Method for culturing cell populations comprising tie2-positive cells of cartilage origin and uses thereof

Abstract

The present application provides a means for efficiently producing a cell population rich in functional chondrocytes that produce extracellular matrix such as type II collagen. The culture method of the present application is a culture method of a cell population containing chondrocyte-derived cells positive for Tie2 expression (chondrocyte-derived Tie2-positive cells), which comprises the step of culturing a cell population containing the chondrocyte-derived Tie2-positive cells in a culture medium to which at least one Tie2 expression enhancer other than a growth factor (e.g., an extract derived from a plant of the genus Cinnamomum or the like) is added. The culture method is preferably performed in a culture vessel having a culture surface coated with a coating agent (e.g., a coating agent containing polylysine).

Description

TECHNICAL FIELD

[0001] The present application relates to a culture method of a cell population containing cells expressing a cell surface marker Tie2 (tyrosine kinase with Igand EGF homology domain-2) contained in cartilage (referred to as "chondrogenic Tie2-positive cells" in the present specification) and the like. More specifically, the present application relates to a culture method of a cell population containing chondrogenic Tie2-positive cells, which can be used in a process for inducing differentiation from the chondrogenic Tie2-positive cells into mature functional chondrocytes, such as Col2 (type II collagen)-expressing chondrocytes. BACKGROUND

[0002] Articular cartilage is a tissue that covers the end of a bone and absorbs the impact applied when a wrist or knee joint is bent and the like. Normal articular cartilage is referred to as "hyaline cartilage" and has a characteristic in which an extracellular matrix (ECM) mainly containing type II collagen and type XI collagen is formed around the periphery of cells. When hyaline cartilage is worn out with age or damaged due to exercise, traffic accidents, and the like, it is sometimes denatured into fibrous cartilage. Once the cartilage is fibrotic, it cannot be restored, the joint becomes difficult to move freely, and sometimes causes pain and inflammation.

[0003] Articular cartilage is a tissue that lacks blood vessels, nerves, and lymphatic vessels, and thus it is difficult to heal the damage as in other tissues once the cartilage is damaged, and it is significantly lacking in self-repairing ability. Therefore, a method of regenerating (repairing) the cartilage by transplanting allogenic (autologous or allogenic) chondrocytes to the affected site is expected, and research and development of a cell preparation containing chondrocytes and ECM and the like for the transplantation is also being conducted. In order to produce such a cell preparation, it is necessary to collect a certain amount of allogenic chondrocytes. In the case of autologous transplantation, for example, for a patient with a knee cartilage damage, sometimes chondrocytes are isolated from healthy articular cartilage of the patient and used. However, since a cartilage collection surgery is required in addition to the transplantation surgery, the healthy cartilage is damaged. Therefore, there is a problem that the burden on the patient is heavy. On the other hand, in the case of allogenic transplantation, for example, a cartilage tissue surgically removed from a patient with polydactyly among infants and young children can be used as a supply source of chondrocytes, but the number of chondrocytes collected by this is small. However, a method in which chondrocytes derived from a plurality of patients (donors) with polydactyly and the like are mixed and used in order to secure the number of chondrocytes cannot completely avoid the risk of viral infection, and thus this method is not desirable. In either the case of autologous transplantation or the case of allogenic transplantation, it is important to culture a cell population containing mature chondrocytes contained in a small amount of cartilage tissue and produce a cell population containing a sufficient number of chondrocytes for treatment.

[0004] To address the aforementioned issues related to the culture and proliferation of chondrocytes contained in collected tissues, a method has been proposed for using chondrocytes obtained by differentiating and inducing differentiation of pluripotent stem cells such as iPS cells, ES cells, or somatic stem cells. For example, Patent Document 1 describes a method for producing chondrocytes from pluripotent stem cells, comprising the following steps: (i) culturing pluripotent stem cells under adhesion conditions in a culture medium containing one or more substances selected from the group consisting of BMP2, TGFβ, and GDF5, and an HMG-CoA reductase inhibitor; and (ii) culturing the cells obtained in step (i) under suspension conditions in a culture medium containing one or more substances selected from the group consisting of BMP2, TGFβ, and GDF5, and an HMG-CoA reductase inhibitor.

[0005] Furthermore, Patent Document 2 discloses a spheroid for cell transplantation therapy and a method for manufacturing the same, wherein the spheroid is formed from a mixture of cells exhibiting a phenotype similar to cartilage tissue. An embodiment of Patent Document 2 describes a method in which chondrocytes contained in cartilage tissue and synovial-derived cells contained in synovial tissue are passaged separately in culture dishes; the obtained passaged cells are mixed in a predetermined ratio; and the resulting cell mixture is further cultured with shaking to manufacture the spheroid for cell transplantation therapy.

[0006] On the other hand, Patent Document 3 and Non-Patent Document 1 describe the following: Cells in the intervertebral disc tissue (nucleus pulposus) that are positive for Tie2 and / or GD2 as cell surface markers are stem cells or progenitor cells that should be called nucleus pulposus cells; in particular, cells that are positive for both Tie2 and GD2 (nucleus pulposus stem cells in an active state) have the ability to form spherical colonies and eventually differentiate into mature nucleus pulposus cells through a series of differentiation cascades (in addition, they also have the ability to differentiate into adipocytes, osteocytes, chondrocytes and nerve cells); and by transplanting nucleus pulposus stem / progenitor cells into the intervertebral disc (nucleus pulposus), extracellular matrix such as type II collagen can be produced in the tissue, and there is a possibility of maintaining or reconstructing the intervertebral disc tissue and preventing or treating intervertebral disc degenerative diseases; etc. As a more specific implementation, Patent Document 3 and Non-Patent Document 1 describe the formation of spherical colonies (along with adhesive colonies) by suspending and culturing cell populations contained in intervertebral disc tissue (nucleus pulposus) in methylcellulose medium; such spherical colonies are derived from the aforementioned Tie2-positive (and GD2-positive) cells; type II collagen and proteoglycans are expressed in the spherical colonies (a portion of their cells); etc. (for example, see the embodiments, paragraphs 0067, 0070, etc. of Patent Document 3).

[0007] However, none of Patent Documents 1 to 3 and Non-Patent Document 1 discloses or suggests that Tie2-positive cells are contained in cartilage and that the Tie2-positive cells can be efficiently proliferated or differentiation-induced into chondrocytes under specific conditions.

[0008] In addition, Patent Document 3 and Non-Patent Document 1 describe that, in order to maintain Tie2-positive nucleus pulposus cells (intervertebral disc nucleus pulposus stem / progenitor cells), a signal transduction mechanism between Tie2 (receptor) and Ang-1 (angiopoietin-1, ligand) is required, the Tie2-positive cells can be expanded by culturing in the presence of Ang-1 (co-culturing with AHESS5 which forcibly expresses Ang-1), and therefore, Ang-1 is considered to be a niche factor which controls the differentiation hierarchy of the nucleus pulposus cells, and the like (Examples: paragraphs 0049, 0069, 0075, and the like).

[0009] However, Tie2 is also expressed in vascular endothelial cells, and activation of Tie2 can also achieve maturation, normalization or stabilization of blood vessels, and is known to be able to inhibit the expansion of vascular disorder observed in, for example, tumors, rheumatoid arthritis, diabetic retinopathy, hyperlipidemia, hypertension, and the like (angiogenesis), or prevent, improve wrinkles, and the like. As a Tie2 activator having such an effect, for example, extracts from plants of the genus Cinnamomum (so-called cinnamon powder, Patent Literature 4), olive fruit extract (Patent Literature 5), and extracts from various plants such as Quillaja, Engelhardtia, Ginkgo, Ostrea, Curcuma, Chrysanthemum, Jujube, Lycium, Chamomile, Myrothamnus, Crataegus, Averrhoa, Alpinia, Nelumbo, ASPALATHUS LINEARIS, Tamarindus indica, Chaenomeles, Psidium, Eleutherococcus, Mangifera indica, Red Ginseng, Elaeagnus pungens, Sarcocornia, Salix, Hemerocallis fulva, Colocasia esculenta, Zanthoxylum, Clerodendrum, Morinda, Astragalus, Momordica, Morinda officinalis, and the like (Patent Literatures 6 to 10) are proposed. Furthermore, as a component that brings about a Tie2 activation effect, for example, ursolic acid, corosolic acid, 3-O-galloyl procyanidin B-1, linolenic acid, 13-hydroxy-9Z, 11E, 15E-octadecatrienoic acid, procyanidin B-2, epicatechin-(4β-6)-epicatechin(4β-8)-epicatechin, procyanidin C-1, Astragalus saponin VIII, soyasaponin I, 3'-O-methylgallocatechin, elembopin, syringaresinol, 2-methoxycinnamaldehyde, Eleutherococcus acuminatus glycoside E, Eleutherococcus acuminatus glycoside El, sesamin, Eudistomins, forsythoside, pinoresinol, Larix gmelinii resinol B dimethyl ether, forsythinol, coumarin, and the like (Patent Literatures 7, 12 to 14) are proposed.

