JP6446222B2 - Cartilage differentiation culture medium and method for producing cartilage tissue - Google Patents
Cartilage differentiation culture medium and method for producing cartilage tissue Download PDFInfo
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Description
本発明は、軟骨分化培養液、及び軟骨組織の製造方法に関する。 The present invention relates to a cartilage differentiation culture solution and a method for producing a cartilage tissue.
近年、幹細胞を用いた再生医療の研究が盛んに行われている。幹細胞は分化能を有し、培地組成の選択により分化をコントロールできることから、各種分野における応用が期待されている。 In recent years, research on regenerative medicine using stem cells has been actively conducted. Stem cells have differentiation potential and can be controlled by selection of medium composition, and thus are expected to be applied in various fields.
そして、例えば補綴歯科治療の分野においては、従来から骨造成の要求が高かった。そこで、間葉系幹細胞を軟骨分化させ、培養軟骨による骨造成技術の検討がなされるようになっている。 For example, in the field of prosthetic dental treatment, there has been a high demand for bone formation. Thus, a mesenchymal stem cell is differentiated into cartilage, and a bone formation technique using cultured cartilage has been studied.
間葉系幹細胞の軟骨分化は、軟骨分化培養液を用いた培地中で間葉系幹細胞を培養することによりなされており、例えば非特許文献１には、α−ＭＥＭに、ＩＴＳ、ＴＧＦβ３、デキザメサゾン、アスコルビン酸を添加した培養液を用いた例が開示されている。 Cartilage differentiation of mesenchymal stem cells is achieved by culturing mesenchymal stem cells in a medium using a cartilage differentiation culture solution. For example, Non-Patent Document 1 discloses α-MEM, ITS, TGFβ3, dexamethasone. An example using a culture solution added with ascorbic acid is disclosed.
しかしながら、非特許文献１に開示されているような従来の培養液では軟骨分化が十分に促進できていなかった。 However, the conventional culture solution disclosed in Non-Patent Document 1 cannot sufficiently promote cartilage differentiation.
本発明は上記従来技術が有する問題に鑑みてなされたものであって、本発明の一側面では、間葉系幹細胞の軟骨分化を促進することができる軟骨分化培養液を提供することを目的とする。 The present invention has been made in view of the above-described problems of the prior art, and an object of the present invention is to provide a cartilage differentiation culture medium that can promote cartilage differentiation of mesenchymal stem cells. To do.
本発明の一態様によれば、ヘパリンと、ｂＦＧＦと、を含み、前記ｂＦＧＦの含有量が、１ｎｇ／ｍＬ以上である、軟骨分化培養液を提供する。 According to one aspect of the present invention, seen containing heparin, and bFGF, the content of the bFGF is at 1 ng / mL or more, to provide a cartilage differentiation culture.
本発明の一態様によれば、間葉系幹細胞の軟骨分化を促進することができる軟骨分化培養液を提供することができる。 According to one embodiment of the present invention, a cartilage differentiation culture medium that can promote cartilage differentiation of mesenchymal stem cells can be provided.
以下、本発明を実施するための形態について説明するが、本発明は、下記の実施形態に制限されることはなく、本発明の範囲を逸脱することなく、下記の実施形態に種々の変形及び置換を加えることができる。 DESCRIPTION OF EMBODIMENTS Hereinafter, embodiments for carrying out the present invention will be described. However, the present invention is not limited to the following embodiments, and various modifications and changes can be made to the following embodiments without departing from the scope of the present invention. Substitutions can be added.
本実施形態では軟骨分化培養液の一構成例について説明する。 In the present embodiment, a configuration example of a cartilage differentiation culture solution will be described.
本実施形態の軟骨分化培養液は、ヘパリンと、ｂＦＧＦ（ｂａｓｉｃ Ｆｉｂｒｏｂｌａｓｔ Ｇｒｏｗｔｈ Ｆａｃｔｏｒ：塩基性線維芽細胞増殖因子）とを含むことができる。 The cartilage differentiation culture medium of this embodiment can contain heparin and bFGF (basic fibroblast growth factor).
