell division. For example, when mesenchymal stem cells are cultured as a single cell, they may or may not exhibit proliferative potential. When a single mesenchymal stem cell exhibits proliferative potential, it has the ability to produce two or more cells by cell division and form two or more cells derived from that cell.

[0027] The isolation method of this embodiment includes, as essential steps, a first culturing step, a recovery step, and an isolation step, and may include, as optional steps, a repeating step, an introduction step, and / or a second culturing step. Each step in the isolation method of this embodiment will be specifically described below.

[0028] (First culture step) In the method of the present invention, the "first culture step" is a step of culturing a cell population containing mesenchymal stem cells in a first medium containing human platelet lysate. The culture method in this step is not limited, and may be, for example, adhesion culture, suspension culture, or spheroid culture. Because mesenchymal stem cells are adherent cells, adhesion culture is preferred as the culture method in this step. However, suspension culture, in which mesenchymal stem cells are cultured by suspending microcarriers attached thereto, or spheroid culture, in which mesenchymal stem cells are formed into spheroids and cultured, can also be used.

[0029] The "first medium" used in this step is a medium prepared by adding human platelet lysate at an arbitrary concentration as an essential component to the above-mentioned "medium," and may contain other components as necessary. In this first medium, single cells can be cultured immediately before the isolation step described below.

[0030] As used herein, "platelet lysate" refers to a cell culture additive that is a lysate of blood-derived platelets and contains large amounts of multiple growth factors and chemokines as components. The animal species from which the platelet lysate is derived is not particularly limited, but is preferably human. As used herein, human-derived platelet lysate is often abbreviated as "hPL." Examples of growth factors that can be contained in human platelet lysate include platelet-derived growth factor isoforms (PDGF-AA, -AB, -BB), transforming growth factor-b (TGF-b), insulin-like growth factor-1 (IGF-1), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (FBGF), and fibroblast growth factor (FGF). Examples of such growth factors include hepatocyte growth factor (bFGF or FGF-2), hepatocyte growth factor (HGF), connective tissue growth factor (CTGF), and morphogenetic protein-2, -4, and -6 (BMP-2, -4, and -6). Examples of chemokines that can be contained in the human platelet lysate include interleukin-8 (IL-8), neutrophil-activating peptide-2 (NAP-2), regulated on activation, normal T cell expressed and secreted (TANTES), monocyte chemotactic protein-1, -3 (MCP-1, 3), macrophage inflammatory protein-1 alpha (MIP-1α), and beta-thromboglobulin.

[0031] The method for producing the human platelet lysate used in the method of the present invention is not particularly limited. Platelet lysate is usually produced by repeatedly freezing and thawing platelets extracted from whole blood, followed by purification by removing cell fragments and the like, but any production method is not particularly limited and may be used. Specific preparation methods can be found, for example, in the following literature: Schallmoser k et al., Methods Mol Biol. 2013; 946: 349-62.

[0032] The human platelet lysate used in the present invention is preferably subjected to bacterial and viral inactivation and / or sterilization treatment.

[0033] The method of the present invention can also use commercially available human platelet lysates, such as Stemulate (Cook Regenetec), PLTMax (Mill Creek Life Science), UltraGRO (AventaCell BioMedical), and PLUS (Compass Biomedical).

[0034] The method of the present invention can also use clarified human platelet lysate. Clarification, as used herein, refers to a process for removing insoluble fractions from human platelet lysate that have formed during long-term storage or freeze-thawing. Clarification methods include, but are not limited to, centrifugation, filtration, sedimentation, and sedimentation (e.g., spontaneous sedimentation), as long as they are capable of removing the insoluble fraction.

[0035] The concentration of the human platelet lysate in the first medium is not particularly limited. The first medium may be, for example, 0.1 v / v% or more, 0.2 v / v% or more, 0.3 v / v% or more, 0.4 v / v% or more, 0.5 v / v% or more, 0.6 v / v% or more. Above, 0.7 v / v% or more, 0.8 v / v% or more, 0.9 v / v% or more, 1 v / v% or more, 1.2 v / v% or more, 1.5 v / v% or more, 2 v / v% or more, 2 .5v / v% or more, 3v / v% or more, 3.5v / v% or more, 4v / v% or more, 4.5v / v% or more, or 5v / v% or more, and / or 40v / v% or less, 35v / v% or less, 30v / v% or less, 25v / v% or less, 20v / v% or less, 15v / v% or less, or 10v / v% or less of human platelet lysate. As used herein, when the concentration of human platelet lysate is expressed in units of "v / v %," it refers to the dilution rate of human platelet lysate in terms of volume ratio (volume of hPL solution before dilution / volume of solution after dilution), more specifically, it refers to the volume ratio at which crude platelet lysate or commercially available platelet lysate is diluted with a medium such as the first medium. Note that commercially available platelet lysate may be, for example, human platelet lysate having a total protein concentration of 40 to 80 mg / mL, which may be diluted and used.

