WO2011111787A1 - 間葉系幹細胞を含む細胞製剤及びその製造方法 - Google Patents
間葉系幹細胞を含む細胞製剤及びその製造方法 Download PDFInfo
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- C12N2501/15—Transforming growth factor beta (TGF-β)
Definitions
- the present invention relates to a cell preparation containing mesenchymal stem cells and a production method thereof, and further to a culture medium additive, a culture medium and a kit used in the production method, and a culture method using these.
- Mesenchymal stem cells are isolated not only from bone marrow, fat, synovium, alveolar bone, periodontal ligament and other adult tissues but also from placenta, umbilical cord blood, umbilical cord cells, etc. And can be cultured and amplified in vitro. Furthermore, mesenchymal stem cells can be differentiated not only into multiple mesenchymal cells (osteoblasts, adipocytes, chondrocytes) but also into cells of non-mesenchymal (neural progenitor cells, hepatocytes) lineage. It is expected to be used as a cell source for regenerative medicine and cell therapy.
- MSC Mesenchymal Stem Cell
- FBS fetal bovine serum
- Patent Document 1 and Non-Patent Document 1 describe serum-free culture of mesenchymal stem cells. As described in Patent Document 1 and Non-Patent Document 1, by performing serum-free culture of mesenchymal stem cells, a growth promoting effect superior to that of culturing mesenchymal stem cells in a medium containing 5 to 15% FBS can be obtained. This culture yields mesenchymal stem cells that maintain (promote) pluripotency.
- Non-Patent Documents 2 to 5 mesenchymal stem cells have been shown not only to have low immunogenicity but also affect the function of various immune effector cells (T cells, B cells, NK cells, dendritic cells). Therefore, it is expected that mesenchymal stem cells are applied to the treatment of various diseases involving immune responses (Non-patent Document 6).
- Non-Patent Document 7 describes that mesenchymal stem cells suppress the proliferation of T cells by mouse lymphocyte mixed reaction (MLR).
- MLR mouse lymphocyte mixed reaction
- mesenchymal stem cells having immunosuppressive ability can be obtained by serum-free or low-serum culture with a low risk of contamination with a heterologous protein or synthetic medium.
- the FBS-containing medium is problematic not only from the amount of bovine serum albumin but also from other soluble substances having allergenicity, and the use of a culture medium not containing this is indispensable.
- the present invention has been made in view of the above problems, and its object is to provide a production method for producing a cell preparation containing mesenchymal stem cells having maintained immunosuppressive ability by serum-free or low-serum culture. It is to provide.
- the present inventors diligently studied the influence of serum-free culture on the immunosuppressive ability of mesenchymal stem cells, and as a result, mesenchyme cultured in a serum-free medium containing a specific additive.
- the present inventors have found that the stem cells maintain immunosuppressive ability and that the immunosuppressive effect is improved, and have completed the present invention.
- the method for producing a cell preparation containing mesenchymal stem cells comprises FGF, PDGF, TGF- ⁇ , HGF, EGF, at least one phospholipid, and at least one fatty acid in serum-free medium A. And a proliferating step for proliferating mesenchymal stem cells, and a screening step for screening mesenchymal stem cells whose immunosuppressive ability is maintained or improved from mesenchymal stem cells after the proliferating step.
- the cell preparation containing mesenchymal stem cells according to the present invention is characterized by being produced by the production method described above.
- the medium additive according to the present invention is a serum-free medium additive for producing a cell preparation containing mesenchymal stem cells whose immunosuppressive ability is maintained or improved, and includes FGF, PDGF, TGF- ⁇ , It is characterized by containing HGF, EGF, at least one phospholipid, and at least one fatty acid.
- the culture medium according to the present invention is a serum-free culture medium for producing a cell preparation containing mesenchymal stem cells whose immunosuppressive ability is maintained or improved, and contains the above-mentioned medium additive It is a feature.
- the culture method according to the present invention is a culture method for producing a cell preparation containing mesenchymal stem cells whose immunosuppressive ability is maintained or improved, and includes a step of culturing mesenchymal stem cells in the culture medium. It is characterized by that.
- the kit according to the present invention is a kit for producing a cell preparation containing mesenchymal stem cells whose immunosuppressive ability is maintained or improved, and is characterized by comprising at least the above-mentioned medium additive.
- the method for producing a cell preparation containing mesenchymal stem cells includes a method for producing a cell preparation in serum-free medium A containing FGF, PDGF, TGF- ⁇ , HGF, EGF, at least one phospholipid, and at least one fatty acid. Since it includes a proliferation step for growing mesenchymal stem cells, and a screening step for screening mesenchymal stem cells whose immunosuppressive ability is maintained or improved from mesenchymal stem cells after the above proliferation step, serum-free or low It is possible to produce a cell preparation containing mesenchymal stem cells whose immunosuppressive ability is maintained or improved by serum culture.
- Patent Document 1 and Non-Patent Document 1 describe a medium for culturing cells without losing growth in a serum-free medium (serum-free medium).
- This medium is a medium in which a complex of a specific growth factor group, phospholipid and fatty acid is added to the basal medium. By using this medium, even under serum-free conditions, it is equivalent to or better than 10% serum. It leads to the effect of promoting cell proliferation. Further, in this medium, mesenchymal stem cells can be cultured without serum while maintaining the differentiation ability and enhancing the maintenance ability. Further, in this medium, serum-free culture of bone marrow-derived MSC, fat-derived MSC, and synovium-derived MSC is possible.
- the present invention provides a method for producing a cell preparation containing mesenchymal stem cells whose immunosuppressive ability is maintained or improved under serum-free conditions.
- the method for producing a cell preparation according to the present invention allows mesenchymal stem cells to grow in serum-free medium A containing FGF, PDGF, TGF- ⁇ , HGF, EGF, at least one phospholipid, and at least one fatty acid. It includes a proliferation step and a screening step for screening mesenchymal stem cells having maintained or improved immunosuppressive ability from mesenchymal stem cells after the proliferation step.
- mesenchymal stem cells are cultured in serum-free medium A containing FGF, PDGF, TGF- ⁇ , HGF, EGF, at least one phospholipid, and at least one fatty acid. And expand mesenchymal stem cells.
- serum-free medium is intended to be a serum-free medium
- serum-free culture is intended to be a serum-free culture.
- low serum culture is a culture using a medium containing less serum than a general serum-containing medium (for example, a medium containing 10% FBS) and a general serum-containing medium. It is intended that the culture period using the serum-containing medium is shorter than the culture.
- the “cell preparation” is a therapeutic agent formulated into a cell, which is used for regenerative medicine as a material for regenerative medicine, and the cell is formulated without changing its function in its original state. It includes not only those but also those obtained by formulating cells whose functions such as differentiation ability and immunosuppressive ability are improved by culturing and proliferating under specific conditions.
- mesenchymal stem cells are not only derived from adult tissues such as bone marrow, adipocytes, synovial cells, alveolar bone, periodontal ligament, but also various cells of placenta, umbilical cord blood, fetus Although it is preferable that it is a human mesenchymal stem cell including those isolated from humans, it may be a mesenchymal stem cell derived from a non-human animal such as a rat or a mouse.
- the basal medium for constituting the serum-free medium A used in the growth step is not particularly limited as long as it is a well-known animal cell medium in the art.
- Preferred basal media include, for example, Ham's F12 medium and DMEM medium. RPMI-1640 medium, MCDB medium, and the like. These basal media may be used alone or in combination.
- the basal medium for constituting the serum-free medium A is preferably a medium in which MCDB and DMEM are mixed at a ratio of 1: 1.
- serum-free medium A in which FGF, PDGF, TGF- ⁇ , HGF, EGF, at least one phospholipid, and at least one fatty acid are added to the basal medium may be used in the growth step.
- the content of FGF with respect to the basal medium is preferably 0.1 to 100 ng / ml, more preferably 3 ng / ml at the final concentration.
- the PDGF content in the basal medium is preferably 0.5 to 100 ng / ml, more preferably 10 ng / ml, at the final concentration.
- the content of TGF- ⁇ with respect to the basal medium is preferably 0.5 to 100 ng / ml, more preferably 10 ng / ml, at the final concentration.
- the content of HGF with respect to the basal medium is preferably 0.1 to 50 ng / ml, more preferably 5 ng / ml, at the final concentration.
- the EGF content in the basal medium is preferably 0.5 to 200 ng / ml, more preferably 20 ng / ml, at the final concentration.
- the total content of phospholipids with respect to the basal medium is preferably 0.1 to 30 ⁇ g / ml, more preferably 10 ⁇ g / ml in the final concentration.
- the total content of fatty acids in the basal medium is preferably 1/1000 to 1/10 of the basal medium, more preferably 1/100.
- examples of the phospholipid contained in the serum-free medium A include phosphatidic acid, lysophosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphati Examples thereof include dilethanolamine, phosphatidylcholine, and phosphatidylglycerol, and these phospholipids may be contained alone or in combination.