本発明の発明者らは、間葉系幹細胞の軟骨分化を促進することができる軟骨分化培養液について検討を行った。そして、ヘパリンと、ｂＦＧＦとを含む軟骨分化培養液を用いることで間葉系幹細胞の軟骨分化を特に促進できることができることを見出し、本発明を完成させた。 The inventors of the present invention examined a cartilage differentiation culture medium that can promote cartilage differentiation of mesenchymal stem cells. And it discovered that the cartilage differentiation of a mesenchymal stem cell can be especially accelerated | stimulated by using the cartilage differentiation culture solution containing heparin and bFGF, and completed this invention.
本発明の発明者らの検討によると、ｂＦＧＦは、間葉系幹細胞の軟骨分化を促進する働きを有している。しかしながら、ｂＦＧＦは短期間に分解してしまうため、軟骨分化培養液がｂＦＧＦを含有するのみでは、間葉系幹細胞の軟骨分化を促進する観点では十分な効果を発揮することができなかった。 According to the study by the inventors of the present invention, bFGF has a function of promoting cartilage differentiation of mesenchymal stem cells. However, since bFGF is degraded in a short period of time, if the cartilage differentiation culture solution only contains bFGF, a sufficient effect cannot be exerted in terms of promoting cartilage differentiation of mesenchymal stem cells.
そこで、本発明の発明者らがさらに検討を行ったところ、ヘパリンは、ｂＦＧＦを安定化する働きを有することが見出された。このため、軟骨分化培養液がヘパリンとｂＦＧＦとを併せて含有することにより、ｂＦＧＦによる間葉系幹細胞の軟骨分化促進の効果を継続させることができ、間葉系幹細胞の軟骨分化を促進する機能を特に高めることができる。 Then, when the inventors of the present invention further examined, it was found that heparin has a function of stabilizing bFGF. Therefore, when the cartilage differentiation culture medium contains heparin and bFGF in combination, the effect of promoting the cartilage differentiation of mesenchymal stem cells by bFGF can be continued, and the function of promoting the cartilage differentiation of mesenchymal stem cells Can be particularly enhanced.
軟骨分化培養液中のヘパリン、及びｂＦＧＦの含有量は特に限定されるものではなく、任意に選択することができる。例えば、軟骨分化培養液中のヘパリンの含有量は１Ｕｎｉｔ／ｍＬ以上１００００Ｕｎｉｔ／ｍＬ以下であることが好ましく、１０Ｕｎｉｔ／ｍＬ以上１０００Ｕｎｉｔ／ｍＬ以下であることがより好ましい。 The contents of heparin and bFGF in the cartilage differentiation culture medium are not particularly limited and can be arbitrarily selected. For example, the heparin content in the cartilage differentiation culture medium is preferably 1 Unit / mL or more and 10,000 Unit / mL or less, and more preferably 10 Unit / mL or more and 1000 Unit / mL or less.
また、軟骨分化培養液中のｂＦＧＦの含有量は１ｎｇ／ｍＬ以上１００００ｎｇ／ｍＬ以下であることが好ましく、１０ｎｇ／ｍＬ以上１０００ｎｇ／ｍＬ以下であることがより好ましい。 In addition, the content of bFGF in the cartilage differentiation culture solution is preferably 1 ng / mL or more and 10,000 ng / mL or less, and more preferably 10 ng / mL or more and 1000 ng / mL or less.
本実施形態の軟骨培養液には上述の成分に限られず、さらに任意の成分を含むことができる。 The cartilage culture solution of the present embodiment is not limited to the above-described components, and can further include arbitrary components.
本実施形態の軟骨分化培養液には、例えばさらに、ＴＧＦ−β３（ｔｒａｎｓｆｏｒｍｉｎｇ ｇｒｏｗｔｈ ｆａｃｔｏｒ−β３）と、インスリンと、ＩＴＳ＋（Ｉｎｓｕｌｉｎ−Ｔｒａｎｓｆｅｒｒｉｎ−Ｓｅｌｅｎｉｔｅ）と、を含むことが好ましい。 The cartilage differentiation culture medium of the present embodiment preferably further contains, for example, TGF-β3 (transforming growth factor-β3), insulin, and ITS + (Insulin-Transferrin-Selenite).
ＴＧＦ−β３、インスリン、及びＩＴＳ＋についも間葉系幹細胞の軟骨分化を促進する働きを有しており、これらの成分を含有する軟骨分化培養液は間葉系幹細胞の軟骨分化を促進することができる。 TGF-β3, insulin, and ITS + also have a function of promoting cartilage differentiation of mesenchymal stem cells, and a cartilage differentiation culture medium containing these components can promote cartilage differentiation of mesenchymal stem cells. it can.
