Method for producing mesenchymal stem cells from a biological cell sample containing mesenchymal stem cells

Abstract

The present invention provides a method for producing mesenchymal stem cells from a living body-derived cell sample containing mesenchymal stem cells, the method comprising: (1) a step for culturing a living body-derived cell sample containing mesenchymal stem cells in a serum‐free medium in the presence of vitronectin or a partial peptide of the vitronectin capable of bonding mesenchymal stem cells; and (2) a step for collecting a cell aggregate of the mesenchymal stem cells.

Description

[Technical Field]

[0001] The present invention relates to a method for producing mesenchymal stem cells from a biologically derived cell sample containing mesenchymal stem cells, which comprises the step of culturing the biologically derived cell sample containing mesenchymal stem cells in a serum-free medium or a xenogeneic component-free medium in the presence of vitronectin or a partial peptide thereof capable of adhering mesenchymal stem cells. [Background technology]

[0002] In recent years, advances in pharmaceutical development and regenerative medicine using living cells or tissues have attracted considerable attention. Research using pluripotent embryonic stem cells (ESCs) and induced pluripotent stem cells (IPS) has accelerated as organ regeneration technologies and drug discovery screening tools. However, the generation of embryonic stem cells requires the destruction of embryos developed from fertilized eggs, raising ethical concerns. Although IPS cells are derived by reprogramming somatic cells and thus avoid the aforementioned issues, concerns remain about the potential for cancer cell development from IPS cells due to factors such as the use of c-MYC as a reprogramming factor, random gene transfer into chromosomes using retroviral vectors, and residual undifferentiated cells after differentiation. On the other hand, mesenchymal stem cells (MSCs) possess the pluripotency to differentiate into cells of multiple mesenchymal lineages (osteoblasts, adipocytes, and chondrocytes) as well as non-mesenchymal lineages (neural progenitor cells and hepatocytes). Because these stem cells lack the challenges inherent in embryonic stem cells and IPS cells, they are expected to serve as a cell source for regenerative medicine and cell therapy.

[0003] Mesenchymal stem cells can be produced not only from adult tissues such as bone marrow, adipose tissue, synovial membrane, alveolar bone, and periodontal ligament, but also from various tissues such as placenta, umbilical cord blood, and umbilical cord. Furthermore, they can be cultured and expanded ex vivo. Conventional methods for obtaining mesenchymal stem cells involve culturing bone marrow mononuclear cells in a fetal bovine serum (FBS)-containing medium, since bone marrow mononuclear cells contain small amounts of mesenchymal stem cells. The mesenchymal stem cells are then produced by utilizing their adhesive properties to culture vessels. However, when using mesenchymal stem cells as a cell source for regenerative medicine, contamination of the mesenchymal stem cells with heterologous components is inconvenient. Therefore, a method for culturing mesenchymal stem cells using a serum-free medium has been devised (Patent Document 1). However, in order to proliferate mesenchymal stem cells, they must be produced from bone marrow mononuclear cells containing mesenchymal stem cells. A method for efficiently producing mesenchymal stem cells from bone marrow mononuclear cells containing mesenchymal stem cells using a serum-free medium has yet to be developed. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2011 / 111787 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a method for efficiently producing mesenchymal stem cells from a biologically derived cell sample containing mesenchymal stem cells. [Means for solving the problem]

[0006] The present inventors conducted extensive research to achieve the above-mentioned objectives and successfully produced cell aggregates formed by mesenchymal stem cells adhering to the culture vessel via vitronectin when bone marrow mononuclear cells containing mesenchymal stem cells were cultured in serum-free medium on vitronectin-coated culture vessels. The resulting aggregates were dissociated to obtain single mesenchymal stem cell populations. The mesenchymal stem cells obtained in serum-free medium were then cultured again in the presence of vitronectin, and the resulting cell numbers were significantly higher than those obtained under the same conditions except that fibronectin was used instead of vitronectin. Furthermore, we confirmed that TGFβ receptor inhibitors further increased the production efficiency of mesenchymal stem cells. Furthermore, we found that mesenchymal stem cells could also be produced from adipocytes containing mesenchymal stem cells by using vitronectin and a TGFβ receptor inhibitor. These findings led to the completion of the present invention.

[0007] That is, the present invention is as follows. [1] A method for producing mesenchymal stem cells from a biological cell sample containing mesenchymal stem cells, comprising the steps of: (1) culturing a biologically derived cell sample containing mesenchymal stem cells in a serum-free medium in the presence of vitronectin or a partial peptide thereof capable of adhering mesenchymal stem cells; (2) A step of recovering cell aggregates of mesenchymal stem cells. [2] The method according to [1], wherein the culture in the presence of vitronectin or a partial peptide thereof capable of adhering mesenchymal stem cells is culture on a culture vessel to which vitronectin or a partial peptide thereof capable of adhering mesenchymal stem cells is solidified. [3] The method according to [1] or [2], further comprising the following steps: (3) dissociating the collected cell aggregates; (4) culturing the dissociated mesenchymal stem cells in a serum-free medium in the presence of an extracellular matrix protein or a partial peptide thereof capable of adhering mesenchymal stem cells; (5) A step of recovering mesenchymal stem cells proliferated on a culture vessel via an extracellular matrix protein or a partial peptide thereof capable of adhering mesenchymal stem cells. [4] The method according to [3], wherein the culturing in the presence of an extracellular matrix protein or a partial peptide thereof capable of adhering mesenchymal stem cells is culturing on a culture vessel onto which an extracellular matrix protein or a partial peptide thereof capable of adhering mesenchymal stem cells is immobilized. [5] The method according to any one of [1] to [4], wherein the partial peptide of vitronectin contains an RGD domain. [6] The method according to [5], wherein the partial peptide of vitronectin further comprises a somatomedin B domain. [7] The method according to [6], wherein the partial peptide of vitronectin is a polypeptide consisting of amino acids 1 to 379 of the amino acid sequence represented by SEQ ID NO: 1. [8] The method according to any one of [1] to [7], wherein the serum-free medium in step (1) contains a TGF-β receptor inhibitor. [9] A method for producing mesenchymal stem cells from a biological cell sample containing mesenchymal stem cells, comprising the steps of: (1) culturing a biological cell sample containing mesenchymal stem cells in a xenogeneic component-free medium in the presence of vitronectin or a partial peptide thereof capable of adhering mesenchymal stem cells; (2) A step of recovering cell aggregates of mesenchymal stem cells.

[10] The method according to [9], wherein the culture in the presence of vitronectin or a partial peptide thereof capable of adhering mesenchymal stem cells is culture on a culture vessel onto which vitronectin or a partial peptide thereof capable of adhering mesenchymal stem cells is solidified.

[11] The method according to [9] or

[10] , further comprising the steps of: (3) dissociating the collected cell aggregates; (4) culturing the dissociated mesenchymal stem cells in a xenogeneic component-free medium in the presence of an extracellular matrix protein or a partial peptide thereof capable of adhering mesenchymal stem cells; (5) A step of recovering mesenchymal stem cells proliferated on a culture vessel via an extracellular matrix protein or a partial peptide thereof capable of adhering mesenchymal stem cells.

[12] The method according to

[11] , wherein the culturing in the presence of an extracellular matrix protein or a partial peptide thereof capable of adhering mesenchymal stem cells is culturing on a culture vessel onto which an extracellular matrix protein or a partial peptide thereof capable of adhering mesenchymal stem cells is solidified.

[13] The method according to any one of [9] to

[12] , wherein the partial peptide of vitronectin contains an RGD domain.

[14] The method according to

[13] , wherein the partial peptide of vitronectin further comprises a somatomedin B domain.

[15] The method according to

[14] , wherein the partial peptide of vitronectin is a polypeptide consisting of amino acid numbers 1 to 379 of the amino acid sequence represented by SEQ ID NO: 1.

[16] The method according to any one of [9] to

[15] , wherein the xenogeneic component-free medium in step (1) contains a TGF-β receptor inhibitor.

