WO2016125582A1 - Agent for promoting proliferation of mesenchymal stem cells, agent for promoting cartilage differentiation of mesenchymal stem cells, mesenchymal-stem-cell preparation method, chondrocytic-cell preparation method and mesenchymal-stem-cell culture medium - Google Patents

Agent for promoting proliferation of mesenchymal stem cells, agent for promoting cartilage differentiation of mesenchymal stem cells, mesenchymal-stem-cell preparation method, chondrocytic-cell preparation method and mesenchymal-stem-cell culture medium Download PDF

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WO2016125582A1
WO2016125582A1 PCT/JP2016/051447 JP2016051447W WO2016125582A1 WO 2016125582 A1 WO2016125582 A1 WO 2016125582A1 JP 2016051447 W JP2016051447 W JP 2016051447W WO 2016125582 A1 WO2016125582 A1 WO 2016125582A1
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mesenchymal stem
stem cells
mesenchymal
culture medium
interleukin
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PCT/JP2016/051447
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French (fr)
Japanese (ja)
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一郎 関矢
宗田　大
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国立大学法人東京医科歯科大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses

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  • the present invention relates to a mesenchymal stem cell growth promoter, a mesenchymal stem cell cartilage differentiation promoter, a mesenchymal stem cell preparation method, a chondrogenic cell preparation method, and a mesenchymal stem cell culture medium.
  • Suture and excision are known as treatment methods for meniscus injury. Suture may cause re-tearing, and may not be applicable depending on the damaged site. On the other hand, excision can be applied to places where the teared part cannot be excised by suturing, and resecting can be prevented by excision, but the function of the excised part is impaired.
  • mesenchymal stem cells are known to differentiate into various cells and are also known to differentiate into cartilage.
  • Patent Documents 1 and 2 disclose such mesenchymal systems.
  • a medium that can be used for culturing stem cells is disclosed.
  • Patent Document 3 discloses a method for preparing mesenchymal stem cells with good culture efficiency using dexamethasone.
  • Patent Document 4 discloses a cartilage that can induce differentiation of mesenchymal stem cells into cartilage without using a medium component for differentiation induction or a differentiation induction medium by modifying the culture surface with a dendrimer compound. A method for preparing cells is disclosed.
  • the present invention replaces the conventional method for preparing mesenchymal stem cells and the method for preparing chondrocytes from mesenchymal stem cells, as described above, and a novel mesenchymal system excellent in the efficiency of promoting proliferation of mesenchymal stem cells.
  • Preparation method of stem cells preparation method of chondrocytes excellent in efficiency of promoting differentiation of mesenchymal stem cells into chondrocytes, proliferation promoter of mesenchymal stem cells, promoter of cartilage differentiation of mesenchymal stem cells, and such
  • An object is to provide a culture medium for mesenchymal stem cells that can be used for the preparation of mesenchymal stem cells.
  • the present inventors have found that interleukin promotes the proliferation of mesenchymal stem cells and promotes the differentiation of mesenchymal stem cells into chondrocytes, and has completed the present invention. More specifically, the present invention provides the following.
  • a method for preparing mesenchymal stem cells comprising a step of culturing synovial stem cells derived from synovium in a culture medium containing IL-1 and / or TNF- ⁇ .
  • a method for preparing mesenchymal stem cells comprising adding interleukin to a culture medium for mesenchymal stem cells, and culturing the mesenchymal stem cells in the mesenchymal stem cell culture medium after the addition.
  • a method for preparing mesenchymal stem cells comprising adding inflammatory cytokines to a mesenchymal stem cell culture medium, and culturing the mesenchymal stem cells in the mesenchymal stem cell culture medium after the addition.
  • inflammatory cytokine includes one or more selected from the group consisting of IL-1 and TNF- ⁇ .
  • a mesenchymal stem cell proliferation promoter comprising interleukin.
  • An agent for promoting cartilage differentiation of mesenchymal stem cells comprising interleukin.
  • a culture medium for mesenchymal stem cells for addition of interleukin (13) A culture medium for mesenchymal stem cells for addition of interleukin.
  • a mesenchymal stem cell proliferation promoter comprising inflammatory cytokines.
  • An agent for promoting cartilage differentiation of mesenchymal stem cells comprising inflammatory cytokines.
  • a culture medium for mesenchymal stem cells comprising interleukin and / or inflammatory cytokine.
  • the preparation of a novel mesenchymal stem cell excellent in the efficiency of promoting proliferation of mesenchymal stem cells is provided.
  • Method, preparation method of chondrocytes excellent in efficiency of promoting differentiation of mesenchymal stem cells into chondrocytes, agent for promoting proliferation of mesenchymal stem cells, agent for promoting cartilage differentiation of mesenchymal stem cells, and such mesenchymal system A culture medium for mesenchymal stem cells that can be used for the preparation of stem cells can be provided.
  • FIG. (A) is a figure which shows the image after crystal violet dyeing
  • FIG. (B) is a figure which shows the image after crystal violet dyeing
  • FIG. It is a figure which shows the graph of the cell number of the mesenchymal stem cell which concerns on Example 3 and the control example 3.
  • FIG. FIG. 6 is a view showing images after crystal violet staining of mesenchymal stem cells according to Examples 4 to 6 and Control Example 4.
  • FIG. 10 is a view showing images after crystal violet staining of mesenchymal stem cells according to Examples 7 to 10 and Control Example 5.
  • FIG. 11 It is a figure which shows the image after crystal violet dyeing
  • FIG. It is a figure which shows the graph of the doubling number of the cell about the mesenchymal stem cell which concerns on Example 11, 12, Reference example 1, and the control example 6, and a cell number.
  • FIG. It is a figure which shows the image of the chondrocyte which concerns on Example 13 and the control example 7.
  • FIG. It is a figure which shows the image of the chondrocyte which concerns on Examples 14-16.
  • FIG. 1 It is a figure which shows the image after crystal violet dyeing
  • FIG. It is a figure which shows the image after crystal violet dyeing
  • FIG. It is a figure which shows the ratio which the number of colonies after culture
  • FIG. It is a figure which shows the image of the chondrocyte which concerns on Example 20 and the control example 11.
  • the method for preparing mesenchymal stem cells of the present invention comprises a step of adding interleukin to a culture medium for mesenchymal stem cells and culturing the mesenchymal stem cells in the culture medium for mesenchymal stem cells after the addition.
  • cell proliferation includes one or both of increasing the colony formation rate and increasing the colony size in the medium.
  • interleukin is not particularly limited, and examples include IL-1 (IL-1 ⁇ , IL-1 ⁇ , etc.), IL-6, IL-10, IL-17, and the like. Of these, IL-1 (IL-1 ⁇ , IL-1 ⁇ , etc.), IL-6, etc., which are inflammatory cytokines, are particularly preferred. IL-1 includes IL-1 ⁇ , IL-1 ⁇ and the like. Of these, IL-1 ⁇ and IL-1 ⁇ are particularly preferred. Interleukin may be used independently and may use 2 or more types together.
  • inflammatory cytokines have been conventionally thought to inhibit the proliferation of chondrocytes, but the present inventors have unexpectedly used mesenchymal stem cells by using inflammatory cytokines. It was found that proliferation was promoted. This is presumably because mesenchymal stem cells exhibit proliferative ability when they become inflamed upon contact with interleukins.
  • the origin of the interleukin species is not particularly limited, and interleukins derived from humans, pigs, cows, rabbits, rats, mice, etc. can be used, and the homology between them and amino acid sequences is 90% or more.
  • the mesenchymal stem cells are not particularly limited, and mesenchymal stem cells of various mesenchymal tissues including mesenchymal stem cells can be used.
  • mesenchymal stem cells of various mesenchymal tissues including mesenchymal stem cells can be used.
  • mesenchymal stem cells for example, synovial membrane, periosteum, muscle, fat (subcutaneous fat, etc.) ), Mesenchymal stem cells such as bone tissue, ligament, tendon, meniscus and the like.
  • synovial-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells because the rate of proliferation of mesenchymal stem cells is promoted.
  • any medium can be used as long as it is a known medium that can be used for culturing conventional mesenchymal stem cells.
  • Examples of such a culture medium for mesenchymal stem cells include ⁇ MEM ( ⁇ -Minimum Essential Medium), DMEM (Doulbecco's Modified Eagle's Medium), and EMEM (Eagle's Minimum Essential Medium).
  • Medium ⁇ MEM ( ⁇ -Minimum Essential Medium), DMEM (Doulbecco's Modified Eagle's Medium), and EMEM (Eagle's Minimum Essential Medium).
  • Medium Ham F12, Ham F10, F12K (Kaighns modified of Ham's F12), Leibovitz's L-15 medium, McCoy's 5A, and Medium 199 (199 medium) are not particularly limited. .
  • the use of interleukin facilitates the proliferation of mesenchymal stem cells, and is particularly suitable for culturing in a serum-free medium. From such a viewpoint, it is preferable to use a serum-free medium as the medium.
  • the serum-free medium is not particularly limited, and examples thereof include STEMPRO (registered trademark) MSC SFM (Life Technologies) and STK2 (Tucell).
  • the culture process in the present invention is a process in which interleukin is added to the culture medium for mesenchymal stem cells and the mesenchymal stem cells are cultured in the culture medium for mesenchymal stem cells after the addition
  • the method is particularly It is not limited.
  • interleukins may be added after culturing for a certain period of time in a state containing mesenchymal stem cells in the culture medium for mesenchymal stem cells. Stem cells and interleukins may be added simultaneously.
  • mesenchymal stem cells may be added in a state where interleukin is added to the culture medium for mesenchymal stem cells and interleukin is added to the medium in advance. That is, the culturing step in the present invention may include a step of culturing mesenchymal stem cells in a culture medium containing interleukin.
  • the culture conditions in the culture step can be appropriately set according to the types of mesenchymal stem cells and interleukins, the number of target mesenchymal stem cells, and the like. For example, at a temperature at which normal mesenchymal stem cells are cultured (eg, 37 ° C.), the total time is 0.5 to 28 days (eg, 1 to 24 days, 2 to 20 days, 4 to 18 days, and 6 to 16 days). 8-14 days, 10-12 days, etc.).
  • the use of interleukin promotes the growth of mesenchymal stem cells and is therefore suitable for short-term culture. For example, a large amount of mesenchymal stem cells can be obtained even by culturing for 3 days or less after the addition of interleukin.
  • interleukin when interleukin is added after the start of culture is not particularly limited. For example, even when interleukin is added during 0 to 24 days However, it is preferable to add interleukin between 0 and 20 days after the start of culture, and 0 to 14 days (for example, 0 to 7, 7 to 14, 2 to 12, 4 to 10 days, More preferably, interleukin is added during 6 to 8 days).
  • the amount of interleukin added to the culture medium for mesenchymal stem cells is not particularly limited.
  • the concentration of interleukin in the medium after addition is 0.00001 to 100 ng / ml (for example, 0 .0001-90 ng / ml, 0.001-70 ng / ml, 0.01-60 ng / ml, 0.05-50 ng / ml, 0.1-30 ng / ml, 0.5-10 ng / ml, 1-5 ng / Ml, etc.), but proliferation of mesenchymal stem cells is promoted when the amount of interleukin added is large.
  • the interleukin concentration in the medium after addition is preferably 0.005 ng / ml or more, more preferably 0.05 ng / ml or more, and 1.0 ng / ml. More preferably, it is more preferably 10 ng / ml or more.
  • the culture medium for mesenchymal stem cells may or may not contain interleukins in advance, but it may be used for mesenchymal stem cells even if it contains interleukins in advance. If the concentration of interleukin in the culture medium is too low, the growth promoting effect is not sufficiently exhibited. Therefore, regardless of whether or not it contains interleukins, the method for preparing mesenchymal stem cells of the present invention uses a mesenchymal stem cell culture medium in which the interleukin concentration is 0.00001 ng / ml or more.
  • the mesenchymal stem cells are cultured in a culture medium for mesenchymal stem cells of 001 ng / ml or more (0.005 ng / ml or more), and the interleukin concentration is 0.01 ng / ml or more (0.05 ng).
  • the interleukin concentration is 0.01 ng / ml or more (0.05 ng).
  • Ml or more, 1.0 ng / ml or more, 10 ng / ml or more, etc. in the mesenchymal stem cell culture medium It is most preferable to culture the cells.
  • the mesenchymal stem cells are preferably cultured in a culture medium for mesenchymal stem cells in which the concentration of IL-1 ⁇ of at least interleukin is 0.01 ng / ml or more, and is 0.1 ng / ml or more. More preferably, the mesenchymal stem cells are cultured in the mesenchymal stem cell culture medium, and the mesenchymal stem cells are cultured in the mesenchymal stem cell culture medium in which the concentration of IL-1 ⁇ is 1 ng / ml or more.
  • the mesenchymal stem cells are more preferably cultured in a culture medium for mesenchymal stem cells in which the concentration of IL-1 ⁇ is 10 ng / ml or more, while the concentration of IL-1 ⁇ is 50 ng / ml or more.
  • the mesenchymal stem cells are cultured in a culture medium for mesenchymal stem cells.
  • the upper limit of the interleukin concentration (including the case where at least the upper limit of the concentration of IL-1 ⁇ among the interleukins) is not particularly limited, for example, 90 ng / Ng or less, 70 ng / ml or less, 60 ng / ml or less, 50 ng / ml or less, 30 ng / ml or less, 10 ng / ml, 5 ng / ml or less.
  • the method for preparing mesenchymal stem cells of the present invention may further include a step of adding a cell growth factor. This further promotes the proliferation of mesenchymal stem cells.
  • the cell growth factor is not particularly limited, and examples thereof include FGF-2 and PDGF. Of these, FGF-2 is preferably used because it has a particularly high growth promoting effect when used in combination with interleukins.
  • the amount of the cell growth factor to be added is not particularly limited, and may be, for example, 0.01 to 100 ng / ml, but the concentration of the cell growth factor in the medium is 0.1 to 50 ng / ml. Preferably, it is added so as to be 1 to 50 ng / ml, more preferably 10 to 50 ng / ml.
  • the method for preparing mesenchymal stem cells of the present invention may have steps other than the above steps.
  • such a process includes a recovery process of recovering mesenchymal stem cells after the culture process.
  • the above-described culturing step may be a step of adding inflammatory cytokines to the mesenchymal stem cell culture medium and culturing the mesenchymal stem cells in the mesenchymal stem cell culture medium after the addition. . Further, it may be a step of culturing mesenchymal stem cells in a culture medium containing interleukin and / or inflammatory cytokine.
  • Such inflammatory cytokines include, but are not limited to, IL-1 (IL-1 ⁇ , IL-1 ⁇ , etc.), TNF- ⁇ , IL-6, and the like.
  • IL-1 (IL-1 ⁇ , IL-1 ⁇ , etc.) and TNF- ⁇ are preferably used as inflammatory cytokines.
  • Inflammatory cytokines may be used alone or in combination of two or more.
  • inflammatory cytokines When inflammatory cytokines are used to promote the proliferation of mesenchymal stem cells, they can be used under the same conditions as when interleukins are used.
  • the method for preparing chondrocytes of the present invention comprises a step of contacting mesenchymal stem cells and interleukin ex vivo, and a step of inducing differentiation of mesenchymal stem cells into cartilage.
  • the contact step in the chondrocyte preparation method of the present invention is a step of contacting mesenchymal stem cells and interleukins ex vivo.
  • the present invention can improve the efficiency of promoting the differentiation of mesenchymal cells into chondrocytes.
  • the reason for this is presumed to be that interleukins repeatedly destroy and regenerate mesenchymal stem cells and, as a result, promote differentiation into chondrocytes.
