Method for isolating single mesenchymal stem cells

Abstract

To provide a new method for isolating mesenchymal stem cells in a single cell form with good efficiency and proliferating the single cells with good efficiency. A method for isolating a single mesenchymal stem cell from a cell population containing mesenchymal stem cells, the method comprising: a first culture step of culturing the cell population in a first culture medium containing human platelet lysate; a recovery step of recovering the cell population after the first culture step; and an isolation step of isolating a single mesenchymal stem cell from the cell population after the recovery step.

Description

Technical Field

[0001] This invention relates to methods for isolating and manufacturing single mesenchymal stem cells. Background Technology

[0002] Pluripotent stem cells are cells capable of differentiating into various somatic cells. Examples of pluripotent stem cells include iPS cells obtained by introducing reprogramming factors into somatic cells, ES cells derived from internal cell masses, and mesenchymal stem cells as somatic stem cells.

[0003] Mesenchymal stem cells (MSCs) are known to differentiate into osteoblasts, chondrocytes, and adipocytes, and their existence in various tissues, including bone marrow, fat, dental pulp, and fetal appendages (placenta, umbilical cord, fetal membranes, etc.), is well-established (Patent Document 1). Based on their pluripotency, MSCs are being practically applied as regenerative medicine materials. In fact, due to their immunosuppressive capabilities, they are being used clinically for a wide range of diseases, including acute graft-versus-host disease (GVHD), Crohn's disease, spinal cord injury, and knee osteoarthritis. Furthermore, because MSCs 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 and fetal appendages, it is necessary to remove cells other than mesenchymal stem cells from the same tissues. For example, bone marrow fluid contains a large number of hematopoietic stem cells in addition to mesenchymal stem cells, and fat contains a large number of adipocytes in addition to mesenchymal stem cells; therefore, it is desirable to remove these cells. Furthermore, the amnion, a type of fetal appendage, has attracted attention as a promising biological tissue containing a large number of mesenchymal stem cells, but it also contains a large number of epithelial cells in addition to mesenchymal stem cells; therefore, it is desirable to remove these epithelial cells.

[0005] Furthermore, from a cell quality management perspective, it is preferable to isolate mesenchymal stem cells as single cells and then expand and culture these single cells to create a highly homogeneous cell population. For example, when mesenchymal stem cells transformed with plasmid vectors or viral vectors are used for therapeutic purposes, differences in vector copy number and genome insertion sites can lead to variations in cell quality. To obtain a highly homogeneous cell population, it is necessary to isolate single cells from gene-introduced mesenchymal stem cells and then expand and culture them.

[0006] Therefore, there is a need for new methods to efficiently isolate mesenchymal stem cells as single cells and to efficiently proliferate these single cells.

[0007] Existing technical documents

[0008] Patent documents

[0009] Patent Document 1: WO2015 / 025810 Summary of the Invention

[0010] The problem that the invention aims to solve

[0011] The object of this invention is to provide a new method for efficiently isolating mesenchymal stem cells in single-cell form and efficiently proliferating such single cells.

[0012] Problem Solving Methods

[0013] In single-cell clones, which are isolated from and proliferated from a cell population containing multiple cells, after gene introduction as needed, cells are separated one by one using a cell sorting instrument, and then seeded and cultured in each well of a multi-well plate to obtain a cell population derived from a single cell. Figure 4 ).

[0014] In order to isolate mesenchymal stem cells (MSCs) in single-cell form, the inventors cultured MSCs collected from the amnion in a commonly used culture medium containing fetal bovine serum (FBS). After isolating and seeding the cultured amniotic MSCs one by one in each well of a 96-well plate using a cell sorting instrument, cell proliferation was attempted through further culture. However, no cell proliferation was observed in any of the 96 wells of the amniotic MSCs cultured in the FBS-containing medium after isolation as single cells.

[0015] Next, the inventors cultured amniotic mesenchymal stem cells in a medium containing human platelet lysate (hPL) instead of fetal bovine serum (FBS), and isolated and cultured the cells one by one in each 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.

[0016] Furthermore, the inventors conducted the same experiment using bone marrow mesenchymal stem cells as the target cells. After culturing bone marrow mesenchymal stem cells in a medium containing FBS, the cells were individually separated and cultured in each well of a 96-well plate using a cell sorting instrument. Cell proliferation was observed in only 20 wells out of the 96. On the other hand, when bone marrow mesenchymal stem cells were cultured in a medium containing hPL and the cells were individually separated and cultured, cell proliferation was observed in 45 wells out of the 96.

[0017] Based on the above results, it is clear that by culturing cells before and / or after cell sorting in a medium containing hPL, single cells of mesenchymal stem cells with proliferative capacity can be obtained with good efficiency. This invention is based on the above insights and provides the following method.

[0018] (1) A method for isolating a single mesenchymal stem cell from a cell population containing mesenchymal stem cells, the method comprising:

[0019] The first culture step involves culturing the aforementioned cell population in a first culture medium containing human platelet lysate;

[0020] The recovery process involves recovering the cell population from the first culture step; and

[0021] The separation process involves isolating a single mesenchymal stem cell from the cell population following the recovery process described above.

[0022] (2) According to the method described in (1), wherein,

[0023] The aforementioned cell populations originated from the amnion or bone marrow.

[0024] (3) According to the method described in (1) or (2), wherein,

[0025] The first culture medium mentioned above contains more than 3 v / v% of the aforementioned human platelet lysate.

[0026] (4) According to the method described in (3), wherein,

[0027] The first culture medium mentioned above contains 3 to 20 v / v% of the aforementioned human platelet lysate.

[0028] (5) The method according to any one of (1) to (4), wherein,

[0029] The aforementioned first culture medium further contains heparin or a heparin substitute.

[0030] (6) The method according to any one of (1) to (5), wherein,

[0031] The first culture medium mentioned above is either the basal culture medium or the serum-free culture medium.