[17] The method according to any one of [9] to

[16] , wherein the xenogeneic component-free medium contains allogeneic serum.

[18] The method according to

[17] , wherein the allogeneic serum is autologous serum.

[19] The method according to any one of [1] to

[18] , wherein the biological cell sample containing mesenchymal stem cells is bone marrow-derived cells.

[20] The number of bone marrow-derived cells cultured was 0.5 × 10 5 ~25×10 5 cells / cm 2 The method according to

[19] ,

[21] The method according to

[19] or

[20] , wherein the culture period of the bone marrow-derived cells is 4 to 14 days.

[22] The method according to any one of [1] to

[18] , wherein the biological cell sample containing mesenchymal stem cells is adipose tissue-derived cells.

[23] The number of adipose tissue-derived cells cultured was 1 × 10 3 ~1×10 6 cells / cm 2 The method according to

[22] .

[24] The method according to

[22] or

[23] , wherein the adipose tissue-derived cells are cultured for 1 to 14 days. [Effects of the Invention]

[0008] By culturing a biologically derived cell sample containing mesenchymal stem cells in a serum-free medium or a xenogeneic component-free medium in the presence of vitronectin or its partial peptide, mesenchymal stem cells can be efficiently produced from the biologically derived cell sample containing mesenchymal stem cells. By employing this method, the obtained mesenchymal stem cells can be used as a cell source for regenerative medicine. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows photographs of MSC aggregates 5 days after MNC seeding. [Figure 2] FIG. 1 shows the results of measuring the cell count 13 days after MNC seeding. [Figure 3] FIG. 1 shows photographs of cells 13 days after MNC seeding. [Figure 4] FIG. 1 shows the results of measuring the number of MSCs 13 days after MNC seeding when different vitronectins were used. [Figure 5] FIG. 1 shows the results of measuring the number of MSCs 12 days after seeding with MNCs when different vitronectins were used. [Figure 6] FIG. 1 shows the results of measuring the number of MSCs 15 days after MNC seeding when a TGFβ inhibitor was used. [Figure 7] FIG. 1 shows the results of measuring the number of MSCs 12 days after MNC seeding when different TGFβ receptor inhibitors were used. [Figure 8] FIG. 1 shows the results of measuring the cell number 5 days after seeding of cells isolated from mouse adipose tissue. [Figure 9] FIG. 1 shows photographs of cells isolated from mouse adipose tissue 5 days after seeding. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention provides a method for producing mesenchymal stem cells from a biologically derived cell sample containing mesenchymal stem cells (hereinafter referred to as the production method of the present invention).

[0011] As used herein, a biological cell sample containing mesenchymal stem cells refers to a cell sample isolated from biological tissue containing mesenchymal stem cells, such as bone marrow, adipose tissue, synovial membrane, alveolar bone, periodontal ligament, placenta, umbilical cord blood, and umbilical cord.

[0012] As used herein, a cell sample refers to a cell population contained in biological tissue. A cell population refers to two or more cells of the same or different types. It also refers to a mass of cells of the same or different types. The cell population may be primary cells directly isolated from biological tissue, or cells subcultured from primary cells. Here, "directly" means not via a process of ex vivo culture and / or proliferation.

[0013] As used herein, mesenchymal stem cells are somatic stem cells derived from mesodermal tissue (mesenchyme). Mesenchymal stem cells express positive markers on the cell surface but do not express negative markers. By detecting both markers on the cell surface, it is possible to determine whether a cell is a mesenchymal stem cell. Positive markers include CD73, CD90, and CD105. Negative markers include CD11b, CD14, CD19, CD34, CD45, CD79a, and HLA-Class II (DR). The expression of these markers can be examined by known immunological methods (e.g., flow cytometry using antibodies).

[0014] In one embodiment, the production method of the present invention includes the following steps. (1a) A step of culturing a biologically derived cell sample containing mesenchymal stem cells in a serum-free medium in the presence of vitronectin or a partial peptide thereof capable of adhering mesenchymal stem cells (step (1a) of the present invention). (2a) A step of recovering cell aggregates of mesenchymal stem cells (step (2a) of the present invention). In another embodiment, the production method of the present invention includes the following steps: (1b) A step of culturing a biological cell sample containing mesenchymal stem cells in a xenogeneic component-free medium in the presence of vitronectin or a partial peptide thereof capable of adhering mesenchymal stem cells (step (1b) of the present invention). (2b) A step of recovering cell aggregates of mesenchymal stem cells (step (2b) of the present invention).

[0015] In step (1a) or (1b) of the present invention, the culture is carried out in the presence of vitronectin or a partial peptide thereof capable of adhering mesenchymal stem cells (hereinafter referred to as a "partial peptide of vitronectin"). Vitronectin may be, for example, a protein isolated and purified from mammalian cells (e.g., human, mouse, rat, rabbit, sheep, pig, cow, horse, cat, dog, monkey, chimpanzee, etc.) or any tissue or organ in which such cells exist. Alternatively, it may be a protein chemically synthesized or biochemically synthesized in a cell-free translation system, or a recombinant protein produced from a transformant into which a nucleic acid having a nucleotide sequence encoding vitronectin has been introduced.

[0016] The amino acid sequence of vitronectin is disclosed in publicly known databases, such as NCBI Reference Sequence Nos. NP_000629 (human vitronectin) and NP_035837 (mouse vitronectin). Because mesenchymal stem cells produced by the production method of the present invention preferably do not contain xenogeneic components, the vitronectin is preferably derived from the organism from which the cell sample to be cultured is derived. Therefore, when the cell sample to be cultured is derived from a human, the vitronectin used in the production method of the present invention is preferably a protein containing an amino acid sequence identical or substantially identical to SEQ ID NO: 1.

[0017] An amino acid sequence that is substantially identical to the amino acid sequence represented by SEQ ID NO: 1 includes an amino acid sequence that has a homology of about 60% or more, preferably about 70% or more, more preferably about 80% or more, and particularly preferably about 90% or more with the amino acid sequence represented by SEQ ID NO: 1. Here, "homology" refers to the percentage (%) of identical and similar amino acid residues out of all overlapping amino acid residues in optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm can take into account the introduction of gaps into one or both of the sequences for optimal alignment). The amino acid sequence homology herein can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectation value = 10; gaps allowed; matrix = BLOSUM62; filtering = OFF). More preferably, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 is an amino acid sequence that has an identity of about 60% or more, preferably about 70% or more, even more preferably about 80% or more, and particularly preferably about 90% or more with the amino acid sequence represented by SEQ ID NO: 1.

[0018] A preferred example of a protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 is a protein that contains an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 and has substantially the same activity as a protein containing the amino acid sequence represented by SEQ ID NO: 1.

[0019] An example of a substantially equivalent activity is mesenchymal stem cell adhesion activity. "Substantially the same" means that the activity is qualitatively (e.g., physiologically or pharmacologically) the same. Therefore, it is preferable that the mesenchymal stem cell adhesion activity is equivalent (e.g., about 0.5 to about 2 times), but the level of these activities and quantitative factors such as the molecular weight of the protein may differ.

[0020] Human vitronectin also includes, for example, (1) an amino acid sequence in which one or two or more (preferably about 1 to 10) amino acids are deleted from the amino acid sequence represented by SEQ ID NO: 1; (2) an amino acid sequence in which one or two or more (preferably about 1 to 10) amino acids are added to the amino acid sequence represented by SEQ ID NO: 1; (3) an amino acid sequence in which one or two or more (preferably about 1 to 10) amino acids are inserted into the amino acid sequence represented by SEQ ID NO: 1; (4) an amino acid sequence in which one or two or more (preferably about 1 to 10) amino acids in the amino acid sequence represented by SEQ ID NO: 1 are substituted with other amino acids; or (5) proteins containing amino acid sequences that are a combination of these. When an amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited as long as the activity of the protein is maintained.