従来の培養液において、インスリン、またはＩＴＳ＋のいずれか一方を添加することは知られていたが、両成分を同時に添加することは行われていなかった。ところが、本発明の発明者らの検討によると、インスリンと、ＩＴＳ＋と、を同時に添加することにより、間葉系幹細胞の軟骨分化を特に促進することができる。 Although it has been known to add either insulin or ITS + in a conventional culture solution, the addition of both components has not been performed at the same time. However, according to the study of the inventors of the present invention, the cartilage differentiation of mesenchymal stem cells can be particularly promoted by simultaneously adding insulin and ITS +.
軟骨分化培養液中のＴＧＦ−β３、インスリン、及びＩＴＳ＋の含有量は特に限定されるものではなく、任意にその含有量を選択することができる。 The contents of TGF-β3, insulin, and ITS + in the cartilage differentiation culture medium are not particularly limited, and the contents can be arbitrarily selected.
例えば軟骨分化培養液中のＴＧＦ−β３の含有量は０．１ｎｇ／ｍＬ以上１０００ｎｇ／ｍＬ以下の割合であることが好ましく、１ｎｇ／ｍＬ以上１００ｎｇ／ｍＬ以下であることがより好ましい。 For example, the content of TGF-β3 in the cartilage differentiation culture solution is preferably in the range of 0.1 ng / mL to 1000 ng / mL, more preferably 1 ng / mL to 100 ng / mL.
また、軟骨分化培養液中のインスリンの含有量は、１μｇ／ｍＬ以上１００００μｇ／ｍＬ以下であることが好ましく、１０μｇ／ｍＬ以上１０００μｇ／ｍＬ以下であることがより好ましい。 In addition, the content of insulin in the cartilage differentiation culture solution is preferably 1 μg / mL or more and 10,000 μg / mL or less, and more preferably 10 μg / mL or more and 1000 μg / mL or less.
軟骨分化培養液中のＩＴＳ＋の含有量は、体積比で０．１％以上１０％以下であることが好ましく、０．５％以上５％以下であることがより好ましい。 The content of ITS + in the cartilage differentiation culture medium is preferably 0.1% or more and 10% or less, and more preferably 0.5% or more and 5% or less by volume.
本実施形態の軟骨分化培養液は、さらに任意の成分を含むことができる。例えば、脂肪酸濃縮液、ビタミン液、ＥＡＡ（Ｅｓｓｅｎｔｉａｌ Ａｍｉｎｏ Ａｃｉｄｓ）、エリスロポエチン、コンドロイチン硫酸、ヒアルロン酸、及び活性型ビタミンＤ３を含むことができる。 The cartilage differentiation culture medium of this embodiment can further contain arbitrary components. For example, fatty acid concentrate, vitamin liquid, EAA (Essential Amino Acids), erythropoietin, chondroitin sulfate, hyaluronic acid, and active vitamin D 3 can be included.
上述した成分のうち、脂肪酸濃縮液、ビタミン液、ＥＡＡは、細胞の培養に必要な脂肪酸、ビタミン、アミノ酸を供給するために添加しているものである。 Among the components described above, fatty acid concentrate, vitamin liquid, and EAA are added to supply fatty acids, vitamins, and amino acids necessary for cell culture.
上述した成分のうち、エリスロポエチンは赤血球の分泌を促進する成分として知られているが、本発明の発明者らの検討によると、間葉系幹細胞の軟骨分化を促進する機能も有することが見出された。 Among the components described above, erythropoietin is known as a component that promotes the secretion of erythrocytes, but according to the study of the inventors of the present invention, it has been found that it also has a function of promoting cartilage differentiation of mesenchymal stem cells. It was done.
また、コンドロイチン硫酸、ヒアルロン酸は軟骨の基質に含まれる成分であり、間葉系幹細胞の軟骨分化を補助する機能を有する。 In addition, chondroitin sulfate and hyaluronic acid are components contained in the cartilage matrix and have a function of assisting the cartilage differentiation of mesenchymal stem cells.