  • the interleukin is not particularly limited, and examples thereof include IL-1 (IL-1 ⁇ , IL-1 ⁇ , etc.), IL-6, IL-10, and the like. Of these, IL-1 (IL-1 ⁇ , IL-1 ⁇ , etc.), IL-6, etc., which are inflammatory cytokines, are particularly preferred. IL-1 includes IL-1 ⁇ , IL-1 ⁇ and the like. Of these, IL-1 ⁇ and IL-1 ⁇ are particularly preferred. Interleukin may be used independently and may use 2 or more types together.
  • inflammatory cytokines have hitherto been considered to have a negative effect on differentiation into chondrocytes, but the present inventors have unexpectedly realized by using inflammatory cytokines. And found to promote differentiation into chondrocytes. This is presumed to be because the destruction and regeneration of mesenchymal stem cells are further repeated by using inflammatory cytokines, and as a result, differentiation into chondrocytes is further promoted.
  • the origin of the interleukin species is not particularly limited, and interleukins derived from humans, pigs, cows, rabbits, rats, mice, etc. can be used, and the homology between them and amino acid sequences is 90% or more.
  • the mesenchymal stem cells are not particularly limited, and mesenchymal stem cells of various mesenchymal tissues including mesenchymal stem cells can be used.
  • mesenchymal stem cells of various mesenchymal tissues including mesenchymal stem cells can be used.
  • mesenchymal stem cells for example, synovial membrane, periosteum, muscle, fat (subcutaneous fat, etc.) ), Mesenchymal stem cells such as bone tissue, ligament, tendon, meniscus and the like.
  • synovial-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells it is particularly preferable to use synovial-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells in order to promote proliferation of mesenchymal stem cells to chondrocytes.
  • the contact process in the present invention is not particularly limited as long as it uses means for contacting mesenchymal stem cells and interleukins ex vivo.
  • mesenchymal stem cells may be added to a medium for inducing cartilage differentiation containing interleukin, and after adding mesenchymal stem cells to a medium for inducing cartilage differentiation, interleukin may be added to the medium. May be simultaneously added to the medium for inducing cartilage differentiation.
  • interleukins may be added to the culture medium during preculture of mesenchymal stem cells. These promote the differentiation of mesenchymal stem cells into cartilage.
  • the concentration of interleukin added to the above-mentioned medium for induction of cartilage differentiation is not particularly limited, but for example, interleukin can be added so as to be 0.01 to 100 ng / ml.
  • interleukin can be added so as to be 0.01 to 100 ng / ml.
  • the amount of interleukin added to the medium is larger, differentiation of mesenchymal stem cells into chondrocytes is promoted. From this point of view, it is preferable to add interleukin to the cartilage differentiation induction medium so that the interleukin concentration is 0.1 ng / ml or more, and interleukin is added to the cartilage so that the interleukin concentration is 1 ng / ml or more.
  • the interleukin is added to the medium for induction of cartilage differentiation so that the concentration of interleukin is 10 ng / ml or more, and the concentration of interleukin is 50 ng / ml or more.
  • the upper limit of the concentration of interleukin added to the cartilage differentiation induction medium is 90 ng / ml or less, 70 ng / ml or less, 50 ng / ml or less, 30 ng / ml or less, 10 ng / ml, 5 ng / ml or less, 1 ng / ml or less.
  • the amount is not particularly limited, but a larger amount is preferred.
  • the concentration of interleukin in the medium during the pre-culture after the addition is preferably 0.01 ng / ml or more, more preferably 0.1 ng / ml or more, and 1.0 ng / ml More preferably, it is more than ml, and most preferably more than 10 ng / ml.
  • the culture medium used for preculture can use the same thing as the culture medium for mesenchymal stem cells used in the preparation method of the above-mentioned mesenchymal stem cells.
  • interleukin When interleukin is added to the medium during the preculture of mesenchymal stem cells, interleukin may be added at any time, but 0 to 14 days (2 to 12 days, 4 to 10 days) after the start of the mesenchymal stem cell culture. Day, 6-8 days, etc.). Further, the period of culture after the addition of interleukin to the medium is not particularly limited. For example, the culture may be performed for 0 to 28 days (2 to 24 days, 5 to 20 days, 7 to 14 days, etc.).
  • the cartilage differentiation induction step in the chondrocyte preparation method of the present invention is a step of inducing differentiation of mesenchymal stem cells into cartilage.
  • conditions for inducing cartilage differentiation in the medium for inducing cartilage differentiation are not particularly limited.
  • a normal cartilage differentiation induction temperature eg, 37 ° C.
  • 0 to 21 days 2 to 18 days, Differentiation from mesenchymal stem cells to cartilage can be induced for 4-14 days, 7-10 days, etc.
  • the use of interleukin promotes the induction of cartilage differentiation of mesenchymal stem cells, which is suitable for induction in a short period.
  • a large amount of chondrocytes can be obtained even by induction for 14 days or less.
  • the contact step and the cartilage differentiation induction step may be the same step or different steps.
  • the above-described cartilage differentiation induction step is performed using the mesenchymal stem cells after pre-culture.
  • interleukin may not be added to the cartilage differentiation-inducing medium (that is, the contact process may not be further performed in the cartilage differentiation induction process), and interleukin may be added (that is, cartilage differentiation induction process). In this case, a contact step may be further performed).
  • interleukin may or may not be added in the preculture, or the preculture itself may not be performed.
  • the chondrocyte preparation method of the present invention may have other steps other than the contact step, the cartilage differentiation induction step, and the pre-culture.
  • a process includes a recovery process of recovering chondrocytes after induction of cartilage differentiation.
  • inflammatory cytokines are effective in terms of promoting the differentiation of mesenchymal stem cells into cartilage. Therefore, it is not limited to interleukins, and by using inflammatory cytokines, the rate of mesenchymal stem cell differentiation into cartilage can be promoted. That is, the above-mentioned contact step may be a step of contacting mesenchymal stem cells with interleukins and / or inflammatory cytokines ex vivo.
  • Such inflammatory cytokines include, but are not limited to, IL-1 (IL-1 ⁇ , IL-1 ⁇ , etc.), TNF- ⁇ , IL-6, and the like.
  • IL-1 (IL-1 ⁇ , IL-1 ⁇ , etc.) and TNF- ⁇ are preferably used as inflammatory cytokines. Inflammatory cytokines may be used alone or in combination of two or more.
  • the agent for promoting proliferation of mesenchymal stem cells of the present invention comprises interleukin or inflammatory cytokine.
  • the same one as used in the above-described method for preparing mesenchymal stem cells can be used.
  • the mesenchymal stem cell cartilage differentiation promoting agent of the present invention is composed of interleukin or inflammatory cytokine.
  • the same one as used in the above-described method for preparing chondrocytes can be used.
  • the culture medium for mesenchymal stem cells of the present invention can be used for interleukin addition.
  • the culture medium for mesenchymal stem cells can promote the growth of mesenchymal stem cells by adding interleukin. Therefore, the culture medium for mesenchymal stem cells is suitable for being used for interleukin addition.
  • the culture medium for mesenchymal stem cells can be the same as that used in the above-described method for preparing mesenchymal stem cells, and in particular, the growth of mesenchymal stem cells is promoted even in a serum-free medium. Therefore, when a serum-free medium is used, it is particularly suitable for use as an interleukin-added product.
  • the culture medium of the present invention may contain interleukin and / or inflammatory cytokine.
  • the mesenchymal stem cells to be cultured are not particularly limited, and examples thereof include mesenchymal stem cells such as synovium, periosteum, muscle, fat (subcutaneous fat, etc.), bone tissue, ligament, tendon, meniscus and the like. It is done.
  • the culture medium of the present invention is particularly preferably used as a medium for culturing synovial-derived mesenchymal stem cells and fat-derived mesenchymal stem cells.
  • Example 1 Using 83-year-old male synovial stem cell derived mesenchymal stem cells, ⁇ MEM medium supplemented with 10% FBS was used for 4 passages at 37 ° C. and pre-cultured mesenchymal stem cells for 2 weeks. . Thereafter, the number of mesenchymal stem cells was adjusted to 100 / dish with a 10 cm 2 dish, IL-1 ⁇ was added to a final concentration of 5 ng / ml, and cultured at 37 ° C. for 2 weeks. Mesenchymal stem cells according to Example 1 were prepared.
  • Example 2 After the preculture, the mesenchymal stem cells according to Example 2 were prepared under the same conditions as in Example 1 except that the number of mesenchymal stem cells was adjusted to 200 / dish.
  • Control 1 mesenchymal stem cells according to Control Example 1 were prepared under the same conditions as in Example 1 except that IL-1 ⁇ was not added.
  • Control 2 mesenchymal stem cells according to Control Example 2 were prepared under the same conditions as in Example 2 except that IL-1 ⁇ was not added.
  • FIG. 1 shows an image after crystal violet staining of mesenchymal stem cells according to Example 1 and Control Example 1 and (b) shows mesenchymal according to Example 2 and Control Example 2.
  • staining about a stem cell is shown.
  • Example 1 As shown in FIG. 1, in both Example 1 and Example 2, the colony formation rate and the colony size were increased as compared with Control Examples 1 and 2 in which IL-1 ⁇ was not added. From these results, it was shown that IL-1 ⁇ promotes cell proliferation.
  • Example 3 the number of cells was significantly increased as compared to Control Example 3 in which IL-1 ⁇ was not added.
  • Example 4 Using a mesenchymal stem cell derived from synovium of a 72-year-old female, primary mesenchymal stem cells were pre-cultured at 37 ° C. for 2 weeks using an ⁇ MEM medium supplemented with 10% FBS. Thereafter, the number of mesenchymal stem cells was adjusted to 1000 / dish with a 60 cm 2 dish, IL-1 ⁇ was added to a final concentration of 0.5 ng / ml, and cultured at 37 ° C. for 2 weeks. Mesenchymal stem cells according to Example 4 were prepared.
  • Example 5 A mesenchymal stem cell according to Example 5 was prepared under the same conditions as in Example 4 except that IL-1 ⁇ was added to a final concentration of 5 ng / ml.
  • Example 6 A mesenchymal stem cell according to Example 6 was prepared under the same conditions as in Example 4 except that IL-1 ⁇ was added to a final concentration of 50 ng / ml.
  • Control 4 mesenchymal stem cells according to Control Example 4 were prepared under the same conditions as in Example 4 except that IL-1 ⁇ was not added.
  • Example 7 Using mesenchymal stem cells derived from synovial membrane of a 72-year-old woman, the cells were subcultured once at 37 ° C. using an ⁇ MEM medium supplemented with 10% FBS, and mesenchymal stem cells were precultured for 2 weeks. Thereafter, the number of mesenchymal stem cells was adjusted to 1000 cells / dish with a 60 cm 2 dish, IL-1 ⁇ was added to a final concentration of 0.01 ng / ml, and cultured at 37 ° C. for 2 weeks. A mesenchymal stem cell according to Example 7 was prepared.
  • Example 8 A mesenchymal stem cell according to Example 8 was prepared under the same conditions as in Example 7 except that IL-1 ⁇ was added to a final concentration of 0.1 ng / ml.
  • Example 9 A mesenchymal stem cell according to Example 9 was prepared under the same conditions as in Example 7 except that IL-1 ⁇ was added to a final concentration of 1 ng / ml.
  • Example 10 A mesenchymal stem cell according to Example 10 was prepared under the same conditions as in Example 7 except that IL-1 ⁇ was added to a final concentration of 10 ng / ml.
  • Example 11 Using mesenchymal stem cells derived from synovium, primary culture of mesenchymal stem cells was performed at 37 ° C. for 14 days using ⁇ MEM medium supplemented with 10% FBS. Thereafter, the number of mesenchymal stem cells was adjusted to 100 / dish with a 60 cm 2 dish, IL-1 ⁇ was added to a final concentration of 0.3 ng / ml, and cultured at 37 ° C. for 12 days. Then, mesenchymal stem cells according to Example 11 were prepared.
  • Example 12 The mesenchymal stem cell according to Example 12 was prepared under the same conditions as in Example 11 except that FGF (fibroblast growth factor) -2 was added together with IL-1 ⁇ to a final concentration of 10 ng / ml. Prepared.
  • FGF fibroblast growth factor
  • Reference Example 1 A mesenchymal stem cell according to Reference Example 1 was prepared under the same conditions as in Example 12 except that IL-1 ⁇ was not added.
  • the results shown in FIG. 5 indicate that the proliferation of mesenchymal stem cells is further promoted by using FGF-2, which is a cell growth factor, and IL-1 ⁇ in combination. Further, from the results shown in FIG. 6, it was confirmed that the combined use of FGF-2 and IL-1 ⁇ significantly promoted cell proliferation compared to the case where each was used alone. Thus, it was shown that the combined use of FGF-2 and IL-1 ⁇ synergistically promotes the proliferation of mesenchymal stem cells.
  • Example 13 Using 83-year-old male synovial-derived mesenchymal stem cells, 10% FBS-added ⁇ MEM medium was used for 3 passages at 37 ° C., and mesenchymal stem cells were cultured for 2 weeks. Thereafter, the cells were recovered, and the number of cells was matched with that of Control Example 7 to prepare a pellet.
  • Cartilage differentiation medium (DMEM supplemented with 1000 ng / ml BMP7, 10 ng / ml TGF-beta, 10 ⁇ 7 M dexamethasone) 5 ng / ml IL-1 ⁇ was added thereto and cultured for 3 weeks to prepare chondrocytes according to Example 13.
  • Example 7 After the preculture, mesenchymal stem cells according to Control Example 7 were prepared under the same conditions as in Example 13 except that IL-1 ⁇ was not added. In addition, when producing a pellet, Example 13 and the cell number were match
  • Example 13 As shown in FIG. 7, the chondrocytes of Example 13 were larger than Control Example 7 in which IL-1 ⁇ was not added. From these results, it was shown that IL-1 ⁇ promotes differentiation induction of mesenchymal stem cells into cartilage.
  • ⁇ Preparation 2 of chondrocytes> (Example 14) Using mesenchymal stem cells derived from synovium of a 72-year-old woman, the cells were subcultured once at 37 ° C. using an ⁇ MEM medium supplemented with 10% FBS, and mesenchymal stem cells were precultured for 14 days. Thereafter, the number of mesenchymal stem cells was adjusted to 1000 cells / dish with a 60 cm 2 dish, and IL-1 ⁇ was added to a final concentration of 0.5 ng / ml, and the culture was performed at 37 ° C. for 14 days. Culture was performed.
  • a pellet was prepared by combining the cells with Examples 15 and 16, and using a cartilage differentiation medium (DMEM supplemented with 1000 ng / ml BMP7, 10 ng / ml TGF-beta, 10 ⁇ 7 M dexamethasone). After culturing for days, chondrocytes according to Example 14 were prepared.
  • DMEM cartilage differentiation medium
  • Example 15 Chondrocytes according to Example 15 were prepared under the same conditions as in Example 14 except that IL-1 ⁇ was added to a final concentration of 5 ng / ml.
  • Example 16 Chondrocytes according to Example 16 were prepared under the same conditions as in Example 14 except that IL-1 ⁇ was added to a final concentration of 50 ng / ml.
  • Example 17 and 18 and Control Examples 8 and 9 were subjected to crystal violet staining after preparation.
  • the results for Example 17 and Control Example 8 are shown in FIG. 9, and the results for Example 18 and Control Example 9 are shown in FIG.
  • Example 19 significantly (* p ⁇ 0.05) increased mesenchymal stem cells compared to Control Example 10. I understood.
  • ⁇ Preparation 3 of chondrocytes> (Example 20) Using mesenchymal stem cells derived from synovium, the cells were subcultured once at 37 ° C. using an ⁇ MEM medium supplemented with 10% FBS, and the mesenchymal stem cells were precultured for 2 weeks. Thereafter, the number of mesenchymal stem cells was adjusted to 10 4 / dish in a 60 cm 2 dish, IL-1 ⁇ was added to a final concentration of 25 ng / ml, and cultured at 37 ° C. for 14 days. Went.