- the serum-free medium A may contain a combination of phosphatidic acid and phosphatidylcholine, and these phospholipids may be derived from animals or plants. Good.
- the fatty acid contained in the serum-free medium A examples include linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, stearic acid, and the like.
- the additive may contain these fatty acids alone or in combination.
- the serum-free medium A according to the present embodiment may further contain cholesterol in addition to the fatty acid.
- FGF is intended to be a growth factor selected from the fibroblast growth factor (FGF) family and is preferably FGF-2 (bFGF). May be selected from other FGF families such as -1.
- FGF fibroblast growth factor
- PDGF is intended to be a growth factor selected from the platelet derived growth factor (PDGF) family, and is preferably PDGF-BB or PDGF-AB.
- TGF- ⁇ is a growth factor selected from the transforming growth factor- ⁇ (TGF- ⁇ ) family, and is TGF- ⁇ 3. Preferably, it may be selected from other TGF- ⁇ families.
- HGF is intended to be a growth factor selected from the hepatocyte growth factor family
- EGF is selected from the epidermal growth factor (EGF) family. Growth factors are contemplated.
- the serum-free medium A is at least selected from the group consisting of a connective tissue growth factor (CTGF), a vascular endothelial growth factor (VEGF), and an ascorbic acid compound. Two factors may further be contained.
- CTGF connective tissue growth factor
- VEGF vascular endothelial growth factor
- ascorbic acid compound Two factors may further be contained.
- ascorbic acid compound is intended to be ascorbic acid (vitamin C) or ascorbic acid diphosphate, or a similar compound.
- the above-mentioned growth factors contained in the serum-free medium A may be natural or may be produced by genetic recombination.
- the serum-free medium A preferably contains a lipid antioxidant.
- the lipid antioxidant contained in serum-free medium A can be DL- ⁇ -tocopherol acetate (vitamin E).
- the serum-free medium A may further contain a surfactant.
- the surfactant contained in serum-free medium A can be Pluronic F-68 or Tween 80.
- Serum-free medium A may further contain insulin, transferrin and selenate.
- insulin may be an insulin-like growth factor and may be derived from natural cells or produced by genetic recombination.
- the culture medium additive according to the present invention may further contain dexamethasone or other glucocorticoids.
- the above-described serum-free medium A is seeded with mesenchymal stem cells isolated from animal tissues or cells such as humans by a conventionally known method and cultured until they grow to a desired number.
- mesenchymal stem cells isolated from animal tissues or cells such as humans by a conventionally known method and cultured until they grow to a desired number.
- 1 to 2 ⁇ 10 4 mesenchymal stem cells are preferably seeded per 1 ml of medium
- the culture temperature is 37 ° C. ⁇ 1 ° C.
- the culture time is 48 to 96 hours
- mesenchymal stem cells having maintained or improved immunosuppressive ability can be obtained efficiently and in large quantities.
- the culture vessel used for the culture is not particularly limited as long as the mesenchymal stem cells can grow.
- the mesenchymal stem cells can grow.
- cell proliferation may be affected by the type of culture vessel used.
- a culture vessel suitable for proliferation is used for each mesenchymal stem cell to be proliferated in the proliferation process (hereinafter also referred to as “proliferation target cell”). It is preferable to perform the proliferation step.
- Examples of a method for selecting a culture vessel suitable for the growth of cells to be proliferated include a method for causing the cells to be proliferated to select an optimal culture vessel. Specifically, multiple types of culture containers are prepared, the cells to be proliferated are grown under the same culture conditions except that the types of culture containers are different, and the number of cells after 2 weeks from the start of the culture is measured by a known method. And it can be judged that it is a culture container suitable for the proliferation of a proliferation object cell in an order from a thing with many cells.
- the cells to be proliferated are proliferated in order from the shortest period in which the number of cells reaching 80 to 90% of the confluent state is reached even before two weeks have passed since the start of the culture. It can be determined that the culture vessel is suitable.
- the culture vessel may be used.
- the manufacturing method according to the present invention is for selecting a culture container suitable for the proliferation of cells to be proliferated.
- a “culture vessel selection step” (described later) may be further included before the growth step.
- cell adhesion molecule examples include fibronectin, collagen, gelatin and the like. One type of these cell adhesion molecules may be used alone, or a plurality of types may be used in combination.
- the content of the cell adhesion molecule with respect to the serum-free medium A is preferably 1 to 50 ⁇ g / ml, more preferably 5 ⁇ g / ml in the final concentration.
- the adhesion efficiency of the cells to be proliferated to the culture vessel can be improved by adding the fibronectin to the serum-free medium A so that the final concentration is 5 ⁇ g / ml. it can.
- mesenchymal stem cells may be passaged at least once. Since mesenchymal stem cells proliferate in an anchorage-dependent manner, when the mesenchymal stem cells are locally biased and proliferated, the culture conditions can be changed by substituting the mesenchymal stem cells during the growth process. Can be improved.
- the method for subculturing mesenchymal stem cells is not particularly limited, and it can be performed using a conventionally known method for subculturing mesenchymal stem cells. Since the state of mesenchymal stem cells after passage is good, in the above proliferation step, when the passage is performed, the above mesenchymal stem cells are used using a cell exfoliant that does not contain a component derived from mammals or microorganisms. Is preferably peeled off. Examples of the above-mentioned “cell detachment agent that does not contain components derived from mammals and microorganisms” include ACCUTASE (Innovative Cell Technologies, Inc.).
- mesenchymal stem cells after the first passage (P1) after collection from animal tissues such as humans for the proliferation step.
- mesenchymal stem cells having maintained or improved immunosuppressive ability are screened from the mesenchymal stem cells after the above-described proliferation step.
- the mesenchymal stem cells grown in the above-described serum-free medium A after the growth step are maintained at least in the immunosuppressive ability, and the immunosuppressive ability is further enhanced. Therefore, by screening such mesenchymal stem cells on the basis of immunosuppressive ability, mesenchymal stem cells having maintained or improved immunosuppressive ability can be selected.
- the above-mentioned serum-free medium A does not contain a heterologous protein, and mesenchymal stem cells cultured in the medium maintain the function as stem cells.
- the mesenchymal stem cells cultured in the above-described serum-free medium A have a high proliferative ability and a high activity showing an immunosuppressive effect as compared with mesenchymal stem cells cultured in a fetal bovine serum-containing medium. Therefore, when the mesenchymal stem cells cultured in the above-described serum-free medium A are used for transplantation treatment, synergistic effects can be expected because the number of cells that retain the characteristics increases and the activity of individual cells is high.
- the inventors of the present invention have investigated the tumorigenicity that is concerned from the effect of the growth ability of the above-mentioned serum-free medium A using the in vitro vitro agar culture method and the insensitive in vivo immunodeficient mouse (NOG mouse). Each was examined by a neoplastic test method. Specifically, 3 lots of synovial membrane-derived human mesenchymal stem cells and 1 lot of bone marrow-derived human mesenchymal stem cells were cultured in the above-described serum-free medium A to obtain 1,000,000 cultured cells. Were transplanted into the medium for soft agar culture and 10 subcutaneous sites of NOG mice.
- immunosuppressive ability refers to the ability to suppress immune rejection that occurs when allogeneic or allogeneic cells are transplanted to various immunity, such as suppressing the proliferation of T cells. It is intended to have the ability to suppress immune responses caused by these by affecting the function of effector cells. This is part of the anti-inflammatory ability.
- “maintaining immunosuppressive ability” means that the immunosuppressive ability originally possessed by mesenchymal stem cells is not lost by the above-described proliferation step, and “improves immunosuppressive ability” "Is intended to improve the immunosuppressive ability of mesenchymal stem cells before and after the above-described proliferation step.
- the immunosuppressive ability is obtained by co-culturing the proliferated mesenchymal stem cells and immune cells in the serum-free medium A after the above proliferation step, and evaluating the number of immune cells in the medium after the co-culture.
- the present invention is not limited to this.
- T cells were used as immune cells, as shown in Examples described later, the amount of T cells increased or the amount of cytokine production was evaluated, and B cells or activated NK cells were used as immune cells.
- mesenchymal stem cells having maintained or improved immunosuppressive ability can be screened by evaluating the increased amount of these cells.
- dendritic cells when used as immune cells, mesenchymal stem cells having maintained or improved immunosuppressive ability can be screened by evaluating their differentiation, maturation, activation and the like.
- the immune cells used for such screening are preferably T cells, B cells, or activated NK cells, more preferably T cells or B cells, and most preferably T cells.
- the production method according to the present invention may further include a second screening step of screening mesenchymal stem cells not having tumorigenicity from mesenchymal stem cells after the above-described proliferation step.