[0036] The concentration of the human platelet lysate in the first medium can also be expressed in terms of dry weight of the human platelet lysate. The concentration of the human platelet lysate in the first medium, in terms of dry weight, may be, for example, 0.06 mg / mL or more, 0.3 mg / mL or more, 0.6 mg / mL or more, 1.2 mg / mL or more, 1.8 mg / mL or more, or 3 mg / mL or more, and / or 24 mg / mL or less, 21 mg / mL or less, 18 mg / mL or less, 15 mg / mL or less, 12 mg / mL or less, 9 mg / mL or less, or 6 mg / mL or less.

[0037] The concentration of human platelet lysate in the first medium can also be specified by the human platelet lysate protein concentration per mL of the first medium. For example, the human platelet lysate protein concentration per mL of the first medium may be 0.02 mg / mL to 32 mg / mL, 0.04 mg / mL to 28 mg / mL, 0.2 mg / mL to 24 mg / mL, 0.4 mg / mL to 20 mg / mL, 0.8 mg / mL to 16 mg / mL, 1.2 mg / mL to 12 mg / mL, 1.6 mg / mL to 8 mg / mL, or 2 mg / mL to 4 mg / mL, for example, 3 mg / mL.

[0038] The first medium may contain other components in addition to the essential component, human platelet lysate. For example, the first medium may contain albumin, blood-derived components, and / or growth factors in addition to human platelet lysate. When the first medium contains albumin in addition to human platelet lysate, the albumin concentration may be, for example, 0.05% to 5% by mass. Examples of blood-derived components include various types of serum (animal-derived serum such as fetal bovine serum (FBS) or FCS) and human serum) and / or plasma. When the first medium contains blood-derived components in addition to human platelet lysate, the concentration of the blood-derived components may be, for example, 2% to 40% by volume, 3% to 30% by volume, or 5% to 20% by volume, or may be, for example, 10% by volume. When the first medium contains growth factors in addition to human platelet lysate, a reagent for stabilizing the growth factors in the medium (e.g., an anticoagulant such as heparin, a gel, a polysaccharide, etc.) may be added in addition to the growth factors. Examples of growth factors that can be used include, but are not limited to, fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor (TGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and factors belonging to these families.

[0039] In one embodiment, the first medium further contains heparin or a heparin substitute. For example, the human platelet lysate contained in the first medium can contain heparin or a heparin substitute. Heparin has anticoagulant activity and can stabilize the human platelet lysate in the medium. As used herein, a "heparin substitute" refers to any substance that has anticoagulant activity similar to that of heparin. Specific types of heparin substitutes are known in the art, and specific examples include fucoidan, sulfated fucan, and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK). The concentration of heparin or heparin substitute in the first medium is, for example, 0.1 U / mL or more, 1 U / mL or more, or 2 U / mL or more, for example, 2 U / mL.

[0040] The culture temperature in this step is not limited as long as it is a temperature at which mesenchymal stem cells can grow. For example, the culture can be performed at 30 to 42°C, 32 to 40°C, 35 to 39°C, or 36 to 38°C, for example, 37°C.

[0041] The culture time in this step is not particularly limited, and may be, for example, 1 hour or more, 2 hours or more, 4 hours or more, 12 hours or more, 24 hours or more, 2 days or more, or 3 days or more, and / or 2 weeks or less, 1 week or less, 6 days or less, 5 days or less, or 4 days or less. For example, the culture may be carried out until the mesenchymal stem cells become subconfluent.

[0042] (Recovery step) In the method of the present invention, the "recovery step" is a step of recovering a cell population. The recovery method in this step can be selected appropriately depending on the type of culture method in the first culture step. For example, if the culture method in the first culture step is adhesion culture, mesenchymal stem cells can be recovered by detaching the cells from the culture vessel after adhesion culture. Furthermore, if the culture method in the first culture step uses microcarriers, the cells can be detached from the microcarriers, and if spheroid culture is used, the mesenchymal stem cells can be recovered by dispersing the spheroids.