[0021] The partial peptide of vitronectin may be any peptide having the partial amino acid sequence of vitronectin described above and having substantially the same activity as vitronectin. Here, "substantially the same activity" has the same meaning as above. Furthermore, "substantially the same activity" can be measured in the same manner as in the case of vitronectin. Such a partial peptide of vitronectin includes a protein containing an RGD domain. More preferably, the partial peptide of vitronectin is a protein containing a somatomedin B domain and an RGD domain. Specifically, the somatomedin B domain may be, for example, the region of amino acids 1 to 40 in the amino acid sequence represented by SEQ ID NO: 1. The RGD domain may be, for example, the region of amino acids 41 to 52 in the amino acid sequence represented by SEQ ID NO: 1. The size of the vitronectin partial peptide is not particularly limited as long as it has mesenchymal stem cell adhesive activity. Preferably, the partial peptide contains a partial amino acid sequence of 100 or more amino acids, more preferably a partial amino acid sequence of 200 or more amino acids, and even more preferably a partial amino acid sequence of 300 or more amino acids. The partial amino acid sequence may be a single continuous partial amino acid sequence, or a plurality of discontinuous partial amino acid sequences linked together. The most preferred partial peptide of vitronectin that satisfies these conditions is a polypeptide consisting of amino acids 1 to 379 in the amino acid sequence represented by SEQ ID NO: 1. Alternatively, commercially available vitronectin partial peptides may be used, such as Vitronectin (20-398 aa) (Wako), Vitronectin (VTN-N, 62-478 aa) (Thermo Fisher Scientific), Vitronectin (Full length, 20-478 aa) (Sigma), and Synthemax II (Corning Incorporated).

[0022] In step (1a) or (1b) of the present invention, the culture of a biologically derived cell sample containing mesenchymal stem cells in the presence of vitronectin or a vitronectin partial peptide may be carried out by any method that brings mesenchymal stem cells into contact with vitronectin or a vitronectin partial peptide. For example, culture may be carried out in a state in which vitronectin or a vitronectin partial peptide is present either in the culture medium or on the surface of the culture vessel. The presence of vitronectin or a vitronectin partial peptide in the culture medium refers to an embodiment in which the vitronectin or vitronectin partial peptide is directly contained in the culture medium. When vitronectin or a vitronectin partial peptide is contained in the culture medium, the concentration of the vitronectin or vitronectin partial peptide in the culture medium is 0.1 μg / ml to 4.0 μg / ml, preferably 1.0 μg / ml to 4.0 μg / ml.

[0023] Furthermore, the presence of vitronectin or a partial peptide of vitronectin on the surface of a culture vessel refers to an embodiment in which vitronectin or a partial peptide of vitronectin is immobilized on the surface of the culture vessel. When vitronectin or a partial peptide of vitronectin is immobilized on the surface of the culture vessel, for example, a vessel or carrier (e.g., microbeads) used for cell culture can be used as the culture vessel. Any material or shape can be used for the culture vessel as long as it does not inhibit cell maintenance, survival, differentiation, maturation, or self-replication. Examples of materials for the culture vessel include glass, synthetic resins including nonwoven fabrics, natural resins, and metals. The shape of the culture vessel can also be any shape, such as a polygonal prism (e.g., triangular prism, cube, or rectangular parallelepiped), a cylinder, a polygonal pyramid (e.g., triangular pyramid, square pyramid), a cone, or a gourd, as well as a sphere, hemisphere, circle, oval, or semicircle. Commercially available culture flasks, culture dishes, culture bags, hollow fiber culture devices, and the like can also be used. Gas-permeable culture bags are preferred. When a large number of cells are required, a large culture tank may be used. The culture can be carried out in either an open system or a closed system, but when the obtained mesenchymal stem cells are intended for administration to humans, it is preferable to carry out the culture in a closed system.

[0024] Immobilization of vitronectin or a vitronectin partial peptide on a culture vessel can be carried out by known methods. For example, vitronectin or a vitronectin partial peptide can be immobilized on a culture vessel by dissolving the vitronectin or vitronectin partial peptide in a solvent (e.g., sterile distilled water, buffer solution, or physiological saline), adding the solution to the culture vessel, and allowing it to stand overnight at 4°C. The concentration of the vitronectin or vitronectin partial peptide solution used for immobilizing the vitronectin or vitronectin partial peptide on a culture vessel can be determined appropriately by those skilled in the art. For example, the concentration can be set so that 0.5 μg to 10.0 μg of vitronectin or vitronectin partial peptide is typically immobilized per unit area of ​​the culture vessel.

[0025] Culture vessels on which vitronectin or a partial peptide of vitronectin has been immobilized can be stored at low temperatures, for example, at 4° C. Immediately before use, the solution containing vitronectin or a partial peptide of vitronectin is removed by suction from the culture vessels, and the vessels are washed once with PBS and then once with culture medium before use.

[0026] In step (1a) of the present invention, the serum-free medium is not particularly limited as long as it does not contain serum. Therefore, as long as the serum-free medium does not contain serum, it may contain components derived from the same species (allogeneic components) as the species from which the biological cell sample containing mesenchymal stem cells to be cultured is derived, or components derived from a different species (xenogeneic components). Examples of allogeneic components include platelet lysate and serum-derived proteins (e.g., albumin). Examples of xenogeneic components include animal-derived lipids.

[0027] In step (1b) of the present invention, the xenogeneic component-free medium is not particularly limited as long as it does not contain xenogeneic components. Therefore, the xenogeneic component-free medium may contain allogeneic serum as long as it does not contain xenogeneic components. The allogeneic serum is preferably autologous serum. Here, autologous serum and autologous plasma, which will be described later, refer to serum and plasma obtained from blood collected from the same donor as the biological cell sample to be cultured, respectively.

[0028] Since plasma contains serum components, a medium containing autologous plasma may be used. Preferably, inactivated autologous plasma is added to the medium. For example, cells are cultured in a culture medium containing 10% (V / V) or less, preferably 5% (V / V) or less, and more preferably 2% (V / V) or less of inactivated autologous plasma. By using autologous plasma, xenogeneic components are excluded from the production method of the present invention, providing a highly safe method for producing mesenchymal stem cells.

[0029] Serum-free or xenogeneic component-free media can be prepared using media typically used for culturing animal cells as basal media, such as Dulbecco's medium (e.g., IMDM), Eagle's medium (e.g., DMEM, EMEM, BME, MEM, αMEM), Ham's medium (e.g., F10 medium, F12 medium), RPMI medium (e.g., RPMI-1640 medium, RPMI-1630 medium), MCDB medium (e.g., MCDB104, 107, 131, 151, 153 medium), Fischer's medium, 199 medium, primate ES cell medium (primate ES / iPS cell culture medium, ReproCell), mouse ES cell medium (TX-WES culture medium, ThromboX), serum-free medium (mTeSR, Stemcell), and others. Examples of suitable media include, but are not limited to, ESF-B medium, ESF-C medium, CSTI-7 medium, Neurobasal medium (Life Technologies), StemPro-34 medium, and StemFit (registered trademark) (e.g., StemFit AK03N, StemFit AK02N). These media can be mixed and used as needed, for example, to produce DMEM / F12 medium. Furthermore, known or commercially available media may be used as serum-free or xeno-free media, either directly or after modification. Examples of commercially available xeno-free media include DEF-CS500 XF (Cellartis) and DXF (PromoCell).