[0032] (7) The method according to any one of (1) to (6), wherein,

[0033] The above-described recovery process recovers the cell population from the first culture medium.

[0034] (8) The method according to any one of (1) to (7), wherein,

[0035] After the above-mentioned recycling process, dead or live cells are marked, and only live cells are separated in the above-mentioned separation process.

[0036] (9) The method according to any one of (1) to (8) further comprises:

[0037] In the introduction process, the gene expression vector is introduced into the cell population mentioned above before the separation process.

[0038] (10) According to the method described in (9), wherein,

[0039] The gene expression vectors mentioned above contain marker genes.

[0040] (11) The method according to (9) or (10), wherein,

[0041] The above-mentioned introductory process is performed before the first culture process.

[0042] (12) According to the method described in (10) or (11), wherein,

[0043] The above separation process uses the expression of the marker protein encoded by the above marker gene as an indicator to separate the above single mesenchymal stem cells expressing the above marker protein.

[0044] (13) The method according to any one of (10) to (12), wherein,

[0045] The aforementioned marker genes are antibiotic resistance genes that confer resistance to antibiotics.

[0046] (14) The method according to any one of (1) to (13), the method further comprising:

[0047] The second culture step involves culturing the single mesenchymal stem cell obtained after the above separation step in a second culture medium that does not contain other mesenchymal stem cells.

[0048] (15) According to the method described in (14), wherein,

[0049] The second culture medium mentioned above contains human platelet lysate.

[0050] (16) The method described according to (14) of (13), or (15) of (13) and (14), wherein,

[0051] The second culture medium mentioned above contains the antibiotics mentioned above.

[0052] (17) A method for manufacturing a single mesenchymal stem cell line, the method comprising:

[0053] The above-mentioned single mesenchymal stem cells were produced using the methods described in (1) to (16).

[0054] (18) A method for producing a population of mesenchymal stem cells derived from a single mesenchymal stem cell line, the method comprising:

[0055] The mesenchymal stem cell population is generated by proliferating the single mesenchymal stem cell in the second culture step using any one of the methods in (14) to (16).

[0056] (19) A method for culturing a single mesenchymal stem cell line, the method comprising:

[0057] The first culture step involves culturing a cell population containing mesenchymal stem cells in a first culture medium.

[0058] The recovery process involves recovering the cell population obtained after the first culture process.

[0059] The separation process involves isolating single mesenchymal stem cells from the cell population following the aforementioned recovery process; and

[0060] The second culture step involves culturing the single mesenchymal stem cell obtained after the above separation step in a second culture medium that does not contain other mesenchymal stem cells.

[0061] The second culture medium mentioned above contains human platelet lysate.

[0062] This specification includes the disclosure of Japanese Patent Application No. 2024-003123, which forms the basis of the priority claim of this application.

[0063] The effects of the invention

[0064] According to the present invention, a new method is provided for isolating mesenchymal stem cells in single-cell form with good efficiency and for proliferating such single cells with good efficiency. Attached Figure Description

[0065] Figure 1This is a schematic diagram illustrating the outline of an experiment in which pores containing proliferating cells are identified after isolating mesenchymal stem cells from FBS or hPL cultures as single cells.

[0066] Figure 2 This figure shows the results of counting the number of wells containing proliferating cells in 96 wells after isolating bone marrow mesenchymal stem cells (bone marrow MSCs) or amniotic mesenchymal stem cells (amniotic MSCs) in single-cell form after FBS or hPL culture.

[0067] Figure 3 This is a graph showing the doubling number of bone marrow mesenchymal stem cells and amniotic mesenchymal stem cells in long-term FBS culture.

[0068] Figure 4 This is a schematic diagram illustrating a method for isolating single cells after introducing a marker gene in one embodiment of the present invention. Detailed Implementation

[0069] 1. Methods for isolating single mesenchymal stem cells

[0070] 1-1. Overview

[0071] A first aspect of the present invention relates to a method for isolating a single mesenchymal stem cell from a cell population containing mesenchymal stem cells (hereinafter, sometimes simply referred to as the "isolation method"). The isolation method of this aspect includes the following steps as essential steps: a first culture step, culturing the cell population containing mesenchymal stem cells in a first culture medium containing human platelet lysate; a recovery step, recovering the cell population; and an isolation step, isolating a single mesenchymal stem cell. The mesenchymal stem cells isolated by the isolation method of this aspect proliferate with good efficiency under single-cell culture conditions. Therefore, it is possible to produce a highly homogeneous mesenchymal stem cell population with good efficiency.

[0072] 1-2. Definition of Terms

[0073] The following terms, which are frequently used in this specification, are defined.

[0074] In this specification, "fetal appendages" refers to the tissues or organs within the uterus that support fetal development, excluding the fetus's body; specifically, the fetal membranes, placenta, umbilical cord, and amniotic fluid. "Fetal membranes" refers to the amniotic sac containing the fetus's amniotic fluid, and is composed of the amnion, chorion, and decidua from the inside out. The amnion is the innermost, bloodless, transparent membrane of the fetal membranes. The inner layer of the amnion (also called the epithelial cell layer) is covered by a layer of secretory epithelial cells that secrete amniotic fluid, while the outer layer of the amnion (also called the extracellular matrix layer, equivalent to the mesenchyme) contains mesenchymal stem cells.