- the mesenchymal stem cells that have grown in the above-described serum-free medium A after the growth step are selectively increased in mesenchymal stem cells that do not have tumorigenicity. Therefore, mesenchymal stem cells that do not have tumorigenicity can be selected by screening such mesenchymal stem cells on the basis of tumorigenicity. And the transplantation treatment of the mesenchymal stem cell which does not have tumorigenicity is implement
- a mesenchymal stem cell that does not have tumorigenicity means that while the above-described method for confirming the presence or absence of tumorigenicity is confirmed to have no tumorigenicity. Intended for leaf stem cells.
- the order of performing the first screening step and the second screening step is not particularly limited. That is, the first screening step may be performed before the second screening step, or the first screening step may be performed after the second screening step.
- the production method according to the present invention may further include a serum culture step of culturing mesenchymal stem cells after the proliferation step in a serum-containing medium before the screening step.
- a serum culture step of culturing mesenchymal stem cells after the proliferation step in a serum-containing medium before the screening step.
- the mesenchymal stem cells cultured without serum in the proliferation step are cultured in a serum-containing medium.
- the mesenchymal stem cells after the serum culture process are subjected to a screening process.
- the serum-containing medium used in the serum culture step a conventionally known serum-containing medium can be used, and a 10% FBS-containing medium obtained by adding 10% FBS to the above-described basal medium is used as the serum-containing medium. May be.
- the mesenchymal stem cells obtained in the growth step are seeded and cultured in such a serum-containing medium.
- the number of mesenchymal stem cells is preferably 1 to 2 ⁇ 10 4 per 1 ml of medium
- the culture temperature is 37 ° C. ⁇ 1 ° C.
- the culture time is 48 to 96 hours
- 5% CO 2. 2 is preferable.
- mesenchymal stem cells cultured in the serum-containing medium are subjected to a screening step, and mesenchymal stem cells whose immunosuppressive ability is maintained or improved are selected.
- mesenchymal stem cells are pre-cultured in serum-free medium A and then main-cultured in a serum-containing medium, so that both the pre-culture and the main culture have immunosuppressive effects equivalent to or higher than those cultured in a serum-containing medium.
- the immunosuppressive effect of mesenchymal stem cells can be exerted more quickly.
- serum since serum is used only during main culture, the serum content is low and low serum culture can be realized.
- the production method according to the present invention is a pre-growth method for proliferating mesenchymal stem cells in a serum-free medium B containing FGF, PDGF, EGF, at least one phospholipid, and at least one fatty acid before the proliferation step.
- a process may be further performed.
- the “serum-free medium B” is different from the serum-free medium A described in the “Proliferation step” above in that it does not contain HGF and TGF- ⁇ .
- the components other than HGF and TGF- ⁇ (FGF, PDGF, EGF, at least one phospholipid, and at least one fatty acid) and the basal medium are as described for the serum-free medium A in the “Proliferation step” above. Since there is, explanation is omitted here.
- the serum-free medium B preferably contains a lipid antioxidant, like the serum-free medium A.
- the serum-free medium B may further contain a surfactant.
- the serum-free medium B may further contain insulin, transferrin, and selenate.
- the serum-free medium B may further contain dexamethasone or other glucocorticoids. Since these components are also as described for the serum-free medium A in the above-mentioned “Proliferation step”, the description thereof is omitted here.
- the content of the above-mentioned components contained in the serum-free medium B is contained in the serum-free medium A as long as it is within the content range described for the serum-free medium A in the above “Proliferation step”.
- the content of each component may be the same or different.
- the above-described serum-free medium B is seeded with mesenchymal stem cells isolated from animal tissues such as humans by a conventionally known method, and cultured until they grow to a desired number.
- mesenchymal stem cells isolated from animal tissues such as humans by a conventionally known method
- the culture temperature is 37 ° C. ⁇ 1 ° C.
- the culture time is 3 to 14 day between, and is preferably below 5% CO 2.
- the mesenchymal stem cells subjected to the pre-proliferation step there is no particular limitation on the mesenchymal stem cells subjected to the pre-proliferation step, but the initial mesenchymal stem cells, that is, cells that have not been subcultured after being collected from animal tissues such as humans. preferable. As shown in the examples described later, the number of mesenchymal stem cells obtained in the proliferation step is significantly increased by proliferating the initial mesenchymal stem cells in serum-free medium B before being subjected to the proliferation step. It becomes possible.
- the method for culturing mesenchymal stem cells in the pre-proliferation step is, for example, seeding mesenchymal stem cells in a serum-free medium B at a seeding concentration of 2 ⁇ 10 5 cells / cm 2 , and then one week To the extent, 10% of the serum-free medium B of the culture medium at the time of seeding is added every 2 days, and the cells are grown until the number of cells reaches 70-80% of the confluent state.
- the mesenchymal stem cells previously cultured in the serum-free medium B for the proliferation step it is possible to efficiently obtain a large amount of mesenchymal stem cells whose immunosuppressive ability is maintained or improved.
- each mesenchymal stem cell to be proliferated in the pre-proliferation step (hereinafter also referred to as “pre-proliferation target cell”) is suitable for proliferation. It is preferable to perform the pre-growth step using the cultured vessel.
- the method for selecting a culture vessel suitable for the growth of the pre-proliferation target cells is as described in the above-mentioned “Proliferation step”, and thus the description thereof is omitted here.
- the serum-free medium B may further contain a cell adhesion molecule.
- the cell adhesion molecule is the same as described in the above-mentioned “Proliferation step”, and the description thereof is omitted here.
- the mesenchymal stem cells may be passaged at least once in the pre-growth step.
- the culture conditions can be improved by passage of mesenchymal stem cells in the middle of the pre-growth step.
- the pre-growth step is preferably performed during the period from the primary culture (P0) to the third passage (P3).
- the method for subculturing mesenchymal stem cells in the middle of the pre-proliferation step and the method for substituting cells after the pre-proliferation step for the proliferation step are as described above in the section of “Proliferation step”. Then, explanation is omitted.
- the production method according to the present invention may further include a culture vessel selection step of selecting a culture vessel suitable for the growth of mesenchymal stem cells before the growth step (or before the pre-growth step). .
- the method for selecting a culture vessel suitable for the growth of mesenchymal stem cells is the same as described in the above-mentioned “Proliferation step”, and the description thereof is omitted here.
- the present invention relates to a serum-free medium additive for producing a cell preparation containing mesenchymal stem cells whose immunosuppressive ability is maintained or improved.
- the culture medium additive according to the present invention contains FGF, PDGF, TGF- ⁇ , HGF, EGF, at least one phospholipid, and at least one fatty acid.
- the medium additive according to the present invention is added to a conventionally known basal medium, and a serum-free medium (serum-free medium A) for producing a cell preparation containing mesenchymal stem cells whose immunosuppressive ability is maintained or improved.
- serum-free medium serum-free medium A
- Examples of the phospholipid contained in the culture medium additive according to the present invention include phosphatidic acid, lysophosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and phosphatidyl. Examples thereof include choline and phosphatidylglycerol, and the culture medium additive according to the present invention may contain these phospholipids alone or in combination. In one embodiment, the culture medium additive according to the present invention contains phosphatidic acid and phosphatidylcholine in combination. Moreover, these phospholipids may be derived from animals or plants.
- Examples of the fatty acid contained in the culture medium additive according to this embodiment include linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, and stearic acid.
- the culture medium additive according to the embodiment may contain these fatty acids alone or in combination.
- the culture-medium additive which concerns on this embodiment may contain cholesterol further in addition to the said fatty acid.
- FGF is intended to be a growth factor selected from the fibroblast growth factor (FGF) family and is preferably FGF-2 (bFGF). May be selected from other FGF families such as -1.
- FGF fibroblast growth factor
- PDGF is intended to be a growth factor selected from the platelet derived growth factor (PDGF) family, and is preferably PDGF-BB or PDGF-AB.
- TGF- ⁇ is a growth factor selected from the transforming growth factor- ⁇ (TGF- ⁇ ) family, and is TGF- ⁇ 3. Preferably, it may be selected from other TGF- ⁇ families.
- HGF is intended to be a growth factor selected from the hepatocyte growth factor family
- EGF is selected from the epidermal growth factor (EGF) family. Growth factors are contemplated.
- the serum-free medium A is at least selected from the group consisting of a connective tissue growth factor (CTGF), a vascular endothelial growth factor (VEGF), and an ascorbic acid compound. Two factors may further be contained.
- CTGF connective tissue growth factor
- VEGF vascular endothelial growth factor
- ascorbic acid compound Two factors may further be contained.
- ascorbic acid compound is intended to be ascorbic acid (vitamin C) or ascorbic acid diphosphate, or a similar compound.
- the growth factor contained in the culture medium additive according to the present invention may be natural or produced by genetic recombination.
- the culture medium additive according to the present invention preferably contains a lipid antioxidant.
- the lipid antioxidant contained in the medium additive according to the present embodiment may be DL- ⁇ -tocopherol acetate (vitamin E).
- the culture medium additive according to the present invention may further contain a surfactant.
- the surfactant contained in the culture medium additive according to the present embodiment may be Pluronic F-68 or Tween 80.