[0030] In step (1a) or (1b) of the present invention, the serum-free medium or xenogeneic component-free medium may contain a TGF-β receptor inhibitor. TGF-β is a peptide factor secreted in an inactive form by almost all normal cells, activated under specific conditions, and exhibiting various functions, such as inhibiting the proliferation of epithelial cells and lymphocytes. Furthermore, examples of TGF-β family molecules with structures similar to TGF-β include bone morphogenetic proteins (BMPs) that induce osteoblast differentiation and activins that promote the secretion of follicle-stimulating hormone and the differentiation of erythrocytes. As used herein, TGF-β also includes TGF-β family molecules. Specifically, examples of TGF-β include TGF-β, activins, Nodal, BMPs, growth / differentiation factors (GDFs), anti-Mollerian hormones (AMHs), and Mullerian inhibitory substances (MISs), with TGF-β being preferred. TGF-β receptors consist of type I and type II receptors present on the cell membrane. Both type I and type II receptors have serine / threonine kinase activity, and the substrate of type II receptor is type I receptor. When TGF-β family molecules bind to the TGF-β receptor, type II receptor phosphorylates type I receptor, and the activated type I receptor further phosphorylates Smad, an intracellular signaling molecule, and transmits a signal into the cell. Specifically, combinations of type I and type II TGF-β receptors include, for TGF-β, the combination of TGF-β type I receptor (TGFBR1, activin-like receptor kinase (ALK5)) or ALK1 and TGF-β type II receptor (TGFBR2); for activin and Nodal, the combination of ALK4 or ALK7 and ActR-II or ActR-IIB; for BMP, the combination of ALK2, ALK3, or ALK6 and BMPR-II; for GDF, the combination of ALK2, ALK3, or ALK6 and ActR-II or ActR-IIB; and for AMH or MIS, the combination of ALK2, ALK3, or ALK6 and ActR-II or ActR-IIB.A preferred combination of type I and type II TGF-β receptors is the combination of ALK5 or ALK1 and TGFBR2. The inhibitor of the TGF-β receptor may be any substance as long as it suppresses the above-mentioned function of the TGF-β receptor, and examples thereof include substances that inhibit the formation of a complex between TGF-β and the TGF-β receptor.

[0031] Specific examples of TGF-β receptor inhibitors include neutralizing antibodies against TGF-β receptors. The antibodies may be either polyclonal or monoclonal. These antibodies can be produced according to publicly known methods for producing antibodies or antisera. The antibody isotype is not particularly limited, but is preferably IgG, IgM, or IgA, with IgG being particularly preferred. Furthermore, the antibody is not particularly limited as long as it has at least a complementarity-determining region (CDR) for specifically recognizing and binding to a target antigen. It may be a complete antibody molecule, or a fragment such as Fab, Fab', or F(ab')2; a genetically engineered conjugate molecule such as scFv, scFv-Fc, a minibody, or a diabody; or a derivative thereof modified with a molecule having a protein-stabilizing effect, such as polyethylene glycol (PEG). Since the neutralizing antibody is contained in a serum-free medium or a xenogeneic component-free medium, when the biological cell sample is of human origin, it is preferable to obtain a human antibody by (i) immunizing a human antibody-producing animal (e.g., a mouse), (ii) producing a chimeric antibody, a humanized antibody, or a fully human antibody, or (iii) combining an in vitro immunization method with viral cell immortalization, human-human (or mouse) hybridoma production technology, phage display, etc. The concentration of the neutralizing antibody against the TGF-β receptor in the serum-free medium or xenogeneic component-free medium is not limited as long as it is a concentration that can inhibit intracellular signal transduction of the TGF-β receptor, and is, for example, 0.01 μg / mL to 10 μg / mL, preferably 0.05 μg / mL to 5 μg / mL, and more preferably 0.1 μg / mL to 2.5 μg / mL.

[0032] In another preferred embodiment, the TGF-β receptor inhibitor is a small molecule compound that exhibits antagonist activity against the TGF-β receptor, where "antagonist activity" refers to the activity of binding to the TGF-β receptor and inhibiting the binding of TGF-β to the TGF-β receptor. Examples of such compounds include SB431542 (Stemgent), sc-203294, RepSox, Vactosertib (TEW-7197), SB525334, GW788388, SB505124, SD-208, LDN-193189, Galunisertib (LY2157299), LY2109761, LY364947, K02288, LDN-214117, ML347, LDN-212854, DMH1, Pirfenidone, LY 3200882, Alantolactone, SIS3, Hesperetin, and A-83-01. The concentration of a low molecular weight compound that exhibits antagonist activity against TGF-β receptor in a serum-free medium or a heterologous component-free medium is not limited as long as it is a concentration that can inhibit intracellular signal transduction of the TGF-β receptor, and is, for example, 0.1 μM to 100 μM, preferably 1 μM to 50 μM, and more preferably 5 μM to 25 μM.

[0033] Serum-free media or xenogeneic component-free media may further contain, as appropriate, insulin, transferrin, selenium, various vitamins, L-glutamine, various amino acids such as non-essential amino acids, 2-mercaptoethanol, various cytokines (interleukins (IL-2, IL-7, IL-15, etc.), stem cell factor (SCF), activin, etc.), various hormones, various growth factors (leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), etc.), antibiotics such as penicillin / streptomycin and puromycin, pH indicators such as phenol red, and the like.

[0034] The biologically derived cell sample containing mesenchymal stem cells cultured in step (1a) or (1b) of the present invention is not particularly limited as long as it contains mesenchymal stem cells. In one embodiment of the present invention, the biologically derived cell sample containing mesenchymal stem cells is bone marrow-derived cells. Bone marrow-derived cells can be separated from bone marrow by known methods. For example, they can be separated by density gradient centrifugation using a density-adjusted separation medium to remove interstitial cells from collected bone marrow fluid. Specifically, bone marrow fluid diluted with saline is layered on top of the separation medium in a tube and centrifuged, resulting in a layer of bone marrow-derived cells containing mesenchymal stem cells and mononuclear cells at the interface between the separation medium and the bone marrow fluid. The bone marrow-derived cells thus obtained contain a trace amount of mesenchymal stem cells. The proportion of mesenchymal stem cells in the bone marrow-derived cells is not particularly limited, but is approximately 0.01% to 1%, preferably approximately 0.01% to 0.1%, of the total number of bone marrow-derived cells.

[0035] The number of bone marrow-derived cells, including mesenchymal stem cells, to be cultured in step (1a) or (1b) of the present invention is not particularly limited, but is usually 0.5 × 10 5 cells / cm 2 ~25×10 5 cells / cm 2 may be 2 x 10 5 cells / cm 2 ~13×10 5 cells / cm 2 is preferred.

[0036] The culture conditions for bone marrow-derived cells, including mesenchymal stem cells, are not particularly limited, and conventional cell culture conditions can be used. Examples of such culture conditions include a temperature of 37°C, humidity of 95%, and a CO2 concentration of 5%, but the present invention is not limited to these conditions. For example, culture at a temperature of 30-40°C, humidity of 90-98%, and a CO2 concentration of 3-7% is exemplified. However, the temperature, humidity, and CO2 concentration may be outside of these ranges as long as the desired cell proliferation is achieved.

[0037] It is preferable to change the medium at appropriate intervals during culture. Medium changes include complete replacement of the medium, partial replacement of the medium, addition of medium, and combinations thereof. In a preferred embodiment of the present invention, the medium is completely replaced with a medium of the same composition the day after the start of culture, and then 20% of the medium is added on the third and fifth days after the start of culture.

[0038] The culture period is, for example, 4 to 14 days, preferably 7 days. This culture allows mesenchymal stem cells contained in bone marrow-derived cells to selectively adhere to the culture vessel via vitronectin or a partial peptide of vitronectin. The adhered mesenchymal stem cells form cell aggregates. Here, cell aggregates encompass cell populations that grow parallel to the adhesive surface of the culture vessel, cell populations that grow perpendicularly to the adhesive surface of the culture vessel, and cell populations that share characteristics of both. If the culture period is shorter than 4 days, the number of cell aggregates formed is too small to ensure the number of cells required for culturing in step (4) of the present invention, as described below. Furthermore, if the culture period exceeds 14 days, the cell aggregates collapse, resulting in a decrease in the number of cells, making it impossible to ensure the number of cells required for culturing in step (4) of the present invention.

[0039] In another embodiment, the biologically derived cell sample containing mesenchymal stem cells cultured in step (1a) or (1b) of the present invention is adipose tissue-derived cells. Adipose tissue-derived cells may be isolated from adipose tissue by known methods. For example, adipose tissue-derived cells containing mesenchymal stem cells can be isolated from adipose tissue by shredding collected adipose tissue, incubating the tissue in a collagenase solution, and filtering through a mesh sheet. The adipose tissue-derived cells thus obtained contain a trace amount of mesenchymal stem cells. The proportion of mesenchymal stem cells contained in the adipose tissue-derived cells is not particularly limited, but is approximately 0.01% to approximately 1%, preferably approximately 0.1% to approximately 1%, of the total number of adipose tissue-derived cells.