[0043] As used herein, "detachment" refers to the physical separation of cells from the surface of a culture substrate such as a culture vessel or microcarrier. Cells may be detached in cell clumps or single cells. Examples of detachment include physical detachment (e.g., mechanical detachment by scraping the vessel surface with a cell scraper, splashing, shaking, or vibration, and ultrasonic treatment), enzymatic detachment using enzymes such as proteases, chemical detachment using compounds such as chelating agents, and any combination thereof.

[0044] The method for recovering the detached mesenchymal stem cells from the culture substrate such as the culture vessel or microcarrier is not limited, and for example, the cell suspension containing the detached mesenchymal stem cells may be recovered by suction or decantation.

[0045] The number of mesenchymal stem cells collected in this step is not limited, but may be, for example, 1 x 10 １ pcs or more, 1×10 ２ pcs or more, 1×10 ３ pcs or more, 1×10 ４ pcs or more, 1×10 ５ 1 x 10 or more ６ The viability of the cells recovered in this step is not limited, but is, for example, 50% or more, 70% or more, 80% or more, 90% or more, or 95% or more.

[0046] In one embodiment, this step involves recovering a cell population from the first medium used in the first culture step described above. According to this embodiment, mesenchymal stem cells immediately after culture in the first medium are isolated in the isolation step described below.

[0047] (Repeating step) In the method of this embodiment, the "repeating step" is a selective step in which the above-mentioned first culturing step to the recovery step are repeated. The number of times this step is repeated is not limited. The method of this embodiment includes repeating the step, for example, once, twice, three times, or four or more times.

[0048] (Introduction Step) In the method of the present invention, the "introduction step" is a selective step of introducing a gene expression vector into a cell population before the isolation step described below.

[0049] As used herein, the term "gene expression vector" refers to a vector that contains a gene or gene fragment of interest in an expressible state and includes an expression unit that can control the expression of the gene. The gene expression vector may be a plasmid vector or a viral vector. The gene expression vector may contain a marker gene (selection marker gene) as needed.

[0050] The gene or gene fragment of interest contained in the gene expression vector is not particularly limited and may be a gene sequence encoding a protein of interest or a fragment thereof, or an RNA molecule of interest. The protein of interest or a fragment thereof is not particularly limited and can be appropriately selected depending on the purpose. Specific examples include gene sequences encoding IL-10 (Interleukin-10), HGF (Hepatocyte Growth Factor), VEGF (Vascular Endothelial Growth Factor), BDNF (Brain-Derived Neurotrophic Factor), and miRNA (microRNA) for the purpose of functional enhancement.

[0051] As used herein, the term "expressible state" refers to the state in which a gene to be expressed is placed downstream of a promoter under the control of the promoter.

[0052] The plasmid vector may be, for example, a commercially available expression vector for mammalian cells such as Promega's pCI vector or pSI vector, or a shuttle vector that can replicate between mammalian cells and bacteria such as E. coli.

[0053] Viral vectors that can be used include, for example, retroviral vectors (including oncoretroviral vectors, lentiviral vectors, and pseudotyped vectors), adenoviral vectors, adeno-associated virus (AAV) vectors, simian virus vectors, vaccinia virus vectors, Sendai virus vectors, Epstein-Barr virus (EBV) vectors, and HSV vectors. Replication-deficient viral vectors that do not autonomously replicate in infected cells may also be used.

[0054] In this step, the method for introducing the gene expression vector into the cell population before the isolation step is not particularly limited. For example, a gene transfer method (transformation method) known in the art, such as that described in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, may be used. Specific examples include the heat shock method, lipofection, electroporation, microinjection, calcium phosphate method, DEAE-dextran method, introduction using cationic lipids, introduction using cationic polymers (e.g., polyethyleneimine (PEI)), introduction using nanoparticles, introduction using viruses, and particle bombardment.

[0055] As used herein, a "label gene" or "marker gene" refers to a gene that encodes a label protein, also known as a selectable marker or reporter protein. The term "label protein" or "marker protein" is not limited as long as it can label and / or select cells into which a gene expression vector has been introduced based on its activity. Examples include drug (e.g., antibiotic) resistance proteins, chromoproteins, fluorescent proteins, and luminescent proteins.