- the medium additive according to the present invention may further contain insulin, transferrin, and selenate.
- insulin may be an insulin-like growth factor and may be derived from natural cells or produced by genetic recombination.
- the culture medium additive according to the present invention may further contain dexamethasone or other glucocorticoids.
- the present invention provides a serum-free medium additive for producing a cell preparation containing mesenchymal stem cells whose immunosuppressive ability is maintained or improved.
- the culture medium additive (medium additive A) according to the present invention contains FGF, PDGF, TGF- ⁇ , HGF, EGF, at least one phospholipid, and at least one fatty acid.
- the culture medium additive according to the present invention may further contain a cell adhesion molecule.
- the culture medium additive kit according to the present invention includes FGF, PDGF, TGF- ⁇ , HGF, EGF, at least one phospholipid, and at least one fatty acid, even if they are contained in the same container. You may be prepared for. Moreover, the culture medium additive kit according to the present invention may further contain a cell adhesion molecule.
- the cell adhesion molecule is the same as described in the “Proliferation step” in the section of “(1) Method for producing cell preparation containing mesenchymal stem cells” in the present specification, and therefore, the description thereof is omitted here.
- the medium additive kit according to the present invention is added to a conventionally known basal medium to produce a serum-free medium (serum-free medium A) for producing a cell preparation containing mesenchymal stem cells whose immunosuppressive ability is maintained or improved. ) Can be used.
- composition is a form in which each main component is contained in one substance
- kit is a form in which at least one of each main ingredient is contained in another substance. It is intended to be in form. Therefore, it is easily understood that the growth factor, phospholipid and fatty acid provided in the culture medium additive kit according to the present invention are the same as those described above for the culture medium additive.
- the kit according to the present invention is a kit for producing a cell preparation containing mesenchymal stem cells whose immunosuppressive ability is maintained or improved, and the culture medium additive (medium additive A) according to the present invention. At least.
- the kit according to the present invention may further include a medium additive B containing FGF, PDGF, EGF, at least one phospholipid, and at least one fatty acid.
- the description of “serum-free medium B” can be read as the description of “medium additive B”.
- the present invention provides a serum-free culture medium for producing a cell preparation containing mesenchymal stem cells whose immunosuppressive ability is maintained or improved.
- the culture medium according to the present invention contains FGF, PDGF, TGF- ⁇ , HGF, EGF, at least one phospholipid, and at least one fatty acid.
- the culture medium according to the present invention can be used as a serum-free medium (serum-free medium A) for producing a cell preparation containing mesenchymal stem cells whose immunosuppressive ability is maintained or improved.
- the culture medium according to the present invention only needs to contain FGF, PDGF, TGF- ⁇ , HGF, EGF, at least one phospholipid, and at least one fatty acid, and these components are added simultaneously to the basal medium. May also be added separately. That is, it can be said that the culture medium according to the present invention only needs to contain the components contained in the above-described medium additive or the components provided in the medium additive kit.
- the basal medium for constituting the culture medium according to the present invention is not particularly limited as long as it is a well-known medium for animal cells in the art.
- Preferred basal media include, for example, Ham's F12 medium, DMEM medium, and RPMI. -1640 medium, MCDB medium and the like. These basal media may be used alone or in combination.
- the basal medium for constituting the culture medium according to the present invention is preferably a medium in which MCDB and DMEM are mixed at a ratio of 1: 1.
- the present invention provides a culture method for producing a cell preparation containing mesenchymal stem cells whose immunosuppressive ability is maintained or improved.
- the culture method according to the present invention comprises a mesenchymal system in a serum-free medium (serum-free medium A) containing FGF, PDGF, TGF- ⁇ , HGF, EGF, at least one phospholipid, and at least one fatty acid. It includes a step of culturing stem cells (culturing step A).
- serum-free medium A serum-free medium containing FGF, PDGF, TGF- ⁇ , HGF, EGF, at least one phospholipid, and at least one fatty acid. It includes a step of culturing stem cells (culturing step A).
- the above-described serum-free culture medium may be used when culturing mesenchymal stem cells.
- the culture method according to the present invention cultivates mesenchymal stem cells in the serum-free medium B containing FGF, PDGF, EGF, at least one phospholipid, and at least one fatty acid before the culture step A.
- the process (culture process B) to perform may be further included.
- the “culturing step A” and the “culturing step B” correspond to the “proliferation step” and the “pre-proliferation step” in the method for producing a cell preparation containing mesenchymal stem cells according to the present invention, respectively.
- the description of the “proliferation step” and the “pre-proliferation step” in the section “(1) Method for producing cell preparation containing mesenchymal stem cells” in the present specification is referred to as “culture step A” and It can be read as an explanation for “culture step B”.
- the culture method according to the present invention includes the step of simultaneously adding FGF, PDGF, TGF- ⁇ , HGF, EGF, at least one phospholipid, and at least one fatty acid to the basal medium.
- the culture method according to the present invention may include a step of simultaneously adding FGF, PDGF, EGF, at least one phospholipid, and at least one fatty acid to the basal medium.
- the basal medium is not particularly limited as long as it is a medium for animal cells well known in the art as described above.
- mesenchymal stem cells can be grown at a rate equal to or higher than that in a serum-containing medium.
- the immunosuppressive ability of the proliferated mesenchymal stem cells is maintained or improved.
- the mesenchymal stem cells proliferated in the present invention are selectively increased in mesenchymal stem cells not having tumorigenicity.
- the mesenchymal stem cells proliferated in the present invention maintain or improve the differentiation ability (see Patent Document 1).
- the mesenchymal stem cells contained in the cell preparation produced by the present invention can be said to maintain the function of affecting the immune effector cells inherent in the mesenchymal stem cells, the mesenchymal stem cells further
- the cell preparation produced according to the present invention can be expected to be applied to treatments that have an immunomodulatory action and an immunotolerant action and that expect these actions.
- a function as an anti-inflammatory agent in transplantation treatment can be expected due to the anti-inflammatory action of mesenchymal stem cells.
- the cell preparation is also expected to have an anti-aging effect due to anti-inflammatory action.
- the cell preparation produced according to the present invention is not only for treatment of local diseases administered to sites requiring transplantation of mesenchymal stem cells (local administration), but also systemically administered by being administered intravenously etc. Therefore, it is expected that treatment for strong immune rejection caused by acute GVHD caused by bone marrow transplantation and the like will be more effectively performed, and the survival rate of human beings will be significantly improved.
- the cell preparation produced by the present invention is produced by serum-free culture, useful growth factors and differentiation factors are not non-specifically adsorbed to serum and other transplant materials such as ceramics. . Therefore, the tissue regeneration ability by transplantation of the cell preparation produced according to the present invention is much higher, and as a result, the therapeutic effect is also high. Furthermore, it goes without saying that treatment can be performed by combining local administration and systemic administration.
- the mesenchymal stem cells contained in the cell preparation produced according to the present invention maintain or improve immunosuppressive ability and suppress immune rejection at the time of transplantation, so that tissues and cells other than self are transplanted to others. It can be applied to the treatment by autologous transplantation (different donor and recipient). Furthermore, it can be said that it can be suitably used not only for allotransplantation (allogeneic transplantation) using human tissues or human cells but also for xenotransplantation (xenotransplantation) using animal tissues or animal cells other than humans.
- the cell preparation produced by the present invention exerts an immunosuppressive effect earlier than mesenchymal stem cells obtained using only conventional serum-containing media, the therapeutic effect can be expressed early at the time of transplantation. Expected and expected to increase healing rate.
- the method for producing a cell preparation containing mesenchymal stem cells according to the present invention further includes a serum culture step of culturing the mesenchymal stem cells after the above-described proliferation step in a medium containing serum before the above-described screening step. You may do it.
- the method for producing a cell preparation containing mesenchymal stem cells according to the present invention includes a serum-free medium B containing FGF, PDGF, EGF, at least one phospholipid, and at least one fatty acid before the above-described proliferation step. It is preferable that the method further includes a pre-growth step for growing mesenchymal stem cells.
- a second screening step of screening mesenchymal stem cells not having tumorigenicity from mesenchymal stem cells after the above-described proliferation step may be further included.
- the method for producing a cell preparation containing mesenchymal stem cells it is preferable to proliferate the mesenchymal stem cells using a culture container suitable for the proliferation of the mesenchymal stem cells in the proliferation step. .
- the serum-free medium A further contains a cell adhesion molecule in the growth step.
- the mesenchymal stem cells may be passaged at least once in the proliferation step.
- the method for producing a cell preparation containing mesenchymal stem cells uses the cell peeling agent that does not contain a component derived from a mammal or a microorganism, when performing subculture in the growth step. It is preferable to exfoliate leaf stem cells.
- the method for producing a cell preparation containing mesenchymal stem cells according to the present invention further includes a culture vessel selection step of selecting a culture vessel suitable for the growth of mesenchymal stem cells before the above-described proliferation step. Also good.