[0040] The number of adipose tissue-derived cells containing mesenchymal stem cells to be cultured in step (1a) or (1b) of the present invention is not particularly limited, but is usually 1×10 3 cells / cm 2 ~1×10 6 cells / cm 2 may be 1 x 10 4 cells / cm 2 ~1×10 5 cells / cm 2 is preferred.

[0041] There are no particular limitations on the culture conditions for adipose tissue-derived cells including mesenchymal stem cells, and culture conditions similar to those for bone marrow-derived cells including mesenchymal stem cells can be used.

[0042] It is preferable to change the medium at appropriate intervals during the culture. The change of the medium may be a complete change of the medium, a partial change of the medium, addition of the medium, or a combination thereof. In a preferred embodiment of the present invention, the culture is carried out while changing the entire medium with a medium of the same composition on the day after and on the second day after the start of the culture.

[0043] The culture period is, for example, 1 to 14 days, preferably 5 days. This culture allows mesenchymal stem cells contained in adipose tissue-derived cells to selectively adhere to the culture vessel via vitronectin or a partial peptide of vitronectin. The adhered mesenchymal stem cells form cell aggregates, particularly cell populations that proliferate and spread parallel to the adhesion surface of the culture vessel.

[0044] In step (2a) or (2b) of the present invention, cell aggregates formed by mesenchymal stem cells adhered to a culture vessel via vitronectin or a partial peptide of vitronectin are recovered by known means. For example, cells other than mesenchymal stem cells contained in a biological cell sample do not adhere to the culture vessel via vitronectin or a partial peptide of vitronectin, and are therefore removed from the culture vessel along with the serum-free or xenogeneic component-free medium by a complete medium change. As a result, mesenchymal stem cell aggregates remain in the culture vessel during culture after a complete medium change. Although mesenchymal stem cell aggregates adhere to the culture vessel via vitronectin or a partial peptide of vitronectin, the cell aggregates exhibit weak intercellular adhesion and are easily separated from the cell aggregates and float in the medium. Therefore, recovery of mesenchymal stem cell aggregates may include two steps: (i) recovery of mesenchymal stem cell aggregates floating in serum-free or xenogeneic component-free medium, and (ii) recovery of mesenchymal stem cell aggregates adhered to the culture vessel. (i) Collection of mesenchymal stem cell aggregates suspended in serum-free or xenogeneic component-free medium can be achieved, for example, by recovering the entire serum-free or xenogeneic component-free medium and centrifuging it. (ii) Collection of mesenchymal stem cell aggregates adhered to a culture vessel can be achieved, for example, by simply pipetting them off the culture vessel, recovering the entire volume together with the medium and PBS, and centrifuging them. In another embodiment, collection of mesenchymal stem cell aggregates adhered to a culture vessel can be achieved by treating them with a detachment agent to break down the adhesion between vitronectin or a partial peptide of vitronectin and the mesenchymal stem cell aggregates, thereby recovering single-celled mesenchymal stem cells. The detachment agent can be a mixture of trypsin and EDTA (usually 0.001-0.5% trypsin / 0.1-5 mM EDTA, preferably approximately 0.1% trypsin / 1 mM EDTA), or a commercially available product (e.g., TrypLE (Thermo Fisher Scientific)).

[0045] Vitronectin or a partial peptide of vitronectin has a higher adhesive activity for mesenchymal stem cells than other extracellular matrices and can efficiently adhere mesenchymal stem cells contained in a biological cell sample. As a result, mesenchymal stem cells can be efficiently produced from a biological cell sample as described above. However, when cell aggregates of mesenchymal stem cells adhered in step (1a) or (1b) of the present invention are continuously cultured for a long period of time, proliferation of mesenchymal stem cells is not observed, depending on the origin of the cell sample. For example, when bone marrow-derived cells are cultured in step (1a) or (1b) of the present invention and the adhered mesenchymal stem cell aggregates are continuously cultured for a long period of time, proliferation of mesenchymal stem cells is not observed. On the other hand, when adipose tissue-derived cells are cultured in step (1a) or (1b) of the present invention and the adhered mesenchymal stem cell aggregates are confirmed to proliferate. This is thought to be due to the presence of cells in the bone marrow-derived cells that inhibit the proliferation of mesenchymal stem cells. Therefore, when it is desired to obtain a large amount of mesenchymal stem cells produced from a biological cell sample containing mesenchymal stem cells by steps (1a) and (2a) or steps (1b) and (2b) of the present invention, it is preferable to replated, proliferate, and recover the produced mesenchymal stem cells again. Therefore, the production method of the present invention may further include the following steps. That is, the production method of the present invention including steps (1a) and (2a) may further include the following steps. (3a) A step of dissociating the collected cell aggregates (step (3a) of the present invention). (4a) A step of culturing dissociated mesenchymal stem cells in a serum-free medium in the presence of an extracellular matrix protein or a partial peptide thereof capable of adhering mesenchymal stem cells (step (4a) of the present invention). (5a) A step of recovering mesenchymal stem cells proliferated on a culture vessel via an extracellular matrix protein or a partial peptide thereof capable of adhering mesenchymal stem cells (step (5a) of the present invention). Furthermore, the production method of the present invention including steps (1b) and (2b) may further include the following steps. (3b) A step of dissociating the collected cell aggregates (step (3b) of the present invention). (4b) A step of culturing dissociated mesenchymal stem cells in a xenogeneic component-free medium in the presence of an extracellular matrix protein or a partial peptide thereof capable of adhering mesenchymal stem cells (step (4b) of the present invention). (5b) A step of recovering mesenchymal stem cells proliferated on a culture vessel via an extracellular matrix protein or a partial peptide thereof capable of adhering mesenchymal stem cells (step (5b) of the present invention).

[0046] In step (3a) or (3b) of the present invention, the recovered cell aggregates are dissociated by known means. Because cell-to-cell adhesion in cell aggregates is weak, the intercellular adhesion of the cell aggregates can be easily broken down by, for example, simple pipetting, to prepare a single cell population of mesenchymal stem cells. Alternatively, dissociation can be performed by treating with the above-mentioned detachment agent.

[0047] In step (4a) or (4b) of the present invention, the culture vessel, serum-free medium, xenogeneic component-free medium used for the culture, and the manner in which the mesenchymal stem cells come into contact with the extracellular matrix protein during the culture may be the same as those in step (1a) or (1b) of the present invention.

[0048] In step (4a) or (4b) of the present invention, the culture is carried out in the presence of an extracellular matrix protein or a partial peptide thereof capable of adhering mesenchymal stem cells (hereinafter referred to as a "partial peptide of an extracellular matrix protein"). The extracellular matrix protein is not particularly limited, as long as it is capable of adhering mesenchymal stem cells to a culture vessel. Examples of such extracellular matrix proteins include vitronectin, fibronectin, laminin, and collagen. Examples of partial peptides of extracellular matrix proteins include iMatrix-511 (a partial peptide of laminin-511).

[0049] Like vitronectin, extracellular matrix proteins may be proteins isolated and purified from mammalian cells, biochemically synthesized proteins, or recombinant proteins produced from transformants into which nucleic acids having a base sequence encoding the extracellular matrix protein have been introduced.

[0050] Furthermore, the partial peptide of an extracellular matrix protein may be any peptide having a partial amino acid sequence of the extracellular matrix protein and having mesenchymal stem cell adhesion activity. Examples of such partial peptides of extracellular matrix proteins include proteins containing at least one domain selected from the group consisting of an RGD domain and a heparin-binding domain.