[0075] In this specification, "mesenchymal stem cells" refers to stem cells that (i) exhibit adhesion to the surface of plastic culture containers under standard culture conditions (a basal medium supplemented with serum, serum substitutes, or growth factors); and (ii) are positive for surface antigens CD105, CD73, and CD90, and negative for CD45. In this specification, "mesenchymal stem cells" is also referred to as "MSC," which has the same meaning as "mesenchymal stromal cells." The source species of mesenchymal stem cells can be any species possessing mesenchymal stem cells; there are no particular limitations. For example, they can be fish, birds, or mammals. Examples of mesenchymal stem cells include mouse cells, chimpanzee cells, or human cells. In addition, sources of mesenchymal stem cells include bone marrow, hematopoietic stem cells, umbilical cord blood, umbilical cord, amnion, amniotic fluid, placental villi, nerves, adipose tissue, pancreas, synovium, dental pulp, deciduous teeth, sperm, testes, and cornea, but are not limited to these. Furthermore, mesenchymal stem cells can also be immortalized mesenchymal stem cell lines.

[0076] In this specification, "cell population containing mesenchymal stem cells" refers, for example, to a cell population containing mesenchymal stem cells derived from any of the aforementioned tissues (e.g., amnion or bone marrow). The morphology of the cell population containing mesenchymal stem cells is not particularly limited; for example, it can be cell granules, cell aggregates, cell floats, or cell suspensions.

[0077] In this specification, "amniotic mesenchymal stem cells" refers to mesenchymal stem cells derived from the amnion. In this specification, amniotic mesenchymal stem cells are also referred to as "amniotic MSCs".

[0078] In addition, in this specification, "bone marrow mesenchymal stem cells" refers to mesenchymal stem cells derived from bone marrow. In this specification, bone marrow mesenchymal stem cells are also referred to as "bone marrow MSCs".

[0079] In this specification, "culture medium" refers to any culture medium capable of maintaining mesenchymal stem cells and other cells in a viable cellular form; there is no limitation. For example, any culture medium commonly used in cell culture and known in the art is acceptable. The culture medium can be any of the following: basal medium, serum-free medium, low-serum medium, and serum-added medium. It can typically be a basal medium (e.g., standard cell culture medium) or a serum-free medium, and other components can be added as needed. The term "standard cell culture medium" as used herein refers to a highly versatile basal medium primarily used for culturing various cell types derived from mammals. Specific examples include: 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 their mixtures (e.g., DMEM / F12 medium (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 Ham)), etc., without particular limitation. Additionally, various commercially available serum-free media can also be used. Other components that can be added to the above-mentioned basic culture medium include, for example, albumin, blood-derived components, and growth factors.

[0080] In this specification, "single cell" refers to a single cell that does not contain any other cells. In this specification, a single cell is also referred to as "monocellular".

[0081] In this specification, "single-cell cloning" refers to the isolation of a single cell (i.e., a single cell) capable of proliferation from a cell population containing two or more cells, followed by the culture of the isolated single cell. For example, in the case of single-cell cloning of mesenchymal stem cells, one isolated mesenchymal stem cell is cultured in a culture medium that does not contain other mesenchymal stem cells or other cells.

[0082] In this specification, "proliferative capacity" refers to the ability of a cell to increase its number of cells through cell division. For example, when mesenchymal stem cells are cultured as single cells, cases showing proliferative capacity and cases not showing proliferative capacity are identified. When a single-cell mesenchymal stem cell shows proliferative capacity, it has the ability to generate two or more cells through cell division, forming two or more cells derived from that cell.

[0083] 1-3. Methods

[0084] The separation method of this invention includes a first culture step, a recovery step, and a separation step as essential steps, and may include a repeating step, an introduction step, and / or a second culture step as optional steps. The following is a detailed description of each step in the separation method of this invention.

[0085] (First cultivation process)

[0086] In the method of this invention, the "first culture step" refers to the step of culturing a cell population containing mesenchymal stem cells in a first culture medium containing human platelet lysate. The culture method in this step is not limited; for example, it can be adherent culture, suspension culture, or spheroid culture. Since mesenchymal stem cells are adhesive cells, adherent culture is preferred in this step, but suspension culture (where microcarriers containing attached mesenchymal stem cells are suspended and cultured) or spheroid culture (where mesenchymal stem cells are spheroidized and cultured) can also be used.

[0087] The "first culture medium" used in this process is a culture medium containing human platelet lysate as an essential component, added at any concentration to the aforementioned "culture medium," and may also contain other components as needed. In this first culture medium, single cells can be cultured before the separation process described later.

[0088] In this specification, "platelet lysate" refers to the lysate of platelets derived from blood, and is a cell culture additive containing a large number of growth factors and chemokines as components. In this specification, there is no particular limitation on the animal species that can be the source of platelet lysate, but humans are preferred. In this specification, human platelet lysate (human platelet lysate) is often abbreviated as "hPL". Examples of growth factors that can be contained in human platelet lysates include: platelet-derived growth factor subtypes (PDGF-AA, -AB, -BB), transforming growth factor-β (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 (bFGF or FGF-2), hepatocyte growth factor (HGF), connective tissue growth factor (CTGF), morphogenetic proteins-2, -4, and -6 (BMP-2, -4, and -6), etc. In addition, examples of chemokines that can be contained in the aforementioned human platelet lysates include interleukin-8 (IL-8), neutrophil activation peptide-2 (NAP-2), activating normal T-cell secretion factor (TANTES), monocyte chemoattractant proteins-1 and-3 (MCP-1 and 3), macrophage inflammatory protein-1α (MIP-1α), and β-thromboglobulin.

[0089] The method for manufacturing the human platelet lysate used in the method of this invention is not particularly limited. Platelet lysate is usually manufactured by repeatedly freezing and thawing platelets extracted from whole blood to remove cell fragments and purify them, but the manufacturing method is not particularly limited and can be any method. Specific preparation methods can be found in, for example, the following literature: Schallmoser k et al., Methods Mol Biol. 2013; 946: 349-62.

[0090] The human platelet lysate used in this invention is preferably treated with bacteria, viruses and / or sterilization.

[0091] The method of the present invention can also use commercially available human platelet lysate. Examples of commercially available human platelet lysate include Stemulate (Cook Regentec), PLTMax (Mill Creek Life Science), UltraGRO (AventaCell BioMedical), and PLUS (Compass Biomedical).