- the phospholipid is phosphatidic acid, lysophosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine. , Phosphatidylcholine, and phosphatidylglycerol.
- the fatty acid is a group consisting of linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, and stearic acid. It is characterized by being more selected.
- the medium additive according to the present invention may further contain a cell adhesion molecule.
- kit according to the present invention may further include a medium additive B containing FGF, PDGF, EGF, at least one phospholipid, and at least one fatty acid.
- ⁇ 2-2 Stimulation with anti-CD3 / anti-CD28>
- Mouse spleen cells (1 ⁇ 10 5 ) were seeded in each well of a 96-well plate and stimulated with 2.5 ⁇ g / ml anti-CD3 and 0.5 ⁇ g / ml anti-CD28.
- FIG. 1 is a graph showing the immunosuppressive effect of bone marrow-derived hMSCs on anti-CD3 and anti-CD28 stimulated T cell proliferation responses when mouse-derived activated T cells and bone marrow-derived hMSCs are co-cultured in MSCGM medium.
- 2 is a graph showing the immunosuppressive effect of bone marrow-derived hMSCs on anti-CD3 and anti-CD28 stimulated T cell proliferation responses when mouse-derived activated T cells and bone marrow-derived hMSCs are co-cultured in medium 1.
- FIG. 3 is a graph showing the immunosuppressive effect of bone marrow-derived hMSCs on PMA and ionomycin-stimulated (mitogen-stimulated) T cell proliferation responses when mouse-derived activated T cells and bone marrow-derived hMSCs were co-cultured in MSCGM medium.
- FIG. 4 is a graph showing the immunosuppressive effect of bone marrow-derived hMSCs on PMA and ionomycin-stimulated (mitogen-stimulated) T cell proliferation responses when mouse-derived activated T cells and bone marrow-derived hMSCs were co-cultured in medium 1. is there.
- bone marrow-derived hMSCs cultured in medium 1 were stimulated by mitogen-stimulated T cells and anti-CD3 / anti-CD28 stimulation, as were bone marrow-derived hMSCs cultured in serum-containing MSCGM medium. Inhibited the proliferation of activated T cells. That is, it was shown that the bone marrow-derived hMSC cultured using the serum-free medium 1 maintains the immunosuppressive effect.
- Example 2 The immunosuppressive effect of bone marrow-derived hMSC on T cell proliferation by mouse lymphocyte mixed reaction (MLR) was examined.
- the experimental method and the cells and medium used are the same as in Example 1.
- 2 ⁇ 10 4 bone marrow-derived hMSCs were seeded in each well of a 96-well plate and performed in the same manner as in Example 1.
- mouse spleen cells (2 ⁇ 10 5 ) and mouse bone marrow-derived dendritic cells (BMDC) (3.3 ⁇ 10 4 ) are co-cultured in each well of a 96-well plate. (MLR stimulation).
- BMDC collects bone marrow cells from C3H (H-2k) purchased from Charles River Japan Co., Ltd., hemolyzes them, and then uses 1% FBS / Advanced PRMI (GIBCO) containing GM-CSF for one day. The medium was changed every other day, stimulated with 100 ng / ml LPS on the 6th day of culture, cultured overnight, washed, irradiated with gamma rays using a Gamma cell 40 executor, and used for experiments in which cell division was inhibited. .
- C3H H-2k
- GEBCO FBS / Advanced PRMI
- FIGS. 5 and 6 are graphs showing the immunosuppressive effect of bone marrow-derived hMSCs on T cell proliferation by MLR. Values are shown as mean ⁇ standard deviation. Similar results were obtained from three or more independent experimental results.
- FIG. 5 shows the results on the third day of culture
- FIG. 6 shows the results on the fourth day of culture.
- M shows the result when pre-cultured with MSCGM medium and then cultured with serum-containing medium
- S shows the result of pre-culture with medium 1 and then cultured with serum-containing medium.
- bone marrow-derived hMSC pre-cultured in medium 1 maintained the effect of inhibiting activated T cell proliferation in the same manner as bone marrow-derived hMSC pre-cultured in MSCGM medium.
- bone marrow-derived hMSC pre-cultured in MSCGM medium showed an immunosuppressive effect on the 4th day from the start of co-culture, whereas bone marrow-derived hMSC pre-cultured in medium 1 was 3 days after the start of co-culture.
- the bone marrow origin hMSC pre-cultured with the culture medium 1 showed the same result also with respect to mitogen stimulation T cell proliferation and anti-CD3 / anti-CD28 stimulation T cell proliferation.
- bone marrow-derived hMSCs cultured in medium 1 not only maintained the cytostatic effect of activated T cells, but were also cultured in MSCGM medium (serum-containing medium). It was confirmed that the cell proliferation inhibitory effect of activated T cells was exhibited earlier than bone marrow-derived hMSC.
- MSCGM medium serum-containing medium
- the cell proliferation inhibitory effect of activated T cells was exhibited earlier than bone marrow-derived hMSC.
- MSCGM medium serum-containing medium
- culturing bone marrow-derived hMSCs in medium 1 is not only advantageous for growing a necessary number of cells in a short period of time, but also has an immunosuppressive effect maintained. From this, it can be said that the culture medium 1 is particularly effective for culturing bone marrow-derived hMSC aimed at clinical application.
- adipose tissue-derived mesenchymal stem cells (adipose tissue-derived hMSC) were isolated and cultured by the following procedures (i) to (vii).
- Adipose tissue was collected from a human and the adipose tissue was washed 2 to 3 times using serum-free DMEM medium.
- the washed adipose tissue was finely cut with scissors (1 mm 3 ).
- Adipose tissue was treated using a 0.1-0.2% collagenase (GIBCO 17100-017) solution with stirring with a stir bar (37 ° C., 30-60 minutes).
- the washed cells are seeded on a culture plate (BECTON, DICKINSON (Falcon) 353047), and a serum-free medium 1 shown in Table 1 or a MEM medium (sigma D6046) containing 10% FBS (“ 10% FBS-MEM ").
- FIG. 7 is a diagram showing the growth state of adipose tissue-derived hMSC on the 8th day from the start of culture.
- the adipose tissue-derived hMSC was observed at a magnification of 40 times.
- the number of cells significantly increased (2 to 3 times) in the adipose tissue-derived hMSC cultured in medium 1 as compared with the adipose tissue-derived hMSC cultured in 10% FBS-MEM. It was shown that.
- the initial synovium-derived hMSC obtained by centrifugation was cultured using the following medium.
- the medium 2 as the serum-free medium B is a medium obtained by removing HGF and TGF- ⁇ from the medium 1 shown in Table 1.
- -DMEM containing 10% FBS (10% FBS-DMEM) (sigma D6046, FBS; Hyclone, PS (+)) -Medium 1 -Medium 2
- the culture was performed in a carbon dioxide incubator (95% air and 5% CO 2 ) maintained at 37 ° C.
- FIG. 8 is a graph showing the growth-promoting effect of medium 1 and medium 2 on the initial synovial-derived hMSC
- (a) is a graph showing the number of synovial-derived hMSC cells on the 12th day from the start of culture.
- (B) is a figure which shows the proliferation state of synovium origin hMSC of the 12th day from culture
- synovial membrane-derived hMSCs were observed at a magnification of 10 times.
- the growth promoting effect of the medium 2 is remarkable for the initial synovial membrane-derived hMSC (8 times the case of using the medium 1 and 10% FBS-containing DMEM). It was confirmed that it was higher (40 times or more than when using).
- the initial synovium-derived hMSCs obtained by centrifugation were cultured for 11 days using medium 2 and then cultured using the following medium.
- -DMEM containing 10% FBS (10% FBS-DMEM) (sigma D6046, FBS; Hyclone, PS (+)) -Medium 1 -Medium 2
- the culture was performed in a carbon dioxide incubator (95% air and 5% CO 2 ) maintained at 37 ° C.
- FIG. 9 is a graph showing changes over time in the number of synovial cell-derived hMSCs from day 0 to day 68 of the start of culture.
- the synovial-derived hMSC is cultured by using the medium 2 and then culturing the synovial-derived hMSC using the medium 1 for the initial synovial-derived hMSC.
- synovial membrane-derived hMSCs obtained by centrifugation were transferred to a flask, and culture medium 2 was used from the first culture (P0) to the third passage (P3).
- the culture medium 1 shown in Table 1 was used between the first round (P1) and the fourth passage (P4).
- the culture was performed in a carbon dioxide incubator (95% air and 5% CO 2 ) maintained at 37 ° C.
- -Flask 1 75 cm 2 flask made of Falcon-Flask 2: 75 cm 2 flask made of Sumitomo Bakelite (culture solution) The following culture solutions were used.
- -DMEM containing 10% FBS (10% FBS-DMEM) (sigma D6046, FBS; Hyclone, PS (+))
- -Medium 1 Medium 1 + fibronectin (final concentration 5 ⁇ g / mL)
- the following bone marrow-derived mesenchymal stem cells (bone marrow-derived hMCS) were used.