[0051] The tissue from which the mesenchymal stem cells seeded in step (4a) or (4b) of the present invention are derived is not particularly limited, but tissues from which the mesenchymal stem cells in the cell aggregates do not proliferate sufficiently in step (1a) or (1b) of the present invention are preferred. Examples of such tissues include bone marrow and umbilical cord blood. Furthermore, even if the mesenchymal stem cells in the cell aggregates proliferate in step (1a) or (1b) of the present invention, the mesenchymal stem cells may be cultured in step (4a) or (4b) of the present invention to further increase the number of mesenchymal stem cells.

[0052] The number of mesenchymal stem cells to be seeded in step (4a) or (4b) of the present invention is not particularly limited, but is usually 2×10 5 cells / cm 2 ~26×10 5 cells / cm 2 may be 8 x 10 5 cells / cm 2 ~13×10 5 cells / cm 2 is preferred.

[0053] The culture conditions for mesenchymal stem cells are not particularly limited and may be the same as the culture conditions for biologically derived cell samples containing mesenchymal stem cells. Conventional cell culture conditions can be used.

[0054] It is preferable to change the medium at appropriate intervals during culture. Medium changes include complete change of the medium, partial change of the medium, addition of medium, and combinations thereof. In a preferred embodiment of the present invention, the medium is completely changed with a medium of the same composition every two or three days from the start of culture.

[0055] The culture period is, for example, 1 to 14 days, preferably 1 to 8 days. This culture initiates proliferation of mesenchymal stem cells.

[0056] In step (5a) or (5b) of the present invention, mesenchymal stem cells proliferated in a culture vessel via an extracellular matrix protein or a partial peptide of an extracellular matrix protein are collected by known means, which may be the same as the method described in step (2a) or (2b) of the present invention.

[0057] The present invention will be explained in more detail below by way of examples, but these are merely illustrative and the present invention is not limited to these examples. [Example]

[0058] Example 1: Examination of the effect of vitronectin on promoting mesenchymal stem cell (MSC) production Due to limitations in purification technology, bone marrow mononuclear cells (MNCs) isolated from bone marrow are contaminated with small amounts of mesenchymal stem cells (MSCs). In this example, we examine a method for producing MSCs from MNCs using serum-free medium. Bone marrow mononuclear cells (MNCs) (Lonza) were induced to sleep using the following seeding medium: Fibronectin (Sigma) or Vitronectin (Wako) at 1.5 μg / cm . 2 2.6 x 10 cells onto a coated 24-well plate at a concentration of 6 The cells were seeded at a density of 100 cells / well and cultured at 37°C in 5% CO. The day after seeding, the entire medium in the plate was replaced with seeding medium, and on days 3 and 5 after seeding, additional seeding medium was added in an amount equivalent to 20% of the medium volume in the plate. Seeding medium: StemFit® AK03N medium (Ajinomoto Co., Inc.) solution A, StemFit® AK03N medium (Ajinomoto Co., Inc.) solution B, 3 ng / mL bFGF (peprotech), 10 μM SB431542 (Stemgent), 1 / 100 Lipid Concentrate (Life Technologies), 10 nM Dexamethasone (Sigma-Aldrich), 10 ng / mL PDGF-BB (Fujifilm Wako Pure Chemical Industries), 1 mM Lithium Chloride (Sigma-Aldrich) Figure 1 shows a photograph of the cells 5 days after seeding. More cell aggregates were formed in the vitronectin-coated well plate than in the fibronectin-coated well plate. Seven days after seeding, the aggregates were collected, and the cells were replated using the following growth medium. Specifically, after collecting the culture supernatant, DPBS (Nacalai Tesque) was added to the plate, and the aggregates were detached from the plate by pipetting, and all of the aggregates were collected together with the DPBS. The collected culture supernatant and DPBS were then centrifuged together to collect only the aggregates. The collected aggregates were dissociated into single cells by resuspending them in the growth medium. Fibronectin (Sigma), Vitronectin (Wako), and iMatrix-511 (Nippi) were used, and fibronectin and Vitronectin were 1.5 μg / cm. 2 concentration of iMatrix-511 is 0.5 μg / cm 2 The entire amount of collected cells was seeded onto 24-well plates coated with each of the following concentrations and cultured at 37°C and 5% CO2. Thereafter, the entire medium in the plate was replaced with growth medium every 2-3 days until the cells became subconfluent. Growth medium: StemFit® AK03N medium (Ajinomoto Co., Inc.) solution A, 1 / 4 StemFit® AK03N medium (Ajinomoto Co., Inc.) solution B, StemFit® AK03N medium (Ajinomoto Co., Inc.) solution C, 1 / 100 Lipid Concentrate (Life Technologies), 10 nM Dexamethasone (Sigma-Aldrich), 10 ng / mL PDGF-BB (Fujifilm Wako Pure Chemical Industries, Ltd.), 1 mM Lithium Chloride (Sigma-Aldrich) Once the cells were confirmed to be subconfluent, they were passaged 13 days after seeding and the cell count was measured. Figure 2 shows the results of the cell count measurement, and Figure 3 shows a photograph of the cells. It was found that the difference in the extracellular matrix coating the culture vessel during replated cells did not affect the number of cells obtained. These results indicate that when vitronectin-coated culture vessels are used for seeding cells, the number of cell aggregates obtained and the number of cells obtained by subsequent reseeding are greater than when fibronectin-coated culture vessels are used. Furthermore, cells detached from the vitronectin-coated plates were expanded and subjected to surface antigen analysis. Using FACS, surface antigen analysis was performed for three MSC-positive markers (CD105, CD90, CD73) and two MSC-negative markers (CD45 and CD34). The analysis results are shown in Table 1. The obtained cells were positive for CD105, CD90, and CD73, but negative for CD45 and CD34, confirming their identity as MSCs.

[0059] [Table 1]

[0060] Example 2: Examination of differences in MSC production promotion effects depending on the type of vitronectin MNCs (Lonza) were induced to sleep using the following seeding medium (serum-free), and then inoculated with Vitronectin (20-398 aa) (wako) (corresponding to amino acids 1 to 379 of SEQ ID NO: 1), Vitronectin (VTN-N, 62-478 aa) (Life Technologies) (corresponding to amino acids 43 to 459 of SEQ ID NO: 1), and Vitronectin (Full length, 20-478 aa) (Sigma) (corresponding to SEQ ID NO: 1) at 1.5 μg / cm . 2 2.6 x 10 cells / well of a 24-well plate coated with 100 µL of PBS at a concentration of 0.05%. 6 The cells were seeded at a density of 1.5 μg / cm and cultured at 37°C under 5% CO conditions. Vitronectin (20-398 aa) (Wako) and Synthemax II (Corning) were used at 1.5 μg / cm respectively. 2 and 5.0 μg / cm 2 Coated 24-well plates at a concentration of 1.6 x 10 6 Cells were seeded at a density of 100 cells / well and cultured at 37°C under 5% CO2. Synthemax II is a vitronectin-based synthetic peptide containing an RGD motif and flanking sequences. The culture medium in the plate was completely replaced with seeding medium the day after seeding. On days 3 and 5 after seeding, additional seeding medium was added in an amount equivalent to 20% of the culture medium volume in the plate. Seeding medium: StemFit® AK03N medium (Ajinomoto Co., Inc.) solution A, 1 / 4 StemFit® AK03N medium (Ajinomoto Co., Inc.) solution B, 3 ng / mL bFGF (peprotech), 10 μM SB431542 (Stemgent), 1 / 100 Lipid Concentrate (Life Technologies), 10 nM Dexamethasone (Sigma-Aldrich), 10 ng / mL PDGF-BB (Fujifilm Wako Pure Chemical Industries), 1 mM Lithium Chloride (Sigma-Aldrich). Seven days after seeding, aggregates were collected and replated using the growth medium described below. Specifically, after collecting the culture supernatant, DPBS (Nacalai Tesque) was added to the plate, and the aggregates were detached from the plate by pipetting. All aggregates were then collected together with the DPBS. The collected culture supernatant and DPBS were then centrifuged to collect only the aggregates. The collected aggregates were dissociated into single cells by resuspending them in growth medium. The entire amount of collected cells was seeded into a 24-well plate and cultured at 37°C and 5% CO2. The medium in the plate was then completely replaced with growth medium every 2–3 days until the cells became subconfluent. Growth medium: StemFit® AK03N medium (Ajinomoto Co., Inc.) Solution A, 1 / 4 StemFit® AK03N medium (Ajinomoto Co., Inc.) Solution B, StemFit® AK03N medium (Ajinomoto Co., Inc.) Solution C, 1 / 100 Lipid Concentrate (Life Technologies), 10 nM Dexamethasone (Sigma-Aldrich), 10 ng / mL PDGF-BB (Fujifilm Wako Pure Chemical Industries, Ltd.), 1 mM Lithium Chloride (Sigma-Aldrich), 0.2 μg / mL iMatrix 511 (Nippi) After confirming that the cells had reached subconfluence, they were passaged on days 13 and 12 after seeding and cell counts were measured. Figure 4 shows the results of cell count measurements using Vitronectin (20-398 aa) (Wako), Vitronectin (VTN-N, 62-478 aa) (Life Technologies), and Vitronectin (Full length, 20-478 aa) (Sigma). MSCs were produced using any of the vitronectins used when seeding MNCs, but Vitronectin (20-398 aa) (Wako) produced MSCs most efficiently from MNCs. Figure 5 shows the results of cell count measurements using Vitronectin (20-398 aa) (Wako) and Synthemax II (CORNING). MSCs were also efficiently produced from MNCs when Synthemax II (CORNING) was used when seeding MNCs.