[0056] This step may be performed either before or after the first culture step, as long as it is performed before the isolation step. For example, when this step is performed before the first culture step, mesenchymal stem cells containing the gene expression vector can be selected in a medium (e.g., a first medium used for culture before the isolation step described below, or a second medium used for culture after the isolation step) containing a drug (e.g., an antibiotic) corresponding to the gene encoding the drug resistance protein in the gene expression vector. Furthermore, when this step is performed after the first culture step, mesenchymal stem cells containing the gene expression vector can also be selected in the isolation step described below based on fluorescence detection using a cell sorter or the like, based on the gene encoding a fluorescent protein in the gene expression vector.

[0057] (Isolation Step) In the method of the present invention, the "isolation step" is a step of isolating a single mesenchymal stem cell from the cell population after the recovery step.

[0058] As used herein, "isolating a single mesenchymal stem cell" means that the single mesenchymal stem cell has been separated from other mesenchymal stem cells, and may or may not be separated from cell types other than mesenchymal stem cells; however, it is more preferable that the single mesenchymal stem cell has been separated from living cells other than mesenchymal stem cells (e.g., cells having the ability to proliferate).

[0059] The isolation method used in this step is not limited as long as it can separate mesenchymal stem cells into single cells after the recovery step described above. For example, a suspension containing mesenchymal stem cells can be diluted and dispensed into a multi-well plate such as a 96-well plate at one cell per well, allowing one cell to be separated into each well. When mesenchymal stem cells are labeled with a fluorescent substance or dye, they can be separated one cell at a time using a cell sorter or the like, using flow cytometry with the fluorescent substance or dye as an indicator. Mesenchymal stem cells can be labeled, for example, by introducing a marker gene (marker gene) into mesenchymal stem cells using a gene expression vector, or by binding a fluorescently labeled antibody to the mesenchymal stem cells. Alternatively, single cells can be separated using a micromanipulator, micromesh filter, or the like.

[0060] In one embodiment, this step can isolate only viable cells by labeling either dead or viable cells after the recovery step described above. Examples of labeling methods include staining dead cells with 7-AAD (7-amino-actinomycin D) staining solution or PI (propidium iodide).

[0061] When isolating a plurality of single mesenchymal stem cells by this step, the plurality of single mesenchymal stem cells can be sorted into a plurality of culture vessels or a plurality of wells of a multiwell plate. The shape of the culture vessel or multiwell plate is not limited, and a plurality of cells can be isolated as single cells by sorting a single cell into each well of a multiwell plate such as a 6-well plate, a 12-well plate, or a 96-well plate.

[0062] (Second culture step) In the method of the present invention, the "second culture step" is a selective step in which single cells of mesenchymal stem cells obtained after the isolation step are cultured in a second medium that does not contain other mesenchymal stem cells.

[0063] The "second medium" used in this step is a medium for culturing single mesenchymal stem cells as single cells. In this second medium, single cells can be cultured immediately after the isolation step described above.

[0064] As used herein, "single-cell culture" refers to culturing under conditions in which only one cell is contained in the same culture medium. The second medium used in the second culture step does not contain any cells other than the single mesenchymal stem cell obtained after the isolation step.

[0065] The specific composition of the second medium is the same as that of the "medium" described above. For example, the second medium may be any of a basal medium, a serum-free medium, a low-serum medium, and a serum-added medium. It is usually a basal medium (e.g., a standard cell culture medium) or a serum-free medium, and may be a medium to which other components such as albumin, blood-derived components, and growth factors have been added.