- -Cell 1 (P2) 1 ⁇ 10 6 cells that were generated and passaged on day 12 of culture.
- -Cell 2 (P3) 1 ⁇ 10 6 cells, which were raised and passaged on the 30th day of culture. The growth speed is slow.
- -Cell 3 (P1) 1 ⁇ 10 6 cells that were generated and passaged on day 12 of culture.
- ⁇ Cell 2> At the fourth passage, cells were seeded in a flask and culture was started. On the fifth day from the start of culture, the growth state of the cells was visually observed using an optical microscope.
- ⁇ Cell 3> At the second passage, cells were seeded in a flask and culture was started. On the fifth day from the start of culture, the growth state of the cells was visually observed using an optical microscope.
- FIG. 10 is a diagram showing the proliferation state of bone marrow-derived hMSC (cell 1) on the fifth day from the start of culture.
- FIG. 10 bone marrow-derived hMSCs were observed with a magnification of 40 times.
- FIG. 11 is a diagram showing the state of proliferation of bone marrow-derived hMSC (cell 2) on the fifth day from the start of culture.
- bone marrow-derived hMSCs were observed with a magnification of 40 times.
- condition B cell 2 proliferated to about 70 to 80% of the confluent state
- condition E it proliferated to about 90% of the confluent state and was almost confluent.
- condition B and condition E compared to condition A and condition D, the cells were atrophied and a hole-like space was formed.
- condition C and condition F cell 2 grew to about 80% of the confluent state.
- the cell adhesion state was the same as in Condition B and Condition E, but the number of cells was slightly smaller.
- FIG. 12 is a diagram showing the proliferation state of bone marrow-derived hMSC (cell 3) on the fifth day from the start of culture.
- FIG. 12 bone marrow-derived hMSCs were observed with a magnification of 40 times.
- condition A and D the cells 3 grew to about 80 to 90% of the confluent state and were in a slightly confluent state. Further, in condition B and condition E, part of the cells was detached in condition B, but in other cases, the cells proliferated to about 80 to 90% of the confluent state. Furthermore, in condition C and condition F, cell detachment was not observed as compared with condition B and condition E, and the cells grew to about 90% of the confluent state.
- condition B and condition E cell proliferation ability was higher in condition E regardless of which cell line of cells 1 to 3 was used. That is, it was confirmed that flask 2 was more suitable than flask 1 for the growth of cells 1 to 3.
- a soft agar medium (DMEM-10% FCS-0.6% agar) was added to each well, and after gelation, cells were suspended (DMEM-10% FCS-0. 4% agar) was added.
- the cells were human chondrosarcoma cell line (OUMS-27, purchased from JCRB cell bank), normal human dermal fibroblasts (NHDF, purchased from Lonza), and the number of cells seeded per well was 0-10000. did.
- the human chondrosarcoma cell line is a cancerous mesenchymal cell. Groups with 10% and 20% serum content of the culture solution added on the soft agar medium were prepared. The reason why serum was added to the culture solution in this experiment was to make the condition that the cells would proliferate more actively.
- -DMEM containing 4.5 g / L glucose (DMEM 4.5 g / L glucose) Medium 1 (see Table 1)
- DMEM 4.5 g / L glucose
- OUMS-27 was considered to have poor colony formation in a low concentration glucose medium (1 g / ml), and a high concentration glucose-containing medium (commercially available product) was selected and used for culture.
- FIG. 13 is a graph showing the effect of medium 1 on the growth of normal human dermal fibroblasts and human chondrosarcoma cell lines, where (a) shows the number of colonies of normal human dermal fibroblasts on day 14 from the start of culture. (B) is a graph showing the number of colonies of the human chondrosarcoma cell line on the 14th day from the start of culture. FIG. 13 shows the number of colonies having a size of 25 ⁇ m or more.
- the culture medium 1 was used as the culture solution when 4.5 g / L glucose-containing DMEM was used as the culture solution.
- the number of colonies formed is significantly larger than that of the case where the cells were formed, the cells were judged to be tumorigenic, and the culture solution used DMEM containing 4.5 g / L glucose.
- the culture solution used DMEM containing 4.5 g / L glucose was used as the culture solution.
- the present invention can provide a safer and more useful transplantation treatment material using mesenchymal stem cells, it can be suitably used for regenerative medicine such as transplantation treatment using mesenchymal stem cells. .
Abstract
Description