[0010] For example, in Patent Literature 4, as an experiment related to "Tie2 activator", it is confirmed by Western blotting that, when "Baf3 cells which forcibly express Tie2" or "normal human umbilical vein vascular endothelial cells (HUVEC)" are cultured in a medium to which a hot water extract of Cinnamomum is added, the Tie2 protein expressed in these cells is phosphorylated at a higher level than a control (paragraphs 0024 to 0027, Figures 1 to 3

[0011] However, in Patent Literatures 4 to 14, there is no description or suggestion that a Tie2 activator is used in the culture of Tie2-positive cells contained in a cell group derived from cartilage, rather than blood cells (hemocytes) and vascular endothelial cells, and what kind of effect can be brought about thereby. ​

[0012] Prior Art Documents

[0013] Patent Documents

[0014] Patent Document 1: WO2016 / 133208

[0015] Patent Document 2: Japanese Patent Application Publication No. 2011-41472

[0016] Patent Document 3: Japanese Patent No. 5863639 (corresponding to WO2011 / 122601)

[0017] Patent Document 4: WO2009 / 123211

[0018] Patent Document 5: WO2016 / 060249

[0019] Patent Document 6: WO2012 / 073627

[0020] Patent Document 7: Japanese Patent Application Publication No. 2012-236795

[0021] Patent Document 8: Japanese Patent Application Publication No. 2011-201811

[0022] Patent Document 9: Japanese Patent Application Publication No. 2011-102275

[0023] Patent Document 10: Japanese Patent Application Publication No. 2011-102274

[0024] Patent Document 11: Japanese Patent Application Publication No. 2011-102273

[0025] Patent Document 12: Japanese Patent Application Publication No. 2014-97977

[0026] Patent Document 13: Japanese Patent Application Publication No. 2013-241356

[0027] Patent Document 14: Japanese Patent Application Publication No. 2011-102272

[0028] Non-Patent Documents

[0029] Non-Patent Document 1: Sakai D et al., Nat Commun. 2012; 3: 1264 SUMMARY

[0030] Problem to be solved by the invention

[0031] In order to produce a cell preparation containing homologous (autologous or allogeneic) chondrocytes for treating articular cartilage damage and the like, a certain amount of functional chondrocytes that produce extracellular matrix such as type II collagen and proteoglycans is required to be collected. In particular, for improving the therapeutic effect when the cell preparation is administered to a patient, it is important to prepare a cell population of functional chondrocytes that produce a high yield of extracellular matrix such as type II collagen as efficiently as possible. Furthermore, even chondrocytes collected from hyaline cartilage have a problem that they are easily denatured into fibroblast-like cells due to culture. If such fibroblast-like chondrocytes are transplanted, fibrocartilage is formed in the repaired tissue, and there is a concern that the damaged site cannot be treated with hyaline cartilage.

[0032] An object of the present application is to provide a means for efficiently preparing a cell population enriched with functional chondrocytes that produce extracellular matrix such as type II collagen.

[0033] Solution to the problem

[0034] The present inventors have found that Tie2-positive cells, which include stem / progenitor cells having the ability to differentiate into chondrocytes, are also contained in cartilage. On this basis, the present inventors focused on the state of the cell population to be cultured and the culture conditions when culturing a cell population containing such cartilage-derived Tie2-positive cells (chondroblast stem / progenitor cells) in the course of their research, and as a result, found the characteristic technical matters described below that are useful for solving the above-described problems.

[0035] The culture method of the cell population containing cartilage-derived Tie2-positive cells of the present application includes the step of culturing a cell population containing cartilage-derived Tie2-positive cells in a culture medium to which at least one Tie2 expression enhancer other than a growth factor is added.

[0036] It is known that Tie2-positive cells such as hematopoietic stem cells originally make the activation (phosphorylation) of Tie2 excessive by binding to Angiopoietin-1 (Ang-1), which is a ligand for Tie2 (receptor tyrosine kinase), and a method of culturing Tie2-positive cells in a culture medium to which Ang-1 is added (to enhance the expression of Tie2) has become known as a result of prior art (Prior Art Document 3, etc.). In addition, it is known that FGF2 (bFGF) also has the same effect of enhancing the expression of Tie2, and a method of culturing Tie2-positive cells in a culture medium to which FGF2 is added is also known.

[0037] However, the inventors have discovered that when Tie2 expression enhancers of different types than growth factors such as Ang-1 and FGF2, such as extracts of cinnamon powder, are used as plant-derived Tie2 expression enhancers for culturing previously unreported cell populations containing cartilage-derived Tie2-positive cells, especially when Tie2 expression enhancers of plant-derived extracts are used in combination with growth factors such as FGF2, the resulting cell populations are rich in functional chondrocytes differentiated from cartilage-derived Tie2-positive cells, and contain cartilage-derived Tie2-positive stem / progenitor cells themselves at a higher rate than in previous methods. In other words, when a cell population containing cartilage-derived Tie2-positive cells was cultured in a medium supplemented with a Tie2 expression enhancer, the following opposite objectives were unexpectedly achieved in a balanced manner: enhancing Tie2 expression in the cartilage-derived Tie2-positive cells, maintaining immature Tie2-positive cells (proliferative capacity and ability to differentiate into chondrocytes, and globule-forming ability) in the cell population, and promoting differentiation into functional chondrocytes expressing type II collagen (Col2), thereby increasing the number of Col2-positive cells in the cell population.

[0038] In a preferred embodiment, the cell population comprising cartilage-derived Tie2-positive cells (cartilage stem / progenitor cells) of the present invention, as described above, is cultured in a two-dimensional (planar) culture environment using a culture vessel having a culture surface coated with a coating containing extracellular matrix or other biorelated molecules, such as polylysine. Further three-dimensional culture using scaffold materials such as gels or porous materials, or suspension culture using non-adhesive culture vessels, may also be performed.

[0039] Based on the above-described respective culture methods, the present inventors have constructed a preferred method for preparing a cell population containing chondrocytes differentiated from the stem / progenitor cells from the cell population containing the chondrocyte-derived Tie2-positive cells (chondrocyte stem / progenitor cells, etc.). That is, the method for preparing a cell population of the present application at least includes: a culture stage (differentiation culture stage) for differentiating into functional chondrocytes expressing Col2, etc. while maintaining the Tie2 expression of the chondrocyte-derived Tie2-positive cells in the cell population is enhanced according to the above-described culture method of the chondrocyte-derived Tie2-positive cells of the present application. Such a method for preparing a cell population can greatly increase the absolute number of functional chondrocytes, particularly chondrocytes positive for cell markers such as Col2, or the ratio of the same in the cell population, compared to the conventionally known preparation methods. In the above-described differentiation culture stage, a cell population in which the Tie2-positive cells do not completely disappear but remain at a certain level of number or ratio can be obtained even at a stage where the number or ratio of Col2-positive cells, etc. reaches a certain level. Such a cell population also contains a certain degree of chondrocyte stem / progenitor cells having the ability to produce functional chondrocytes after administration, and is thus considered to be more excellent in therapeutic or prophylactic effects.

[0040] If the above-described technical concept is combined with and embodied in the (preferred) embodiments described below, the present application can be expressed, for example, as an invention including at least the following items. [1]

[0042] A culture method of a cell population containing cells positive for expression of Tie2 (tyrosine kinase with Ig and EGF homology domain-2) derived from cartilage (hereinafter referred to as "chondrocyte-derived Tie2-positive cells").