脂肪酸濃縮液、ビタミン液、ＥＡＡ、エリスロポエチン、コンドロイチン硫酸、ヒアルロン酸、及び活性型ビタミンＤ３は、それぞれ単独で本実施形態の軟骨分化培養液に含まれていても上述の効果を発揮できるが、全てが同時に含まれていることが好ましい。本実施形態の軟骨分化培養液が、上述の成分を全て同時に含む場合、特に間葉系幹細胞の軟骨分化を促進することができる。 Fatty acid concentrate, vitamin liquid, EAA, erythropoietin, chondroitin sulfate, hyaluronic acid, and active vitamin D 3 can each exhibit the above-described effects even when included in the cartilage differentiation culture medium of this embodiment, It is preferred that all are included simultaneously. When the cartilage differentiation culture medium of this embodiment contains all the above-mentioned components at the same time, cartilage differentiation of mesenchymal stem cells can be particularly promoted.
軟骨分化培養液中のこれらの成分の含有量についても特に限定されるものではなく、任意にその含有量を選択することができる。 The content of these components in the cartilage differentiation culture solution is not particularly limited, and the content can be arbitrarily selected.
例えば、軟骨分化培養液中の脂肪酸濃縮液の含有量は、体積比で０．０１％以上１０％以下であることが好ましく、０．１％以上５％以下であることがより好ましい。 For example, the content of the fatty acid concentrate in the cartilage differentiation culture solution is preferably 0.01% or more and 10% or less, and more preferably 0.1% or more and 5% or less by volume.
軟骨分化培養液中のビタミン液の含有量は、体積比で０．０１％以上１０％以下であることが好ましく、０．１％以上５％以下であることがより好ましい。 The content of the vitamin solution in the cartilage differentiation culture solution is preferably 0.01% or more and 10% or less, and more preferably 0.1% or more and 5% or less by volume ratio.
軟骨分化培養液中のＥＡＡの含有量は、体積比で０．０１％以上１０％以下であることが好ましく、０．１％以上５％以下であることがより好ましい。 The content of EAA in the cartilage differentiation culture medium is preferably 0.01% or more and 10% or less, more preferably 0.1% or more and 5% or less by volume ratio.
軟骨分化培養液中のエリスロポエチンの含有量は、０．１ＩＵ／ｍＬ以上１０００ＩＵ／ｍＬ以下であることが好ましく、１ＩＵ／ｍＬ以上１００ＩＵ／ｍＬ以下であることがより好ましい。 The content of erythropoietin in the cartilage differentiation culture medium is preferably 0.1 IU / mL or more and 1000 IU / mL or less, and more preferably 1 IU / mL or more and 100 IU / mL or less.
軟骨分化培養液中のコンドロイチン硫酸の含有量は、０．１μｇ／ｍＬ以上１０００μｇ／ｍＬ以下であることが好ましく、１μｇ／ｍＬ以上１００μｇ／ｍＬ以下であることがより好ましい。 The chondroitin sulfate content in the cartilage differentiation culture medium is preferably 0.1 μg / mL or more and 1000 μg / mL or less, and more preferably 1 μg / mL or more and 100 μg / mL or less.
軟骨分化培養液中のヒアルロン酸の含有量は、０．０１μｇ／ｍＬ以上１００μｇ／ｍＬ以下であることが好ましく、０．１μｇ／ｍＬ以上１０μｇ／ｍＬ以下であることがより好ましい。 The content of hyaluronic acid in the cartilage differentiation culture solution is preferably 0.01 μg / mL or more and 100 μg / mL or less, and more preferably 0.1 μg / mL or more and 10 μg / mL or less.
軟骨分化培養液中の活性型ビタミンＤ３の含有量は、０．０１ｎｇ／ｍＬ以上１００ｎｇ／ｍＬ以下であることが好ましく、０．１ｎｇ／ｍＬ以上１０ｎｇ／ｍＬ以下であることがより好ましい。 The content of active vitamin D 3 in the cartilage differentiation culture medium is preferably 0.01 ng / mL or more and 100 ng / mL or less, and more preferably 0.1 ng / mL or more and 10 ng / mL or less.
また、上述の成分に加えて抗生物質等も含むことができる。 In addition to the above components, antibiotics and the like can also be included.