[0066] In one embodiment, the second culture medium contains human platelet lysate. The concentration of human platelet lysate in the second culture medium is not particularly limited, and may be 0.1 v / v% or more, 0.2 v / v% or more, 0.3 v / v% or more, 0.4 v / v% or more, 0.5 v / v% or more, 0.6 v / v% or more, 0.7 v / v% or more, 0.8 v / v% or more, 0.9 v / v% or more, 1 v / v% or more, 1.2 v / v% or more, 1.5 v / v% or more, 2 v / v% or more, 2.5 v / v% or more, 3 v / v% or more, 4 v / v% or more, 5 v / v% or more, 6 v / v% or more, 7 v / v% or more, 8 v / v% or more, 9 v / v% or more, 10 v / v% or more, 11 v / v% or more, 12 v / v% or more, 13 v / v% or more, 14 v / v% or more, 15 v / v% or more, 16 v / v% or more, 17 v / v% or more, 18 v / v% or more, 19 v / v% or more, 20 v / v% or more, 21 v / v% or more, 22 v / v% or more, 23 v / v% or more, 24 v / v% or more, 25 v / v% or more, 26 v / v% or more, 27 v / v% or more, 28 v / v% or more, 29 v / v% or more, 30 v / v% or more, 31 v / v% or more, 32 v / v% or more, 33 v / v% or more, 34 v / v% or more, 35 v / v% or The concentration may be 40 v / v% or more, 3.5 v / v% or more, 4 v / v% or more, 4.5 v / v% or more, or 5 v / v% or more, and / or 40 v / v% or less, 35 v / v% or less, 30 v / v% or less, 25 v / v% or less, 20 v / v% or less, 15 v / v% or less, or 10 v / v% or less, and may be any of the above concentrations expressed in dry weight terms or the above human platelet lysate protein concentrations.

[0067] In another embodiment, the second culture medium does not contain human platelet lysate. For example, the second culture medium may be a basal medium or a serum-free medium optionally supplemented with a blood-derived component such as serum (animal serum such as fetal bovine serum (FBS or FCS) or human serum). Even when the second culture medium does not contain human platelet lysate, single cells of mesenchymal stem cells having the ability to proliferate can be obtained with high efficiency as long as the first culture medium contains human platelet lysate.

[0068] In one embodiment, the second medium contains an antibiotic. By culturing in this step in the presence of the antibiotic, mesenchymal stem cells containing the gene encoding the antibiotic resistance protein introduced in the above-mentioned introduction step can be selected.

[0069] The time from the culture in the first culture step to the start of the culture in this step is not particularly limited, but the culture in this step is carried out, for example, within 24 hours, within 18 hours, within 12 hours, within 6 hours, within 4 hours, within 3 hours, within 2 hours, or within 1 hour from the culture in the first culture step.

[0070] 1-4. Effects According to the isolation method of the present invention, single mesenchymal stem cells having proliferation potential can be efficiently obtained by culturing mesenchymal stem cells in a medium containing hPL in the first culture step prior to the isolation step.

[0071] 2. Method for producing a mesenchymal stem cell population derived from a single mesenchymal stem cell 2-1. Overview A second aspect of the present invention is a method for producing a mesenchymal stem cell population derived from a single mesenchymal stem cell (hereinafter sometimes abbreviated as "production method"). The production method of this aspect comprises producing a single cell based on isolating a single mesenchymal stem cell using the isolation method described in the first aspect above, and can efficiently produce a cell population consisting of a single mesenchymal stem cell or multiple mesenchymal stem cells derived from a single mesenchymal stem cell.

[0072] 2-2. Method The production method of this embodiment involves producing single mesenchymal stem cells using the method described in the first embodiment. Therefore, the production method of this embodiment also includes the above-mentioned first culture step, recovery step, and isolation step as essential steps, and can include the above-mentioned repeat step, introduction step, and / or second culture step as optional steps. The configuration of each step is similar to that of the first embodiment, so detailed description here will be omitted.

[0073] In one embodiment, the production method of this aspect uses the method described in the first aspect, which includes the second culture step described above, and can produce a cell population consisting of a plurality of mesenchymal stem cells by proliferating a single mesenchymal stem cell in the second culture step.

[0074] According to the present invention, there are also provided a single mesenchymal stem cell produced by the production method of this embodiment, a culture medium containing the single mesenchymal stem cell, and a culture supernatant of the single mesenchymal stem cell, as well as a cell population consisting of mesenchymal stem cells derived from a single mesenchymal stem cell produced by the production method of this embodiment, a culture medium containing the cell population, and a culture supernatant of the cell population.

[0075] Also provided is a method for evaluating mesenchymal stem cells into which any exogenous gene has been introduced, using a single mesenchymal stem cell produced by the production method of this embodiment, or a cell population consisting of mesenchymal stem cells derived from a single mesenchymal stem cell produced by the production method of this embodiment.