[0043] The culture method includes a step of culturing a cell population containing the chondrocyte-derived Tie2-positive cells in a culture medium to which at least one plant-derived extract having a Tie2 expression-enhancing effect is added. [2]

[0045] The culture method according to item 1, wherein the plant is a plant of the genus Cinnamomum. [3]

[0047] The culture method according to item 1 or 2, which is performed in a culture vessel having a culture surface coated with a coating agent containing an extracellular matrix and / or a polyamino acid. [4]

[0049] The culture method according to item 3, wherein the extracellular matrix is collagen type IV and / or fibronectin. [5]

[0051] The culture method according to item 3 or 4, wherein the polyamino acid is polylysine. [6]

[0053] A method for producing a cell population containing chondrocytes differentiated from chondrocyte-derived Tie2-positive cells,

[0054] The method for producing includes a culture stage (hereinafter referred to as "differentiation culture stage") for differentiation induction into chondrocytes while enhancing Tie2 expression of chondrocyte-derived Tie2-positive cells in the cell population, including a process of the culture method according to any one of items 1 to 5. [7]

[0056] The method for producing according to item 6, wherein the chondrocytes contain cells positive for expression of Col2 (collagen type II). [8]

[0058] The method for producing according to item 6 or 7, wherein the differentiation culture stage further includes a process of culturing a cell population containing chondrocyte-derived Tie2-positive cells in a culture medium to which at least one growth factor selected from the group consisting of FGF (fibroblast growth factor), EGF (epidermal growth factor), and Ang-1 (angiopoietin-1) is added. [9]

[0060] The method for producing according to any one of items 6 to 8, wherein, before the differentiation culture stage, a process of:

[0061] a culture stage for proliferation of a cell population containing chondrocyte-derived Tie2-positive cells (hereinafter referred to as "proliferation culture stage").

[10]

[0063] The method for producing according to any one of items 6 to 9, wherein a cell population in which chondrocyte-derived Tie2-positive cells remain is obtained by the differentiation culture stage.

[11]

[0065] A cell population obtained by the culture method according to any one of items 1 to 5.

[12]

[0067] A culture containing a culture medium used in the culture method according to any one of items 1 to 5 and a cell population subjected to, cultured in, or obtained by the culture method.

[13]

[0069] A cell population obtained by the differentiation culture stage in the production method according to any one of items 6 to 10.

[14]

[0071] A culture medium containing the medium for the differentiation culture stage in the production method according to any one of items 6 to 10 and the cell population cultured or obtained for the differentiation culture stage.

[15]

[0073] A composition for cell therapy containing the cell population according to item 10 or 13.

[16]

[0075] The composition for cell therapy according to item 15 for use in the treatment or prevention of a disorder, condition or disease of cartilage.

[0076] Effects of the invention

[0077] The culture method of cartilage-derived Tie2-positive cells according to the present application can promote differentiation of the cells into functional (positive for cell markers such as Col2) chondrocytes while enhancing Tie2 expression of the cartilage-derived Tie2-positive cells and maintaining their immaturity. Furthermore, a cell population rich in functional chondrocytes can be produced by a differentiation culture stage including a process of the culture method of the present application. By using a cell population obtained based on the culture method and production method of the present application, a cell preparation effective for the treatment or prevention of a disorder, condition or disease of cartilage can be efficiently produced.

[0078] According to a representative embodiment of the present application, by using only a small amount of cartilage that can be collected and efficiently proliferating and differentiating Tie2-positive cells (chondroblast / progenitor cells) contained therein, a cell population rich in functional chondrocytes (further containing a small number of Tie2-positive cells) having a high production ability of extracellular matrix such as type II collagen, which can be expected to have a high therapeutic effect upon transplantation, can be simply, reproducibly and in a large amount. Such a suitable cell population has been difficult to produce in the past, but can be efficiently produced by the present application, and thus it is easy to perform a regenerative therapy of cartilage such as joints by administering a cell population (a cell preparation containing the cell population), and industrialization becomes easy to achieve. BRIEF DESCRIPTION OF DRAWINGS

[0079] Figure 1is a graph showing Tie2-positive rate of cell population finally obtained from commercially available knee joint cartilage cells (NHAC2) in the case where the culture method of the present application (2% cinnamon) was performed in the differentiation culture stage (first step of differentiation culture) and in the case where the culture method as a control (comparative example) was performed (10 ng / ml bFGF) in Example 1.

[0080] Figure 2 is a graph showing the number of Tie2-positive cells (Tie2+) and Tie2-negative cells (Tie2-) contained in commercially available knee joint cartilage cells (NHAC2) in the case where the culture method of the present application was performed in the differentiation culture stage in Example 2.

[0081] Figure 3 In [a], [a] is a graph showing the number of spherical colony-forming units (CFU-S) and fibroblast-like colony-forming units (CFU-F) in the case where the differentiation culture step based on the present application was performed (cinnamon 2%) and in the case where the differentiation culture step as a control (comparative example) was performed (cinnamon 0%) in the differentiation culture stage in Example 3, and [b] is an optical microscope photograph of a spherical colony (spheroid).

[0082] Figure 4 is a graph showing the positive rate of proteoglycan (PG), Type 1 collagen (Type 1 col), and Type 2 collagen (Type 2 col) of cell population finally obtained from commercially available knee joint cartilage cells (NHAC2) in the case where the differentiation culture step based on the present application was performed (2% cinnamon) and in the case where the differentiation culture step as a control (comparative example) was performed (10 ng / ml bFGF) in the differentiation culture stage in Example 4. DETAILED DESCRIPTION

[0083] -TERMS-

[0084] "Stem cell" means a cell having self-replication ability and differentiation ability (totipotency, pluripotency, multipotency, or unipotency). "Progenitor cell" means a cell which, although it eventually becomes a terminally differentiated cell and does not have strict self-replication ability, has a differentiation ability to gradually differentiate into a prescribed cell while proliferating relatively actively. The (particular) cell which a person skilled in the art generally understands as including the name of "stem cell" or "progenitor cell" corresponds to "stem cell" or "progenitor cell" in the present specification.

[0085] In the present specification, "stem cells and / or progenitor cells" is an expression including stem cells, progenitor cells, or both, and is also sometimes expressed as "stem / progenitor cells". In addition, in the present specification, a cell population including stem cells and / or progenitor cells is sometimes expressed as "stem / progenitor cell population", and a cell population including cells (terminally differentiated cells) obtained by differentiation, maturation of stem cells and / or progenitor cells is expressed as "mature cell population".

[0086] "Stem cells" and "progenitor cells" are generally distinguished from other cells in terms of the expression of one or more than two specific genes (marker genes, cell markers). That is, "stem cells" and "progenitor cells" having the self-replication ability and / or differentiation ability as described above are sometimes defined as cells in which the expression of each specific marker gene is positive or negative.

[0087] Whether the expression of a marker gene (cell marker) is "positive" or "negative" is determined quantitatively or qualitatively by measuring the expression amount of mRNA transcribed from the gene (genome) or protein translated from the mRNA according to a general method, and it can be determined as positive when the expression amount is a certain level or more (or exceeds a certain level), and as negative when it is a certain level or less (or less than a certain level). The expression amount of protein can be quantitatively or qualitatively determined by an immunological method using a specific antibody and a labeling agent for the protein, such as flow cytometry, immunostaining, ELISA, and the like. Note that Tie2 protein is a protein expressed on the cell surface, and Col2 is a protein expressed inside the cell, and each can be measured using a suitable method among methods for detecting proteins present on the cell surface and inside the cell (immunofluorescence staining method, etc.). The expression amount of mRNA can be quantitatively or qualitatively determined by analysis using a specific (complementary) nucleic acid and a labeling agent for the mRNA, a nucleic acid amplification method (means), such as RT-PCR, microarray, biochip, and the like. The ratio of cells in which the expression of a prescribed marker gene (cell marker) is positive or negative (positive rate or negative rate) in a cell population can be calculated by measuring the total number of cells in a cell population using various methods as described above, for example, flow cytometry, and the number of cells determined as positive or negative using the methods as described above, respectively.

[0088] In the present specification, "stem cells and / or progenitor cells in which expression of Tie2 is positive" or "Tie2-positive cells" mean cells in which expression of Tie2 (tyrosine kinase with Ig and EGF homology domain-2), which is known as one of cell markers, is determined to be positive, for example, cells in which expression of protein is determined to be positive using a flow cytometer. The Tie2-positive cells in the present application are cells that are equivalent to "chondrogenic stem / progenitor cells" described below (presumably containing) as "chondrogenic Tie2-positive cells" that are Tie2-positive cells present in cartilage (that can be collected from cartilage) or Tie2-positive cells obtained by subculturing the same.

[0089] In the present application, "chondrocytes" mean cells that are mature and reach terminal differentiation, or cultured cells having equivalent properties thereto, which occupy a large portion of the cell population contained in cartilage. Chondrocytes are preferably functional chondrocytes that are positive for at least collagen type II (Col2) in the extracellular matrix as a cell marker, and further generally positive for proteoglycans (aggrecan). For example, cells determined to be positive for Col2 (and also positive for aggrecan and the like) as a cell marker (preferably a protein) using a flow cytometer are equivalent to functional chondrocytes in the present application. Note that, for the extracellular matrix such as Col2, aggrecan and the like, the amount of production of these proteins can be measured using a flow cytometer, or instead of this, the amount of expression of mRNA of these can be measured using real-time PCR and the like. In the present application, the chondrocytes as described above, particularly chondrocytes positive for the prescribed markers such as Col2 and aggrecan, become cells having a function appropriate for the use, i.e., target cells, obtained from chondrogenic Tie2-positive cells by prescribed differentiation induction (differentiation culture stage). With respect to chondrocytes, in addition, CD81, CD90, CD44, CD105, CD106, GD2 are known to be positive, and one or two or more of these can be further used in combination with Col2 and the like described above as a cell marker for chondrocytes.