軟骨分化培養液の基材としては例えば基礎培地を用いることができ、基材に上記成分を添加、混合することにより軟骨分化培養液とすることができる。この際用いる基礎培地の種類は特に限定されるものではなく、一般的に市販されている各種基礎培地を用いることができる。特に基礎培地としては例えばＤＭＥＭ等を好ましく用いることができる。また基礎培地は、市販品に依らなくとも、水にアミノ酸、ビタミン、ｐＨ調整剤等の各種成分を添加して作製することもできる。このとき用いる水は、微粒子、イオン微粒金属や有機物を除去した超純水であることが好ましい。勿論、水に基礎培地の成分及び、上記成分を混合することによっても本実施形態の軟骨分化培養液を作製することができる。 As the base material of the cartilage differentiation culture solution, for example, a basal medium can be used, and a cartilage differentiation culture solution can be obtained by adding and mixing the above components to the base material. The kind of basal medium used at this time is not particularly limited, and various commercially available basal media can be used. In particular, for example, DMEM can be preferably used as the basal medium. Further, the basal medium can be prepared by adding various components such as amino acids, vitamins and pH adjusters to water without depending on commercial products. The water used at this time is preferably ultrapure water from which fine particles, ionic fine metals and organic substances have been removed. Of course, the cartilage differentiation culture medium of this embodiment can also be prepared by mixing the components of the basal medium and the above components in water.
そして、上述の軟骨分化培養液を用いた培地中で、分化した軟骨細胞からなる軟骨組織を生成することができる。 And the cartilage tissue which consists of the differentiated chondrocyte can be produced | generated in the culture medium using the above-mentioned cartilage differentiation culture solution.
上述の軟骨分化培養液を用いた培地中で、間葉系幹細胞を分化した軟骨細胞からなる軟骨組織を生成する場合、従来の培養液を用いた培地により間葉系幹細胞を分化した軟骨細胞からなる軟骨組織を生成する場合と比較して、軟骨基質の産生量を多くすることができる。 When producing a cartilage tissue composed of chondrocytes obtained by differentiating mesenchymal stem cells in a medium using the above-described cartilage differentiation culture medium, from a chondrocyte obtained by differentiating mesenchymal stem cells using a medium using a conventional culture medium The amount of cartilage matrix produced can be increased as compared with the case of generating cartilage tissue.
まず、以下の各実験例において調製した軟骨分化培養液の評価手順について説明する。
（移植細胞の準備）
移植細胞としては、以下の手順によりヒト腸骨骨髄液から採取した間葉系幹細胞を用いた。
First, the evaluation procedure of the cartilage differentiation culture solution prepared in each of the following experimental examples will be described.
(Preparation of transplanted cells)
As transplanted cells, mesenchymal stem cells collected from human iliac bone marrow fluid by the following procedure were used.
ヒト腸骨骨髄液を採取し、αＭＥＭ培地（１０％ＦＢＳ、３２単位／ｍｌ ペンシリン、５０μｇ／ｍｌ ストレプトマイシン）でよく懸濁し，骨髄液をほぐした後、３００ｇ、５分間遠心分離して、細胞を分離した。 Human iliac bone marrow fluid is collected, suspended well in αMEM medium (10% FBS, 32 units / ml pencillin, 50 μg / ml streptomycin), and the bone marrow fluid is loosened, followed by centrifugation at 300 g for 5 minutes to obtain cells. separated.
前記骨髄液から約７×１０７個の有核細胞を得た。骨髄液から採取した細胞を有核細胞数３．７５×１０７細胞個／７５ｃｍ２となるように培養フラスコへ播種し、３７℃にて５％炭酸ガス存在下で培養した。３日目で培地を交換し、以後３日に１回培地を交換した。ｂＦＧＦは５日目から３ｎｇ／ｍｌで培地に添加した。１０日前後でほぼ集密的にまで増殖した。これらの培養皿をトリプシン（０．０５％）＋ＥＤＴＡ（０．２ｍＭ）で５分間インキュベートして、細胞を単離した。細胞数をＣｏｕｌｔｅｒカウンター（Ｚ１シングル，コールター社製）で計測し、５，０００細胞個／ｃｍ２の密度で細胞を播種した。この操作を繰り返して、ほぼ集密的（コンフルエント）になった二代目の継代培養皿から得た三代目の細胞を試験に用いた。
（軟骨への分化誘導）
増殖させた細胞を２×１０５細胞個／１ｍｌの密度で、各実験例で調製した軟骨分化培養液を用いた軟骨分化培地に懸濁させ、１５ｍｌ遠枕管に移し、５００Ｇで遠心し、ペレット（細胞塊）を得た。
About 7 × 10 7 nucleated cells were obtained from the bone marrow fluid. Cells collected from the bone marrow were seeded in a culture flask so that the number of nucleated cells was 3.75 × 10 7 cells / 75 cm 2 and cultured at 37 ° C. in the presence of 5% carbon dioxide gas. The medium was changed on the third day, and thereafter the medium was changed once every three days. bFGF was added to the medium at 3 ng / ml from day 5. It grew to almost confluence around 10 days. These culture dishes were incubated with trypsin (0.05%) + EDTA (0.2 mM) for 5 minutes to isolate the cells. The number of cells was counted with a Coulter counter (Z1 single, manufactured by Coulter), and the cells were seeded at a density of 5,000 cells / cm 2 . This operation was repeated, and the third-generation cells obtained from the second-generation subculture dish that became almost confluent were used for the test.