[0076] 3. Method for Culturing Single Mesenchymal Stem Cells 3-1. Overview A third aspect of the present invention is a method for culturing single mesenchymal stem cells (hereinafter sometimes abbreviated as "culture method"). The culture method of this aspect comprises a first culture step of culturing in a first medium containing a cell population containing mesenchymal stem cells, a recovery step of recovering the cell population, an isolation step of isolating a single mesenchymal stem cell, and a second culture step of culturing the single mesenchymal stem cell after the isolation step in a second medium not containing other mesenchymal stem cells, wherein the second medium contains human platelet lysate. The culture method of this aspect allows single mesenchymal stem cells to proliferate efficiently, and makes it possible to efficiently produce a population of highly homogeneous mesenchymal stem cells.

[0077] 3-2. Method The culture method of this embodiment includes, as essential steps, a first culture step, a recovery step, an isolation step, and a second culture step, and may include, as optional steps, a repeat step and / or an introduction step.

[0078] The configuration of each step in this embodiment is similar to that of the first embodiment. However, in the culture method of this embodiment, the first medium used in the first culture step may or may not contain human platelet lysate, and the configuration of the components other than the human platelet lysate is similar to that of the first medium in the first embodiment. For example, the first medium used in the first culture step in this embodiment can be configured similar to that of the second medium in the first embodiment.

[0079] Furthermore, in the culture method of this embodiment, the second medium used in the second culture step is a medium containing human platelet lysate as an essential component, to which human platelet lysate has been added at an arbitrary concentration, and the composition other than the human platelet lysate conforms to the composition of the second medium of the first embodiment. For example, the second medium used in the second culture step in the culture method of this embodiment can be composed similarly to the first medium of the first embodiment.

[0080] In a further aspect of the present invention, there is provided a method for isolating and culturing a single mesenchymal stem cell, comprising: a first culturing step of culturing a cell population in a first culture medium; a recovering step of recovering the cell population after the first culturing step; an isolation step of isolating a single mesenchymal stem cell from the cell population after the recovery step; and a second culturing step of culturing the single mesenchymal stem cell after the isolation step in a second culture medium that does not contain other mesenchymal stem cells, wherein the first culture medium and / or the second culture medium (e.g., both the first culture medium and the second culture medium, or only either the first culture medium or the second culture medium) comprises human platelet lysate. More preferably, there is provided a method in which both the first culture medium and the second culture medium comprise human platelet lysate.

[0081] The present invention will be specifically described below with reference to examples. Note that these examples are for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0082] Example 1: Isolation of single cells after FBS culture or hPL culture (Objective) Bone marrow- or amniotic membrane-derived mesenchymal stem cells are cultured in a medium containing fetal bovine serum (FBS) or human platelet lysate (hPL). After culture, the cells are isolated using a cell sorter, and single cells are cultured in each well of a 96-well plate, and the number of wells containing proliferating cells is counted.

[0083] (Method) (1) FBS Culture of Bone Marrow Mesenchymal Stem Cells Bone marrow aspirate was collected from adult donors who provided informed consent. Alpha Modification of Minimum Essential Medium Eagle (αMEM) containing 10% final concentration of fetal bovine serum (FBS (Moregate, 599-04425)) was mixed with the bone marrow aspirate at a 5:1 ratio and seeded into a culture vessel. The bone marrow mesenchymal stem cells were then cultured in an adherent medium until subconfluent, with medium changes every 3-4 days using FBS-containing medium. Hereinafter, the method of culturing in FBS-containing medium is referred to as "FBS culture," and bone marrow mesenchymal stem cells were subcultured under similar conditions as needed.

[0084] (2) hPL Culture of Bone Marrow Mesenchymal Stem Cells Bone marrow aspirate was collected from adult donors who provided informed consent. αMEM containing 5% final concentration of human platelet lysate (hPL (AventaCell, HPCFDCGLI50)) was mixed with the bone marrow aspirate at a 5:1 ratio and seeded into a culture vessel. The bone marrow mesenchymal stem cells were then cultured as adherent cells until they became subconfluent, with medium changes every 3-4 days using medium containing hPL. Hereinafter, the method of culturing in medium containing hPL is referred to as "hPL culture," and bone marrow mesenchymal stem cells were subcultured under similar conditions as needed.