[0092] The method of the present invention can also use clarified human platelet lysate. Clarification, as described herein, refers to the process of removing insoluble components generated during long-term storage or freeze-thaw cycles from the human platelet lysate. Examples of clarification methods include centrifugation, filtration, precipitation, and sedimentation (e.g., natural sedimentation), as long as the insoluble components can be removed, and are not limited thereto.

[0093] There is no particular limitation on the concentration of human platelet lysate in the first culture medium. The first culture medium may contain, for example, human platelet lysate at concentrations of ≥0.1 v / v%, ≥0.2 v / v%, ≥0.3 v / v%, ≥0.4 v / v%, ≥0.5 v / v%, ≥0.6 v / v%, ≥0.7 v / v%, ≥0.8 v / v%, ≥0.9 v / v%, ≥1 v / v%, ≥1.2 v / v%, ≥1.5 v / v%, ≥2 v / v%, ≥2.5 v / v%, ≥3 v / v%, ≥3.5 v / v%, ≥4 v / v%, ≥4.5 v / v%, or ≥5 v / v%, and / or ≤40 v / v%, ≤35 v / v%, ≤30 v / v%, ≤25 v / v%, ≤20 v / v%, ≤15 v / v%, or ≤10 v / v%. In this specification, when the concentration of human platelet lysate is expressed in "v / v%", the volume ratio (hPL volume before dilution / volume after dilution) represents the dilution rate of the human platelet lysate. More specifically, it represents the volume ratio of crude platelet lysate or commercially available platelet lysate diluted with a culture medium such as a first culture medium. It should be noted that commercially available platelet lysate may be, for example, human platelet lysate with a total protein concentration of 40-80 mg / mL, which can be diluted for use.

[0094] The concentration of human platelet lysate in the first culture medium can also be expressed by converting the dry weight of human platelet lysate. The concentration of human platelet lysate in the first culture medium, converted by dry weight, can be, for example, ≥0.06 mg / mL, ≥0.3 mg / mL, ≥0.6 mg / mL, ≥1.2 mg / mL, ≥1.8 mg / mL, or ≥3 mg / mL, and / or ≤24 mg / mL, ≤21 mg / mL, ≤18 mg / mL, ≤15 mg / mL, ≤12 mg / mL, ≤9 mg / mL, or ≤6 mg / mL.

[0095] In addition, the concentration of human platelet lysate in the first culture medium can also be specified by the average concentration of human platelet lysate protein per 1 mL of the first culture medium. For example, the average concentration of human platelet lysate protein per 1 mL of the first culture medium can 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.

[0096] In addition to human platelet lysate, which is an essential component, the first culture medium may also contain other components. For example, besides human platelet lysate, the first culture medium may also contain albumin, blood-derived components, and / or growth factors. When the first culture medium contains albumin in addition to human platelet lysate, the albumin concentration may, for example, be 0.05% to 5% by mass. Other blood-derived components include various serums (fetal bovine serum (FBS, FCS, etc.), human serum), and / or plasma. When the first culture medium contains blood-derived components in addition to human platelet lysate, the concentration of these components may, for example, be 2% to 40% by volume, 3% to 30% by volume, or 5% to 20% by volume, for example, 10% by volume. When the first culture medium contains growth factors in addition to human platelet lysate, reagents for stabilizing the growth factors in the culture medium (anticoagulants such as heparin, gelling agents, polysaccharides, etc.) may be further added. Examples of growth factors may include 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 their families, without particular limitation.

[0097] In one embodiment, the first culture medium further comprises heparin or a heparin substitute. For example, the human platelet lysate contained in the first culture medium may contain heparin or a heparin substitute. Heparin has anticoagulant activity and can stabilize the human platelet lysate in the culture medium. In this specification, "heparin substitute" means any substance having anticoagulant activity similar to 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 the heparin substitute in the first culture medium is, for example, 0.1 U / mL or more, 1 U / mL or more, or 2 U / mL or more, such as 2 U / mL.

[0098] The culture temperature for this step can be any temperature at which mesenchymal stem cells can grow; there are no restrictions. For example, the culture temperature can be 30~42℃, 32~40℃, 35~39℃, or 36~38℃, such as 37℃.

[0099] There is no specific limitation on the culture time in this process. For example, it can be more than 1 hour, more than 2 hours, more than 4 hours, more than 12 hours, more than 24 hours, more than 2 days, or more than 3 days, and / or less than 2 weeks, less than 1 week, less than 6 days, less than 5 days, or less than 4 days. For example, it can be cultured until the mesenchymal stem cells reach subconfluent.

[0100] (Recycling process)

[0101] In the method of this invention, the "recovery step" refers to the step of recovering the cell population. The recovery method in this step can be appropriately selected according to the type of culture method in the first culture step. For example, if the culture method in the first culture step is adherent culture, the mesenchymal stem cells can be recovered by peeling the adherent cultured cells from the culture container. In addition, if the culture method in the first culture step uses microcarriers, it is sufficient to peel the cells from the microcarriers; if it is spheroid culture, it is sufficient to disperse the spheroids and recover the mesenchymal stem cells.

[0102] In this specification, "peeling" refers to the physical separation of cells from the surface of culture media such as culture containers and microcarriers. Cells can be peeled off in the form of cell clumps or single-cell units. Examples of peeling include physical peeling (e.g., mechanical peeling by rubbing the container surface with a cell scraper, rinsing, shaking, vibration, and ultrasonic treatment), enzymatic peeling using enzymes such as proteases, chemical peeling using compounds such as chelating agents, and any combination thereof.

[0103] There are no limitations on the method for recovering the dissected mesenchymal stem cells from culture media such as culture containers and microcarriers. For example, the cell suspension containing the dissected mesenchymal stem cells can be recovered by aspiration or decanting.