  • a pellet was prepared by combining the number of cells with Control Example 11 described later, and using a cartilage differentiation medium (DMEM supplemented with 1000 ng / ml BMP7, 10 ng / ml TGF-beta, 10 ⁇ 7 M dexamethasone). Cultured for a day, chondrocytes according to Example 20 were prepared.
  • DMEM cartilage differentiation medium
  • the results shown in FIG. 13 indicate that the addition of TNF- ⁇ promotes differentiation induction of mesenchymal stem cells into cartilage.
  • TNF- ⁇ when the synovial stem cells added with TNF- ⁇ were evaluated for their ability to differentiate into fat, as with the cartilage differentiation ability, TNF- ⁇ was added more than the medium without TNF- ⁇ added. The added one had higher ability to differentiate into fat. From this result, it was also found that TNF- ⁇ promotes differentiation of mesenchymal stem cells into fat.

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Abstract

As alternatives to conventional mesenchymal-stem-cell preparation methods and methods for preparing chondrocytic cells from mesenchymal stem cell, provided are a novel mesenchymal-stem-cell preparation method that achieves excellent efficiency of promoting proliferation of mesenchymal stem cells, a chondrocytic-cell preparation method that achieves excellent efficiency of promoting differentiation of mesenchymal stem cells into chondrocytic cells, an agent for promoting proliferation of mesenchymal stem cells, an agent for promoting cartilage differentiation of mesenchymal stem cells, and a mesenchymal-stem-cell culture medium that can be used for preparation of such mesenchymal stem cells. The mesenchymal-stem-cell preparation method includes a step in which a mesenchymal-stem-cell culture medium is supplemented with interleukin, and mesenchymal stem cells are cultured in the interleukin-supplemented mesenchymal-stem-cell culture medium. Furthermore, the chondrocytic-cell preparation method includes a step in which mesenchymal stem cells and interleukin are brought into contact with each other, and a step in which cartilage differentiation of the mesenchymal stem cells is induced.

Description

間葉系幹細胞の増殖促進剤、間葉系幹細胞の軟骨分化促進剤、間葉系幹細胞の調製方法、軟骨細胞の調製方法、及び間葉系幹細胞用培養培地Mesenchymal stem cell growth promoter, mesenchymal stem cell cartilage differentiation promoter, mesenchymal stem cell preparation method, chondrocyte preparation method, and mesenchymal stem cell culture medium
　本発明は、間葉系幹細胞の増殖促進剤、間葉系幹細胞の軟骨分化促進剤、間葉系幹細胞の調製方法、軟骨分化細胞の調製方法、及び間葉系幹細胞用培養培地に関する。 The present invention relates to a mesenchymal stem cell growth promoter, a mesenchymal stem cell cartilage differentiation promoter, a mesenchymal stem cell preparation method, a chondrogenic cell preparation method, and a mesenchymal stem cell culture medium.
　軟骨欠損や半月板損傷等の軟骨に関する障害が、従来より問題となっており、そのための様々な治療法が開発されている。 Disorders related to cartilage, such as cartilage defects and meniscal damage, have been a problem, and various treatment methods have been developed.
　半月板損傷の治療法として、縫合術と切除術とが知られている。縫合術は、再断裂のおそれがあり、また、損傷した箇所によっては縫合術が適用できない場合がある。他方、切除術は、縫合術により断裂部を切除できない箇所にも適用可能であり、切除術によると再断裂を防ぐことができるが、切除部の機能を損ねてしまう。 Suture and excision are known as treatment methods for meniscus injury. Suture may cause re-tearing, and may not be applicable depending on the damaged site. On the other hand, excision can be applied to places where the teared part cannot be excised by suturing, and resecting can be prevented by excision, but the function of the excised part is impaired.
　そこで、近年、再生医療が着目されており、軟骨や半月板に関する障害を治療するための様々な研究が盛んに行われている。 Therefore, in recent years, regenerative medicine has attracted attention, and various studies for treating disorders related to cartilage and meniscus have been actively conducted.
　例えば、間葉系幹細胞は、様々な細胞に分化することが知られており、軟骨にも分化することが知られているが、特許文献１や特許文献２には、そのような間葉系幹細胞の培養に用いることができる培地が開示されている。 For example, mesenchymal stem cells are known to differentiate into various cells and are also known to differentiate into cartilage. Patent Documents 1 and 2 disclose such mesenchymal systems. A medium that can be used for culturing stem cells is disclosed.
　また、特許文献３には、デキサメタゾンを利用した、培養効率の良い間葉系幹細胞の調製方法が開示されている。特許文献４には、培養面をデンドリマー化合物で修飾することで、分化誘導のための培地成分又は分化誘導培地を使用しなくても、間葉系幹細胞の軟骨への分化を誘導可能な、軟骨細胞の調製方法が開示されている。 Patent Document 3 discloses a method for preparing mesenchymal stem cells with good culture efficiency using dexamethasone. Patent Document 4 discloses a cartilage that can induce differentiation of mesenchymal stem cells into cartilage without using a medium component for differentiation induction or a differentiation induction medium by modifying the culture surface with a dendrimer compound. A method for preparing cells is disclosed.
特開２００７－３７４２６号公報JP 2007-37426 A 特開２００７－３１４４３２号公報JP 2007-314432 A 特許第４６４６６００号Patent No. 4646600 特許第５６４１４６８号Japanese Patent No. 5641468
　本発明は、上述したような、従来の間葉系幹細胞の調製方法、間葉系幹細胞からの軟骨細胞の調製方法に替わる、新規な、間葉系幹細胞の増殖促進効率に優れた間葉系幹細胞の調製方法、間葉系幹細胞の軟骨細胞への分化促進効率に優れた軟骨細胞の調製方法、間葉系幹細胞の増殖促進剤、間葉系幹細胞の軟骨分化促進剤、及び、このような間葉系幹細胞の調製に使用可能な間葉系幹細胞用培養培地を提供することを目的とする。 The present invention replaces the conventional method for preparing mesenchymal stem cells and the method for preparing chondrocytes from mesenchymal stem cells, as described above, and a novel mesenchymal system excellent in the efficiency of promoting proliferation of mesenchymal stem cells. Preparation method of stem cells, preparation method of chondrocytes excellent in efficiency of promoting differentiation of mesenchymal stem cells into chondrocytes, proliferation promoter of mesenchymal stem cells, promoter of cartilage differentiation of mesenchymal stem cells, and such An object is to provide a culture medium for mesenchymal stem cells that can be used for the preparation of mesenchymal stem cells.
　本発明者らは、インターロイキンにより、間葉系幹細胞の増殖が促進すること、及び間葉系幹細胞の軟骨細胞への分化が促進することを見出し、本発明を完成するに至った。より具体的には、本発明は以下のようなものを提供する。 The present inventors have found that interleukin promotes the proliferation of mesenchymal stem cells and promotes the differentiation of mesenchymal stem cells into chondrocytes, and has completed the present invention. More specifically, the present invention provides the following.
　（１）　ＩＬ－１および／またはＴＮＦ－αを含む培養培地において滑膜由来の間葉系幹細胞を培養する工程を含む、間葉系幹細胞の調製方法。 (1) A method for preparing mesenchymal stem cells, comprising a step of culturing synovial stem cells derived from synovium in a culture medium containing IL-1 and / or TNF-α.
　（２）　前記ＩＬ－１がＩＬ－１βである、（１）に記載の間葉系幹細胞の調製方法。 (2) The method for preparing a mesenchymal stem cell according to (1), wherein the IL-1 is IL-1β.
　（３）　間葉系幹細胞用培養培地に、インターロイキンを添加し、添加後の前記間葉系幹細胞用培養培地において、間葉系幹細胞を培養する工程を有する、間葉系幹細胞の調製方法。 (3) A method for preparing mesenchymal stem cells, comprising adding interleukin to a culture medium for mesenchymal stem cells, and culturing the mesenchymal stem cells in the mesenchymal stem cell culture medium after the addition.
　（４）　前記インターロイキンは、ＩＬ－１を含む、（３）に記載の間葉系幹細胞の調製方法。 (4) The method for preparing mesenchymal stem cells according to (3), wherein the interleukin contains IL-1.
　（５）　間葉系幹細胞用培養培地に、炎症性サイトカインを添加し、添加後の前記間葉系幹細胞用培養培地において、間葉系幹細胞を培養する工程を有する、間葉系幹細胞の調製方法。 (5) A method for preparing mesenchymal stem cells, comprising adding inflammatory cytokines to a mesenchymal stem cell culture medium, and culturing the mesenchymal stem cells in the mesenchymal stem cell culture medium after the addition. .
　（６）　前記炎症性サイトカインは、ＩＬ－１及びＴＮＦ－αからなる群から選択される１種以上を含む、（５）に記載の間葉系幹細胞の調製方法。 (6) The method for preparing mesenchymal stem cells according to (5), wherein the inflammatory cytokine includes one or more selected from the group consisting of IL-1 and TNF-α.
　（７）　前記間葉系幹細胞は、滑膜由来又は脂肪由来の間葉系幹細胞である、（３）から（６）のいずれかに記載の間葉系幹細胞の調製方法。 (7) The method for preparing a mesenchymal stem cell according to any one of (3) to (6), wherein the mesenchymal stem cell is a synovial or fat-derived mesenchymal stem cell.
　（８）　前記間葉系幹細胞用培養培地が、無血清培地である、（１）から（７）のいずれかに記載の間葉系幹細胞の調製方法。 (8) The method for preparing mesenchymal stem cells according to any one of (1) to (7), wherein the culture medium for mesenchymal stem cells is a serum-free medium.
　（９）　細胞増殖因子を培地中に添加する工程を更に含有する、（１）から（８）のいずれかに記載の間葉系幹細胞の調製方法。 (9) The method for preparing a mesenchymal stem cell according to any one of (1) to (8), further comprising a step of adding a cell growth factor to the medium.
　（１０）　間葉系幹細胞とインターロイキンおよび／または炎症性サイトカインとをｅｘ　ｖｉｖｏで接触させる工程と、
　間葉系幹細胞の軟骨への分化を誘導する工程と、を有する、軟骨細胞の調製方法。 (10) contacting the mesenchymal stem cell with interleukin and / or inflammatory cytokine ex vivo;
And a step of inducing differentiation of mesenchymal stem cells into cartilage.
　（１１）　インターロイキンからなる、間葉系幹細胞の増殖促進剤。 (11) A mesenchymal stem cell proliferation promoter comprising interleukin.
　（１２）　インターロイキンからなる、間葉系幹細胞の軟骨分化促進剤。 (12) An agent for promoting cartilage differentiation of mesenchymal stem cells, comprising interleukin.
　（１３）　インターロイキン被添加用である、間葉系幹細胞用培養培地。 (13) A culture medium for mesenchymal stem cells for addition of interleukin.
　（１４）　炎症性サイトカインからなる、間葉系幹細胞の増殖促進剤。 (14) A mesenchymal stem cell proliferation promoter comprising inflammatory cytokines.
　（１５）　炎症性サイトカインからなる、間葉系幹細胞の軟骨分化促進剤。 (15) An agent for promoting cartilage differentiation of mesenchymal stem cells, comprising inflammatory cytokines.
　（１６）　インターロイキンおよび／または炎症性サイトカインを含有することを特徴とする、間葉系幹細胞用培養培地。 (16) A culture medium for mesenchymal stem cells, comprising interleukin and / or inflammatory cytokine.
　（１７）　前記インターロイキンがＩＬ－１βである、（１６）に記載の間葉系幹細胞用培養培地。 (17) The culture medium for mesenchymal stem cells according to (16), wherein the interleukin is IL-1β.
　（１８）　前記炎症性サイトカインがＴＮＦ－αである、（１６）に記載の間葉系幹細胞用培養培地。 (18) The culture medium for mesenchymal stem cells according to (16), wherein the inflammatory cytokine is TNF-α.
　（１９）　前記間葉系幹細胞が滑膜由来の間葉系幹細胞である、（１６）から（１８）のいずれかに記載の間葉系幹細胞用培養培地。 (19) The culture medium for mesenchymal stem cells according to any one of (16) to (18), wherein the mesenchymal stem cells are synovial stem cells.
　本発明によれば、従来の間葉系幹細胞の調製方法、間葉系幹細胞からの軟骨細胞の調製方法に替わる、新規な、間葉系幹細胞の増殖促進効率に優れた間葉系幹細胞の調製方法、間葉系幹細胞の軟骨細胞への分化促進効率に優れた軟骨細胞の調製方法、間葉系幹細胞の増殖促進剤、間葉系幹細胞の軟骨分化促進剤、及び、このような間葉系幹細胞の調製に使用可能な間葉系幹細胞用培養培地を提供することができる。 According to the present invention, in place of the conventional method for preparing mesenchymal stem cells and the method for preparing chondrocytes from mesenchymal stem cells, the preparation of a novel mesenchymal stem cell excellent in the efficiency of promoting proliferation of mesenchymal stem cells is provided. Method, preparation method of chondrocytes excellent in efficiency of promoting differentiation of mesenchymal stem cells into chondrocytes, agent for promoting proliferation of mesenchymal stem cells, agent for promoting cartilage differentiation of mesenchymal stem cells, and such mesenchymal system A culture medium for mesenchymal stem cells that can be used for the preparation of stem cells can be provided.
（ａ）は、実施例１、対照例１に係る間葉系幹細胞についてのクリスタル・バイオレット染色後の画像を示す図である。（ｂ）は、実施例２、対照例２に係る間葉系幹細胞についてのクリスタル・バイオレット染色後の画像を示す図である。(A) is a figure which shows the image after crystal violet dyeing | staining about the mesenchymal stem cell which concerns on Example 1 and the control example 1. FIG. (B) is a figure which shows the image after crystal violet dyeing | staining about the mesenchymal stem cell which concerns on Example 2 and the control example 2. FIG. 実施例３、対照例３に係る間葉系幹細胞の細胞数のグラフを示す図である。It is a figure which shows the graph of the cell number of the mesenchymal stem cell which concerns on Example 3 and the control example 3. FIG. 実施例４～６、対照例４に係る間葉系幹細胞についてのクリスタル・バイオレット染色後の画像を示す図である。FIG. 6 is a view showing images after crystal violet staining of mesenchymal stem cells according to Examples 4 to 6 and Control Example 4. 実施例７～１０、対照例５に係る間葉系幹細胞についてのクリスタル・バイオレット染色後の画像を示す図である。FIG. 10 is a view showing images after crystal violet staining of mesenchymal stem cells according to Examples 7 to 10 and Control Example 5. 実施例１１、１２、参考例１、対照例６に係る間葉系幹細胞についてのクリスタル・バイオレット染色後の画像を示す図である。It is a figure which shows the image after crystal violet dyeing | staining about the mesenchymal stem cell which concerns on Example 11, 12, Reference example 1, and the control example 6. FIG. 実施例１１、１２、参考例１、対照例６に係る間葉系幹細胞についての細胞の倍増数及び細胞数のグラフを示す図である。It is a figure which shows the graph of the doubling number of the cell about the mesenchymal stem cell which concerns on Example 11, 12, Reference example 1, and the control example 6, and a cell number. 実施例１３、対照例７に係る軟骨細胞の画像を示す図である。It is a figure which shows the image of the chondrocyte which concerns on Example 13 and the control example 7. FIG. 実施例１４～１６に係る軟骨細胞の画像を示す図である。It is a figure which shows the image of the chondrocyte which concerns on Examples 14-16. 実施例１７、対照例８に係る間葉系幹細胞についてのクリスタル・バイオレット染色後の画像を示す図である。It is a figure which shows the image after crystal violet dyeing | staining about the mesenchymal stem cell which concerns on Example 17 and the control example 8. FIG. 実施例１８、対照例９に係る間葉系幹細胞についてのクリスタル・バイオレット染色後の画像を示す図である。It is a figure which shows the image after crystal violet dyeing | staining about the mesenchymal stem cell which concerns on Example 18 and the control example 9. FIG. 実施例１９、対照例１０に係る間葉系幹細胞についてのクリスタル・バイオレット染色後の画像を示す図である。It is a figure which shows the image after crystal violet dyeing | staining about the mesenchymal stem cell which concerns on Example 19 and the control example 10. FIG. 実施例１９、対照例１０に係る間葉系幹細胞についての培養前のコロニー数に対する培養後のコロニー数の増加した割合を示す図である。It is a figure which shows the ratio which the number of colonies after culture | cultivation increased with respect to the number of colonies before culture | cultivation about the mesenchymal stem cell which concerns on Example 19 and the control example 10. FIG. 実施例２０、対照例１１に係る軟骨細胞の画像を示す図である。It is a figure which shows the image of the chondrocyte which concerns on Example 20 and the control example 11. FIG.