[0090] The "cartilage stem / progenitor cell" in the present application is a collective term for progenitor cells (chondrocyte progenitor cells) having at least the ability to differentiate into chondrocytes (preferably functional chondrocytes positive for Col2), and stem cells (chondrocyte stem cells) having the ability to differentiate into the progenitor cells and the ability to self-replicate, which occupy a part of the cell population contained in cartilage, or cultured cells having equivalent properties thereto. Specifically, the chondrocyte stem / progenitor cell at least includes a cell positive for Tie2 as a marker gene. For example, a cell determined to be positive for Tie2 as a protein (cell marker) by flow cytometry (presumably including) corresponds to the chondrocyte stem / progenitor cell in the present application. In an embodiment of the present application, a Tie2-positive cell can also be substantially regarded as a chondrocyte stem / progenitor cell (as a majority thereof), and for example, in the description of the present specification, "Tie2-positive cell" can be replaced with "Tie2-positive chondrocyte stem / progenitor cell".

[0091] The "spherical colony" in the present application is a spherical cell aggregate including stem cells and / or progenitor cells, and further can include cells differentiated from the cells. The "spherical colony" is also an object generally referred to as "spheroid" and the like by those skilled in the art.

[0092] The "expression of Tie2 is enhanced" (Tie2 expression enhancement) in the present application means that the expression of the Tie2 gene is enhanced in each Tie2-positive cell, that is, the expression is increased compared to usual, and the expression amount of mRNA or protein is increased. Even if the expression of the Tie2 gene is almost lost under usual conditions, the expression is not lost but maintained at a certain level, that is, the expression of Tie2 is maintained, which also corresponds to "enhancement of the expression of Tie2". In addition, the number of cells in the cell population determined to be positive for the expression of mRNA or protein of Tie2 (expression amount exceeding a certain level) is increased by enhancing the expression of Tie2 in each Tie2-positive cell, that is, the ratio of Tie2-positive cells in the cell population is higher than usual, which can also be understood as a description of "Tie2 expression enhancement".

[0093] More specifically, for example, for a cell population subjected to the Tie2 expression enhancement treatment (Tie2 expression enhancement treatment group) and a cell population not subjected to the treatment (control group), a treatment of fluorescently labeling the Tie2 protein on the cell surface is performed and measured by flow cytometry, and when the fluorescence intensity of the Tie2 expression enhancement treatment group is higher than a predetermined level compared to the control group, the ratio of cells determined to be positive is high, and / or the average fluorescence intensity per cell is high, it can be said that the Tie2 expression of the cell population of the Tie2 expression enhancement treatment group (Tie2-positive cells included therein) is enhanced (in other words, the Tie2 expression enhancement treatment exerts a predetermined effect).

[0094] In the present invention, the agent that exerts the above-mentioned "Tie2 expression-enhancing" effect is referred to as a "Tie2 expression-enhancing agent" in the present invention. Note that a part of growth factors (FGF2 and the like) have a Tie2 expression-enhancing effect, and can be said to be equivalent to a "Tie2 expression-enhancing agent", and therefore a case other than such growth factors is referred to as a "Tie2 expression-enhancing agent other than growth factors".

[0095] - Culture method -

[0096] The culture method of the present invention, which is a cell population containing cartilage-derived Tie2-positive cells, includes a step of culturing a cell population containing cartilage-derived Tie2-positive cells in a culture medium to which at least one Tie2 expression-enhancing agent other than a growth factor is added. In the present specification, the above-mentioned culture method is sometimes referred to simply as "the culture method of the present invention".

[0097] The culture method of the present invention can enhance Tie2 expression of cartilage-derived Tie2-positive cells while maintaining the naivety of cartilage-derived Tie2-positive cells, i.e., maintaining the replicative ability and (at least) the differentiation ability into myeloblasts, and can promote differentiation of cartilage-derived Tie2-positive cells into chondrocytes, particularly functional chondrocytes positive for expression of Col2 and the like. The culture method of the present invention can maintain cartilage-derived Tie2-positive cells that have maintained naivety for a longer period of time in a cell population (improving the number and / or ratio of Tie2-positive cells compared to conventional culture methods), and thus can produce a large number of chondrocytes (particularly functional chondrocytes as described later). The step of the culture method of the present invention performed in the differentiation culture stage of the production method of the present invention described below is sometimes referred to as "the differentiation culture step of the present invention".

[0098] - Production method (culture step) -

[0099] The production method of the present invention, which is a cell population containing chondrocytes differentiated from Tie2-positive cells, includes at least a culture stage (hereinafter referred to as "differentiation culture stage") for differentiation induction into chondrocytes while enhancing Tie2 expression of cartilage-derived Tie2-positive cells in a cell population, which includes the differentiation culture step of the present invention. In the present specification, the above-mentioned production method is sometimes referred to simply as "the production method of the present invention".

[0100] The differentiation culture stage can be composed of a single differentiation culture step, or can contain a plurality of differentiation culture steps. In addition, the differentiation culture step can be a step that does not include passaging, or can be a step that includes one or two or more passages as needed.

[0101] The differentiation culture stage refers to a stage in which the chondrocyte-derived Tie2-positive cells are differentiated into chondrocytes, and in particular, functional chondrocytes positive for Col2 and the like, by culture under prescribed conditions, and in which the effects of achieving this purpose are exerted. That is, if the number and / or ratio of chondrocytes, particularly functional chondrocytes, in the cell population after culture is higher than that before culture, the culture stage can be referred to as a differentiation culture stage.

[0102] In addition, the differentiation culture process performed in the differentiation culture stage can also exhibit the following effects in addition to the above-mentioned effects: the number and / or ratio of chondrocyte-derived Tie2-positive cells is increased, maintained, or the decrease in the number and / or ratio is slowed down.

[0103] The differentiation culture stage can further include, as needed, a process (a culture process based on culture conditions different from the differentiation culture process of the present application) other than the differentiation culture process of the present application, in accordance with the main purpose of the stage of differentiating chondrocyte-derived Tie2-positive cells into chondrocytes, particularly functional chondrocytes. As such a process, for example, a process of culturing a cell population containing chondrocyte-derived Tie2-positive cells in a culture medium to which no Tie2 expression enhancer (extracts of animal or plant origin, preferably extracts of plant origin) other than a growth factor has been added (referred to herein as an "additional differentiation culture process") can be cited. As a growth factor that can be added to the culture medium of the additional differentiation culture process, at least one selected from the group consisting of FGF, EGF, and Ang-1 can be cited. The additional differentiation culture process can be performed before or after the differentiation culture process of the present application, and is preferably performed after. The additional differentiation culture process can be a process that does not include passaging, or can be a process that includes one or two or more passages, as needed.

[0104] The production method of the present application can further include a culture stage other than the differentiation culture stage, as needed. As a further culture stage that can be included in the production method of the present application, for example, a culture stage performed before the differentiation culture stage for pre-propagating a cell population (containing chondrocyte-derived Tie2-positive cells, chondrocytes differentiated therefrom, and the like) to be supplied to the differentiation culture stage can be cited. In the present application, the culture stage performed for this purpose is sometimes referred to as a "propagation culture stage", and the process (method) performed in the propagation culture stage is referred to as a "propagation culture process (method)".

[0105] Note that in the proliferation culture stage (process, method), differentiation of the chondrocyte-derived Tie2-positive cells into other cells (chondrocytes) is also allowed to occur, but differs from the differentiation culture stage (process, method) of the present application in that no Tie2 expression enhancer (extracts from animal or plant sources, etc.) other than growth factors is added to the culture medium. On the other hand, in the proliferation culture stage (process, method), it is preferable to further add growth factors such as FGF as needed. The proliferation culture process can be set to a process that does not include subculturing, but can also be a process that includes subculturing once or twice or more as needed.