(Induction of differentiation into cartilage)
The proliferated cells were suspended at a density of 2 × 10 5 cells / ml in a cartilage differentiation medium using the cartilage differentiation medium prepared in each experimental example, transferred to a 15 ml far pillow, centrifuged at 500 G, A pellet (cell mass) was obtained.
遠枕管のフタをゆるめ、３７℃、５％炭酸ガス存在下で培養した。２日目で軟骨分化培地を交換し、以後２日に１回培地を交換し、４週間培養して軟骨へと分化誘導した。 The lid of the far pillow tube was loosened and cultured at 37 ° C. in the presence of 5% carbon dioxide gas. On the second day, the cartilage differentiation medium was changed, and thereafter, the medium was changed once every two days, and cultured for 4 weeks to induce differentiation into cartilage.
なお、培地を交換する際にも、各実験例で調製した軟骨分化培養液を用いている。
（評価方法）
上述のように各実験例の軟骨分化培養液を用いて軟骨へと分化誘導した後、以下の２つの手法により評価を行った。
＜病理組織学的評価＞
各実験例の軟骨分化培養液により培養した組織を１０％ホルマリン中性緩衝溶液で２日間固定した後にパラフィン包埋し、ミクロトームにて厚さ５μｍに切り出し、トルイジンブルー染色を行った。そして、光学顕微鏡下にて観察を行い染色の有無を確認した。
When exchanging the medium, the cartilage differentiation culture solution prepared in each experimental example is used.
(Evaluation method)
After induction of differentiation into cartilage using the cartilage differentiation culture medium of each experimental example as described above, evaluation was performed by the following two methods.
<Histopathological evaluation>
Tissues cultured in the cartilage differentiation culture medium of each experimental example were fixed with 10% formalin neutral buffer solution for 2 days, embedded in paraffin, cut out to a thickness of 5 μm with a microtome, and stained with toluidine blue. And it observed under the optical microscope and confirmed the presence or absence of dyeing | staining.
後述する表に示した各実験例の評価結果のうち×は染色が無かったことを、すなわち軟骨組織が確認されなかったことを示している。 Among the evaluation results of each experimental example shown in the table described later, × indicates that there was no staining, that is, no cartilage tissue was confirmed.
そして、後述する表に示した各実験例の評価結果のうち、〇については染色があったことを、すなわち軟骨組織が確認されたことを示している。染色があった試料については、トルイジンブルー染色にて赤紫色に染まるメタクロマジー（異調性）陽性の軟骨基質が多く沈着した成熟した軟骨組織である事を確認した。
＜生物学的評価＞
培養した組織をパパイン水溶液で消化したサンプルをＧＡＧ定量キット及びＤＮＡ定量キットで定量し、単位ＤＮＡあたりのグリコサミノグリカン量を算定し、別途細胞数とＤＮＡ量の関係を計測した結果より単位細胞あたりのグリコサミノグリカン量を定量した。ＩＩ型コラーゲン、Ｉ型コラーゲンはＥＬＩＳＡにより定量し、同様に単位細胞あたりの数値として算出した。
And among the evaluation results of each experimental example shown in the table which will be described later, ◯ indicates that there was staining, that is, that the cartilage tissue was confirmed. The stained sample was confirmed to be a mature cartilage tissue in which a large amount of metachromamy (heterotic) positive cartilage matrix that was stained purplish red by toluidine blue staining was deposited.