　以下、本発明の実施形態について説明するが、本発明はこれに限定されるものではない。 Hereinafter, although an embodiment of the present invention will be described, the present invention is not limited to this.
　＜間葉系幹細胞の調製方法＞
　本発明の間葉系幹細胞の調製方法は、間葉系幹細胞用培養培地に、インターロイキンを添加し、添加後の前記間葉系幹細胞用培養培地において、間葉系幹細胞を培養する工程を有する。なお、本発明において、「細胞の増殖」とは、培地中においてコロニー形成率が増えること、コロニーサイズが増大することのいずれか一方、又は両方を含む。 <Method for preparing mesenchymal stem cells>
The method for preparing mesenchymal stem cells of the present invention comprises a step of adding interleukin to a culture medium for mesenchymal stem cells and culturing the mesenchymal stem cells in the culture medium for mesenchymal stem cells after the addition. . In the present invention, “cell proliferation” includes one or both of increasing the colony formation rate and increasing the colony size in the medium.
　［培養工程］
　本発明の間葉系幹細胞の調製方法における培養工程は、間葉系幹細胞用培養培地に、インターロイキンを添加し、添加後の前記間葉系幹細胞用培養培地において、間葉系幹細胞を培養する工程である。本発明は、これにより、間葉系幹細胞の増殖が促進される。このように、本発明の間葉系幹細胞の調製方法によると、間葉系幹細胞用培養培地中にインターロイキンが添加されることで、間葉系幹細胞の増殖が促進される。その理由は、間葉系幹細胞が、インターロイキンと接触して炎症状態となった際に、増殖能を発揮するからであると推測される。 [Culture process]
In the culturing step in the method for preparing mesenchymal stem cells of the present invention, interleukin is added to the culture medium for mesenchymal stem cells, and the mesenchymal stem cells are cultured in the culture medium for mesenchymal stem cells after the addition. It is a process. Thus, the present invention promotes the proliferation of mesenchymal stem cells. Thus, according to the method for preparing mesenchymal stem cells of the present invention, proliferation of mesenchymal stem cells is promoted by adding interleukin to the culture medium for mesenchymal stem cells. The reason for this is presumed to be that mesenchymal stem cells exhibit proliferative ability when in contact with interleukin and become inflamed.
　インターロイキンの種類は、特に限定されないが、ＩＬ－１（ＩＬ－１α、ＩＬ－１β等）、ＩＬ－６、ＩＬ－１０、ＩＬ－１７等が挙げられる。これらのうち、特に、炎症性サイトカインである、ＩＬ－１（ＩＬ－１α、ＩＬ－１β等）、ＩＬ－６等を用いることが好ましい。ＩＬ－１は、ＩＬ－１α、ＩＬ－１β等が上げられる。これらのうち、ＩＬ－１α、ＩＬ－１βが特に好ましい。インターロイキンは、単独で使用してもよく、２種以上を併用してもよい。ここで、炎症性サイトカインは、従来、軟骨細胞の増殖を阻害するものと考えられてきたが、本発明者らは、炎症性サイトカインを用いることによって、予想外なことに、間葉系幹細胞の増殖が促進することを見出した。これは、間葉系幹細胞が、インターロイキンと接触して炎症状態となった際に、増殖能を発揮するからと推測される。 The type of interleukin is not particularly limited, and examples include IL-1 (IL-1α, IL-1β, etc.), IL-6, IL-10, IL-17, and the like. Of these, IL-1 (IL-1α, IL-1β, etc.), IL-6, etc., which are inflammatory cytokines, are particularly preferred. IL-1 includes IL-1α, IL-1β and the like. Of these, IL-1α and IL-1β are particularly preferred. Interleukin may be used independently and may use 2 or more types together. Here, inflammatory cytokines have been conventionally thought to inhibit the proliferation of chondrocytes, but the present inventors have unexpectedly used mesenchymal stem cells by using inflammatory cytokines. It was found that proliferation was promoted. This is presumably because mesenchymal stem cells exhibit proliferative ability when they become inflamed upon contact with interleukins.
　また、インターロイキンの種の由来も特に限定されず、ヒト、ブタ、ウシ、ウサギ、ラット、マウス等の由来のインターロイキンを用いることができ、また、これらとアミノ酸配列の相同性が９０％以上であるタンパク等を用いることができる。 Also, the origin of the interleukin species is not particularly limited, and interleukins derived from humans, pigs, cows, rabbits, rats, mice, etc. can be used, and the homology between them and amino acid sequences is 90% or more. The protein etc. which are can be used.
　間葉系幹細胞は、特に限定されず、間葉系幹細胞を含む種々の間葉系の組織の間葉系幹細胞を使用可能であるが、例えば、滑膜、骨膜、筋肉、脂肪（皮下脂肪等）、骨組織、靭帯、腱、半月板等の間葉系幹細胞が挙げられる。これらのうち、特に、間葉系幹細胞が増殖する速度が促進されることから、滑膜由来の間葉系幹細胞、脂肪由来の間葉系幹細胞を使用することが好ましい。 The mesenchymal stem cells are not particularly limited, and mesenchymal stem cells of various mesenchymal tissues including mesenchymal stem cells can be used. For example, synovial membrane, periosteum, muscle, fat (subcutaneous fat, etc.) ), Mesenchymal stem cells such as bone tissue, ligament, tendon, meniscus and the like. Among these, it is particularly preferable to use synovial-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells because the rate of proliferation of mesenchymal stem cells is promoted.
　間葉系幹細胞用培養培地は、従来の間葉系幹細胞の培養に用いることが可能な公知の培地であれば、いずれの培地も使用することができる。そのような間葉系幹細胞用培養培地としては、例えば、αＭＥＭ（α‐Ｍｉｎｉｍｕｍ　Ｅｓｓｅｎｔｉａｌ　Ｍｅｄｉｕｍ）、ＤＭＥＭ（Ｄｏｕｌｂｅｃｃｏ’ｓ　Ｍｏｄｉｆｉｅｄ　Ｅａｇｌｅ’ｓ　Ｍｅｄｉｕｍ）、ＥＭＥＭ（Ｅａｇｌｅ’ｓ　Ｍｉｎｉｍｕｍ　Ｅｓｓｅｎｔｉａｌ　Ｍｅｄｉｕｍ）、ＢＭＥ（Ｂａｓａｌ　Ｅａｇｌｅ　Ｍｅｄｉｕｍ）、Ｈａｍ　Ｆ１２、Ｈａｍ　Ｆ１０、Ｆ１２Ｋ（Ｋａｉｇｈｎｓ　ｍｏｄｉｆｉｅｄ　ｏｆ　Ｈａｍ’ｓ　Ｆ１２）、Ｌｅｉｂｏｖｉｔｚ’ｓ　Ｌ－１５　ｍｅｄｉｕｍ、ＭｃＣｏｙ’ｓ　５Ａ、Ｍｅｄｉｕｍ　１９９（１９９培地）等が挙げられるが、特にこれらに限定されない。 As the culture medium for mesenchymal stem cells, any medium can be used as long as it is a known medium that can be used for culturing conventional mesenchymal stem cells. Examples of such a culture medium for mesenchymal stem cells include αMEM (α-Minimum Essential Medium), DMEM (Doulbecco's Modified Eagle's Medium), and EMEM (Eagle's Minimum Essential Medium). Medium), Ham F12, Ham F10, F12K (Kaighns modified of Ham's F12), Leibovitz's L-15 medium, McCoy's 5A, and Medium 199 (199 medium) are not particularly limited. .
　間葉系幹細胞のような再生医療に用いる細胞を培養する場合、安全性の観点からは、無血清培地を用いることが好ましいが、無血清培地では間葉系幹細胞は増殖しにくいため、効率的に間葉系幹細胞を得ることができない。しかしながら、本発明においては、インターロイキンを用いることで、間葉系幹細胞が増殖しやすくなるため、特に、無血清培地での培養に適している。このような観点から、培地は、無血清培地を用いることが好ましい。無血清培地としては、特に限定されないが、例えば、ＳＴＥＭＰＲＯ（登録商標）ＭＳＣ　ＳＦＭ（ライフテクノロジーズ社）、ＳＴＫ２（ツーセル社）等が挙げられる。 When culturing cells used for regenerative medicine such as mesenchymal stem cells, it is preferable to use a serum-free medium from the viewpoint of safety. However, since mesenchymal stem cells are difficult to proliferate in a serum-free medium, it is efficient. I cannot get mesenchymal stem cells. However, in the present invention, the use of interleukin facilitates the proliferation of mesenchymal stem cells, and is particularly suitable for culturing in a serum-free medium. From such a viewpoint, it is preferable to use a serum-free medium as the medium. The serum-free medium is not particularly limited, and examples thereof include STEMPRO (registered trademark) MSC SFM (Life Technologies) and STK2 (Tucell).
　本発明における培養工程は、間葉系幹細胞用培養培地に、インターロイキンを添加し、添加後の間葉系幹細胞用培養培地において、間葉系幹細胞を培養する工程であれば、その方法は特に限定されない。例えば、間葉系幹細胞用培養培地中に、間葉系幹細胞を含むような状態で、一定期間培養した後に、インターロイキンを添加してもよく、間葉系幹細胞用培養培地に、間葉系幹細胞とインターロイキンとを同時に加えてもよい。あるいは、インターロイキンを間葉系幹細胞用培養培地に加え、あらかじめ培地中にインターロイキンが加えられた状態で、間葉系幹細胞を加えてもよい。つまり、本発明における培養工程は、インターロイキンを含む培養培地において間葉系幹細胞を培養する工程を含むものであってもよい。 If the culture process in the present invention is a process in which interleukin is added to the culture medium for mesenchymal stem cells and the mesenchymal stem cells are cultured in the culture medium for mesenchymal stem cells after the addition, the method is particularly It is not limited. For example, interleukins may be added after culturing for a certain period of time in a state containing mesenchymal stem cells in the culture medium for mesenchymal stem cells. Stem cells and interleukins may be added simultaneously. Alternatively, mesenchymal stem cells may be added in a state where interleukin is added to the culture medium for mesenchymal stem cells and interleukin is added to the medium in advance. That is, the culturing step in the present invention may include a step of culturing mesenchymal stem cells in a culture medium containing interleukin.
　培養工程における培養条件は、間葉系幹細胞やインターロイキンの種類、目標とする間葉系幹細胞の数等の応じて、適宜設定することができる。例えば、通常の間葉系幹細胞を培養する温度（例えば、３７℃）において、全体で０．５～２８日間（例えば、１～２４日、２～２０日、４～１８日、６～１６日、８～１４日、１０～１２日等）、培養することができる。しかし、本発明によると、インターロイキンを用いることで、間葉系幹細胞の増殖が促進されるため、短い期間の培養に適している。例えば、インターロイキンの添加後、３日以下の培養でも、多量の間葉系幹細胞を得ることができる。 The culture conditions in the culture step can be appropriately set according to the types of mesenchymal stem cells and interleukins, the number of target mesenchymal stem cells, and the like. For example, at a temperature at which normal mesenchymal stem cells are cultured (eg, 37 ° C.), the total time is 0.5 to 28 days (eg, 1 to 24 days, 2 to 20 days, 4 to 18 days, and 6 to 16 days). 8-14 days, 10-12 days, etc.). However, according to the present invention, the use of interleukin promotes the growth of mesenchymal stem cells and is therefore suitable for short-term culture. For example, a large amount of mesenchymal stem cells can be obtained even by culturing for 3 days or less after the addition of interleukin.
　また、間葉系幹細胞の全体の培養期間のうち、培養を開始してからいつインターロイキンを添加するかは、特に限定されず、例えば、０～２４日の間にインターロイキンを添加してもよいが、培養開始後、０～２０日の間にインターロイキンを添加するのが好ましく、０～１４日（例えば、０～７日、７～１４日、２～１２日、４～１０日、６～８日等）の間にインターロイキンを添加するのがより好ましい。 Further, of the whole culture period of mesenchymal stem cells, when interleukin is added after the start of culture is not particularly limited. For example, even when interleukin is added during 0 to 24 days However, it is preferable to add interleukin between 0 and 20 days after the start of culture, and 0 to 14 days (for example, 0 to 7, 7 to 14, 2 to 12, 4 to 10 days, More preferably, interleukin is added during 6 to 8 days).
　本発明において、インターロイキンを間葉系幹細胞用培養培地に添加する量は、特に限定されず、例えば、添加後の培地中のインターロイキンの濃度が、０．００００１～１００ｎｇ／ｍｌ（例えば、０．０００１～９０ｎｇ／ｍｌ、０．００１～７０ｎｇ／ｍｌ、０．０１～６０ｎｇ／ｍｌ、０．０５～５０ｎｇ／ｍｌ、０．１～３０ｎｇ／ｍｌ、０．５～１０ｎｇ／ｍｌ、１～５ｎｇ／ｍｌ等）となるように添加することができるが、インターロイキンを添加する量が多い方が、間葉系幹細胞の増殖は促進される。この観点から、インターロイキンは、添加後の培地中のインターロイキンの濃度が、０．００５ｎｇ／ｍｌ以上であることが好ましく、０．０５ｎｇ／ｍｌ以上であることがより好ましく、１．０ｎｇ／ｍｌ以上であることが更に好ましく、１０ｎｇ／ｍｌ以上であることが最も好ましい。 In the present invention, the amount of interleukin added to the culture medium for mesenchymal stem cells is not particularly limited. For example, the concentration of interleukin in the medium after addition is 0.00001 to 100 ng / ml (for example, 0 .0001-90 ng / ml, 0.001-70 ng / ml, 0.01-60 ng / ml, 0.05-50 ng / ml, 0.1-30 ng / ml, 0.5-10 ng / ml, 1-5 ng / Ml, etc.), but proliferation of mesenchymal stem cells is promoted when the amount of interleukin added is large. From this viewpoint, the interleukin concentration in the medium after addition is preferably 0.005 ng / ml or more, more preferably 0.05 ng / ml or more, and 1.0 ng / ml. More preferably, it is more preferably 10 ng / ml or more.