[0106] <cell population>

[0107] The ratio and / or number of chondrocyte-derived Tie2-positive cells to other cells, particularly (functional) chondrocytes differentiated from chondrocyte-derived Tie2-positive cells, etc. in each cell population of each culture process included in the culture method of the present application or the production method of the present application (each culture stage) (hereinafter collectively referred to as "pre-culture cell population" in the present specification) is basically arbitrary. In addition, the ratio of stem / progenitor cells to other cells, and the ratio of stem cells to progenitor cells, in the Tie2-positive cells in the pre-culture cell population is also basically arbitrary. The composition of the pre-culture cell population can be appropriately adjusted in accordance with the embodiment of the present application, and taking into account the effects of each culture method or each culture process, etc.

[0108] The cell population before culture can be prepared or prepared according to a conventional method. For example, in the case where a cell population contained in cartilage collected from a living body is used as the cell population before culture, the cartilage is chopped into an appropriate size using a curved surgical knife or the like, and then the cells are dispersed by treatment with a proteolytic enzyme such as collagenase, and the like, and the cell population contained in the cartilage is isolated and recovered by filtration, centrifugation, washing, and the like, as necessary. The cartilage can be collected from various tissues containing cartilage, such as hyaline cartilage of joints (knee joint, elbow joint, and the like), costal cartilage, tracheal cartilage, nasal septal cartilage, and the like; elastic cartilage of auricle cartilage, and the like; fibrous cartilage of meniscus, and the like, and there is no particular limitation on the tissue from which the cartilage is derived, as long as the effects of the present application can be exerted, and for example, hyaline cartilage of joints and the like is preferred. The cell population thus obtained (cell population containing cartilage-derived Tie2-positive cells) can be used as the cell population before culture for the culture method of the present application or the process of the differentiation culture stage (the differentiation culture process of the present application or the additional differentiation culture process, and the like, which is optionally performed), and the process of the stage other than the differentiation culture stage (the proliferation culture stage, and the like), as necessary. By so doing, the cell population (primary cells or subcultured cells) prepared from the articular cartilage (knee joint) or other tissues can be sold as an article (as a kit containing a culture medium, an instruction manual, a reagent, and the like, as necessary), and can be used as the cell population before culture containing cartilage-derived Tie2-positive cells in the present application.

[0109] The cell population isolated from cartilage or the cell population contained in cartilage (cartilage containing the cell population) prepared as described above can be cryopreserved according to a conventional method until use in the subsequent culture method or culture process. In the case of the cell population or cartilage cryopreserved, the cell population or cartilage can be thawed by a conventional method at the start of the subsequent culture method or culture process. At the time of cryopreservation and thawing, the treatment preferred for the cell population or cartilage can be combined. For example, a cryoprotective agent (DMSO or the like) can be added at the time of cryopreservation, and in this case, the cryoprotective agent can be removed under suitable conditions at the time of thawing.

[0110] In the cell population obtained by the culture method of the present application or the preparation method of the present application (each culture stage) (hereinafter referred to as "cell population after culture" in the present specification), the respective ratios and / or amounts of cartilage-derived Tie2-positive cells and other cells, and in particular, (functional) chondrocytes differentiated from cartilage-derived Tie2-positive cells in the present application, and the like, are basically arbitrary. In addition, the ratio of stem / progenitor cells and other cells contained in the Tie2-positive cells in the cell population after culture, and the ratio of stem cells and progenitor cells are also basically arbitrary. The composition of the cell population after culture can be appropriately adjusted according to the embodiment of the present application, and in consideration of the use of the cell population obtained by each culture method or each culture process, and the like.

[0111] The post-culture cell population can be recovered from the culture medium according to a conventional method and supplied to a subsequent culture method or culture process, or to other methods or processes such as preparation of a cell preparation.

[0112] In the cell population in the course of the culture process included in the culture method of the present application or the preparation method of the present application (each culture stage) (hereinafter referred to as "culture cell population" in the present specification), the respective ratios and / or amounts of the cartilage-derived Tie2-positive cells and other cells, particularly (functional) chondrocytes differentiated from the cartilage Tie2-positive cells in the present application, are basically arbitrary. Further, the ratios of the stem / progenitor cells and other cells, and the ratios of the stem cells and the progenitor cells, included in the Tie2-positive cells in the culture cell population are also basically arbitrary. The composition of the culture cell population is the composition in the course of change from the pre-culture cell population to the post-culture cell population, and, for example, the ratio of the cartilage-derived Tie2-positive cells in the culture cell population (hereinafter referred to as "Tie2-positive ratio" in the present specification) is generally a value within the range between the Tie2-positive ratio of the pre-culture cell population and the Tie2-positive ratio of the post-culture cell population, and a value temporarily deviating from the range is also permissible. The composition of the culture cell population varies depending on the embodiment of the present application, and further depending on the days, the number of passages, and the like, in the culture method of the present application or each culture process.

[0113] As for the "human or other animal" (donor) from which each of the above-described cell populations is derived, selection can be made in consideration of the use of the cell population ultimately obtained by the culture method of the present application or the preparation method of the present application, or the use of the cell population obtained by each culture process included in the preparation method of the present application (each culture stage), and the like. In a typical embodiment of the present application, when a cell population for manufacturing a cell preparation for prophylaxis or treatment of a prescribed disease, symptom, or the like is prepared, the "human or other animal" is a living organism of the same species as the administration subject (recipient) of the cell preparation, and is preferably a human. Further, the donor and the recipient can be the same individual (autologous), or can be different individuals (allogeneic).

[0114] • Cell population relating to the differentiation culture stage

[0115] The cell population to be used in the culture method of the present application or the cell population to be contained in the process included in the differentiation culture stage of the present application (the differentiation culture process of the present application or the additional differentiation culture process optionally performed, etc.) (referred to as "pre-differentiation culture cell population" in the present specification) is a primary culture cell population or a subculture cell population derived from a human or other animal, for example, a commercially available subculture cell population of cartilage origin. For example, a cell population contained in cartilage collected from a human or other animal in vivo, a (cell line-derived) cell population obtained by subculturing such a cell population can be used as the pre-differentiation culture cell population, and such a cell population is also commercially available.

[0116] The Tie2-positive rate of the pre-differentiation culture cell population can vary depending on individual differences in the cartilage from which it is derived, etc., although it is not particularly limited, and it is preferable that the ratio and / or the number of Tie2-positive cells of cartilage origin be as high as possible. For example, a cell population derived from a donor (young adult individual, etc.) having a good cartilage niche can be used as such a preferable cell population.

[0117] The use of the cell population obtained by each process included in the differentiation culture stage of the present application (referred to as "post-differentiation culture cell population" in the present specification) is not particularly limited. The composition of the post-differentiation culture cell population, for example, the ratio of Col2-positive (cartilage) cells (referred to as "Col2-positive rate" in the present specification) and the ratio of Tie2-positive cells (Tie2-positive rate), and the number of cells thereof can vary depending on the pre-differentiation culture cell population, individual differences in the cartilage from which it is derived, etc., and can be appropriately adjusted depending on the use. For example, for a cell population used to manufacture a cell preparation for transplantation, it is preferable that the cell population contain as many cartilage cells having a functionality useful for exerting a therapeutic or prophylactic effect by transplantation (for example, collagen type II-producing cartilage cells: Col2-positive cartilage cells) as possible, and also contain a number of cartilage-derived Tie2-positive cells that retain the ability to produce such cartilage cells.

[0118] • Cell populations related to stages other than the differentiation culture stage

[0119] When a stage other than the differentiation culture stage is performed as needed in the present application, with respect to the cell population to be used in the process included in the stage, and the cell population obtained by the process included in the stage, it can be said that it is the same as the above-described pre-differentiation culture cell population and the post-differentiation culture cell population. For example, as a cell population to be used in, for example, a proliferation culture process, the same cell population as the above-described pre-differentiation culture cell population can be used. The cell population obtained by the proliferation culture process can be used after being divided into an appropriate number of cells depending on the embodiment of the differentiation culture stage (the type, size, etc. of the culture vessel).

[0120] < Culture medium >

[0121] The medium used in the culture method of the present application or the production method of the present application (each culture step) can be selected as appropriate, taking into account the purpose of the culture method or the culture step, etc., as long as it is suitable for culturing the cartilage-derived Tie2-positive cells and the chondrocytes differentiated therefrom. For the medium used in the culture method of the present application or the differentiation culture step of the present application, it is sufficient to select an additive ingredient that is mainly intended for differentiation induction of the cartilage-derived Tie2-positive cells into chondrocytes and to use it together with a Tie2 expression enhancer other than a growth factor. The medium used in the additional differentiation culture step performed as needed in the present application, the step (e.g., the proliferation culture step) included in the stage other than the differentiation culture stage can also be prepared by selecting an appropriate base medium and an additive ingredient according to the purpose of the step.