<Biological evaluation>
A sample obtained by digesting a cultured tissue with an aqueous papain solution was quantified with a GAG quantification kit and a DNA quantification kit, the amount of glycosaminoglycan per unit DNA was calculated, and the relationship between the number of cells and the amount of DNA was separately measured. The amount of per glycosaminoglycan was quantified. Type II collagen and type I collagen were quantified by ELISA and similarly calculated as numerical values per unit cell.
なお、評価は以下の表１に示す成分からなる培養液を軟骨分化培養液として用いて、上述の手順と同様にして軟骨組織を培養し、培養した軟骨組織について同様にして分析した結果を基準として、後述する評価式に基づいて行った。なお、表１に示す成分からなる培養液は、既述の非特許文献１（より具体的には非特許文献１の参考文献１９も参照）に開示された培養液の組成を示している。また、表１中ＤＭＥＭの添加量を残部としているが、これはＤＭＥＭが軟骨分化培養液を１ｍｌ調製した場合の残部であることを示している。 Evaluation is based on the result of analyzing the cultured cartilage tissue in the same manner as described above, using the culture medium comprising the components shown in Table 1 below as the cartilage differentiation culture medium. As described below, it was performed based on an evaluation formula described later. In addition, the culture solution which consists of a component shown in Table 1 has shown the composition of the culture solution disclosed by the above-mentioned nonpatent literature 1 (refer also the reference literature 19 of nonpatent literature 1 more specifically). In Table 1, the amount of DMEM added is the balance, which indicates that DMEM is the balance when 1 ml of the cartilage differentiation culture solution is prepared.
評価は具体的には、（１）単位細胞あたりのグリコサミノグリカン量、及び（２）ＩＩ型コラーゲン比率（ＩＩ／（Ｉ＋ＩＩ）×１００（％））を算出し、その結果について、以下の評価式に基づいて基準値と、各実験例の結果とを比較して〇〜××で評価している。 Specifically, (1) glycosaminoglycan amount per unit cell, and (2) type II collagen ratio (II / (I + II) × 100 (%)) were calculated. Based on the evaluation formula, the reference value is compared with the result of each experimental example, and evaluated by O to XX.
なお、単位細胞あたりのグリコサミノグリカン量、及びＩＩ型コラーゲン比率は数値が大きいほど軟骨分化が促進されていることを意味する。 In addition, the amount of glycosaminoglycans per unit cell and the type II collagen ratio means that cartilage differentiation is promoted as the values increase.
以下の評価式中Ｃｔｒｌが表１に示した培養液を用いて培養した軟骨組織についての評価結果を、Ｅｘが各実験例で調製した培養液を用いて培養した軟骨組織についての評価結果を示している。
○ ： Ctrl ＜＜＜ Ex （実験結果（Ｅｘ）がコントロール（Ｃｔｒｌ）の１．５倍以上）
△＋ ： Ctrl ＜＜ Ex （実験結果（Ｅｘ）がコントロール（Ｃｔｒｌ）の１．２倍以上１．５倍未満）
△− ： Ctrl ＜ Ex （実験結果（Ｅｘ）がコントロール（Ｃｔｒｌ）の１．０５倍以上１．２倍未満）
× ： Ctrl ＝ Ex （実験結果（Ｅｘ）がコントロール（Ｃｔｒｌ）の１倍以上１．０５倍未満）
×× ： Ctrl ＞ Ex （実験結果（Ｅｘ）がコントロール（Ｃｔｒｌ）未満）
いずれの実験例においても（１）単位細胞あたりのグリコサミノグリカン量についての評価と、（２）ＩＩ型コラーゲン比率（ＩＩ／（Ｉ＋ＩＩ）×１００（％））についての評価とは同じ評価となった。このため、表７〜表９の生物学的評価の欄で１つの結果として示している。
In the following evaluation formula, Ctrl indicates the evaluation result for the cartilage tissue cultured using the culture solution shown in Table 1, and Ex indicates the evaluation result for the cartilage tissue cultured using the culture solution prepared in each experimental example. ing.
○: Ctrl << Ex (Experimental result (Ex) is 1.5 times greater than control (Ctrl))
Δ +: Ctrl << Ex (Experimental result (Ex) is 1.2 times or more and less than 1.5 times the control (Ctrl))
Δ-: Ctrl <Ex (Experimental result (Ex) is 1.05 times or more and less than 1.2 times the control (Ctrl))
X: Ctrl = Ex (Experimental result (Ex) is 1 time or more and less than 1.05 times of control (Ctrl))
XX: Ctrl> Ex (Experimental result (Ex) is less than control (Ctrl))
In any of the experimental examples, (1) the evaluation for the amount of glycosaminoglycan per unit cell and (2) the evaluation for the type II collagen ratio (II / (I + II) × 100 (%)) are the same evaluation. became. For this reason, it shows as one result in the column of biological evaluation of Table 7-9.