[0085] (3) FBS Culture of Amniotic Mesenchymal Stem Cells Fetal appendages, the amniotic membrane and placenta, were collected aseptically from pregnant women undergoing elective cesarean section after obtaining informed consent. The obtained amniotic membrane and placenta were placed in a sterile tray containing physiological saline, and the amniotic membrane was manually peeled from the stump of the amniotic membrane. The amniotic membrane was washed with Hank's balanced salt solution (Ca / Mg-free) to remove any adhering blood and blood clots.

[0086] An amniotic membrane containing an epithelial cell layer and a mesenchymal stem cell layer was immersed in Hank's balanced salt solution (containing Ca / Mg) containing 480 PU / mL collagenase and 400 PU / mL dispase I, and the amniotic membrane was enzymatically treated by shaking and stirring at 50 rpm for 90 minutes at 37°C. The solution after enzyme treatment was filtered through a nylon mesh with 95 μm openings to remove undigested material from the amniotic membrane, and a cell suspension containing amniotic mesenchymal stem cells was recovered.

[0087] The cell population containing the amniotic mesenchymal stem cells was cultured at a density of 6,000 cells / cm ２ After cell seeding, the cells were cultured in an adherent manner in αMEM containing a final concentration of 10% FBS until they became subconfluent. Thereafter, amniotic mesenchymal stem cells were subcultured under the same conditions as necessary.

[0088] (4) hPL Culture of Amniotic Mesenchymal Stem Cells Fetal appendages, the amniotic membrane and placenta, were collected aseptically from pregnant women undergoing elective cesarean section after obtaining informed consent. The obtained amniotic membrane and placenta were placed in a sterile tray containing physiological saline, and the amniotic membrane was manually peeled off from the stump of the amniotic membrane. The amniotic membrane was washed with Hank's balanced salt solution (free of Ca and Mg) to remove any adhering blood and blood clots.

[0089] An amniotic membrane containing an epithelial cell layer and a mesenchymal stem cell layer was immersed in Hank's balanced salt solution (containing Ca / Mg) containing 480 PU / mL collagenase and 400 PU / mL dispase I, and the amniotic membrane was enzymatically treated by rotating and stirring using a rotator at 37°C for 60 minutes at 10 rpm. The solution after enzyme treatment was filtered through a nylon mesh with 95 μm openings to remove undigested material from the amniotic membrane, and a cell suspension containing amniotic mesenchymal stem cells was recovered.

[0090] The cell population containing the amniotic mesenchymal stem cells was cultured at a density of 1,400 cells / cm ２ After cell seeding, the cells were cultured in αMEM containing 5% hPL until they became subconfluent. After reaching subconfluence, amniotic mesenchymal stem cells were seeded at a seeding density of 1,000 cells / cm. ２ The cells were subcultured at a seeding density of 1000 x g / ml and cultured until they became subconfluent. Thereafter, amniotic mesenchymal stem cells were subcultured under the same conditions as necessary.

[0091] (5) Single Cell Cloning The various mesenchymal stem cells cultured in (1) to (4) above were detached using TrypLE™ Select Enzyme (1X) and diluted to a cell concentration of 1 x 10 in phosphate-buffered saline containing 0.5% bovine serum albumin. ６ The cell suspension was adjusted to 100 cells / mL. 7-AAD (7-Amino-Actinomycin D) staining solution was added to this cell suspension to stain dead cells.

[0092] Using the cell suspension as a sample, only live cells (7-AAD-negative fraction) were sorted into five 96-well plates for each type of mesenchymal stem cell at 1 cell / well using the MA900 cell sorter. The mesenchymal stem cells sorted into each well were cultured in a medium containing human platelet lysate. 13 days after sorting, all wells were stained with crystal violet staining solution, and the number of wells containing proliferated cells was counted (Figure 1).

[0093] (Results) The number of wells in which cell growth was confirmed among 96 wells under each condition was counted, and the results are shown in FIG.

[0094] When bone marrow mesenchymal stem cells (bone marrow MSCs in the figure) were cultured in hPL, then single-cell sorted and further cultured, cell proliferation was observed in more than twice as many wells compared to when they were cultured in FBS, then single-cell sorted and further cultured.

[0095] Furthermore, when amniotic mesenchymal stem cells (amniotic MSCs in the figure) were cultured in FBS, then single-cell sorted and further cultured, no wells showing cell proliferation were observed, whereas when they were cultured in hPL, then single-cell sorted and further cultured, cell proliferation was observed in nearly half of the wells.