[0061] Example 3: Examination of the effect of TGFβ receptor inhibitors on promoting MSC production MNCs (Lonza) were incubated in the following seeding medium (1) or (2). Vitronectin (VTN-N, 62-478 aa) (Life Technologies) was used at a concentration of 1.5 μg / cm. 2 2.6 x 10 cells / well of a 24-well plate coated with 100 µL of PBS at a concentration of 0.05%. 6 The cells were seeded at a density of 100 cells / well and cultured at 37°C under 5% CO. The day after seeding, the entire medium in the plate was replaced with seeding medium (1) or (2), and on days 3 and 5 after seeding, seeding medium (1) or (2) was further added in an amount equivalent to 20% of the medium volume in the plate. Seeding medium (1) (TGFβ inhibitor (-)): StemFit (registered trademark) AK03N medium (Ajinomoto Co., Inc.) Solution A, StemFit (registered trademark) AK03N medium (Ajinomoto Co., Inc.) Solution B, 3 ng / mL bFGF (peprotech), 1 / 100 Lipid Concentrate (Life Technologies), 10 nM Dexamethasone (Sigma-Aldrich), 10 ng / mL PDGF-BB (Fujifilm Wako Pure Chemical Industries, Ltd.), 1 mM Lithium Chloride (Sigma-Aldrich). Seeding medium (2) (TGFβ inhibitor (+)): StemFit (registered trademark) AK03N medium (Ajinomoto Co., Inc.) Solution A, StemFit (registered trademark) AK03N medium (Ajinomoto Co., Inc.) Solution B, 3 ng / mL bFGF (peprotech), 10 μM SB431542 (Stemgent), 1 / 100 Lipid Concentrate (Life Technologies), 10 nM Dexamethasone (Sigma-Aldrich), 10 ng / mL PDGF-BB (Fujifilm Wako Pure Chemical Industries, Ltd.), 1 mM Lithium Chloride (Sigma-Aldrich) Seven days after seeding, aggregates were collected and replated using the growth medium described below. Specifically, after collecting the culture supernatant, DPBS (Nacalai Tesque) was added to the plate, and the aggregates were detached from the plate by pipetting. All aggregates were then collected together with the DPBS. The collected culture supernatant was then centrifuged to collect only the aggregates. The collected aggregates were dissociated into single cells by resuspending them in growth medium. The entire amount of collected cells was seeded into a 24-well plate and cultured at 37°C and 5% CO2. The medium in the plate was then completely replaced with growth medium every 2–3 days until the cells became subconfluent. Growth medium: StemFit® AK03N medium (Ajinomoto Co., Inc.) solution A, 1 / 4 StemFit® AK03N medium (Ajinomoto Co., Inc.) solution B, StemFit® AK03N medium (Ajinomoto Co., Inc.) solution C, 1 / 100 Lipid Concentrate (Life Technologies), 10 nM Dexamethasone (Sigma-Aldrich), 10 ng / mL PDGF-BB (Fujifilm Wako Pure Chemical Industries, Ltd.), 1 mM Lithium Chloride (Sigma-Aldrich), 0.2 μg / mL iMatrix 511 (Nippi) Once the cells were confirmed to be subconfluent, they were passaged 15 days after seeding and the cell count was measured. The results of the cell count measurement are shown in Figure 6. By adding a TGFβ inhibitor to the seeding medium when seeding the MNCs, MSCs could be efficiently produced from the MNCs.

[0062] Example 4: Examination of differences in the MSC production promoting effect depending on the type of TGFβ receptor inhibitor MNCs (Lonza) were incubated using the following seeding media (1), (2), and (3). Vitronectin (20-398 aa) (Wako) was used at a concentration of 1.5 μg / cm 2 2.6 x 10 cells / well of a 24-well plate coated with 100 µL of PBS at a concentration of 0.05%. 6The cells were seeded at a density of 100 cells / well and cultured at 37°C under 5% CO. The day after seeding, the entire medium in the plate was replaced with seeding medium (1), (2), or (3). On days 3 and 5 after seeding, seeding medium (1), (2), or (3) was further added in an amount equivalent to 20% of the medium volume in the plate. Seeding medium (1): StemFit® AK03N medium (Ajinomoto Co., Inc.) solution A, 1 / 4 StemFit® AK03N medium (Ajinomoto Co., Inc.) solution B, 3 ng / mL bFGF (peprotech), 10 μM SB431542 (Stemgent), 1 / 100 Lipid Concentrate (Life Technologies), 10 nM Dexamethasone (Sigma-Aldrich), 10 ng / mL PDGF-BB (Fujifilm Wako Pure Chemical Industries, Ltd.), 1 mM Lithium Chloride (Sigma-Aldrich) Seeding medium (2): StemFit® AK03N medium (Ajinomoto Co., Inc.) solution A, 1 / 4 StemFit® AK03N medium (Ajinomoto Co., Inc.) solution B, 3 ng / mL bFGF (peprotech), 0.5 μM A-83-01 (wako), 1 / 100 Lipid Concentrate (Life Technologies), 10 nM Dexamethasone (Sigma-Aldrich), 10 ng / mL PDGF-BB (Fujifilm Wako Pure Chemical Industries), 1 mM Lithium Chloride (Sigma-Aldrich) Seeding medium (3): StemFit® AK03N medium (Ajinomoto Co., Inc.) solution A, 1 / 4 StemFit® AK03N medium (Ajinomoto Co., Inc.) solution B, 3 ng / mL bFGF (peprotech), 0.5 μM LDN-193189 (Stemgent), 1 / 100 Lipid Concentrate (Life Technologies), 10 nM Dexamethasone (Sigma-Aldrich), 10 ng / mL PDGF-BB (Fujifilm Wako Pure Chemical Industries), 1 mM Lithium Chloride (Sigma-Aldrich) Seven days after seeding, aggregates were collected and replated using the growth medium described below. Specifically, after collecting the culture supernatant, DPBS (Nacalai Tesque) was added to the plate, and the aggregates were detached from the plate by pipetting. All aggregates were then collected together with the DPBS. The collected culture supernatant and DPBS were then centrifuged to collect only the aggregates. The collected aggregates were dissociated into single cells by resuspending them in growth medium. The entire amount of collected cells was seeded into a 24-well plate and cultured at 37°C and 5% CO2. The medium in the plate was then completely replaced with growth medium every 2–3 days until the cells became subconfluent. Growth medium: StemFit® AK03N medium (Ajinomoto Co., Inc.) solution A, 1 / 4 StemFit® AK03N medium (Ajinomoto Co., Inc.) solution B, StemFit® AK03N medium (Ajinomoto Co., Inc.) solution C, 1 / 100 Lipid Concentrate (Life Technologies), 10 nM Dexamethasone (Sigma-Aldrich), 10 ng / mL PDGF-BB (Fujifilm Wako Pure Chemical Industries, Ltd.), 1 mM Lithium Chloride (Sigma-Aldrich), 0.2 μg / mL iMatrix 511 (Nippi) After confirming that the cells had reached subconfluence, they were passaged 12 days after seeding and the cell count was measured. The results of the cell count measurement are shown in Figure 7. MSCs were efficiently produced from MNCs using any of the TGFβ receptor inhibitors SB431542 (ALK5 inhibition), A-83-01 (ALK4, ALK5, and ALK7 inhibition), and LDN-193189 (ALK2 and ALK3 inhibition).