[0104] There is no limit to the number of mesenchymal stem cells recovered in this process; for example, it can be 1 × 10⁻⁶ cells. 1 More than one, 1×10 2 More than one, 1×10 3 More than one, 1×10 4 More than one, 1×10 5 More than one, or 1×10 6 More than one. In addition, there is no limit to the survival rate of the cells recovered in this process, such as 50% or more, 70% or more, 80% or more, 90% or more, or 95% or more.

[0105] In one embodiment, this step recovers the cell population from the first culture medium used in the first culture step described above. Through this embodiment, mesenchymal stem cells freshly cultured in the first culture medium are separated in the separation step described later.

[0106] (Repetitive process)

[0107] In this method, a "repeated step" is an optional step that is repeated from the first cultivation step to the recovery step described above. There is no limit to the number of times this step is repeated. This method may include, for example, one, two, three, or four or more repeated steps.

[0108] (Importing Process)

[0109] In the method of the present invention, "introduction step" refers to an optional step of introducing the gene expression vector into the cell population prior to the separation step described later.

[0110] In this specification, "gene expression vector" refers to a vector that contains a target gene or gene fragment in an expressible state, and is an expression unit containing the ability to control the expression of that gene, etc. Gene expression vectors can be plasmid vectors or viral vectors. Gene expression vectors may contain marker genes (screening marker genes) as needed.

[0111] There are no particular restrictions on the target gene or gene fragment contained in the gene expression vector; it can be a gene sequence encoding a target protein or its fragment, or a target RNA molecule. As for the target protein or its fragment, there are no particular restrictions, and it can be appropriately selected according to 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.

[0112] In this specification, "expressible state" refers to the configuration of the gene to be expressed in the downstream region of the promoter under the control of the promoter.

[0113] Plasmid vectors can be commercially available mammalian cell expression vectors such as Promega's pCI vector and pSI vector, or shuttle vectors that can replicate between mammalian cells and bacteria such as Escherichia coli.

[0114] Viral vectors can be, for example, retroviral vectors (including tumor virus vectors, lentiviral vectors, and pseudotyped vectors), adenovirus vectors, adeno-associated virus (AAV) vectors, simian virus vectors, vaccinia virus vectors, Sendai virus vectors, Epstein-Barr virus (EBV) vectors, and herpes simplex virus (HSV) vectors. Viral vectors lacking replication ability and not capable of autonomously replicating within infected cells can also be used.

[0115] There are no particular limitations on the method used to introduce the gene expression vector into the cell population prior to the separation process in this step. For example, gene introduction methods (transformation methods) known in the art as described in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, etc. Specific examples include heat shock method, lipid transfection method, electroporation method, microinjection method, calcium phosphate method, DEAE dextran method, introduction using cationic lipids, introduction using cationic polymers (e.g., polyethyleneimine (PEI)), introduction using nanoparticles, introduction using viruses, particle impaction, etc.

[0116] In this specification, "marker gene" or "marker gene" is a gene that encodes a marker protein (marker protein), also known as a screening marker or reporter protein. There are no limitations on the term "marker protein" or "marker protein" as long as it can be used to mark and / or screen cells introduced with a gene expression vector based on its activity. Examples include: drug (e.g., antibiotic) resistance proteins, pigment proteins, fluorescent proteins, luminescent proteins, etc.

[0117] This step can be performed before the separation step and can be done at any time before or after the first culture step. For example, if this step is performed before the first culture step, mesenchymal stem cells containing the gene expression vector can be selected in a culture medium containing a drug (e.g., an antibiotic) corresponding to the gene encoding the drug resistance protein in the gene expression vector (e.g., the first culture medium for culture before the separation step described later, or the second culture medium for culture after the separation step). Alternatively, if this step is performed after the first culture step, mesenchymal stem cells containing the gene expression vector can be selected in the separation step described later using a cell sorter or similar device based on fluorescence detection, based on the gene encoding the fluorescent protein in the gene expression vector.

[0118] (Separation process)

[0119] In the method of the present invention, the "separation step" refers to the step of separating a single mesenchymal stem cell from the cell population described above after the recovery step.

[0120] In this specification, "isolation of a single mesenchymal stem cell" simply means isolating a single mesenchymal stem cell from other mesenchymal stem cells. It can be isolated from cell species other than mesenchymal stem cells, or it can be left unisolated. More preferably, the single mesenchymal stem cell is isolated from living cells other than mesenchymal stem cells (e.g., cells with proliferative capacity).

[0121] The separation method used in this process is not limited to any method capable of separating the mesenchymal stem cells (MSCs) after the aforementioned recovery process into individual cells. For example, by diluting the suspension containing MSCs and distributing them as one cell per well to a multi-well plate such as a 96-well plate, cells can be sorted individually in each well. When labeling MSCs with fluorescent substances or dyes, cells can be sorted individually using a flow cytometer or cell sorter that uses fluorescence or dye as an indicator. Labeling of MSCs can be achieved by introducing a marker gene (signature gene) into MSCs using a gene expression vector, or by binding an antibody labeled with a fluorescent substance to MSCs. Alternatively, micromanipulation devices or microfiltration apparatus can be used to sort cells individually.

[0122] In one embodiment, by labeling dead or live cells after the above-described recovery process, this step can separate only live cells. Examples of labeling methods include staining dead cells using 7-AAD (7-amino-radicin D) staining solution or PI (propidium iodide).

[0123] In the case of separating multiple single mesenchymal stem cells through this process, multiple single mesenchymal stem cells can be sorted in multiple wells of multiple culture containers or multi-well plates. The shape of the culture container or multi-well plate is not limited. For example, multiple cells can be separated as single cells by sorting single cells in each well of a multi-well plate such as a 6-well plate, 12-well plate, or 96-well plate.

[0124] (Second cultivation process)

[0125] In the method of the present invention, the "second culture step" refers to an optional step of culturing a single cell of mesenchymal stem cells obtained after the isolation step in a second culture medium that does not contain other mesenchymal stem cells.