[0122] In representative embodiments of the present application, the medium used for culturing the cartilage-derived Tie2-positive cells and / or the chondrocytes differentiated therefrom (a cell population mixed with these), the medium used in each step of the differentiation culture stage and other culture stages, e.g., the medium used in the differentiation culture step of the present application and the medium used in the additional differentiation culture step, the medium used in the proliferation culture step, can be prepared, for example, by using an appropriate amount of the following base medium, additive ingredient, growth factor, other ingredient, respectively.

[0123] As the base medium for planar culture, for example, DMEM (Dulbecco's Modified Eagle's Medium, with or without glucose), aMEM (Alpha Modified Eagle's Minimum Essential Medium), Ham's F-10 medium, Ham's F-12 medium, or a mixture thereof (e.g., a mixed medium of DMEM and F-10 medium or F-12 medium) can be exemplified. In addition, as the base medium for suspension culture (three-dimensional culture), for example, methylcellulose medium (e.g., trade name "MethoCult", STEMCELL Technologies, Inc.) can be exemplified.

[0124] As the additive ingredient added to the base medium, for example, FBS (fetal bovine serum), BSA (bovine serum albumin), L-ascorbic acid (as L-ascorbic acid magnesium phosphate salt, etc.), selenous acid (as insulin-transferrin-sodium selenite (ITS), etc.), and 2-mercaptoethanol can be exemplified. As needed, further, penicillin, streptomycin, etc., antibiotics, other ingredients can be added to the medium.

[0125] As growth factors, for example, FGF (fibroblast growth factor), EGF (Epidermal Growth Factor), Ang-1 (angiopoietin-1) can be listed. In one embodiment of the present application, the growth factor added to the culture medium preferably uses at least FGF, more preferably uses both FGF and EGF, and preferably adds Ang-1 as needed in addition to these.

[0126] As FGF, for example, bFGF (basic fibroblast growth factor. Sometimes also referred to as FGF-2.) can be used. The concentration of FGF in the culture medium can generally be in the range of 1 to 50 ng / mL, preferably in the range of 5 to 15 ng / mL, for example, about 10 ng / mL.

[0127] Ang-1 is preferably added to the serum-free culture medium. In addition, as Ang-1, a water-soluble one (soluble Ang-1, recombinant Ang-1) is preferred. The concentration of Ang-1 (preferably soluble Ang-1) in the culture medium can generally be in the range of 100 to 1000 ng / mL, for example, about 500 ng / mL.

[0128] Note that the above-mentioned growth factors such as FGF, EGF, Ang-1 are "growth factors having a Tie2 expression enhancing effect", and can also be understood as "Tie2 expression enhancers" in a broad sense, but the handling of these growth factors in the present application is described elsewhere in this specification. That is, the culture medium used in the differentiation culture step of the present application contains at least a "Tie2 expression enhancer other than a growth factor" (extracts from animals and plants, etc., preferably extracts from plants), and can further contain a "growth factor having a Tie2 expression enhancing effect" such as FGF, EGF, Ang-1. On the other hand, the culture medium used in the additional differentiation culture step does not contain a "Tie2 expression enhancer other than a growth factor" (extracts from animals and plants, etc.), and can contain (preferably contains) a "growth factor having a Tie2 expression enhancing effect" such as FGF, EGF, Ang-1.

[0129] In one embodiment of the present application, as the culture medium used in the differentiation culture step of the present application, a culture medium obtained by adding 2-mercaptoethanol, sodium selenite, ascorbic acid, and 30% BSA to a mixed culture medium of DMEM and F-10 medium, and adding 30% FBS just before use can be used.

[0130] In one embodiment of the present application, the additional differentiation culture step is performed by suspension culture (three-dimensional culture), and as a culture medium for such an additional differentiation culture step, a methylcellulose culture medium can be used.

[0131] In one embodiment of the present application, as a culture medium for the proliferation culture step, aMEM to which 20% FBS is added and to which a growth factor such as FGF can be further added as needed can be used.

[0132] <Tie2 expression enhancer>

[0133] In the culture method of the present application, at least one "Tie2 expression enhancer" other than a growth factor having a Tie2 expression enhancing effect is added to the culture medium. The culture method of the present application is implemented in the process in the differentiation culture stage (as the differentiation culture step of the present application), and by adding a Tie2 expression enhancer at this time, the following effects and the like are obtained: the juvenile nature (proliferation ability and ability to differentiate into at least chondrocytes) of the cartilage-derived Tie2-positive cells is maintained, and the number or ratio of cells that differentiate into functional chondrocytes is increased. The Tie2 expression enhancer can be used as any one, or two or more can be used in combination, and it is sufficient that it is added to the culture medium in an amount in which the above-mentioned Tie2 activity effect can be observed.

[0134] As the "growth factor having a Tie2 expression enhancing effect", for example, Angiopoietin-1 (Ang-1), FGF2 (bFGF), and the like can be listed. In the culture method of the present application, at least one "Tie2 expression enhancer" other than such a "growth factor having a Tie2 expression enhancing effect" is used, but a growth factor having a Tie2 expression enhancing effect can also be used in combination as needed. The culture method of the present application is implemented in the process in the differentiation culture stage (as the differentiation culture step of the present application), and by using a growth factor having a Tie2 expression enhancing effect and a further Tie2 expression enhancer, such as the animal and plant-derived extracts described below, preferably a plant-derived extract, in combination at this time, a synergistic effect can be exerted.

[0135] As a Tie2 expression enhancer other than growth factors, extracts from various source plants known in the prior art as "Tie2 activators" can be used. Examples of such source plant extracts include extracts of milkweed, winged chrysanthemum, tamarind (Tamarindus indica), turmeric, wolfberry, polygonatum, plantain, wingless pigweed, olive fruit, oyster, chamomile, papaya, trichosanthes seed, morinda root, chrysanthemum, polygonatum rhizome, soapberry, ginkgo, dwarf jasmine, wolfberry, oak, alpinia galanga, red ginseng, oak, hawthorn, small red clover, guava, eleutherococcus senticosus, star fruit, star fruit, soapberry thorn, jujube, camphor, allium macrostemon, lotus, wild taro, prickly ash, long pepper, aralia, mango ginger, ginger oil, five-finger vine, double-flowered daylily, myrica, mountain willow, linear asparagus, etc. (see Patent Documents 4-10 above). Alternatively, components contained in such extracts, such as ursolic acid, corosolic acid, 3-O-galloyl proanthocyanidin B-1, linolenic acid, 13-hydroxy-9Z,11E,15E-octadecanoic acid, proanthocyanidin B-2, epicatechin-(4β-6)-epicatechin-(4β-8)-epicatechin, proanthocyanidin C-1, astragaloside VIII, daidzein I, 3'-O-methylgallocatechin, piperidine nonadienylpiperidine, eugenol, 2-methoxycinnamonaldehyde, eleutheroside E, eleutheroside E1, sesamin, eudustomin, forsythinol, pinoresinol, liriope resin phenol B dimethyl ether, forsythinol, coumarin, etc. (refer to the above patent documents 7, 12-14) as Tie2 expression enhancers other than growth factors. For each extract and component, the dosage for which Tie2 expression enhancement is observed, the suitable plant or animal parts (materials) for preparation and extraction methods, and the purification methods for specific components can also be appropriately determined by those skilled in the art based on existing known methods.

[0136] From an industrial point of view, it is advantageous to use one or more extracts selected from the above-mentioned plant-derived plants that are cheaper than growth factors such as Ang-1 and FGF2, preferably have a better Tie2 expression enhancement effect than these growth factors, and more preferably have a synergistic effect when used in combination with these growth factors, as the Tie2 expression enhancer in the differentiation culture process of the present invention.

[0137] Extracts from Cinnamomum camphora plants

[0138] In a preferred embodiment of the present application, as the Tie2 expression enhancer, an extract from a plant of the genus Cinnamomum can be used. The genus Cinnamomum includes more than 300 species such as Cinnamomum cassia Blume, C. camphora, C. daphnoides, Cinnamomum doederleinii, C. japonicum, Cinnamomum pseudo-pedunculatum, Cinnamomum sieboldii, C. verum, C. zeylanicum, and the like. For example, an extract of a product manufactured and sold in the form of a tender branch of cinnamon, i.e., ramulus cinnamomi, or a bark of cinnamon, i.e., cortex cinnamomi, or a powdered cinnamon, can be used as the extract from a plant of the genus Cinnamomum in the present application.