　間葉系幹細胞用培養培地には、インターロイキンをあらかじめ含むものを用いてもよく、含まないものを用いてもよいが、あらかじめ培地中にインターロイキンを含んでいたとしても、間葉系幹細胞用培養培地中のインターロイキンの濃度が低すぎると、増殖促進効果は十分に発揮されない。したがって、インターロイキンを含んでいるか否かにかかわらず、本発明の間葉系幹細胞の調製方法は、インターロイキンの濃度が０．００００１ｎｇ／ｍｌ以上である間葉系幹細胞用培養培地において、間葉系幹細胞を培養することが好ましく、インターロイキンの濃度が０．０００１ｎｇ／ｍｌ以上である間葉系幹細胞用培養培地において、間葉系幹細胞を培養することがより好ましく、インターロイキンの濃度が０．００１ｎｇ／ｍｌ以上（０．００５ｎｇ／ｍｌ以上等）である間葉系幹細胞用培養培地において間葉系幹細胞を培養することが更に好ましく、インターロイキンの濃度が０．０１ｎｇ／ｍｌ以上（０．０５ｎｇ／ｍｌ以上、１．０ｎｇ／ｍｌ以上、１０ｎｇ／ｍｌ以上等）である間葉系幹細胞用培養培地において間葉系幹細胞を培養することが最も好ましい。特に、少なくともインターロイキンのうちＩＬ－１βの濃度が、０．０１ｎｇ／ｍｌ以上である間葉系幹細胞用培養培地において、間葉系幹細胞を培養することが好ましく、０．１ｎｇ／ｍｌ以上である間葉系幹細胞用培養培地において、間葉系幹細胞を培養することがより好ましく、ＩＬ－１βの濃度が１ｎｇ／ｍｌ以上である間葉系幹細胞用培養培地において、間葉系幹細胞を培養することがより一層好ましく、ＩＬ－１βの濃度が１０ｎｇ／ｍｌ以上である間葉系幹細胞用培養培地において間葉系幹細胞を培養することが更に好ましく、ＩＬ－１βの濃度が５０ｎｇ／ｍｌ以上である間葉系幹細胞用培養培地において間葉系幹細胞を培養することが最も好ましい。なお、インターロイキンの濃度の上限（インターロイキンのうち、少なくともＩＬ－１βの濃度の上限である場合を含む）は、インターロイキンを含んでいるか否かにかかわらず、特に限定されず、例えば、９０ｎｇ／ｍｌ以下、７０ｎｇ／ｍｌ以下、６０ｎｇ／ｍｌ以下、５０ｎｇ／ｍｌ以下、３０ｎｇ／ｍｌ以下、１０ｎｇ／ｍｌ、５ｎｇ／ｍｌ以下等であってよい。 The culture medium for mesenchymal stem cells may or may not contain interleukins in advance, but it may be used for mesenchymal stem cells even if it contains interleukins in advance. If the concentration of interleukin in the culture medium is too low, the growth promoting effect is not sufficiently exhibited. Therefore, regardless of whether or not it contains interleukins, the method for preparing mesenchymal stem cells of the present invention uses a mesenchymal stem cell culture medium in which the interleukin concentration is 0.00001 ng / ml or more. It is preferable to cultivate stem cells, and it is more preferable to culture the mesenchymal stem cells in a culture medium for mesenchymal stem cells having an interleukin concentration of 0.0001 ng / ml or more. More preferably, the mesenchymal stem cells are cultured in a culture medium for mesenchymal stem cells of 001 ng / ml or more (0.005 ng / ml or more), and the interleukin concentration is 0.01 ng / ml or more (0.05 ng). / Ml or more, 1.0 ng / ml or more, 10 ng / ml or more, etc.) in the mesenchymal stem cell culture medium It is most preferable to culture the cells. In particular, the mesenchymal stem cells are preferably cultured in a culture medium for mesenchymal stem cells in which the concentration of IL-1β of at least interleukin is 0.01 ng / ml or more, and is 0.1 ng / ml or more. More preferably, the mesenchymal stem cells are cultured in the mesenchymal stem cell culture medium, and the mesenchymal stem cells are cultured in the mesenchymal stem cell culture medium in which the concentration of IL-1β is 1 ng / ml or more. More preferably, the mesenchymal stem cells are more preferably cultured in a culture medium for mesenchymal stem cells in which the concentration of IL-1β is 10 ng / ml or more, while the concentration of IL-1β is 50 ng / ml or more. Most preferably, the mesenchymal stem cells are cultured in a culture medium for mesenchymal stem cells. The upper limit of the interleukin concentration (including the case where at least the upper limit of the concentration of IL-1β among the interleukins) is not particularly limited, for example, 90 ng / Ng or less, 70 ng / ml or less, 60 ng / ml or less, 50 ng / ml or less, 30 ng / ml or less, 10 ng / ml, 5 ng / ml or less.
　本発明の間葉系幹細胞の調製方法において、更に、細胞増殖因子を添加する工程を有してもよい。これにより、更に、間葉系幹細胞の増殖が促進される。細胞増殖因子は、特に限定されないが、例えば、ＦＧＦ－２、ＰＤＧＦ等が挙げられる。これらのうち、インターロイキンと組み合わせて使用した場合に、増殖促進効果が特に高いことから、ＦＧＦ－２を用いることが、好ましい。 The method for preparing mesenchymal stem cells of the present invention may further include a step of adding a cell growth factor. This further promotes the proliferation of mesenchymal stem cells. The cell growth factor is not particularly limited, and examples thereof include FGF-2 and PDGF. Of these, FGF-2 is preferably used because it has a particularly high growth promoting effect when used in combination with interleukins.
　添加する細胞増殖因子の量は、特に限定されず、例えば、０．０１～１００ｎｇ／ｍｌ等であってもよいが、培地中の細胞増殖因子の濃度が０．１～５０ｎｇ／ｍｌとなるように添加することが好ましく、１～５０ｎｇ／ｍｌとなるように添加することがより好ましく、１０～５０ｎｇ／ｍｌとなるように添加することが更に好ましい。 The amount of the cell growth factor to be added is not particularly limited, and may be, for example, 0.01 to 100 ng / ml, but the concentration of the cell growth factor in the medium is 0.1 to 50 ng / ml. Preferably, it is added so as to be 1 to 50 ng / ml, more preferably 10 to 50 ng / ml.
　また、本発明の間葉系幹細胞の調製方法において、上記の各工程以外の工程を有してもよい。例えば、そのような工程としては、培養工程後に、間葉系幹細胞を回収する回収工程が挙げられる。 Further, the method for preparing mesenchymal stem cells of the present invention may have steps other than the above steps. For example, such a process includes a recovery process of recovering mesenchymal stem cells after the culture process.
　上述のとおり、炎症性サイトカインは、間葉系幹細胞の増殖促進という観点において有効である。そのため、インターロイキンに限定されず、炎症性サイトカインを用いることで、間葉系幹細胞の増殖を促進することができる。つまり、上述の培養工程は、間葉系幹細胞用培養培地に、炎症性サイトカインを添加し、添加後の前記間葉系幹細胞用培養培地において、間葉系幹細胞を培養する工程であってもよい。また、インターロイキンおよび／または炎症性サイトカインを含む培養培地において間葉系幹細胞を培養する工程であってもよい。そのような炎症性サイトカインとしては、特に限定されないが、ＩＬ－１（ＩＬ－１α、ＩＬ－１β等）、ＴＮＦ－α、ＩＬ－６等が挙げられる。炎症性サイトカインとしては、ＩＬ－１（ＩＬ－１α、ＩＬ－１β等）、ＴＮＦ－αを用いることが好ましい。炎症性サイトカインは、１種単独で用いてもよく、２種以上を併用してもよい。 As described above, inflammatory cytokines are effective in terms of promoting proliferation of mesenchymal stem cells. Therefore, it is not limited to interleukins, and proliferation of mesenchymal stem cells can be promoted by using inflammatory cytokines. That is, the above-described culturing step may be a step of adding inflammatory cytokines to the mesenchymal stem cell culture medium and culturing the mesenchymal stem cells in the mesenchymal stem cell culture medium after the addition. . Further, it may be a step of culturing mesenchymal stem cells in a culture medium containing interleukin and / or inflammatory cytokine. Such inflammatory cytokines include, but are not limited to, IL-1 (IL-1α, IL-1β, etc.), TNF-α, IL-6, and the like. IL-1 (IL-1α, IL-1β, etc.) and TNF-α are preferably used as inflammatory cytokines. Inflammatory cytokines may be used alone or in combination of two or more.
　炎症性サイトカインを用いて間葉系幹細胞の増殖を促進する場合、インターロイキンを用いた場合と同様の条件で使用することができる。 When inflammatory cytokines are used to promote the proliferation of mesenchymal stem cells, they can be used under the same conditions as when interleukins are used.
　＜軟骨細胞の調製方法＞
　本発明の軟骨細胞の調製方法は、間葉系幹細胞とインターロイキンとをｅｘ　ｖｉｖｏで接触させる工程と、間葉系幹細胞の軟骨への分化を誘導する工程と、を有する。 <Method for preparing chondrocytes>
The method for preparing chondrocytes of the present invention comprises a step of contacting mesenchymal stem cells and interleukin ex vivo, and a step of inducing differentiation of mesenchymal stem cells into cartilage.
　［接触工程］
　本発明の軟骨細胞の調製方法における接触工程は、間葉系幹細胞とインターロイキンとをｅｘ　ｖｉｖｏで接触させる工程である。本発明は、これにより、間葉系細胞の軟骨細胞への分化の促進効率を向上させることができる。その理由は、インターロイキンにより、間葉系幹細胞の破壊と再生が繰り返され、結果として、軟骨細胞への分化が促進されるからであると推測される。 [Contact process]
The contact step in the chondrocyte preparation method of the present invention is a step of contacting mesenchymal stem cells and interleukins ex vivo. Thereby, the present invention can improve the efficiency of promoting the differentiation of mesenchymal cells into chondrocytes. The reason for this is presumed to be that interleukins repeatedly destroy and regenerate mesenchymal stem cells and, as a result, promote differentiation into chondrocytes.
　インターロイキンは、特に限定されないが、ＩＬ－１（ＩＬ－１α、ＩＬ－１β等）、ＩＬ－６、ＩＬ－１０等が挙げられる。これらのうち、特に、炎症性サイトカインである、ＩＬ－１（ＩＬ－１α、ＩＬ－１β等）、ＩＬ－６等を用いることが好ましい。ＩＬ－１は、ＩＬ－１α、ＩＬ－１β等が上げられる。これらのうち、ＩＬ－１α、ＩＬ－１βが特に好ましい。インターロイキンは、単独で使用してもよく、２種以上を併用してもよい。ここで、炎症性サイトカインは、従来、軟骨細胞への分化に対して、負の影響を与えると考えられてきたが、本発明者らは、炎症性サイトカインを用いることによって、予想外なことに、軟骨細胞への分化を促進することを見出した。これは、炎症性サイトカインを用いることで、間葉系幹細胞の破壊と再生がより一層繰り返され、結果として、軟骨細胞への分化がより促進されるからであるからと推測される。 The interleukin is not particularly limited, and examples thereof include IL-1 (IL-1α, IL-1β, etc.), IL-6, IL-10, and the like. Of these, IL-1 (IL-1α, IL-1β, etc.), IL-6, etc., which are inflammatory cytokines, are particularly preferred. IL-1 includes IL-1α, IL-1β and the like. Of these, IL-1α and IL-1β are particularly preferred. Interleukin may be used independently and may use 2 or more types together. Here, inflammatory cytokines have hitherto been considered to have a negative effect on differentiation into chondrocytes, but the present inventors have unexpectedly realized by using inflammatory cytokines. And found to promote differentiation into chondrocytes. This is presumed to be because the destruction and regeneration of mesenchymal stem cells are further repeated by using inflammatory cytokines, and as a result, differentiation into chondrocytes is further promoted.
　また、インターロイキンの種の由来も特に限定されず、ヒト、ブタ、ウシ、ウサギ、ラット、マウス等の由来のインターロイキンを用いることができ、また、これらとアミノ酸配列の相同性が９０％以上であるタンパク等を用いることができる。 Also, the origin of the interleukin species is not particularly limited, and interleukins derived from humans, pigs, cows, rabbits, rats, mice, etc. can be used, and the homology between them and amino acid sequences is 90% or more. The protein etc. which are can be used.
　間葉系幹細胞は、特に限定されず、間葉系幹細胞を含む種々の間葉系の組織の間葉系幹細胞を使用可能であるが、例えば、滑膜、骨膜、筋肉、脂肪（皮下脂肪等）、骨組織、靭帯、腱、半月板等の間葉系幹細胞が挙げられる。これらのうち、特に、間葉系幹細胞の軟骨細胞への増殖を促進することから、滑膜由来の間葉系幹細胞、脂肪由来の間葉系幹細胞を使用することが好ましい。 The mesenchymal stem cells are not particularly limited, and mesenchymal stem cells of various mesenchymal tissues including mesenchymal stem cells can be used. For example, synovial membrane, periosteum, muscle, fat (subcutaneous fat, etc.) ), Mesenchymal stem cells such as bone tissue, ligament, tendon, meniscus and the like. Among these, it is particularly preferable to use synovial-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells in order to promote proliferation of mesenchymal stem cells to chondrocytes.
　本発明における接触工程は、間葉系幹細胞とインターロイキンとをｅｘ　ｖｉｖｏで接触させる手段を用いれば、その方法は特に限定されない。例えば、間葉系幹細胞を、インターロイキンを含む軟骨分化誘導用培地に加えてもよく、間葉系幹細胞を軟骨分化誘導用培地に加えた後に、培地中にインターロイキンを加えてもよく、これらを同時に軟骨分化誘導用培地に加えてもよい。あるいは、間葉系幹細胞の前培養中に、インターロイキンを培養培地に加えてもよい。これらにより、間葉系幹細胞の軟骨への分化は促進される。 The contact process in the present invention is not particularly limited as long as it uses means for contacting mesenchymal stem cells and interleukins ex vivo. For example, mesenchymal stem cells may be added to a medium for inducing cartilage differentiation containing interleukin, and after adding mesenchymal stem cells to a medium for inducing cartilage differentiation, interleukin may be added to the medium. May be simultaneously added to the medium for inducing cartilage differentiation. Alternatively, interleukins may be added to the culture medium during preculture of mesenchymal stem cells. These promote the differentiation of mesenchymal stem cells into cartilage.
　上記軟骨分化誘導用培地に加えるインターロイキンの濃度は、特に限定されないが、例えば、０．０１～１００ｎｇ／ｍｌとなるように、インターロイキンを加えることができる。培地中に加えるインターロイキンの量が多い方が、間葉系幹細胞の軟骨細胞への分化は促進される。この観点から、インターロイキンの濃度が０．１ｎｇ／ｍｌ以上となるようにインターロイキンを軟骨分化誘導用培地に加えることが好ましく、インターロイキンの濃度が１ｎｇ／ｍｌ以上となるようにインターロイキンを軟骨分化誘導用培地に加えることがより好ましく、インターロイキンの濃度が１０ｎｇ／ｍｌ以上となるようにインターロイキンを軟骨分化誘導用培地に加えることが更に好ましく、インターロイキンの濃度が５０ｎｇ／ｍｌ以上となるようにインターロイキンを軟骨分化誘導用培地に加えることが最も好ましい。また、軟骨分化誘導用培地に加えるインターロイキンの濃度の上限は、９０ｎｇ／ｍｌ以下、７０ｎｇ／ｍｌ以下、５０ｎｇ／ｍｌ以下、３０ｎｇ／ｍｌ以下、１０ｎｇ／ｍｌ、５ｎｇ／ｍｌ以下、１ｎｇ／ｍｌ以下等であってもよい。 The concentration of interleukin added to the above-mentioned medium for induction of cartilage differentiation is not particularly limited, but for example, interleukin can be added so as to be 0.01 to 100 ng / ml. When the amount of interleukin added to the medium is larger, differentiation of mesenchymal stem cells into chondrocytes is promoted. From this point of view, it is preferable to add interleukin to the cartilage differentiation induction medium so that the interleukin concentration is 0.1 ng / ml or more, and interleukin is added to the cartilage so that the interleukin concentration is 1 ng / ml or more. More preferably, the interleukin is added to the medium for induction of cartilage differentiation so that the concentration of interleukin is 10 ng / ml or more, and the concentration of interleukin is 50 ng / ml or more. Thus, it is most preferable to add interleukin to the medium for inducing cartilage differentiation. The upper limit of the concentration of interleukin added to the cartilage differentiation induction medium is 90 ng / ml or less, 70 ng / ml or less, 50 ng / ml or less, 30 ng / ml or less, 10 ng / ml, 5 ng / ml or less, 1 ng / ml or less. Etc.