[0126] The "second culture medium" used in this process is a culture medium for culturing single mesenchymal stem cells in single-cell form. In this second culture medium, single cells that have just undergone the separation process described above can be cultured.

[0127] In this specification, "cultured as a single cell" means culturing under conditions where only one cell is contained in the same culture medium. The second culture medium used in the second culture step does not contain any cells other than the mesenchymal stem cell obtained after the separation step.

[0128] The specific composition of the second culture medium is as described in the "Culture Medium" section above. For example, the second culture medium can be any of the following: basal medium, serum-free medium, low-serum medium, and serum-added medium. It is usually a basal medium (e.g., standard cell culture medium) or a serum-free medium, or it can be a medium obtained by adding albumin, blood-derived components, growth factors, or other components to the basal medium.

[0129] 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 can be 0.1 v / v% or higher, 0.2 v / v% or higher, 0.3 v / v% or higher, 0.4 v / v% or higher, 0.5 v / v% or higher, 0.6 v / v% or higher, 0.7 v / v% or higher, 0.8 v / v% or higher, 0.9 v / v% or higher, 1 v / v% or higher, 1.2 v / v% or higher, 1.5 v / v% or higher, 2 v / v% or higher, or 2.5 v / v% or higher. The concentrations of v / v% or higher, 3v / v% or higher, 3.5v / v% or higher, 4v / v% or higher, 4.5v / v% or higher, or 5v / v% or higher, and / or less than 40v / v%, less than 35v / v%, less than 30v / v%, less than 25v / v%, less than 20v / v%, less than 15v / v%, or less than 10v / v% can be any of the concentrations described above as converted to dry weight or the concentrations of human platelet lysate protein described above.

[0130] In another embodiment, the second culture medium does not contain human platelet lysate. For example, the second culture medium may be a basal culture medium supplemented with blood-derived components such as serum (fetal bovine serum (FBS, FCS, etc.), human serum) or a serum-free culture medium, depending on the circumstances. Thus, even if the second culture medium does not contain human platelet lysate, as long as the first culture medium contains human platelet lysate, single cells of mesenchymal stem cells with proliferative capacity can be obtained with high efficiency.

[0131] In one embodiment, the second culture medium contains an antibiotic. By culturing in the presence of this antibiotic, mesenchymal stem cells containing the gene encoding the antibiotic resistance protein introduced in the above-described introduction step can be selected.

[0132] There is no particular time limit from the cultivation in the first cultivation step mentioned above to the start of the cultivation in this step. The cultivation in this step can be carried out within, for example, within 24 hours, 18 hours, 12 hours, 6 hours, 4 hours, 3 hours, 2 hours, or 1 hour from the start of the cultivation in the first cultivation step.

[0133] 1-4. Effects

[0134] According to the isolation method of the present invention, by culturing mesenchymal stem cells in a culture medium containing hPL in a first culture step prior to the isolation step, single mesenchymal stem cells with proliferative capacity can be obtained with good efficiency.

[0135] 2. Method for manufacturing a population of mesenchymal stem cells derived from a single mesenchymal stem cell line.

[0136] 2-1. Overview

[0137] The second aspect of the present invention relates to a method for manufacturing a population of mesenchymal stem cells derived from a single mesenchymal stem cell (hereinafter, sometimes simply referred to as the "manufacturing method"). This manufacturing method includes: manufacturing a single cell by isolating a single mesenchymal stem cell using the isolation method described in the first aspect above, thereby producing a single mesenchymal stem cell or a cell population composed of multiple mesenchymal stem cells derived from a single mesenchymal stem cell with good efficiency.

[0138] 2-2. Methods

[0139] The manufacturing method of this embodiment includes: manufacturing a single mesenchymal stem cell using the method described in the first embodiment above. Therefore, another manufacturing method of this embodiment is also provided, which includes the first culture step, recovery step, and separation step described above as essential steps, and may include the repeating step, introduction step, and / or second culture step described above as optional steps. The structure of each step is the same as in the first embodiment, therefore detailed descriptions are omitted here.

[0140] In one embodiment, the manufacturing method of this method can be achieved by using the method described in the first method, which includes the second culture step described above, to proliferate a single mesenchymal stem cell in the second culture step, thereby creating a cell population composed of multiple mesenchymal stem cells.

[0141] According to the present invention, a single mesenchymal stem cell manufactured by the manufacturing method of this embodiment is also provided, a culture medium containing the single mesenchymal stem cell, a culture supernatant of the single mesenchymal stem cell, and a cell population containing the single mesenchymal stem cell manufactured by the manufacturing method of this embodiment, a culture medium containing the cell population, and a culture supernatant of the cell population.

[0142] In addition, a method is provided for evaluating mesenchymal stem cells into which any exogenous gene has been introduced, using a single mesenchymal stem cell manufactured by the method of this invention, or a cell population containing a single mesenchymal stem cell manufactured by the method of this invention.

[0143] 3. Methods for culturing single mesenchymal stem cells

[0144] 3-1. Overview

[0145] A third aspect of the present invention relates to a method for culturing a single mesenchymal stem cell line (hereinafter, sometimes simply referred to as a "culturing method"). This culturing method is characterized by comprising: a first culturing step of culturing a cell population containing mesenchymal stem cells in a first culture medium; a recovery step of recovering the cell population; a separation step of separating the single mesenchymal stem cell line; and a second culturing step of culturing the separated single mesenchymal stem cell line in a second culture medium containing no other mesenchymal stem cells, wherein the second culture medium contains human platelet lysate. In this culturing method, the single mesenchymal stem cell line can proliferate at a good efficiency, and a highly homogeneous mesenchymal stem cell population can be produced with good efficiency.

[0146] 3-2. Methods

[0147] The cultivation method of this approach includes a first cultivation step, a recovery step, a separation step, and a second cultivation step as required steps, and may include a repeating step and / or an introduction step as optional steps.