本発明は、無血清条件下において免疫抑制能を維持又は向上した間葉系幹細胞を含む細胞製剤の製造方法を提供する。本発明に係る細胞製剤の製造方法は、FGF、PDGF、TGF-β、HGF、EGF、少なくとも1つのリン脂質、及び少なくとも1つの脂肪酸を含有する無血清培地Aにおいて、間葉系幹細胞を増殖させる増殖工程と、上記増殖工程後の間葉系幹細胞から、免疫抑制能を維持又は向上した間葉系幹細胞をスクリーニングするスクリーニング工程とを包含している。
本発明に係る製造方法の増殖工程においては、間葉系幹細胞を、FGF、PDGF、TGF-β、HGF、EGF、少なくとも1つのリン脂質、及び少なくとも1つの脂肪酸を含有する無血清培地Aにおいて培養し、間葉系幹細胞を増殖させる。
(i)細胞層をPBS(-)5mLを用いて洗浄する。
(ii)ACCUTASEを2mL添加する。
(iii)室温にて2分程度静置し、細胞の剥離を確認のうえ遠心管に細胞浮遊液を移す。
(iv)培養容器にPBS(―)を7mL添加し、フラスコ底面をリンスする。
(v)上記(iii)の遠心管に上記(iv)の溶液を移し、1500rpm(200×g)で5分間遠心する。
(vi)上清を除き、5,000個/cm2の播種濃度にて、無血清培地Aを用いて播種する。
本発明に係る製造方法のスクリーニング工程(「第1スクリーニング工程」ともいう。)においては、上記増殖工程後の間葉系幹細胞から、免疫抑制能を維持又は向上した間葉系幹細胞をスクリーニングする。上述した無血清培地Aにおいて増殖した増殖工程後の間葉系幹細胞は、免疫抑制能が少なくとも維持されており、さらに免疫抑制能がこう進している。したがって、このような間葉系幹細胞を、免疫抑制能を基準としてスクリーニングすることによって、免疫抑制能を維持または向上した間葉系幹細胞を選別することができる。そして、選別した間葉系幹細胞を細胞製剤として使用することによって、免疫抑制剤を併用することなく、免疫学的拒絶反応を抑制した間葉系幹細胞の移植治療が実現する。
本発明に係る製造方法は、上記増殖工程後の間葉系幹細胞から、造腫瘍性を有していない間葉系幹細胞をスクリーニングする第2スクリーニング工程をさらに包含していてもよい。
本発明に係る製造方法は、さらに、増殖工程後の間葉系幹細胞を、スクリーニング工程の前に、血清含有培地において培養する血清培養工程を包含していてもよい。血清培養工程においては、増殖工程において無血清培養した間葉系幹細胞を、血清含有培地において培養する。血清培養工程を行う場合、血清培養工程後の間葉系幹細胞をスクリーニング工程に供する。
本発明に係る製造方法は、上記増殖工程の前に、FGF、PDGF、EGF、少なくとも1つのリン脂質、及び少なくとも1つの脂肪酸を含有する無血清培地Bにおいて、間葉系幹細胞を増殖させる前増殖工程をさらにしていてもよい。
本発明に係る製造方法は、上記増殖工程の前(または上記前増殖工程の前)に、間葉系幹細胞の増殖に適した培養容器を選択する培養容器選択工程をさらに包含していてもよい。間葉系幹細胞の増殖に適した培養容器の選択方法としては、上記「増殖工程」の項で説明したとおりであるのでここでは説明を省略する。
本発明は、免疫抑制能を維持又は向上した間葉系幹細胞を含む細胞製剤を製造するための無血清の培地用添加剤を提供する。本発明に係る培地用添加剤は、FGF、PDGF、TGF-β、HGF、EGF、少なくとも1つのリン脂質、及び少なくとも1つの脂肪酸を含有している。本発明に係る培地用添加剤は、従来公知の基礎培地に添加して、免疫抑制能を維持又は向上した間葉系幹細胞を含む細胞製剤を製造するための無血清培地(無血清培地A)として使用することができる。
本発明は、免疫抑制能を維持又は向上した間葉系幹細胞を含む細胞製剤を製造するための無血清の培地用添加剤を提供する。本発明に係る培地用添加剤(培地用添加剤A)は、FGF、PDGF、TGF-β、HGF、EGF、少なくとも1つのリン脂質、及び少なくとも1つの脂肪酸を含有している。また、本発明に係る培地用添加剤は、細胞接着分子をさらに含有していてもよい。
本発明は、免疫抑制能を維持又は向上した間葉系幹細胞を含む細胞製剤を製造するための無血清の培養培地を提供する。本発明に係る培養培地は、FGF、PDGF、TGF-β、HGF、EGF、少なくとも1つのリン脂質、及び少なくとも1つの脂肪酸を含有している。本発明に係る培養培地は、免疫抑制能を維持又は向上した間葉系幹細胞を含む細胞製剤を製造するための無血清培地(無血清培地A)として使用することができる。
本発明は、免疫抑制能を維持又は向上した間葉系幹細胞を含む細胞製剤を製造するための培養方法を提供する。本発明に係る培養方法は、FGF、PDGF、TGF-β、HGF、EGF、少なくとも1つのリン脂質、及び少なくとも1つの脂肪酸を含有している無血清培地(無血清培地A)中において間葉系幹細胞を培養する工程(培養工程A)を包含している。本発明に係る培養方法は、間葉系幹細胞を培養する際に、上述した無血清の培養培地を用いればよいといえる。
上述したように、本発明によれば、無血清又は低血清培地であっても血清含有培地において培養した場合と同等又はそれ以上の速度で、間葉系幹細胞を増殖させることが可能である上に、増殖させた間葉系幹細胞の免疫抑制能が維持又は向上している。さらに、本発明において増殖させた間葉系幹細胞は、造腫瘍性を有していない間葉系幹細胞が選択的に増加している。さらに、本発明において増殖させた間葉系幹細胞は、分化能が維持又は向上している(特許文献1参照のこと)。したがって、このような間葉系幹細胞を含む細胞製剤を患者に投与した場合、間葉系幹細胞による優れた移植治療が実現できるのみならず、移植治療の大きな問題の1つである免疫拒絶反応を効果的に抑制し、患者の負担を軽減すると共に、安定した治療を実現し得る。また、血清を用いて製造した従来の細胞製剤と比較して、血清のロット差を考慮する必要がなく、移植治療において安定した治癒率を実現できる。
以下の実験を行った。
ヒト骨髄由来間葉系幹細胞(骨髄由来hMSC)は、Lonza Walkersville, Inc.(以下、Lonza社)より購入し、Mesenchymal Stem Cell Basal Medium(MSCBM)(Lonza社)にMesenchymal Cell Growth Supplement(MCGS)(Lonza社)を加えた血清含有培地(MSCGM培地)、又は表1に示す無血清の培地1で培養した。
<2-1:マイトジェンによる刺激>
96ウエルプレートの各ウエルに、マウス脾臓細胞(1×105)を播種し、2.5ng/ml Phorbol 12-myristate 13-acetate(以下、PMAと称する)、125ng/ml ionomycinで刺激した。
96ウエルプレートの各ウエルにマウス脾臓細胞(1×105)を播種し、2.5μg/ml anti-CD3、0.5μg/ml anti-CD28で刺激した。
骨髄由来hMSCの前処理として、96ウエルプレートの各ウエルに、骨髄由来hMSC1×104を播種し、張り付いたのを確認してから(数時間から一晩培養)、ガンマセル40イグザクターを用いてガンマ線照射を行い、細胞分裂を阻害した。その上に、上述したように活性化したマウス脾臓細胞を播種し、骨髄由来hMSCと共培養した。
共培養開始から3日目及び4日目に、各ウエルに[3H]-Thymidineを加え、さらに8時間培養した。培養細胞をガラスフィルターに吸着させた後、液体シンチレーションカウンターでThymidineの取り込みを測定し、細胞増殖を測定した。
骨髄由来hMSCとマウス由来活性化T細胞との共培養を、10%FBS含有のMSCGM培地又は無血清培地Aである培地1(STK2(登録商標))で培養した結果を、図1~4に示す。図1~4おいて、値は平均±標準偏差で示している。同様の結果が、3回以上の独立した実験結果から得られた。#4及び#5は、それぞれ独立した骨髄由来hMSCの結果を示している。
マウスリンパ球混合反応(MLR)によるT細胞増殖に対する骨髄由来hMSCの免疫抑制効果を調べた。実験方法、並びに使用した細胞及び培地は、実施例1と同様である。骨髄由来hMSCの前処理として、96ウエルプレートの各ウエルに2×104の骨髄由来hMSCを播種し、実施例1と同様に行った。また、マウス脾臓細胞の活性化を、96ウエルプレートの各ウエルにおいて、マウス脾臓細胞(2×105)とマウス骨髄由来樹状細胞(BMDC)(3.3×104)とを共培養することによって行った(MLR刺激)。
以下の実験を行った。
ヒト脂肪組織由来間葉系幹細胞(脂肪組織由来hMSC)は、以下の(i)~(vii)の手順にて分離し、培養した。
(i)ヒトから脂肪組織を採取し、無血清DMEM培地を用いて脂肪組織を2~3回洗浄した。
(ii)洗浄後の脂肪組織を、ハサミを用いて細かく切断した(1mm3)。
(iii)0.1~0.2%コラゲナーゼ(GIBCO 17100-017)溶液を用いて、スターラーバーで攪拌しながら脂肪組織を処理した(37℃、30~60分)。
(iv)100mmのフィルターを用いてコラゲナーゼ処理後の脂肪組織を濾過した後、100×g、10分間遠心分離を行い、脂肪組織由来hMSCを分離した。
(v)Red lysis buffer(sigma R7757)を用いて、スターラーバーで攪拌しながら遠心分離後の細胞を処理した(5~10分)。
(vi)Red lysis buffer処理後の細胞を、無血清DMEM培地を用いて2~3回洗浄した。
(vii)洗浄後の細胞を培養用プレート(BECTON, DICKINSON (Falcon)353047)に播種し、表1に示した無血清の培地1、又は10%FBSを含有するMEM培地(sigma D6046)(「10%FBS-MEM」と称する。)を用いて培養した。
培養開始から8日目に、細胞の増殖状態を光学顕微鏡を用いて目視で観察した。
結果を図7に示す。図7は、培養開始から8日目の脂肪組織由来hMSCの増殖状態を示す図である。図7では、脂肪組織由来hMSCを40倍に拡大して観察した。図7に示すように、培地1で培養した脂肪組織由来hMSCは、10%FBS-MEMで培養した脂肪組織由来hMSCと比較して、顕著に(2~3倍)細胞数が増加していることが示された。
以下の実験を行った。
ヒト滑膜由来間葉系幹細胞(滑膜由来hMSC)は、以下の(i)~(iii)の手順にて分離した。
(i)ヒトから滑膜を採取した。
(ii)得られた滑膜細胞をPBSで洗浄後、組織を0.4%コラゲナーゼ溶液10ml中に加えて混和し、37℃で1~4時間反応させた。
(iii)フィルタリング後、遠心分離した。
・10%FBS含有DMEM(10%FBS-DMEM)(sigma D6046、FBS;Hyclone,PS(+))
・培地1
・培地2
培養は、37℃に保った炭酸ガスインキュベータ内(95% air and 5% CO2)で行った。
培養開始から12日目(継代0回目)に、細胞の増殖状態を光学顕微鏡を用いて目視で観察した。また、細胞の数をコールターカウンターによって測定した。
