WO2016125582A1 - Agent destiné à promouvoir la prolifération des cellules souches mésenchymateuses et leur différenciation en cartilage, procédé de préparation de cellules souches mésenchymateuses, procédé de préparation de chondrocytes et milieu de culture pour cellules souches mésenchymateuses - Google Patents

Agent destiné à promouvoir la prolifération des cellules souches mésenchymateuses et leur différenciation en cartilage, procédé de préparation de cellules souches mésenchymateuses, procédé de préparation de chondrocytes et milieu de culture pour cellules souches mésenchymateuses 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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English (en)
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

A titre d'alternatives aux procédés classiques de préparation de cellules souches mésenchymateuses et de préparation de chondrocytes à partir de cellules souches mésenchymateuses, l'invention porte sur un nouveau procédé de préparation de cellules souches mésenchymateuses qui fait preuve d'une excellente efficacité en termes de promotion de la prolifération des cellules souches mésenchymateuses, un procédé de préparation de chondrocytes qui fait preuve d'une excellente efficacité en termes de promotion de la différenciation des cellules souches mésenchymateuses en chondrocytes, un agent destiné à promouvoir la prolifération des cellules souches mésenchymateuses, un agent destiné à promouvoir la différenciation des cellules souches mésenchymateuses en cartilage, et un milieu de culture pour cellules souches mésenchymateuses qui peut être utilisé pour la préparation desdites cellules souches mésenchymateuses. Le procédé de préparation de cellules souches mésenchymateuses selon l'invention comprend une étape qui consiste à compléter le milieu de culture pour cellules souches mésenchymateuses avec de l'interleukine, et à cultiver les cellules souches mésenchymateuses dans ce milieu de culture pour cellules souches mésenchymateuses complété avec de l'interleukine. Un procédé de préparation de chondrocytes qui comprend une étape consistant à mettre les cellules souches mésenchymateuses et l'interleukine en contact, et une étape consistant à induire la différenciation des cellules souches mésenchymateuses en cartilage est en outre décrit.
PCT/JP2016/051447 2015-02-05 2016-01-19 Agent destiné à promouvoir la prolifération des cellules souches mésenchymateuses et leur différenciation en cartilage, procédé de préparation de cellules souches mésenchymateuses, procédé de préparation de chondrocytes et milieu de culture pour cellules souches mésenchymateuses WO2016125582A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106987556A (zh) * 2017-03-31 2017-07-28 四川新生命干细胞科技股份有限公司 一种增强脐带间充质干细胞成骨和成软骨分化特性的培养基及培养方法
WO2017150371A1 (fr) * 2016-02-29 2017-09-08 国立大学法人大阪大学 Agent thérapeutique pour lésion tissulaire
JP2019531281A (ja) * 2016-09-07 2019-10-31 エスシーエム ライフサイエンス カンパニー リミテッド 炎症刺激された間葉系幹細胞を含む免疫疾患または炎症疾患の予防または治療用薬学的組成物
WO2019240296A1 (fr) * 2018-06-15 2019-12-19 有限会社大阪空気機械サービス Agent thérapeutique tissulaire
CN113832099A (zh) * 2021-10-13 2021-12-24 浙江领蔚生物技术有限公司 用于制备治疗类风湿性关节炎药物的间充质干细胞制剂

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ANDO, W. ET AL.: "Ovine synovial membrane- derived mesenchymal progenitor cells retain the phenotype of the original tissue that was exposed to in-vivo inflammation: evidence for a suppressed chondrogenic differentiation potential of the cells.", INFLAMM. RES., vol. 61, no. 6, June 2012 (2012-06-01), pages 599 - 608, ISSN: 1023-3830 *
KAZUNORI SHIMOMURA ET AL.: "Hito Katsumaku Yurai Kansaibo o Mochiita Sanjigen Baiyo Saibo eno Rikigaku Shigeki to IL -1beta Shigeki: Saibo Keitai, Matrix Bunkai Koso Idenshi Hatsugen no Kento", THE JOURNAL OF THE JAPANESE ORTHOPAEDIC ASSOCIATION, vol. 83, no. 8, August 2009 (2009-08-01), pages S1130, ISSN: 0021-5325 *
LI, D. ET AL.: "Biological characteristics of human placental mesenchymal stem cells and their proliferative response to various cytokines.", CELLS TISSUES ORGANS., vol. 186, no. 3, July 2007 (2007-07-01), pages 169 - 179, ISSN: 1422-6405 *
LIU, X. ET AL.: "Rescue of proinflammatory cytokine-inhibited chondrogenesis by the antiarthritic effect of melatonin in synovium mesenchymal stem cells via suppression of reactive oxygen species and matrix metalloproteinases.", FREE RADIC. BIOL. MED., vol. 68, March 2014 (2014-03-01), pages 234 - 246, ISSN: 0891-5849 *
WARNAWIN, E. ET AL.: "Preservation of chondrogenic potential of mesenchymal stem cells isolated from osteoarthritic patients during proliferation in response to platelet- derived growth factor (PDGF).", CENT. EUR. J. IMMUNOL., vol. 30, no. 1-2, 2005, pages 26 - 31, ISSN: 1426-3912 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017150371A1 (fr) * 2016-02-29 2017-09-08 国立大学法人大阪大学 Agent thérapeutique pour lésion tissulaire
JP2019531281A (ja) * 2016-09-07 2019-10-31 エスシーエム ライフサイエンス カンパニー リミテッド 炎症刺激された間葉系幹細胞を含む免疫疾患または炎症疾患の予防または治療用薬学的組成物
JP7212371B2 (ja) 2016-09-07 2023-01-25 エスシーエム ライフサイエンス カンパニー リミテッド 炎症刺激された間葉系幹細胞を含む免疫疾患または炎症疾患の予防または治療用薬学的組成物
CN106987556A (zh) * 2017-03-31 2017-07-28 四川新生命干细胞科技股份有限公司 一种增强脐带间充质干细胞成骨和成软骨分化特性的培养基及培养方法
WO2019240296A1 (fr) * 2018-06-15 2019-12-19 有限会社大阪空気機械サービス Agent thérapeutique tissulaire
JP2019218271A (ja) * 2018-06-15 2019-12-26 有限会社大阪空気機械サービス 組織治癒剤
CN113832099A (zh) * 2021-10-13 2021-12-24 浙江领蔚生物技术有限公司 用于制备治疗类风湿性关节炎药物的间充质干细胞制剂

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