[0139] The extract from a plant of the genus Cinnamomum can be obtained by a conventional method, for example, by impregnation or heating reflux of a plant body (e.g., powdered cinnamon) as a raw material with an extraction solvent at ordinary temperature or with heating, followed by recovery of supernatant or filtration of a filtrate, and concentration as necessary. As the extraction solvent, a solvent generally used for extraction can be used alone or in combination, such as an aqueous solvent, for example, water, physiological saline, a phosphate buffer, a boric acid buffer, or an organic solvent, for example, an alcohol such as ethanol, propylene glycol, 1,3-butanediol, glycerol, and the like; a water-alcohol mixed solvent; chloroform; dichloroethane; carbon tetrachloride; acetone; ethyl acetate; hexane; and the like. Preferably, water is used as the solvent. The extract obtained by extraction with the above solvent can be used directly in the form of an extract solution, but from the aspect of convenience, it is solidified (powdered) by drying or freeze-drying, and the like, and stored, and at the time of use, it is diluted or redissolved (redispersed) as necessary by a suitable solvent, and further, it is treated by filtration, and the like, as necessary, and thus can be used. The extract from a plant of the genus Cinnamomum can be one from which impurities are removed (purified product) by an adsorption method using an ion exchange resin (e.g., a porous polymer such as Amber Light XAD-2), and the like, as necessary.

[0140] The concentration of the extract from a plant of the genus Cinnamomum in the culture medium can be appropriately adjusted depending on the properties of the extract used, and the degree of the effect as a Tie2 expression enhancer, and the like. For example, when an extract solution obtained by extraction of 10 mg of powdered cinnamon with 1 mL of water (distilled water) is used as the extract from a plant of the genus Cinnamomum, the extract solution can be added to the culture medium in an amount of about 0.1 to 10 v / v %, for example, about 2 v / v %. In the case of changing the extraction and addition, the effective component as a Tie2 expression enhancer can be made to be the same degree as in the above-described extraction and addition.

[0141] Note that the extract from Cinnamomum species obtained in the above manner contains various compounds, and the compounds effective for the effects of the present application are not particularly limited, and e.g. sylvatin (one of the compounds belonging to furanofuran-type lignans) can be used as one of the compounds having the Tie2 expression-enhancing effect in the present application.

[0142] <Incubation time, other conditions>

[0143] The time and other conditions (e.g. pH, CO2 concentration, O2 concentration, etc.) of each culture step included in the culture method of the present application and the production method of the present application (each culture stage) can basically be appropriately adjusted according to the purpose of the culture step (including the culture stage of the culture step) to obtain a cell population having a desired cell composition (kind and amount / ratio). The pH can be set to weakly alkaline (e.g. about 7.15). The CO2 concentration can be set to e.g. about 5%. The O2 concentration can be set to 5% or less (e.g. about 2%). The culture medium can be appropriately replaced with a fresh culture medium every prescribed number of days, or the culture medium can be changed by adding a component or increasing or decreasing the concentration of a component, pH, etc. or the atmosphere can be changed as needed during the culture method of the present application and the time of each culture step (stage).

[0144] The time of the culture step in the differentiation culture stage of the present application (the culture time of each step when the differentiation culture stage includes a plurality of culture steps, e.g. when the differentiation culture step of the present application and the additional differentiation culture step are included) is usually about 1 to 3 weeks, and e.g. the differentiation culture step of the present application can be set to about 1 week and the additional differentiation culture step can be set to about 2 weeks. The differentiation culture stage can be ended at the time when a desired differentiation culture after cell population is obtained.

[0145] The time of the culture step in the proliferation culture stage of the present application (the culture time of each step when the proliferation culture stage includes a plurality of culture steps) is usually about 1 to 3 weeks, and e.g. can be set to about 1 week. The proliferation culture stage can be ended at the time when a desired proliferation culture after cell population is obtained.

[0146] <Culture vessel>

[0147] The culture vessel, culture device, etc. used in each culture step included in the culture method of the present application and the production method of the present application (each culture stage) can basically be appropriately selected according to the purpose of the culture method and the culture step (including the culture stage of the culture step) to obtain a cell population having a desired cell composition (kind and amount / ratio).

[0148] The culture vessel can use a flask, a culture dish, a well plate, a bag, or the like, which is a vessel having a general shape, and can be formed with a well capable of housing cells. The culture vessel can use one made of a general material such as glass, plastic, resin, or the like. The size (area, volume) of the culture vessel, and the size (diameter, depth) and number of the wells of the culture vessel when it has wells, and the like can also be appropriately selected.

[0149] The culture method of the present application and the differentiation culture step of the present application, and the proliferation culture step and the like of the present application, which are performed as needed, are typically performed by planar culture of cells by attachment to the culture surface of the culture vessel. Such planar culture is preferably performed using a culture vessel subjected to coating treatment with a prescribed coating agent, and can also be performed using other known means for planar culture, such as a culture vessel that has not been subjected to such coating treatment.

[0150] The additional differentiation culture step of the present application, which is performed as needed, is typically performed by suspension culture of cells suspended in a culture medium. Such suspension culture can be performed, for example, using a methylcellulose culture medium having viscosity as the culture medium, or in a culture vessel having a culture surface subjected to low adhesion treatment (coated with a substance for preventing cell adhesion), with shaking as needed using a culture shaker or the like.

[0151] • Coating agent

[0152] In the culture method of the present application and the differentiation culture step of the present application, a culture vessel having a culture surface subjected to treatment with a coating agent containing extracellular matrix (ECM) or other biologically relevant molecules (hereinafter sometimes referred to simply as a "coated culture vessel" in the present specification) is preferably used. Note that a coated culture vessel can also be used in the proliferation culture step.

[0153] As the ECM contained in the coating agent in the present application, various known ECMs such as collagen (type I, type II, type IV, etc.) or gelatin as a heat-treated product thereof, chondroitin sulfate A, fibronectin, gelatin, laminin, thrombospondin, vitronectin, and proteoglycan (aggregcan, heparan sulfate proteoglycan, etc.) can be exemplified. In addition, as the organism-related molecule other than ECM, polyamino acid such as polylysine (poly-L-lysine or poly-D-lysine) can be exemplified. As the coating agent other than the above, polyglycolic acid, PLGA (poly-lactic acid · glycolic acid copolymer), polyhydroxyalkanoic acid (PHA), poly-ε-caprolactone, polyortho ester, polyanhydride, polyphosphazene, polydimethylsiloxane, polyurethane, polytetrafluoroethylene, polyethylene, polysulfone, polymethyl methacrylate, poly(2-hydroxyethyl methacrylate), polyamide, polypropylene, polyvinyl chloride, polystyrene, polyvinylpyrrolidone, polyornithine, etc. can be exemplified. The coating agent for cell adhesion treatment can contain any one of the above-described substances, or can contain two or more of them. In one example of the preferred embodiment of the present application, the coating agent contains polyamino acid such as polylysine (e.g., poly-L-lysine).

[0154] -Composition for Cell Therapy-

[0155] The composition for cell therapy of the present application can contain a cell population obtained by the culture method or the production method of the present application as described above, i.e., a cell population containing chondrocytes differentiated from chondrocyte-derived Tie2-positive cells (particularly, functional chondrocytes such as Col2-positive cells) and preferably further containing chondrocyte-derived Tie2-positive cells to some extent, and, as needed, other pharmaceutically acceptable ingredients.

[0156] In a typical embodiment of the present application, the composition for cell therapy is a composition for treating or preventing damage, symptoms, or diseases of cartilage. As the applicable subject of the composition for cell therapy of this embodiment, i.e., damage, symptoms, or diseases of cartilage that can be prevented or treated by administering the composition, for example, cartilage injury, osteochondral injury, meniscus injury, arthritis, arthropathy can be exemplified.

[0157] The dosage form of the composition for cell therapy of the present application can be any dosage form that can be transplanted or delivered to a site targeted by the cell population, such as a tissue containing cartilage, e.g., a joint, and can be, for example, an injection, preferably an injection for local administration to cartilage or its vicinity or an injection for vascular administration that can be targeted.

[0158] As the pharmaceutically acceptable ingredient, for example, water for injection or physiological saline when prepared in the form of an injection, a culture solution for a cell population, other appropriate solvents · dispersion media, other additives, etc. can be exemplified.

[0159] The cell therapy composition of the present application can be administered to humans and animals other than humans in an amount sufficient to exert the desired therapeutic or prophylactic effect. Such an effective amount can be appropriately adjusted by the amount of administration per time, the number of administration, and the interval of administration (the number of administration within a certain period of time) and the like, taking into account the components of the cell therapy composition, the dosage form, the subject of administration, the route of administration, and the mode of administration, and the like. The cell therapy composition of the present application can be administered to humans and animals other than humans.