[0148] The structure of each step in this method is the same as that in Method 1. However, in the culture method of this method, the first culture medium used in the first culture step may or may not contain human platelet lysate, and its structure other than human platelet lysate is the same as that of the first culture medium in Method 1. For example, the first culture medium used in the first culture step of this method may be constructed with reference to the second culture medium in Method 1.

[0149] Furthermore, in this method of cultivation, the second culture medium used in the second cultivation step uses human platelet lysate as an essential component. It is a culture medium in which human platelet lysate is added at any concentration, and the composition other than human platelet lysate is the same as that of the second culture medium in the first method. For example, the second culture medium used in the second cultivation step of this method of cultivation can be constructed with reference to the first culture medium in the first method.

[0150] In other aspects of the present invention, a method for isolating and culturing a single mesenchymal stem cell line is provided. The method includes: a first culture step of culturing a cell population in a first culture medium; a recovery step of recovering the cell population after the first culture step; an isolation step of isolating a single mesenchymal stem cell line from the cell population after the recovery step; and a second culture step of culturing the isolated single mesenchymal stem cell line in a second culture medium free of other mesenchymal stem cells, wherein the first culture medium and / or the second culture medium (e.g., both the first and second culture media, or either the first or second culture medium) contain human platelet lysate. More preferably, a method is provided in which both the first and second culture media contain human platelet lysate.

[0151] Example

[0152] The present invention will now be specifically described through embodiments. It should be noted that these embodiments are for illustrative purposes only and do not limit the scope of the invention.

[0153] <Example 1: Isolation of single cells after FBS culture or hPL culture>

[0154] (Purpose)

[0155] Mesenchymal stem cells derived from bone marrow or amnion were cultured in a medium containing fetal bovine serum (FBS) or human platelet lysate (hPL). Cultured cells were separated using a cell sorter, and single cells were cultured in each well of a 96-well plate. The number of wells containing proliferating cells was determined.

[0156] (method)

[0157] (1) FBS culture of bone marrow mesenchymal stem cells

[0158] Bone marrow fluid was collected from adult donors with informed consent. Alpha-MEM (Alpha Modification of Minimum Essential Medium Eagle) containing 10% fetal bovine serum (FBS (Moregate, 599-04425)) was mixed with the bone marrow fluid at a 5:1 ratio and seeded into culture vessels. The culture medium was then changed every 3–4 days using the FBS-containing medium, while the bone marrow mesenchymal stem cells were cultured adherently until subconfluence. The method of culturing in the FBS-containing medium will be referred to below as "FBS culture." Passaging of the bone marrow mesenchymal stem cells was performed under the same conditions as needed.

[0159] (2) hPL culture of bone marrow mesenchymal stem cells

[0160] Bone marrow fluid was collected from adult donors with informed consent. αMEM containing 5% human platelet lysate (hPL (AventaCell, HPCFDCGLI50)) was mixed with the bone marrow fluid at a 5:1 ratio and seeded into culture vessels. The culture medium was then changed every 3–4 days using the hPL-containing medium, while the bone marrow mesenchymal stem cells were cultured adherently until subconfluence. The method of culturing in the hPL-containing medium will be referred to below as "hPL culture." Passaging of the bone marrow mesenchymal stem cells was performed under the same conditions as needed.

[0161] (3) FBS culture of amniotic mesenchymal stem cells

[0162] Amniotic membranes and placenta, which are fetal appendages, were aseptically collected from pregnant women undergoing elective cesarean sections with informed consent. The obtained membranes and placenta were placed in a sterile basin containing physiological saline, and the amniotic membrane was manually detached from its broken ends. The amniotic membrane was then washed with Hank's balanced salt solution (containing no Ca or Mg) to remove any attached blood and clots.

[0163] Amniotic membrane, comprising the epithelial cell layer and the mesenchymal stem cell layer, was immersed in Hank's balanced salt solution (containing Ca and Mg) containing 480 PU / mL collagenase and 400 PU / mL dispersin I, and subjected to enzymatic treatment by shaking and stirring at 50 rpm for 90 minutes at 37°C. The enzyme-treated solution was then filtered through a 95 μm nylon sieve to remove undigested amniotic membrane material, and the cell suspension containing amniotic mesenchymal stem cells was recovered.

[0164] At 6000 cells / cm 2 The cell population containing amniotic mesenchymal stem cells was seeded into a culture vessel at the specified seeding density. After seeding, the cells were cultured in αMEM containing a final concentration of 10% FBS until subconfluence. Subsequently, as needed, the amniotic mesenchymal stem cells were passaged under the same conditions.

[0165] (4) hPL culture of amniotic mesenchymal stem cells

[0166] Amniotic membranes and placenta, which are fetal appendages, were aseptically collected from pregnant women undergoing elective cesarean sections with informed consent. The obtained membranes and placenta were placed in a sterile basin containing physiological saline, and the amniotic membrane was manually detached from its broken ends. The amniotic membrane was then washed with Hank's balanced salt solution (containing no Ca or Mg) to remove any attached blood and clots.

[0167] Amniotic membrane containing both epithelial cell and mesenchymal stem cell layers was immersed in Hank's balanced salt solution (containing Ca and Mg) at 480 PU / mL collagenase and 400 PU / mL dispersin I. The amniotic membrane was enzymatically treated by stirring at 10 rpm for 60 minutes at 37°C. The enzymatically treated solution was then filtered through a 95 μm nylon sieve to remove undigested amniotic membrane material, and the cell suspension containing amniotic mesenchymal stem cells was recovered.