[0063] Example 5: Study of MSC production from adipose tissue Epididymal adipose tissue collected from C57BL / 6J mice (11-week-old, male) was treated with collagenase, and cells were obtained using the following seeding media (1) and (2). 6.0 × 10 cells were plated on a 24-well plate. 4The cells were seeded at a density of 100 cells / well and cultured at 37°C under 5% CO2 conditions. Cells seeded using seeding medium (1) were cultured with Vitronectin (VTN-N, 62-478 aa) (Life Technologies) at 1.5 μg / cm 2 On the day after seeding and on the second day, the entire medium in the plate was replaced with seeding medium (1) or (2). Seeding medium (1): StemFit® AK03N medium (Ajinomoto Co., Inc.) solution A, 1 / 4 StemFit® AK03N medium (Ajinomoto Co., Inc.) solution B, 3 ng / mL bFGF (peprotech), 10 μM SB431542 (Stemgent), 1 / 100 Lipid Concentrate (Life Technologies), 10 nM Dexamethasone (Sigma-Aldrich), 10 ng / mL PDGF-BB (Fujifilm Wako Pure Chemical Industries, Ltd.), 1 mM Lithium Chloride (Sigma-Aldrich) Seeding medium (2): DMEM medium (Sigma), 10% fetal bovine serum (Life Technologies) After confirming that the cells had reached subconfluence, they were passaged 5 days after seeding and the cell count was measured. Figure 8 shows the results of the cell count measurement. Figure 9 shows a photograph of the cells. By using a medium containing a TGFβ receptor inhibitor and vitronectin, MSCs were efficiently produced from adipose tissue. [Industrial Applicability]

[0064] Mesenchymal stem cells can be efficiently produced from a biological cell sample by culturing the biological cell sample containing mesenchymal stem cells in a serum-free medium or a xenogeneic component-free medium in the presence of vitronectin or a partial peptide thereof capable of adhering mesenchymal stem cells. By employing this method, the mesenchymal stem cells obtained can be used directly as a cell source for regenerative medicine. This application is based on Japanese Patent Application No. 2019-156537 (filing date: August 29, 2019) and Japanese Patent Application No. 2020-012333 (filing date: January 29, 2020), the contents of which are incorporated herein in their entirety.

Claims

1. A method for producing mesenchymal stem cells from a biological cell sample containing mesenchymal stem cells, comprising the following steps: (1) culturing a biologically derived cell sample containing mesenchymal stem cells in a serum-free medium in the presence of vitronectin or a partial peptide thereof capable of adhering mesenchymal stem cells, thereby proliferating the mesenchymal stem cells; (2) A step of recovering cell aggregates of mesenchymal stem cells.

2. The method of claim 1, wherein the culturing in the presence of vitronectin or a partial peptide thereof capable of adhering mesenchymal stem cells is culturing on a culture vessel to which vitronectin or a partial peptide thereof capable of adhering mesenchymal stem cells is solidified.

3. 3. The method of claim 1 or 2, further comprising the steps of: (3) dissociating the collected cell aggregates; (4) culturing the dissociated mesenchymal stem cells in a serum-free medium in the presence of an extracellular matrix protein or a partial peptide thereof capable of adhering mesenchymal stem cells; (5) A step of recovering mesenchymal stem cells proliferated on the culture vessel via the extracellular matrix protein or a partial peptide thereof capable of adhering mesenchymal stem cells.

4. The method according to claim 3, wherein the culturing in the presence of an extracellular matrix protein or a partial peptide thereof capable of adhering mesenchymal stem cells is culturing on a culture vessel to which an extracellular matrix protein or a partial peptide thereof capable of adhering mesenchymal stem cells is solidified.

5. The method according to any one of claims 1 to 4, wherein the partial peptide of vitronectin comprises an RGD domain.

6. The method of claim 5, wherein the partial peptide of vitronectin further comprises a somatomedin B domain.

7. The method according to claim 6, wherein the partial peptide of vitronectin is a polypeptide consisting of amino acid numbers 1 to 379 of the amino acid sequence represented by SEQ ID NO:

1.

8. The method according to any one of claims 1 to 7, wherein the serum-free medium in step (1) contains a TGF-β receptor inhibitor.

9. A method for producing mesenchymal stem cells from a biological cell sample containing mesenchymal stem cells, comprising the following steps: (1) culturing a biological cell sample containing mesenchymal stem cells in a serum-free, xenogeneic component-free medium in the presence of vitronectin or a partial peptide thereof capable of adhering mesenchymal stem cells, thereby proliferating the mesenchymal stem cells; (2) A step of recovering cell aggregates of mesenchymal stem cells.

10. The method of claim 9, wherein the culturing in the presence of vitronectin or a partial peptide thereof capable of adhering mesenchymal stem cells is culturing on a culture vessel to which vitronectin or a partial peptide thereof capable of adhering mesenchymal stem cells is solidified.

11. The method of claim 9 or 10, further comprising the steps of: (3) dissociating the collected cell aggregates; (4) culturing the dissociated mesenchymal stem cells in a xenogeneic component-free medium in the presence of an extracellular matrix protein or a partial peptide thereof capable of adhering mesenchymal stem cells; (5) A step of recovering mesenchymal stem cells proliferated on the culture vessel via the extracellular matrix protein or a partial peptide thereof capable of adhering mesenchymal stem cells.

12. The method according to claim 11, wherein the culturing in the presence of an extracellular matrix protein or a partial peptide thereof capable of adhering mesenchymal stem cells is culturing on a culture vessel to which an extracellular matrix protein or a partial peptide thereof capable of adhering mesenchymal stem cells is solidified.

13. The method according to any one of claims 9 to 12, wherein the partial peptide of vitronectin comprises an RGD domain.

14. The method of claim 13, wherein the partial peptide of vitronectin further comprises a somatomedin B domain.

15. The method according to claim 14, wherein the partial peptide of vitronectin is a polypeptide consisting of amino acid numbers 1 to 379 of the amino acid sequence represented by SEQ ID NO:

1.

16. The method according to any one of claims 9 to 15, wherein the xenogeneic component-free medium in step (1) contains a TGF-β receptor inhibitor.

17. The method according to any one of claims 9 to 16, wherein the xeno-free medium comprises allogeneic serum.

18. 18. The method of claim 17, wherein the allogeneic serum is autologous serum.

19. The method according to any one of claims 1 to 18, wherein the biological cell sample containing mesenchymal stem cells is bone marrow-derived cells.

20. The number of bone marrow-derived cells to be cultured is 0.5 × 10 5 ~25×10 5 cells / cm 2 20. The method of claim 19, wherein:

21. The method according to claim 19 or 20, wherein the culture period of the bone marrow-derived cells is 4 to 14 days.

22. The method according to any one of claims 1 to 18, wherein the biological cell sample containing mesenchymal stem cells is adipose tissue-derived cells.

23. The number of adipose tissue-derived cells to be cultured is 1 x 10 3 ~1×10 6 cells / cm 2 23. The method of claim 22, wherein:

24. The method according to claim 22 or 23, wherein the adipose tissue-derived cells are cultured for 1 to 14 days.

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