[0160] Example

[0161] In the following example, as the "cell population containing cartilage-derived Tie2-positive cells" in the present application, commercially available knee joint cartilage cells (cell population derived from knee joint cartilage of normal adult human, passage 2) (hereinafter referred to as "NHAC2") were used.

[0162] As the culture medium for the "proliferation culture stage" in the example (hereinafter referred to as "proliferation culture medium"), a culture medium obtained by adding 20% FBS to αMEM immediately before use was prepared and used.

[0163] As the culture medium for the "first culture step of the differentiation culture stage" in the example (hereinafter referred to as "differentiation culture medium for the first step"), a culture medium obtained by adding 2-mercaptoethanol 1 μL, selenous acid (0.01%) 6 μL, ascorbic acid (5 mg / mL) 1.5 mL, and 30% BSA 5 mL to a mixed culture medium of DMEM (without glucose) 60 mL and F-10 40 mL, and further adding 30% FBS and "2% cinnamon" or "10 ng / mL bFGF" to the culture medium immediately before use was prepared and used. "2% cinnamon" means that "extract (cinnamon extract) obtained by suspending 10 mg of commercially available cinnamon powder in 1 mL of distilled water and extracting overnight at 37°C" was added to the culture medium in an amount of 2%.

[0164] As the culture medium for the "second culture step of the differentiation culture stage" in the example (hereinafter referred to as "differentiation culture medium for the second step"), a methylcellulose culture medium (trade name "MethoCult H4230", STEMCELL Technologies Inc., without growth factors) was used.

[0165] [Example 1]

[0166] NHAC2 was dispersed in the proliferation culture medium and flat culture was started in one well of a 6-well culture dish subjected to coating treatment with poly-L-lysine (PLL) (day 0).

[0167] After 7 days (day 7), the cell population after the proliferation culture step was recovered, dispersed in the medium for the first step of the differentiation culture (with the addition of either "2% cinnamon" or "10 ng / mL bFGF"), and started with planar culture in one well of a 6-well culture dish subjected to coating treatment with PLL.

[0168] After 7 days (day 14), the cell population after the first step of the differentiation culture was recovered, dispersed in the medium for the second step of the differentiation culture, and started with suspension culture in one well of a 6-well culture dish not subjected to coating treatment. After 14 days (day 28), the cell population after the second step of the differentiation culture was recovered, and the number of cells positive for Tie2 expression on the cell surface was measured by flow cytometry (FCM) method, and the ratio to the number of cells in the entire cell population (Tie2-positive rate) was calculated. In the FCM method, a fluorescent labeling agent (R&D Systems, Anti-Tie-2, Human, Mouse-Mono (87315), Allophocyanin, Catalog No. FAB3131A) was used, which is a complex of an anti-human Tie2 antibody and a fluorescent pigment, allophycocyanin. The above culture and measurement were repeated three times.

[0169] The results of the Tie2-positive rate (mean ± standard error, n = 3) are shown in Figure 1 The Tie2-positive rate in the cell population obtained when "2% cinnamon" was added during the differentiation culture step was significantly higher (p < 0.05) than when "10 ng / mL bFGF" was added, and the effect of enhancing Tie2 expression by the differentiation culture step according to the present application was confirmed.

[0170] [Example 2]

[0171] The NHAC2 was separated into cells positive for Tie2 expression and cells negative for Tie2 expression by the FCM method. The Tie2-positive cells and the Tie2-negative cells were each cultured by the same method as in Example 1 (proliferation culture step, first step of differentiation culture, and second step of differentiation culture). For the Tie2-positive cells and the Tie2-negative cells, the spherical (spheroid) colony-forming unit (CFU-S) and the fibroblast-like colony-forming unit (CFU-F) in the cell population after the second step of the differentiation culture were each measured. The above culture and measurement were repeated three times.

[0172] The results of the number of colonies per 1000 cells (mean ± standard error, n = 3) are shown in Figure 2 The Tie2-positive cells had a significantly higher (p < 0.05) number of spherical colonies (CFU-S) than the Tie2-negative cells, and it was considered that the Tie2-positive cells derived from cartilage had stronger properties as stem / progenitor cells to form spherical colonies.

[0173] [Example 3]

[0174] The number of spheroid (spheroid) colony-forming units (CFU-S) and fibroblast-like colony-forming units (CFU-F) in the cell population after the second differentiation culture step when cultured by the same method as in Example 1 (proliferation culture step, first differentiation culture step, and second differentiation culture step) was measured, and the comparison was made by the difference in the added ingredient ("2% cinnamon" and "10 ng / mL bFGF") added to the culture medium in the first differentiation culture step. The above culture and measurement were repeated three times.

[0175] The results of the number of colonies per 1000 cells (mean ± standard error, n = 3) are shown in Figure 3 [a] of FIG. 6. In addition, the optical microscope photograph of the colonies formed when "2% cinnamon" was added is shown in Figure 3 [b] of FIG. 6. The spheroid colony-forming units (CFU-S) when "2% cinnamon" was added were significantly higher (p < 0.05) than when "10 ng / mL bFGF" was added. From the above results, it can be considered that a cell population containing "cells having stronger properties as stem / progenitor cells to form spheroid colonies due to an increase in the Tie2-positive rate of the cell population or an increase in the expression of Tie2-positive cells" was obtained by using the "Tie2 expression enhancer other than a growth factor" (cinnamon as an example thereof) in the present application.

[0176] [Example 4]

[0177] Culturing was performed by the same method as in Example 1 (proliferation culture step, first differentiation culture step, and second differentiation culture step). The cell population after the second differentiation culture step was recovered, and the number of cells in which the expression of each of proteoglycan, type I collagen, and type II collagen on the cell surface was positive was measured by flow cytometry (FCM) method, and the ratio to the number of cells in the entire cell population (positive rate) was calculated.

[0178] The results of the positive rate of each of proteoglycan (PG), type I collagen (Type 1 col), and type II collagen (Type 2 col) (mean ± standard error, n = 3) are shown in Figure 4 . Regarding type II collagen, the positive rate in the cell population obtained when "2% cinnamon" was added in the first differentiation culture step was significantly higher (p < 0.05) than when "10 ng / mL bFGF" was added. It was shown that by performing the differentiation culture step (first differentiation culture step) based on the culture method of the present application, a cell population rich in functional chondrocytes in which the expression of type II collagen was positive could be prepared.

Claims

1. A method for culturing a cell population containing Tie2, a tyrosine kinase derived from human knee cartilage and containing Ig and EGF homologous domain-2, which is positive for expression. The cells described below will be referred to as "Tie2-positive cells derived from human knee cartilage". The culture method includes the following steps: culturing a cell population containing Tie2-positive cells derived from human knee cartilage in a culture medium supplemented with at least one plant-derived extract that enhances Tie2 expression. The plant in question is the Chinese cinnamon tree (Cinnamomumcassia Blume). The culture method is carried out in a culture vessel having a culture surface coated with a cell adhesion treatment agent containing extracellular matrix and / or polyamino acids.

2. The cultivation method according to claim 1, wherein, The extracellular matrix is ​​type IV collagen and / or fibronectin.

3. The cultivation method according to claim 1, wherein, The polyamino acid is polylysine.

4. A method for preparing a cell population comprising chondrocytes differentiated from Tie2-positive cells derived from human knee cartilage. The preparation method includes: A culture stage comprising the steps of the culture method according to any one of claims 1 to 3, for simultaneously differentiating and inducing Tie2 expression in human knee cartilage-derived Tie2-positive cells in a cell population into chondrocytes. The cultivation stage described below will be referred to as the "differentiation cultivation stage".

5. The preparation method according to claim 4, wherein, The chondrocytes include cells that are positive for Col2 (type II collagen).

6. The preparation method according to claim 4, wherein, The differentiation and culture stage further includes the following steps: culturing a cell population containing Tie2-positive cells derived from human knee cartilage in a culture medium supplemented with at least one growth factor selected from the group consisting of FGF (fibroblast growth factor), EGF (epidermal growth factor) and Ang-1 (angiopoietin-1).

7. The preparation method according to any one of claims 4 to 6, wherein, Prior to the differentiation culture stage, the following are included: The culture phase is used to proliferate a cell population containing Tie2-positive cells derived from human knee cartilage. The cultivation stage described below will be referred to as the "proliferation cultivation stage".

8. The preparation method according to any one of claims 4 to 6, wherein, The cell population obtained through the differentiation and culture phase also contained Tie2-positive cells derived from human knee cartilage.

9. The preparation method according to claim 7, wherein, The cell population obtained through the differentiation and culture phase also contained Tie2-positive cells derived from human knee cartilage.

Images