結果を図8に示す。図8は、初期の滑膜由来hMSCに対する培地1および培地2の増殖促進効果を示す図であり、(a)は、培養開始から12日目の滑膜由来hMSCの細胞数を示すグラフであり、(b)は、培養開始から12日目の滑膜由来hMSCの増殖状態を示す図である。図8の(b)では、滑膜由来hMSCを10倍に拡大して観察した。
以下の実験を行った。
ヒト滑膜由来間葉系幹細胞(滑膜由来hMSC)は、実施例4の「1.細胞培養」の項に示した(i)~(iii)の手順によって分離した。
・10%FBS含有DMEM(10%FBS-DMEM)(sigma D6046、FBS;Hyclone,PS(+))
・培地1
・培地2
培養は、37℃に保った炭酸ガスインキュベータ内(95% air and 5% CO2)で行った。
細胞の数をコールターカウンターによって測定した。
結果を図9に示す。図9は、培養開始0日目から68日目までの滑膜由来hMSCの細胞数の経時的な変化を示すグラフである。
以下の実験を行った。
ヒト滑膜由来間葉系幹細胞(滑膜由来hMSC)は、実施例4の「1.細胞培養」の項に示した(i)~(iii)の手順によって分離した。
1gの滑膜組織から培地2と培地1を組み合わせて培養することによって、3~4週間で1000人分(変形性関節症の最大移植細胞数:1.5×109個)の増殖を取得することができた。
以下の実験を行った。
以下のフラスコを用いた。
・フラスコ1:ファルコン製75cm2フラスコ
・フラスコ2:住友ベークライト製75cm2フラスコ
(培養液)
以下の培養液を用いた。
・10%FBS含有DMEM(10%FBS-DMEM)(sigma D6046、FBS;Hyclone,PS(+))
・培地1
・培地1+フィブロネクチン(終濃度5μg/mL)
(細胞)
以下の骨髄由来間葉系幹細胞(骨髄由来hMCS)を用いた。
・細胞1(P2):1×106個、細胞を起して培養12日目に継代したもの。
・細胞2(P3):1×106個、細胞を起して培養30日目に継代したもの。増殖スピードが遅い。
・細胞3(P1):1×106個、細胞を起して培養12日目に継代したもの。
5,000個/cm2の播種密度で、以下の条件にてそれぞれの骨髄由来hMCS(細胞1~細胞3)を培養した。なお、培養液3を用いる場合は、播種時のみフィブロネクチンを添加した。
・条件A:フラスコ1+10%FBS-DMEM(ポジティブコントロール)
・条件B:フラスコ1+培地1
・条件C:フラスコ1+培地1+フィブロネクチン
・条件D:フラスコ2+10%FBS-DMEM(ネガティブコントロール)
・条件E:フラスコ2+培地1
・条件F:フラスコ2+培地1+フィブロネクチン
(2.細胞増殖測定)
<細胞1>
継代3回目になった時点でフラスコに細胞を播種し、培養を開始した。培養開始から5日目に、細胞の増殖状態を光学顕微鏡を用いて目視で観察した。
継代4回目になった時点でフラスコに細胞を播種し、培養を開始した。培養開始から5日目に、細胞の増殖状態を光学顕微鏡を用いて目視で観察した。
継代2回目になった時点でフラスコに細胞を播種し、培養を開始した。培養開始から5日目に、細胞の増殖状態を光学顕微鏡を用いて目視で観察した。
<細胞1>
結果を図10に示す。図10は、培養開始から5日目の骨髄由来hMSC(細胞1)の増殖状態を示す図である。図10では、骨髄由来hMSCを40倍に拡大して観察した。
結果を図11に示す。図11は、培養開始から5日目の骨髄由来hMSC(細胞2)の増殖状態を示す図である。図11では、骨髄由来hMSCを40倍に拡大して観察した。
結果を図12に示す。図12は、培養開始から5日目の骨髄由来hMSC(細胞3)の増殖状態を示す図である。図12では、骨髄由来hMSCを40倍に拡大して観察した。
以下の実験を行った。
96ウェルプレートを用いて、各ウェルに軟寒天培地(DMEM-10%FCS-0.6%寒天)を加え、ゲル化した後に細胞を懸濁した軟寒天培地(DMEM-10%FCS-0.4%寒天)を添加した。細胞は、ヒト軟骨肉腫細胞株(OUMS-27、JCRB細胞バンクから購入)、正常ヒト皮膚線維芽細胞(NHDF、ロンザ社から購入)を用いて、ウェルあたりの細胞播種数は0~10000個とした。ヒト軟骨肉腫細胞株は、間葉系の細胞がガン化したものである。軟寒天培地上に添加する培養液の血清含有量が10%、20%の群を作製した。なお、本実験において培養液に血清を添加したのは、細胞がより活発に増殖すると考えられる条件とするためである。
以下の培養液を用いた。
・4.5g/Lグルコース含有DMEM(DMEM 4.5g/L glucose)
・培地1(表1を参照)
なお、4.5g/Lグルコース含有DMEMにおいて、グルコースは、コロニー形成・増殖促進を目的として添加している。OUMS-27は、低濃度のグルコース培地(1g/ml)ではコロニー形成が悪いと考え、高濃度グルコース含有培地(市販品)を選んで、培養に使用した。
図13は、正常ヒト皮膚線維芽細胞およびヒト軟骨肉腫細胞株の増殖に対する培地1の効果を示すグラフであり、(a)は、培養開始から14日目の正常ヒト皮膚線維芽細胞のコロニー数を示すグラフであり、(b)は、培養開始から14日目のヒト軟骨肉腫細胞株のコロニー数を示すグラフである。なお、図13では、大きさが25μm以上のコロニーの数を示す。
Claims (18)
- FGF、
PDGF、
TGF-β、
HGF、
EGF、
少なくとも1つのリン脂質、及び
少なくとも1つの脂肪酸を
含有する無血清培地Aにおいて、間葉系幹細胞を増殖させる増殖工程と、
上記増殖工程後の間葉系幹細胞から、免疫抑制能を維持又は向上した間葉系幹細胞をスクリーニングするスクリーニング工程と
を包含することを特徴とする間葉系幹細胞を含む細胞製剤の製造方法。 - 上記増殖工程後の間葉系幹細胞を、上記スクリーニング工程の前に、血清を含む培地において培養する血清培養工程をさらに包含することを特徴とする請求項1に記載の製造方法。
- 上記増殖工程の前に、
FGF、
PDGF、
EGF、
少なくとも1つのリン脂質、及び
少なくとも1つの脂肪酸を
含有する無血清培地Bにおいて、間葉系幹細胞を増殖させる前増殖工程をさらに包含することを特徴とする請求項1又は2に記載の製造方法。 - 上記増殖工程後の間葉系幹細胞から、造腫瘍性を有していない間葉系幹細胞をスクリーニングする第2スクリーニング工程をさらに包含することを特徴とする請求項1~3の何れか1項に記載の製造方法。
- 上記増殖工程では、上記間葉系幹細胞の増殖に適した培養容器を用いて当該間葉系幹細胞を増殖させることを特徴とする請求項1~4のいずれか1項に記載の製造方法。
- 上記増殖工程では、上記無血清培地Aに、細胞接着分子をさらに含有させることを特徴とする請求項1~5のいずれか1項に記載の製造方法。
- 上記増殖工程では、上記間葉系幹細胞を少なくとも1回継代することを特徴とする請求項1~6の何れか1項に記載の製造方法。
- 上記増殖工程では、継代を行う場合に、哺乳類および微生物由来の成分を含有していない細胞剥離剤を用いて上記間葉系幹細胞を剥離することを特徴とする請求項7に記載の製造方法。
- 上記増殖工程の前に、間葉系幹細胞の増殖に適した培養容器を選択する培養容器選択工程をさらに包含していることを特徴とする請求項5に記載の製造方法。
- 上記リン脂質が、フォスファチジン酸、リゾフォスファチジン酸、フォスファチジルイノシトール、フォスファチジルセリン、フォスファチジルエタノールアミン、フォスファチジルコリン、及びフォスファチジルグリセロールからなる群より選択されることを特徴とする請求項1~9のいずれか1項に記載の製造方法。
- 上記脂肪酸が、リノール酸、オレイン酸、リノレイン酸、アラキドン酸、ミリスチン酸、パルミトイル酸、パルミチン酸、及びステアリン酸からなる群より選択されることを特徴とする請求項1~10のいずれか1項に記載の製造方法。
- 請求項1~11のいずれか1項に記載の製造方法によって製造された間葉系幹細胞を含む細胞製剤。
- FGF、
PDGF、
TGF-β、
HGF、
EGF、
少なくとも1つのリン脂質、及び
少なくとも1つの脂肪酸を
含有することを特徴とする免疫抑制能を維持又は向上した間葉系幹細胞を含む細胞製剤を製造するための無血清の培地用添加剤。 - 細胞接着分子をさらに含有していることを特徴とする請求項13に記載の培地用添加剤。
- 請求項13又は14に記載の培地用添加剤を含有していることを特徴とする免疫抑制能を維持又は向上した間葉系幹細胞を含む細胞製剤を製造するための無血清の培養培地。
- 請求項15に記載の培養培地において間葉系幹細胞を培養する工程を包含することを特徴とする免疫抑制能を維持又は向上した間葉系幹細胞を含む細胞製剤を製造するための培養方法。
- 請求項13又は14に記載の培地用添加剤Aを少なくとも備えていることを特徴とする免疫抑制能を維持又は向上した間葉系幹細胞を含む細胞製剤を製造するためのキット。
- FGF、
PDGF、
EGF、
少なくとも1つのリン脂質、及び
少なくとも1つの脂肪酸を
含有する培地用添加剤Bをさらに備えていることを特徴とする請求項17に記載のキット。
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US9394521B2 (en) | 2016-07-19 |
CA2792802A1 (en) | 2011-09-15 |
KR101443478B1 (ko) | 2014-09-22 |
EP2545928A4 (en) | 2013-09-04 |
CN102791276B (zh) | 2015-03-04 |
JP5804385B2 (ja) | 2015-11-04 |
CN102791276A (zh) | 2012-11-21 |
KR20120137404A (ko) | 2012-12-20 |
US20120329087A1 (en) | 2012-12-27 |
EP2545928A1 (en) | 2013-01-16 |
WO2011111787A8 (ja) | 2011-12-01 |
AU2011225158C1 (en) | 2014-01-23 |
EP2545928B1 (en) | 2016-07-20 |
JPWO2011111787A1 (ja) | 2013-06-27 |
SG183570A1 (en) | 2012-10-30 |
AU2011225158A1 (en) | 2012-11-01 |
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CA2792802C (en) | 2017-10-31 |
AU2011225158B2 (en) | 2013-09-12 |
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