[0168] At 1400 cells / cm 2The aforementioned cell population containing amniotic mesenchymal stem cells was seeded into a culture vessel at the specified seeding density. After seeding, the cells were cultured in αMEM containing a final concentration of 5% hPL until sub-confluence. Once sub-confluence was achieved, the cells were cultured at a density of 1000 cells / cm². 2 Amniotic mesenchymal stem cells were passaged at the appropriate seeding density and cultured to the subconfluence stage. Subsequently, as needed, amniotic mesenchymal stem cells were passaged under the same conditions.

[0169] (5) Single-cell cloning

[0170] The various mesenchymal stem cells cultured in (1) to (4) above were dissected using TrypLE (trademark) Select Enzyme (1X) and passed through phosphate-buffered saline containing 0.5% bovine serum albumin at a concentration of 1×10⁻⁶ mg / L. 6 Cell concentrations were prepared at cells / mL. Dead cells were stained with 7-AAD (7-amino-radicin D) staining solution added to the cell suspension.

[0171] Using the cell suspensions described above as test samples, a cell sorter MA900 was used to sort each mesenchymal stem cell line into five 96-well plates, selecting only live cells (7-AAD negative fraction) at a rate of 1 unit / well. After sorting, the mesenchymal stem cells added to each well were cultured in a medium containing human platelet lysate. Thirteen days after sorting, all wells were stained with crystal violet, and the wells containing proliferated cells were counted. Figure 1 ).

[0172] (result)

[0173] The number of wells in which cell proliferation was confirmed under each condition was determined, and the results are presented in [the table / data]. Figure 2 .

[0174] Under conditions where bone marrow mesenchymal stem cells (MSCs in the figure) were cultured in hPL followed by single-cell sorting and further culture, cell proliferation in the wells was confirmed to be more than twice that under conditions where single-cell sorting was performed after FBS culture and further culture was performed.

[0175] Furthermore, under the condition of FBS culture followed by single-cell sorting and further culture of amniotic mesenchymal stem cells (MSCs in the figure), no wells showing cell proliferation were identified. In contrast, under the condition of hPL culture followed by single-cell sorting and further culture, cell proliferation was identified in nearly half of the wells.

[0176] The results above indicate that single cells of mesenchymal stem cells with proliferative capacity were obtained with high efficiency by culturing in a medium containing hPL before single-cell sorting.

[0177] <Example 2: Long-term passage culture of mesenchymal stem cells using FBS>

[0178] (Purpose)

[0179] Bone marrow mesenchymal stem cells and amniotic mesenchymal stem cells were not isolated as single cells but passaged for extended periods. This demonstrated that mesenchymal stem cells exhibited proliferative activity in a medium containing FBS.

[0180] (Methods and Results)

[0181] The bone marrow mesenchymal stem cells obtained in Example 1 (1) were repeatedly passaged in αMEM containing a final concentration of 10% FBS until proliferation ceased.

[0182] In addition, the amniotic mesenchymal stem cells obtained in Example 1 (3) were repeatedly passaged and cultured in αMEM containing a final concentration of 10% FBS until proliferation stopped.

[0183] For bone marrow mesenchymal stem cells and amniotic mesenchymal stem cells, the cell number was measured and the doubling number was calculated before each passage during long-term culture. The results are presented as follows: Figure 3 Both bone marrow mesenchymal stem cells and amniotic mesenchymal stem cells showed a doubling time of 40-50 times. This result confirms that both bone marrow mesenchymal stem cells and amniotic mesenchymal stem cells exhibit normal proliferative capacity in FBS-containing culture medium.

[0184] In this embodiment, normal proliferation was confirmed when single cells were cultured for a long period in a medium containing FBS without isolation. Based on this result, it is clear that proliferation is not impaired even when cultured as a cell population, even in a medium containing FBS. Therefore, in Example 1 above, where single cells were isolated and cultured after FBS culture, amniotic mesenchymal stem cells showed no cell proliferation, and bone marrow mesenchymal stem cells exhibited low proliferation; this result is considered to be a phenomenon associated with single-cell culture.

[0185] All publications, patents and patent applications cited in this specification are incorporated herein by reference.

Claims

1. A method for isolating a single mesenchymal stem cell from a cell population containing mesenchymal stem cells, the method comprising: The first culture step involves culturing the cell population in a first culture medium containing human platelet lysate; The recycling process involves recycling the cell population obtained after the first culture process. as well as The separation process involves isolating a single mesenchymal stem cell from the cell population following the recovery process described above.

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

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

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

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

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

7. The method according to claim 1, wherein, The recovery process recovers the cell population from the first culture medium.

8. The method according to claim 1, wherein, After the recycling process, dead or live cells are marked, and only live cells are separated in the separation process.

9. The method of claim 1, further comprising: The introduction process involves introducing the gene expression vector into the cell population prior to the separation process.

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 introductory process is performed before the first culture process.

12. The method according to claim 10, wherein, The separation process uses the expression of the marker protein encoded by the marker gene as an indicator to separate the single mesenchymal stem cell expressing the marker protein.

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

14. The method according to any one of claims 1 to 13, further comprising: The second culture step involves culturing the single mesenchymal stem cell obtained after the isolation step in a second culture medium that does not contain other mesenchymal stem cells.

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

16. A method for manufacturing a single mesenchymal stem cell line, the method comprising: The single mesenchymal stem cell line is produced using the method of claim 1.

17. A method for producing a population of mesenchymal stem cells derived from a single mesenchymal stem cell line, the method comprising: The mesenchymal stem cell population is created by proliferating the single mesenchymal stem cell in the second culture step using the method of claim 14.

18. A method for culturing a single mesenchymal stem cell line, the method comprising: The first culture step involves culturing a cell population containing mesenchymal stem cells in a first culture medium. The recycling process involves recycling the cell population obtained after the first culture process. The separation process separates individual mesenchymal stem cells from the cell population following the recovery process; as well as The second culture step involves culturing the single mesenchymal stem cell obtained after the isolation step in a second culture medium that does not contain other mesenchymal stem cells. The second culture medium contains human platelet lysate.

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