[0096] The above results indicate that single mesenchymal stem cells with proliferation potential can be obtained with high efficiency by culturing in a medium containing hPL before single cell sorting.

[0097] Example 2: Long-term subculture of mesenchymal stem cells with FBS (Objective) Bone marrow mesenchymal stem cells and amniotic mesenchymal stem cells are subcultured for a long period of time without being isolated as single cells. The purpose is to verify that mesenchymal stem cells exhibit proliferation in a medium containing FBS.

[0098] (Method and Results) The bone marrow mesenchymal stem cells obtained in Example 1(1) were repeatedly subcultured in αMEM containing FBS at a final concentration of 10% until proliferation stopped.

[0099] Furthermore, the amniotic mesenchymal stem cells obtained in Example 1 (3) were repeatedly subcultured in αMEM containing a final concentration of 10% FBS until proliferation stopped.

[0100] The cell counts of bone marrow mesenchymal stem cells and amniotic mesenchymal stem cells were measured before each passage during long-term culture, and the doubling times were calculated. The results are shown in Figure 3. Both bone marrow mesenchymal stem cells and amniotic mesenchymal stem cells showed doubling times of 40 to 50. These results demonstrated that bone marrow mesenchymal stem cells and amniotic mesenchymal stem cells exhibit normal proliferation in a medium containing FBS.

[0101] In this example, normal proliferation was observed when single cells were cultured for a long period in a medium containing FBS without isolation, demonstrating that proliferation is not impaired when cells are cultured as a cell mass, even in a medium containing FBS. Therefore, the results of Example 1 above, in which single cells were isolated and cultured after FBS culture, showing no cell proliferation at all for amniotic mesenchymal stem cells and low proliferation for bone marrow mesenchymal stem cells, are considered to be phenomena associated with single-cell culture. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. A method for isolating a single mesenchymal stem cell from a cell population containing mesenchymal stem cells, comprising: a first culturing step of culturing the cell population in a first medium containing human platelet lysate; a recovering step of recovering the cell population after the first culturing step; and an isolating step of isolating a single mesenchymal stem cell from the cell population after the recovering step.

2. The method according to claim 1, wherein the cell population is derived from amnion or bone marrow.

3. The method according to claim 1, wherein the first medium contains 3 v / v% or more of the human platelet lysate.

4. The method according to claim 3, wherein the first medium contains 3 to 20 v / v% of the human platelet lysate.

5. The method according to claim 1, wherein the first medium further contains heparin or a heparin substitute.

6. The method according to claim 1, wherein the first medium is a basal medium or a serum-free medium.

7. The method according to claim 1, wherein the recovering step recovers the cell population from the first medium.

8. The method according to claim 1, wherein after the recovering step, dead cells or live cells are labeled, and only live cells are isolated in the isolating step.

9. The method according to claim 1, further comprising an introducing step of introducing a gene expression vector into the cell population before the isolating step.

10. The method according to claim 9, wherein the gene expression vector contains a marker gene.

11. The method according to claim 9, wherein the introducing step is performed before the first culturing step.

12. The method according to claim 10, wherein the isolating step isolates the single mesenchymal stem cell expressing the marker protein using the expression of the marker protein encoded by the marker gene as an index.

13. The method according to claim 10, wherein the marker gene is an antibiotic resistance gene that confers resistance to an antibiotic.

14. The method according to any one of claims 1 to 13, further comprising a second culturing step of culturing the single mesenchymal stem cell after the isolating step in a second medium not containing other mesenchymal stem cells.

15. The method according to claim 14, which quotes claim 13, wherein the second medium contains the antibiotic.

16. A method for manufacturing a single mesenchymal stem cell, wherein the single mesenchymal stem cell is manufactured using the method according to claim 1.

17. A method for producing a mesenchymal stem cell population derived from a single mesenchymal stem cell, the production method comprising proliferating the single mesenchymal stem cell in the second culture step using the method according to claim 14 to produce the mesenchymal stem cell population.

18. A method for culturing a single mesenchymal stem cell, the method comprising: a first culture step of culturing a cell population containing mesenchymal stem cells in a first medium; a recovery step of recovering the cell population after the first culture step; an isolation step of isolating a single mesenchymal stem cell from the cell population after the recovery step; and a second culture step of culturing the single mesenchymal stem cell after the isolation step in a second medium not containing other mesenchymal stem cells, wherein the second medium contains human platelet lysate.

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