　前培養中の培地にインターロイキンを加える場合、その量は特に限定されないが、量が多い方が好ましい。具体的には、添加後の前培養中の培地中のインターロイキンの濃度が、０．０１ｎｇ／ｍｌ以上であることが好ましく、０．１ｎｇ／ｍｌ以上であることがより好ましく、１．０ｎｇ／ｍｌ以上であることが更に好ましく、１０ｎｇ／ｍｌ以上であることが最も好ましい。なお、前培養に用いる培地は、上述の間葉系幹細胞の調製方法において用いた間葉系幹細胞用培養培地と同様のものを用いることができる。 When interleukin is added to the medium during preculture, the amount is not particularly limited, but a larger amount is preferred. Specifically, the concentration of interleukin in the medium during the pre-culture after the addition is preferably 0.01 ng / ml or more, more preferably 0.1 ng / ml or more, and 1.0 ng / ml More preferably, it is more than ml, and most preferably more than 10 ng / ml. In addition, the culture medium used for preculture can use the same thing as the culture medium for mesenchymal stem cells used in the preparation method of the above-mentioned mesenchymal stem cells.
　間葉系幹細胞の前培養中の培地にインターロイキンを加える場合、インターロイキンは、いつ加えてもよいが、間葉系幹細胞の培養開始後、０～１４日（２～１２日、４～１０日、６～８日等）の間に加えてもよい。また、インターロイキンを培地に添加後、培養を行う期間は特に限定されないが、例えば、０～２８日（２～２４日、５～２０日、７～１４日等）培養を行ってもよい。 When interleukin is added to the medium during the preculture of mesenchymal stem cells, interleukin may be added at any time, but 0 to 14 days (2 to 12 days, 4 to 10 days) after the start of the mesenchymal stem cell culture. Day, 6-8 days, etc.). Further, the period of culture after the addition of interleukin to the medium is not particularly limited. For example, the culture may be performed for 0 to 28 days (2 to 24 days, 5 to 20 days, 7 to 14 days, etc.).
　［軟骨分化誘導工程］
　本発明の軟骨細胞の調製方法における軟骨分化誘導工程は、間葉系幹細胞の軟骨への分化を誘導する工程である。 [Cartilage differentiation induction process]
The cartilage differentiation induction step in the chondrocyte preparation method of the present invention is a step of inducing differentiation of mesenchymal stem cells into cartilage.
　軟骨分化誘導工程において、軟骨分化誘導用培地の軟骨分化誘導条件は、特に限定されないが、例えば、通常の軟骨分化誘導の温度（例えば、３７℃）において、０～２１日間（２～１８日間、４～１４日間、７～１０日間等）、間葉系幹細胞から軟骨への分化を誘導することができる。しかし、本発明によると、インターロイキンを用いることで、間葉系幹細胞の軟骨分化誘導が促進されるため、短い期間の誘導に適している。例えば、１４日以下の誘導でも、多量の軟骨細胞を得ることができる。 In the cartilage differentiation inducing step, conditions for inducing cartilage differentiation in the medium for inducing cartilage differentiation are not particularly limited. For example, at a normal cartilage differentiation induction temperature (eg, 37 ° C.), 0 to 21 days (2 to 18 days, Differentiation from mesenchymal stem cells to cartilage can be induced for 4-14 days, 7-10 days, etc.). However, according to the present invention, the use of interleukin promotes the induction of cartilage differentiation of mesenchymal stem cells, which is suitable for induction in a short period. For example, a large amount of chondrocytes can be obtained even by induction for 14 days or less.
　本発明の軟骨細胞の調製方法において、上記の接触工程と軟骨分化誘導工程とは、同じ工程であってもよく、異なる工程であってもよい。 In the chondrocyte preparation method of the present invention, the contact step and the cartilage differentiation induction step may be the same step or different steps.
　例えば、間葉系幹細胞の前培養中に、インターロイキンを培養培地に加える場合、その後、前培養後の間葉系幹細胞を用いて上記軟骨分化誘導工程を行うが、そのときは、軟骨分化誘導工程中に、軟骨分化誘導用培地にインターロイキンを加えなくてもよく（すなわち、更に軟骨分化誘導工程において接触工程を行わなくてもよく）インターロイキンを加えてもよい（すなわち、軟骨分化誘導工程において更に接触工程を行ってもよい）。軟骨分化誘導工程中に、軟骨分化誘導用培地にインターロイキンを加える場合は、前培養においてインターロイキンを加えてもよく、加えなくてもよく、また、前培養自体を行わなくてもよい。 For example, when interleukin is added to the culture medium during the pre-culture of mesenchymal stem cells, the above-described cartilage differentiation induction step is performed using the mesenchymal stem cells after pre-culture. During the process, interleukin may not be added to the cartilage differentiation-inducing medium (that is, the contact process may not be further performed in the cartilage differentiation induction process), and interleukin may be added (that is, cartilage differentiation induction process). In this case, a contact step may be further performed). When interleukin is added to the cartilage differentiation induction medium during the cartilage differentiation induction step, interleukin may or may not be added in the preculture, or the preculture itself may not be performed.
　また、本発明の軟骨細胞の調製方法において、上記の接触工程や、軟骨分化誘導工程、前培養以外の他の工程を有してもよい。例えば、そのような工程としては、軟骨分化誘導後、軟骨細胞を回収する回収工程が挙げられる。 In addition, the chondrocyte preparation method of the present invention may have other steps other than the contact step, the cartilage differentiation induction step, and the pre-culture. For example, such a process includes a recovery process of recovering chondrocytes after induction of cartilage differentiation.
　上述のとおり、炎症性サイトカインは、間葉系幹細胞の軟骨への分化を促進という観点において有効である。そのため、インターロイキンに限定されず、炎症性サイトカインを用いることで、間葉系幹細胞の軟骨へ分化する速度を促進することができる。つまり、上述の接触工程は、間葉系幹細胞とインターロイキンおよび／または炎症性サイトカインとをｅｘ　ｖｉｖｏで接触させる工程であってもよい。そのような炎症性サイトカインとしては、特に限定されないが、ＩＬ－１（ＩＬ－１α、ＩＬ－１β等）、ＴＮＦ－α、ＩＬ－６等が挙げられる。炎症性サイトカインとしては、ＩＬ－１（ＩＬ－１α、ＩＬ－１β等）、ＴＮＦ－αを用いることが好ましい。炎症性サイトカインは、１種単独で用いてもよく、２種以上を併用してもよい。 As described above, inflammatory cytokines are effective in terms of promoting the differentiation of mesenchymal stem cells into cartilage. Therefore, it is not limited to interleukins, and by using inflammatory cytokines, the rate of mesenchymal stem cell differentiation into cartilage can be promoted. That is, the above-mentioned contact step may be a step of contacting mesenchymal stem cells with interleukins and / or inflammatory cytokines ex vivo. Such inflammatory cytokines include, but are not limited to, IL-1 (IL-1α, IL-1β, etc.), TNF-α, IL-6, and the like. IL-1 (IL-1α, IL-1β, etc.) and TNF-α are preferably used as inflammatory cytokines. Inflammatory cytokines may be used alone or in combination of two or more.
　炎症性サイトカインを用いて間葉系幹細胞の軟骨への分化を促進する場合、インターロイキンを用いた場合と同様の条件で使用することができる。 When promoting the differentiation of mesenchymal stem cells into cartilage using inflammatory cytokines, they can be used under the same conditions as when interleukins are used.
　＜間葉系幹細胞の増殖促進剤＞
　本発明の間葉系幹細胞の増殖促進剤は、インターロイキン、又は、炎症性サイトカインからなる。 <Mesenchymal stem cell proliferation promoter>
The agent for promoting proliferation of mesenchymal stem cells of the present invention comprises interleukin or inflammatory cytokine.
　間葉系幹細胞の増殖促進剤におけるインターロイキン又は炎症性サイトカインは、上述の間葉系幹細胞の調製方法で用いたものと同様のものを用いることができる。 As the interleukin or inflammatory cytokine in the mesenchymal stem cell proliferation promoter, the same one as used in the above-described method for preparing mesenchymal stem cells can be used.
　＜間葉系幹細胞の軟骨分化促進剤＞ <Mechanical stem cell cartilage differentiation promoter>
　本発明の間葉系幹細胞の軟骨分化促進剤は、インターロイキン、又は、炎症性サイトカインからなる。 The mesenchymal stem cell cartilage differentiation promoting agent of the present invention is composed of interleukin or inflammatory cytokine.
　間葉系幹細胞の軟骨分化促進剤におけるインターロイキン又は炎症性サイトカインは、上述の軟骨細胞の調製方法で用いたものと同様のものを用いることができる。 As the interleukin or inflammatory cytokine in the cartilage differentiation promoting agent for mesenchymal stem cells, the same one as used in the above-described method for preparing chondrocytes can be used.
　＜間葉系幹細胞用培養培地＞
　本発明の間葉系幹細胞用培養培地は、インターロイキン被添加用として用いることができる。 <Culture medium for mesenchymal stem cells>
The culture medium for mesenchymal stem cells of the present invention can be used for interleukin addition.
　上述のとおり、間葉系幹細胞用培養培地は、インターロイキンが添加されることで、間葉系幹細胞の増殖を促進することができる。したがって、間葉系幹細胞用培養培地は、インターロイキン被添加用として用いることに適している。 As described above, the culture medium for mesenchymal stem cells can promote the growth of mesenchymal stem cells by adding interleukin. Therefore, the culture medium for mesenchymal stem cells is suitable for being used for interleukin addition.
　間葉系幹細胞用培養培地は、上述の間葉系幹細胞の調製方法で用いたものと同様のものを用いることができるが、特に、無血清培地においても、間葉系幹細胞の増殖の促進が可能であることから、無血清培地を用いた場合は、特に、インターロイキン被添加用として用いることに好適である。 The culture medium for mesenchymal stem cells can be the same as that used in the above-described method for preparing mesenchymal stem cells, and in particular, the growth of mesenchymal stem cells is promoted even in a serum-free medium. Therefore, when a serum-free medium is used, it is particularly suitable for use as an interleukin-added product.
　また、本発明の培養培地は、インターロイキンおよび／または炎症性サイトカインを含有するものであってもよい。また、培養する対象の間葉系幹細胞は、特に限定されず、例えば、滑膜、骨膜、筋肉、脂肪（皮下脂肪等）、骨組織、靭帯、腱、半月板等の間葉系幹細胞が挙げられる。これらのうち、本発明の培養培地は、特に、滑膜由来の間葉系幹細胞、脂肪由来の間葉系幹細胞を培養させるために培地として使用することが好ましい。 Further, the culture medium of the present invention may contain interleukin and / or inflammatory cytokine. Further, the mesenchymal stem cells to be cultured are not particularly limited, and examples thereof include mesenchymal stem cells such as synovium, periosteum, muscle, fat (subcutaneous fat, etc.), bone tissue, ligament, tendon, meniscus and the like. It is done. Among these, the culture medium of the present invention is particularly preferably used as a medium for culturing synovial-derived mesenchymal stem cells and fat-derived mesenchymal stem cells.
　＜間葉系幹細胞の調製１＞
　（実施例１）
　８３歳男性の滑膜由来の間葉系幹細胞を用いて、１０％ＦＢＳを添加したαＭＥＭ培地を用い、３７℃で、４回継代を行い２週間、間葉系幹細胞の前培養を行った。その後、間葉系幹細胞の数を１０ｃｍ２のディッシュで１００個／ディッシュに調節してからＩＬ－１βを、終濃度５ｎｇ／ｍｌとなるように添加し、３７℃で、２週間培養を行い、実施例１に係る間葉系幹細胞を調製した。 <Preparation 1 of mesenchymal stem cells>
(Example 1)
Using 83-year-old male synovial stem cell derived mesenchymal stem cells, αMEM medium supplemented with 10% FBS was used for 4 passages at 37 ° C. and pre-cultured mesenchymal stem cells for 2 weeks. . Thereafter, the number of mesenchymal stem cells was adjusted to 100 / dish with a 10 cm 2 dish, IL-1β was added to a final concentration of 5 ng / ml, and cultured at 37 ° C. for 2 weeks. Mesenchymal stem cells according to Example 1 were prepared.
　（実施例２）
　前培養後、間葉系幹細胞の数を２００個／ディッシュに調節した点以外は、実施例１と同様の条件で、実施例２に係る間葉系幹細胞を調製した。 (Example 2)
After the preculture, the mesenchymal stem cells according to Example 2 were prepared under the same conditions as in Example 1 except that the number of mesenchymal stem cells was adjusted to 200 / dish.
　（対照例１）
　前培養後、ＩＬ－１βを添加しなかった点以外は、実施例１と同様の条件で対照例１に係る間葉系幹細胞を調製した。 (Control 1)
After the preculture, mesenchymal stem cells according to Control Example 1 were prepared under the same conditions as in Example 1 except that IL-1β was not added.
　（対照例２）
　前培養後、ＩＬ－１βを添加しなかった点以外は、実施例２と同様の条件で対照例２に係る間葉系幹細胞を調製した。 (Control 2)
After the preculture, mesenchymal stem cells according to Control Example 2 were prepared under the same conditions as in Example 2 except that IL-1β was not added.
　＜間葉系幹細胞の増殖の評価１＞
　実施例１、２、対照例１、２に係る間葉系幹細胞について、調製後にクリスタル・バイオレット染色を行った。その結果を、図１に示す。図１中、（ａ）は、実施例１、対照例１に係る間葉系幹細胞についてのクリスタル・バイオレット染色後の画像を示し、（ｂ）は、実施例２、対照例２に係る間葉系幹細胞についてのクリスタル・バイオレット染色後の画像を示す。 <Evaluation of proliferation of mesenchymal stem cells 1>
The mesenchymal stem cells according to Examples 1 and 2 and Control Examples 1 and 2 were subjected to crystal violet staining after preparation. The result is shown in FIG. In FIG. 1, (a) shows an image after crystal violet staining of mesenchymal stem cells according to Example 1 and Control Example 1, and (b) shows mesenchymal according to Example 2 and Control Example 2. The image after crystal violet dyeing | staining about a stem cell is shown.
　図１に示すように、実施例１及び実施例２のいずれも、ＩＬ－１βを添加しなかった対照例１、２と比較して、コロニー形成率及びコロニーサイズが増大していた。この結果より、ＩＬ－１βにより、細胞の増殖が促進することが示された。 As shown in FIG. 1, in both Example 1 and Example 2, the colony formation rate and the colony size were increased as compared with Control Examples 1 and 2 in which IL-1β was not added. From these results, it was shown that IL-1β promotes cell proliferation.
　＜間葉系幹細胞の調製２＞
　（実施例３）
　実施例１とは、異なる４つの滑膜由来の間葉系幹細胞を用いた以外は、実施例１と同様の条件で、実施例３に係る間葉系幹細胞を調製した。（ｎ＝４） <Preparation 2 of mesenchymal stem cells>
(Example 3)
A mesenchymal stem cell according to Example 3 was prepared under the same conditions as in Example 1 except that four synovial stem cells derived from synovium different from Example 1 were used. (N = 4)
　（対照例３）
　前培養後、ＩＬ－１βを添加しなかった点以外は、実施例３と同様の条件で対照例３に係る間葉系幹細胞を調製した。 (Control 3)
After the preculture, mesenchymal stem cells according to Control Example 3 were prepared under the same conditions as in Example 3 except that IL-1β was not added.
　＜間葉系幹細胞の増殖の評価２＞
　実施例３、対照例３に係る間葉系幹細胞について、調製後にクリスタル・バイオレット染色を行い、細胞数をカウントした。そのグラフを、図２に示す。 <Evaluation of proliferation of mesenchymal stem cells 2>
The mesenchymal stem cells according to Example 3 and Control Example 3 were subjected to crystal violet staining after preparation, and the number of cells was counted. The graph is shown in FIG.
　図２に示すように、実施例３は、ＩＬ－１βを添加しなかった対照例３と比較して、細胞数が有意に増大していた。 As shown in FIG. 2, in Example 3, the number of cells was significantly increased as compared to Control Example 3 in which IL-1β was not added.
　＜間葉系幹細胞の調製３＞
　（実施例４）
　７２歳女性の滑膜由来の間葉系幹細胞を用いて、１０％ＦＢＳを添加したαＭＥＭ培地を用い、３７℃で、初代間葉系幹細胞の前培養を２週間行った。その後、間葉系幹細胞の数を６０ｃｍ２のディッシュで１０００個／ディッシュに調節し、ＩＬ－１βを終濃度０．５ｎｇ／ｍｌとなるように添加し、３７℃で２週間培養を行い、実施例４に係る間葉系幹細胞を調製した。 <Preparation 3 of mesenchymal stem cells>
Example 4
Using a mesenchymal stem cell derived from synovium of a 72-year-old female, primary mesenchymal stem cells were pre-cultured at 37 ° C. for 2 weeks using an αMEM medium supplemented with 10% FBS. Thereafter, the number of mesenchymal stem cells was adjusted to 1000 / dish with a 60 cm 2 dish, IL-1β was added to a final concentration of 0.5 ng / ml, and cultured at 37 ° C. for 2 weeks. Mesenchymal stem cells according to Example 4 were prepared.
　（実施例５）
　ＩＬ－１βを終濃度５ｎｇ／ｍｌとなるように添加した点以外は、実施例４と同様の条件で、実施例５に係る間葉系幹細胞を調製した。 (Example 5)
A mesenchymal stem cell according to Example 5 was prepared under the same conditions as in Example 4 except that IL-1β was added to a final concentration of 5 ng / ml.
　（実施例６）
　ＩＬ－１βを終濃度５０ｎｇ／ｍｌとなるように添加した点以外は、実施例４と同様の条件で、実施例６に係る間葉系幹細胞を調製した。 (Example 6)
A mesenchymal stem cell according to Example 6 was prepared under the same conditions as in Example 4 except that IL-1β was added to a final concentration of 50 ng / ml.
　（対照例４）
　前培養後、ＩＬ－１βを添加しなかった点以外は、実施例４と同様の条件で対照例４に係る間葉系幹細胞を調製した。 (Control 4)
After the preculture, mesenchymal stem cells according to Control Example 4 were prepared under the same conditions as in Example 4 except that IL-1β was not added.
　＜間葉系幹細胞の増殖の評価３＞
　実施例４～６、対照例４に係る間葉系幹細胞について、調製後にクリスタル・バイオレット染色を行った。その結果を、図３に示す。 <Evaluation of proliferation of mesenchymal stem cells 3>
The mesenchymal stem cells according to Examples 4 to 6 and Control Example 4 were subjected to crystal violet staining after preparation. The result is shown in FIG.
　図３に示す結果から、添加したＩＬ－１βの濃度が増すにしたがって、間葉系幹細胞の増殖が促進することが示された。 The results shown in FIG. 3 showed that the proliferation of mesenchymal stem cells was promoted as the concentration of added IL-1β increased.
　＜間葉系幹細胞の調製４＞
　（実施例７）
　７２歳女性の滑膜由来の間葉系幹細胞を用いて、１０％ＦＢＳ添加αＭＥＭ培地を用い、３７℃で、１回継代を行い２週間、間葉系幹細胞の前培養を行った。その後、間葉系幹細胞の数を６０ｃｍ２のディッシュで１０００個／ディッシュに調節し、ＩＬ－１βを終濃度０．０１ｎｇ／ｍｌとなるように添加し、３７℃で、２週間培養を行い、実施例７に係る間葉系幹細胞を調製した。 <Preparation 4 of mesenchymal stem cells>
(Example 7)
Using mesenchymal stem cells derived from synovial membrane of a 72-year-old woman, the cells were subcultured once at 37 ° C. using an αMEM medium supplemented with 10% FBS, and mesenchymal stem cells were precultured for 2 weeks. Thereafter, the number of mesenchymal stem cells was adjusted to 1000 cells / dish with a 60 cm 2 dish, IL-1β was added to a final concentration of 0.01 ng / ml, and cultured at 37 ° C. for 2 weeks. A mesenchymal stem cell according to Example 7 was prepared.
　（実施例８）
　ＩＬ－１βを終濃度０．１ｎｇ／ｍｌとなるように添加した点以外は、実施例７と同様の条件で、実施例８に係る間葉系幹細胞を調製した。 (Example 8)
A mesenchymal stem cell according to Example 8 was prepared under the same conditions as in Example 7 except that IL-1β was added to a final concentration of 0.1 ng / ml.
　（実施例９）
　ＩＬ－１βを終濃度１ｎｇ／ｍｌとなるように添加した点以外は、実施例７と同様の条件で、実施例９に係る間葉系幹細胞を調製した。 Example 9
A mesenchymal stem cell according to Example 9 was prepared under the same conditions as in Example 7 except that IL-1β was added to a final concentration of 1 ng / ml.
　（実施例１０）
　ＩＬ－１βを終濃度１０ｎｇ／ｍｌとなるように添加した点以外は、実施例７と同様の条件で、実施例１０に係る間葉系幹細胞を調製した。 (Example 10)
A mesenchymal stem cell according to Example 10 was prepared under the same conditions as in Example 7 except that IL-1β was added to a final concentration of 10 ng / ml.
　（対照例５）
　前培養後、ＩＬ－１βを添加しなかった点以外は、実施例７と同様の条件で対照例４に係る間葉系幹細胞を調製した。 (Control 5)
After the preculture, mesenchymal stem cells according to Control Example 4 were prepared under the same conditions as in Example 7 except that IL-1β was not added.
　＜間葉系幹細胞の増殖の評価４＞
　実施例７～１０、対照例５に係る間葉系幹細胞について、調製後にクリスタル・バイオレット染色を行った。その結果を、図４に示す。 <Evaluation of proliferation of mesenchymal stem cells 4>
The mesenchymal stem cells according to Examples 7 to 10 and Control Example 5 were subjected to crystal violet staining after preparation. The result is shown in FIG.
　図４に示す結果から、図３と同様に、添加したＩＬ－１βの濃度が増すにしたがって、間葉系幹細胞の増殖が促進することが示された。 The results shown in FIG. 4 showed that, as in FIG. 3, the proliferation of mesenchymal stem cells was promoted as the concentration of added IL-1β increased.
　＜間葉系幹細胞の調製５＞
　（実施例１１）
　滑膜由来の間葉系幹細胞を用いて、１０％ＦＢＳ添加αＭＥＭ培地を用い、３７℃で、１４日間、間葉系幹細胞の初代培養を行った。その後、間葉系幹細胞の数を６０ｃｍ２のディッシュで１００個／ディッシュに調節し、ＩＬ－１βを終濃度０．３ｎｇ／ｍｌとなるように添加し、３７℃で、１２日間培養を行って、実施例１１に係る間葉系幹細胞を調製した。 <Preparation 5 of mesenchymal stem cells>
(Example 11)
Using mesenchymal stem cells derived from synovium, primary culture of mesenchymal stem cells was performed at 37 ° C. for 14 days using αMEM medium supplemented with 10% FBS. Thereafter, the number of mesenchymal stem cells was adjusted to 100 / dish with a 60 cm 2 dish, IL-1β was added to a final concentration of 0.3 ng / ml, and cultured at 37 ° C. for 12 days. Then, mesenchymal stem cells according to Example 11 were prepared.
　（実施例１２）
　ＩＬ－１βとともに、ＦＧＦ（繊維芽細胞増殖因子）－２を終濃度１０ｎｇ／ｍｌとなるように添加した点以外は、実施例１１と同様の条件で、実施例１２に係る間葉系幹細胞を調製した。 Example 12
The mesenchymal stem cell according to Example 12 was prepared under the same conditions as in Example 11 except that FGF (fibroblast growth factor) -2 was added together with IL-1β to a final concentration of 10 ng / ml. Prepared.
　（参考例１）
　ＩＬ－１βを加えなかった点以外は、実施例１２と同様の条件で、参考例１に係る間葉系幹細胞を調製した。 (Reference Example 1)
A mesenchymal stem cell according to Reference Example 1 was prepared under the same conditions as in Example 12 except that IL-1β was not added.
　（対照例６）
　前培養後、ＩＬ－１βを添加しなかった点以外は、実施例１１と同様の条件で対照例６に係る間葉系幹細胞を調製した。 (Control 6)
After the preculture, mesenchymal stem cells according to Control Example 6 were prepared under the same conditions as in Example 11 except that IL-1β was not added.
　＜間葉系幹細胞の増殖の評価５＞
　実施例１１、１２、参考例１、対照例６に係る間葉系幹細胞について、調製後にクリスタル・バイオレット染色を行った。その結果を、図５に示す。また、実施例１１、１２、参考例１、対照例６に係る間葉系幹細胞についての細胞の倍増数及び細胞数のグラフを、図６に示す。 <Evaluation of proliferation of mesenchymal stem cells 5>
The mesenchymal stem cells according to Examples 11 and 12, Reference Example 1 and Control Example 6 were subjected to crystal violet staining after preparation. The result is shown in FIG. Moreover, the graph of the cell doubling number and the cell number for the mesenchymal stem cells according to Examples 11 and 12, Reference Example 1 and Control Example 6 is shown in FIG.
　図５に示す結果から、細胞増殖因子であるＦＧＦ－２と、ＩＬ－１βを併用することで、間葉系幹細胞の増殖が更に促進することが示された。また、図６に示す結果から、ＦＧＦ－２と、ＩＬ－１βとを併用することで、それぞれを単独で使用したときに比べ有意に細胞の増殖が促進していることが確認された。これにより、ＦＧＦ－２と、ＩＬ－１βとを併用することで、相乗的に、間葉系幹細胞の増殖が促進することが示された。 The results shown in FIG. 5 indicate that the proliferation of mesenchymal stem cells is further promoted by using FGF-2, which is a cell growth factor, and IL-1β in combination. Further, from the results shown in FIG. 6, it was confirmed that the combined use of FGF-2 and IL-1β significantly promoted cell proliferation compared to the case where each was used alone. Thus, it was shown that the combined use of FGF-2 and IL-1β synergistically promotes the proliferation of mesenchymal stem cells.
　＜軟骨細胞の調製１＞
　（実施例１３）
　８３歳男性の滑膜由来の間葉系幹細胞を用いて、１０％ＦＢＳ添加αＭＥＭ培地を用い、３７℃で、３回継代を行い２週間、間葉系幹細胞の培養を行った。その後、細胞を回収して、対照例７と細胞数を合わせてペレットを作製し、軟骨分化培地（ＤＭＥＭに１０００ｎｇ／ｍｌ　ＢＭＰ７，１０ｎｇ／ｍｌ　ＴＧＦ－ｂｅｔａ，　１０－７Ｍ　デキサメタゾンを添加したもの）に５ｎｇ／ｍｌのＩＬ－１βを加え３週間培養し、実施例１３に係る軟骨細胞を調製した。 <Preparation of chondrocytes 1>
(Example 13)
Using 83-year-old male synovial-derived mesenchymal stem cells, 10% FBS-added αMEM medium was used for 3 passages at 37 ° C., and mesenchymal stem cells were cultured for 2 weeks. Thereafter, the cells were recovered, and the number of cells was matched with that of Control Example 7 to prepare a pellet. Cartilage differentiation medium (DMEM supplemented with 1000 ng / ml BMP7, 10 ng / ml TGF-beta, 10 −7 M dexamethasone) 5 ng / ml IL-1β was added thereto and cultured for 3 weeks to prepare chondrocytes according to Example 13.
　（対照例７）
　前培養後、ＩＬ－１βを添加しなかった点以外は、実施例１３と同様の条件で対照例７に係る間葉系幹細胞を調製した。なお、ペレットを作製する際は、実施例１３と細胞数を合わせた。 (Control 7)
After the preculture, mesenchymal stem cells according to Control Example 7 were prepared under the same conditions as in Example 13 except that IL-1β was not added. In addition, when producing a pellet, Example 13 and the cell number were match | combined.
　＜軟骨分化の評価１＞
　実施例１３、対照例７に係る軟骨細胞について、大きさを測定し、軟骨分化がどの程度誘導されたか、評価した。その結果を、図７に示す。図７中、実施例１３と対照例７のそれぞれの画像の下側の数値は、それぞれの軟骨塊の最大径を示す。また、図７中、実施例１３と対照例７のそれぞれの画像の右側の数値は、それぞれの軟骨塊の最大径の平均（ｎ＝３）を示す。 <Evaluation of cartilage differentiation 1>
The size of chondrocytes according to Example 13 and Control Example 7 was measured to evaluate how much cartilage differentiation was induced. The result is shown in FIG. In FIG. 7, the numerical values on the lower side of the images of Example 13 and Control Example 7 indicate the maximum diameter of each cartilage mass. In FIG. 7, the numerical values on the right side of the images of Example 13 and Control Example 7 indicate the average (n = 3) of the maximum diameter of each cartilage mass.
　図７に示すように、実施例１３の方が、ＩＬ－１βを添加しなかった対照例７と比較して、軟骨細胞が大きかった。この結果より、ＩＬ－１βにより、間葉系幹細胞の軟骨への分化誘導が促進することが示された。 As shown in FIG. 7, the chondrocytes of Example 13 were larger than Control Example 7 in which IL-1β was not added. From these results, it was shown that IL-1β promotes differentiation induction of mesenchymal stem cells into cartilage.
　＜軟骨細胞の調製２＞
　（実施例１４）
　７２歳女性の滑膜由来の間葉系幹細胞を用いて、１０％ＦＢＳ添加αＭＥＭ培地を用い、３７℃で、１回継代を行い１４日間、間葉系幹細胞の前培養を行った。その後、間葉系幹細胞の数を６０ｃｍ２のディッシュで１０００個／ディッシュとなるように調節してから、ＩＬ－１βが終濃度０．５ｎｇ／ｍｌとなるように添加し、３７℃で１４日間培養を行った。その後、実施例１５及び１６と細胞数を合わせてペレットを作製し、軟骨分化培地（ＤＭＥＭに１０００ｎｇ／ｍｌ　ＢＭＰ７，１０ｎｇ／ｍｌ　ＴＧＦ－ｂｅｔａ，　１０－７Ｍ　デキサメタゾンを添加したもの）を用いて２１日間培養し、実施例１４に係る軟骨細胞を調製した。 <Preparation 2 of chondrocytes>
(Example 14)
Using mesenchymal stem cells derived from synovium of a 72-year-old woman, the cells were subcultured once at 37 ° C. using an αMEM medium supplemented with 10% FBS, and mesenchymal stem cells were precultured for 14 days. Thereafter, the number of mesenchymal stem cells was adjusted to 1000 cells / dish with a 60 cm 2 dish, and IL-1β was added to a final concentration of 0.5 ng / ml, and the culture was performed at 37 ° C. for 14 days. Culture was performed. Thereafter, a pellet was prepared by combining the cells with Examples 15 and 16, and using a cartilage differentiation medium (DMEM supplemented with 1000 ng / ml BMP7, 10 ng / ml TGF-beta, 10 −7 M dexamethasone). After culturing for days, chondrocytes according to Example 14 were prepared.
　（実施例１５）
　ＩＬ－１βを終濃度５ｎｇ／ｍｌとなるように添加した点以外は、実施例１４と同様の条件で、実施例１５に係る軟骨細胞を調製した。 (Example 15)
Chondrocytes according to Example 15 were prepared under the same conditions as in Example 14 except that IL-1β was added to a final concentration of 5 ng / ml.
　（実施例１６）
　ＩＬ－１βを終濃度５０ｎｇ／ｍｌとなるように添加した点以外は、実施例１４と同様の条件で、実施例１６に係る軟骨細胞を調製した。 (Example 16)
Chondrocytes according to Example 16 were prepared under the same conditions as in Example 14 except that IL-1β was added to a final concentration of 50 ng / ml.
　＜軟骨分化の評価２＞
　実施例１４～１６に係る軟骨細胞について、大きさを測定し、軟骨分化がどの程度誘導されたかを評価した。その結果を、図８に示す。 <Evaluation of cartilage differentiation 2>
The size of the chondrocytes according to Examples 14 to 16 was measured to evaluate how much cartilage differentiation was induced. The result is shown in FIG.
　図８に示す結果から、ＩＬ－１βの量の増加にしたがって、軟骨細胞が大きかったことが確認された。この結果より、添加したＩＬ－１βの濃度が増すにしたがって、間葉系幹細胞の軟骨への分化誘導が促進することが示された。 From the results shown in FIG. 8, it was confirmed that the chondrocytes were larger as the amount of IL-1β increased. From these results, it was shown that differentiation induction of mesenchymal stem cells into cartilage is promoted as the concentration of added IL-1β increases.
　＜間葉系幹細胞の調製６＞
　（実施例１７）
　滑膜由来の間葉系幹細胞を用いて、１０％ＦＢＳを添加したαＭＥＭ培地を用い、３７℃で、継代を行わずに２週間、間葉系幹細胞の前培養を行った。その後、間葉系幹細胞の数を６０ｃｍ２のディッシュで１０００個／ディッシュに調節してからＩＬ－１βを、終濃度１．０ｎｇ／ｍｌとなるように添加し、３７℃で、２週間培養を行い、実施例１７に係る間葉系幹細胞を調製した。（ｎ＝３） <Preparation of mesenchymal stem cells 6>
(Example 17)
Using mesenchymal stem cells derived from synovium, the mesenchymal stem cells were precultured at 37 ° C. for 2 weeks without using subcultures using αMEM medium supplemented with 10% FBS. Thereafter, the number of mesenchymal stem cells was adjusted to 1000 cells / dish with a 60 cm 2 dish, IL-1β was added to a final concentration of 1.0 ng / ml, and cultured at 37 ° C. for 2 weeks. The mesenchymal stem cells according to Example 17 were prepared. (N = 3)
　（実施例１８）
　ＩＬ－１βの代わりにＩＬ－１αを用いた点と、１回継代を行い２週間間葉系幹細胞の前培養を行った点以外は、実施例１７と同様の条件で、実施例１８に係る間葉系幹細胞を調製した。（ｎ＝３） (Example 18)
Example 18 was carried out under the same conditions as in Example 17 except that IL-1α was used instead of IL-1β and that the mesenchymal stem cells were precultured for 2 weeks after being passaged once. Such mesenchymal stem cells were prepared. (N = 3)
　（対照例８）
　前培養後、ＩＬ－１βを添加しなかった点以外は、実施例１７と同様の条件で対照例８に係る間葉系幹細胞を調製した。 (Control 8)
After the preculture, mesenchymal stem cells according to Control Example 8 were prepared under the same conditions as in Example 17 except that IL-1β was not added.
　（対照例９）
　前培養後、ＩＬ－１βを添加しなかった点以外は、実施例１８と同様の条件で対照例９に係る間葉系幹細胞を調製した。 (Control 9)
After the preculture, mesenchymal stem cells according to Control Example 9 were prepared under the same conditions as in Example 18 except that IL-1β was not added.
　＜間葉系幹細胞の増殖の評価６＞
　実施例１７、１８、対照例８、９に係る間葉系幹細胞について、調製後にクリスタル・バイオレット染色を行った。実施例１７、対照例８についての結果を図９に、実施例１８、対照例９についての結果を図１０に示す。 <Evaluation of proliferation of mesenchymal stem cells 6>
The mesenchymal stem cells according to Examples 17 and 18 and Control Examples 8 and 9 were subjected to crystal violet staining after preparation. The results for Example 17 and Control Example 8 are shown in FIG. 9, and the results for Example 18 and Control Example 9 are shown in FIG.
　図９、図１０に示すように、ＩＬ－１βだけでなくＩＬ－１αによっても間葉系幹細胞の増加を促進できることがわかった。 9 and 10, it was found that not only IL-1β but also IL-1α can promote the increase of mesenchymal stem cells.
　＜間葉系幹細胞の調製７＞
　（実施例１９）
　６人の異なる被験者の滑膜由来の間葉系幹細胞（ｎ＝６）を用いて、１０％ＦＢＳを添加したαＭＥＭ培地を用い、３７℃で、１回継代を行い２週間、間葉系幹細胞の前培養を行った。その後、間葉系幹細胞の数を６０ｃｍ２のディッシュで１０４個／ディッシュに調節してからＴＮＦ－αを、終濃度２５ｎｇ／ｍｌとなるように添加し、３７℃で、２週間培養を行い、実施例１９に係る間葉系幹細胞を調製した。 <Preparation of mesenchymal stem cells 7>
(Example 19)
Using mesenchymal stem cells (n = 6) derived from synovial membranes of 6 different subjects and using αMEM medium supplemented with 10% FBS at 37 ° C. for 1 week, the mesenchymal system for 2 weeks. Stem cell pre-culture was performed. Thereafter, the number of mesenchymal stem cells was adjusted to 10 4 / dish with a 60 cm 2 dish, TNF-α was added to a final concentration of 25 ng / ml, and cultured at 37 ° C. for 2 weeks. A mesenchymal stem cell according to Example 19 was prepared.
　（対照例１０）
　前培養後、ＴＮＦ－αを添加しなかった点以外は、実施例１９と同様の条件で対照例９に係る間葉系幹細胞を調製した。 (Control 10)
After the preculture, mesenchymal stem cells according to Control Example 9 were prepared under the same conditions as in Example 19 except that TNF-α was not added.
　＜間葉系幹細胞の増殖の評価７＞
　実施例１９、対照例１０に係る間葉系幹細胞について、調製後にクリスタル・バイオレット染色を行った。その結果を、図１１に示す。また、実施例１９、対照例１０に係る間葉系幹細胞についての細胞の倍増した割合を示すグラフを、図１２に示す。 <Evaluation of proliferation of mesenchymal stem cells 7>
The mesenchymal stem cells according to Example 19 and Control Example 10 were subjected to crystal violet staining after preparation. The result is shown in FIG. Moreover, the graph which shows the cell doubled ratio about the mesenchymal stem cell which concerns on Example 19 and the control example 10 is shown in FIG.
　図１１に示すように、炎症性サイトカインであるＴＮＦ－αによっても、間葉系幹細胞が増加することがわかった。また、図１２に示すように、６人の被験者の間葉系幹細胞の全体において、実施例１９は対照例１０に対して有意に（＊　ｐ＜０．０５）間葉系幹細胞が増加することがわかった。 As shown in FIG. 11, it was found that TNF-α, an inflammatory cytokine, also increased mesenchymal stem cells. In addition, as shown in FIG. 12, in the whole mesenchymal stem cells of 6 subjects, Example 19 significantly (* p <0.05) increased mesenchymal stem cells compared to Control Example 10. I understood.
　＜軟骨細胞の調製３＞
　（実施例２０）
　滑膜由来の間葉系幹細胞を用いて、１０％ＦＢＳ添加αＭＥＭ培地を用い、３７℃で、１回継代を行い２週間間葉系幹細胞の前培養を行った。その後、間葉系幹細胞の数を６０ｃｍ２のディッシュで１０４個／ディッシュとなるように調節してから、ＩＬ－１βが終濃度２５ｎｇ／ｍｌとなるように添加し、３７℃で１４日間培養を行った。その後、後述の対照例１１と細胞数を合わせてペレットを作製し、軟骨分化培地（ＤＭＥＭに１０００ｎｇ／ｍｌ　ＢＭＰ７，１０ｎｇ／ｍｌ　ＴＧＦ－ｂｅｔａ，　１０－７Ｍ　デキサメタゾンを添加したもの）を用いて２１日間培養し、実施例２０に係る軟骨細胞を調製した。 <Preparation 3 of chondrocytes>
(Example 20)
Using mesenchymal stem cells derived from synovium, the cells were subcultured once at 37 ° C. using an αMEM medium supplemented with 10% FBS, and the mesenchymal stem cells were precultured for 2 weeks. Thereafter, the number of mesenchymal stem cells was adjusted to 10 4 / dish in a 60 cm 2 dish, IL-1β was added to a final concentration of 25 ng / ml, and cultured at 37 ° C. for 14 days. Went. Thereafter, a pellet was prepared by combining the number of cells with Control Example 11 described later, and using a cartilage differentiation medium (DMEM supplemented with 1000 ng / ml BMP7, 10 ng / ml TGF-beta, 10 −7 M dexamethasone). Cultured for a day, chondrocytes according to Example 20 were prepared.
　（対照例１１）
　前培養後、ＴＮＦ－αを添加しなかった点以外は、実施例２０と同様の条件で対照例１１に係る軟骨細胞を調製した。 (Control 11)
After pre-culture, chondrocytes according to Control Example 11 were prepared under the same conditions as in Example 20 except that TNF-α was not added.
　＜軟骨分化の評価３＞
　実施例２０及び対照例１１に係る軟骨細胞について、大きさを測定し、軟骨分化がどの程度誘導されたかを評価した。その結果を、図１３に示す。図１３中の１目盛は、１ｍｍを示す。 <Evaluation of cartilage differentiation 3>
About the chondrocytes according to Example 20 and Control Example 11, the size was measured, and how much cartilage differentiation was induced was evaluated. The result is shown in FIG. One scale in FIG. 13 indicates 1 mm.
　図１３に示す結果から、ＴＮＦ－αを添加することで、間葉系幹細胞の軟骨への分化誘導が促進することが示された。 The results shown in FIG. 13 indicate that the addition of TNF-α promotes differentiation induction of mesenchymal stem cells into cartilage.
　また、ＴＮＦ－αを添加した滑膜由来の間葉系幹細胞について脂肪への分化能を評価したところ、軟骨分化能と同様に、培地にＴＮＦ－αを添加しなかったものよりＴＮＦ－αを添加したものの方が、脂肪への分化能が高かった。この結果より、ＴＮＦ－αは、間葉系幹細胞の脂肪への分化を促進することもわかった。 In addition, when the synovial stem cells added with TNF-α were evaluated for their ability to differentiate into fat, as with the cartilage differentiation ability, TNF-α was added more than the medium without TNF-α added. The added one had higher ability to differentiate into fat. From this result, it was also found that TNF-α promotes differentiation of mesenchymal stem cells into fat.

Claims (19)

  1. 　ＩＬ－１および／またはＴＮＦ－αを含む培養培地において滑膜由来の間葉系幹細胞を培養する工程を含む、間葉系幹細胞の調製方法。 A method for preparing mesenchymal stem cells, comprising culturing synovial mesenchymal stem cells in a culture medium containing IL-1 and / or TNF-α.
  2. 　前記ＩＬ－１がＩＬ－１βである、請求項１に記載の間葉系幹細胞の調製方法。 The method for preparing mesenchymal stem cells according to claim 1, wherein the IL-1 is IL-1β.
  3. 　間葉系幹細胞用培養培地に、インターロイキンを添加し、添加後の前記間葉系幹細胞用培養培地において、間葉系幹細胞を培養する工程を有する、間葉系幹細胞の調製方法。 A method for preparing mesenchymal stem cells, comprising a step of adding interleukin to a culture medium for mesenchymal stem cells and culturing the mesenchymal stem cells in the culture medium for mesenchymal stem cells after the addition.
  4. 　前記インターロイキンは、ＩＬ－１を含む、請求項３に記載の間葉系幹細胞の調製方法。 The method for preparing mesenchymal stem cells according to claim 3, wherein the interleukin contains IL-1.
  5. 　間葉系幹細胞用培養培地に、炎症性サイトカインを添加し、添加後の前記間葉系幹細胞用培養培地において、間葉系幹細胞を培養する工程を有する、間葉系幹細胞の調製方法。 A method for preparing mesenchymal stem cells, comprising the step of adding inflammatory cytokines to a culture medium for mesenchymal stem cells and culturing mesenchymal stem cells in the culture medium for mesenchymal stem cells after the addition.
  6. 　前記炎症性サイトカインは、ＩＬ－１及びＴＮＦ－αからなる群から選択される１種以上を含む、請求項５に記載の間葉系幹細胞の調製方法。 The method for preparing mesenchymal stem cells according to claim 5, wherein the inflammatory cytokine comprises one or more selected from the group consisting of IL-1 and TNF-α.
  7. 　前記間葉系幹細胞は、滑膜由来又は脂肪由来の間葉系幹細胞である、請求項３から６のいずれかに記載の間葉系幹細胞の調製方法。 The method for preparing a mesenchymal stem cell according to any one of claims 3 to 6, wherein the mesenchymal stem cell is a synovial or fat-derived mesenchymal stem cell.
  8. 　前記間葉系幹細胞用培養培地が、無血清培地である、請求項１から７のいずれかに記載の間葉系幹細胞の調製方法。 The method for preparing mesenchymal stem cells according to any one of claims 1 to 7, wherein the culture medium for mesenchymal stem cells is a serum-free medium.
  9. 　細胞増殖因子を培地中に添加する工程を更に含有する、請求項１から８のいずれかに記載の間葉系幹細胞の調製方法。 The method for preparing a mesenchymal stem cell according to any one of claims 1 to 8, further comprising a step of adding a cell growth factor to the medium.
  10. 　間葉系幹細胞とインターロイキンおよび／または炎症性サイトカインとをｅｘ　ｖｉｖｏで接触させる工程と、
    　間葉系幹細胞の軟骨への分化を誘導する工程と、を有する、軟骨細胞の調製方法。     Contacting mesenchymal stem cells with interleukins and / or inflammatory cytokines ex vivo;
    And a step of inducing differentiation of mesenchymal stem cells into cartilage.
  11. 　インターロイキンからなる、間葉系幹細胞の増殖促進剤。 A mesenchymal stem cell growth promoter consisting of interleukins.
  12. 　インターロイキンからなる、間葉系幹細胞の軟骨分化促進剤。 An agent for promoting cartilage differentiation of mesenchymal stem cells, comprising interleukin.
  13. 　インターロイキン被添加用である、間葉系幹細胞用培養培地。 A culture medium for mesenchymal stem cells that is supplemented with interleukin.
  14. 　炎症性サイトカインからなる、間葉系幹細胞の増殖促進剤。 A mesenchymal stem cell proliferation promoter composed of inflammatory cytokines.
  15. 　炎症性サイトカインからなる、間葉系幹細胞の軟骨分化促進剤。 A mesenchymal stem cell cartilage differentiation promoter consisting of inflammatory cytokines.
  16. 　インターロイキンおよび／または炎症性サイトカインを含有することを特徴とする、間葉系幹細胞用培養培地。 A culture medium for mesenchymal stem cells, comprising interleukin and / or inflammatory cytokine.
  17. 　前記インターロイキンがＩＬ－１βである、請求項１６に記載の間葉系幹細胞用培養培地。 The culture medium for mesenchymal stem cells according to claim 16, wherein the interleukin is IL-1β.
  18. 　前記炎症性サイトカインがＴＮＦ－αである、請求項１６に記載の間葉系幹細胞用培養培地。 The culture medium for mesenchymal stem cells according to claim 16, wherein the inflammatory cytokine is TNF-α.
  19. 　前記間葉系幹細胞が滑膜由来の間葉系幹細胞である、請求項１６から１８のいずれかに記載の間葉系幹細胞用培養培地。 The culture medium for mesenchymal stem cells according to any one of claims 16 to 18, wherein the mesenchymal stem cells are synovial stem cells.
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