次に、各実験例における軟骨分化培養液の調製手順について説明する。例１、例６〜例２２が実施例、例２〜例５が比較例となる。 Next, the preparation procedure of the cartilage differentiation culture solution in each experimental example will be described. Examples 1 and 6 to 22 are examples, and examples 2 to 5 are comparative examples.
なお、以下の各実験例においてＩＴＳ＋、脂肪酸濃縮液、ビタミン液、ＥＡＡとしては表２〜表５に示した成分を含有する試薬を用いている。表２がＩＴＳ＋、表３が脂肪酸濃縮液、表４がビタミン液、表５がＥＡＡの組成をそれぞれ示している。 In the following experimental examples, reagents containing the components shown in Tables 2 to 5 are used as ITS +, fatty acid concentrate, vitamin liquid, and EAA. Table 2 shows the composition of ITS +, Table 3 shows the fatty acid concentrate, Table 4 shows the vitamin solution, and Table 5 shows the composition of EAA.
［例１〜例５］
以下の表６に示した共通成分と、各実験例について表７に示した各成分とを混合することにより軟骨分化培養液を調製した。
[Examples 1 to 5]
A cartilage differentiation culture solution was prepared by mixing the common components shown in Table 6 below and the components shown in Table 7 for each experimental example.
すなわち、例えば例１の場合には、表６に示した共通成分と、表７に示したヘパリン１００Ｕｎｉｔ／ｍｌと、ｂＦＧＦ１００ｎｇ／ｍＬと、ＴＧＦ−β３を１０ｎｇ／ｍＬと、インスリン１００μｇ／ｍＬと、ＩＴＳ＋を１％とを混合して軟骨分化培養液を調製した。 That is, for example, in the case of Example 1, the common components shown in Table 6, heparin 100 Unit / ml shown in Table 7, bFGF 100 ng / mL, TGF-β3 10 ng / mL, insulin 100 μg / mL, A cartilage differentiation culture solution was prepared by mixing ITS + with 1%.
なお、ＩＴＳ＋の単位は体積比での百分率を示しており、共通成分を含む軟骨分化培養液全体に対する比率を示している。 In addition, the unit of ITS + has shown the percentage by volume ratio, and has shown the ratio with respect to the whole cartilage differentiation culture solution containing a common component.
また、表６においてＤＭＥＭの添加量を残部としているが、これはＤＭＥＭが、軟骨分化培養液を１ｍｌ調製した場合の残部であることを示している。 In Table 6, the amount of DMEM added is the balance, which indicates that DMEM is the balance when 1 ml of the cartilage differentiation culture solution is prepared.
得られた軟骨分化培養液について上述の評価を行った。結果を表７に示す。 The obtained cartilage differentiation culture solution was evaluated as described above. The results are shown in Table 7.
以上に示した実験例のうち、例１と、例２〜例５と、を比較すると明らかなように、例１は生物学的評価についても〇となっており、軟骨分化が促進されていることが確認できた。例１の軟骨分化培養液が、ヘパリンと、ｂＦＧＦとを同時に含んでおり、これを添加した軟骨分化培養液を用いた培地が、間葉系幹細胞の軟骨分化を特に促進できたためと考えられる。 Among the experimental examples shown above, as is clear when Example 1 is compared with Examples 2 to 5, Example 1 is also ◯ for biological evaluation, and cartilage differentiation is promoted. I was able to confirm. This is probably because the cartilage differentiation culture medium of Example 1 contains heparin and bFGF at the same time, and the medium using the cartilage differentiation culture medium added with this could particularly promote the cartilage differentiation of mesenchymal stem cells.
また、ヘパリンと、ｂＦＧＦのうち、いずれか一方のみを含む例２、例３についても両方を含まない例４と同様に生物学的評価は×となることが確認できた。従って、ヘパリンと、ｂＦＧＦのうちいずれか一方のみを添加するのではなく、例１のようにヘパリンとｂＦＧＦとを同時に添加することで軟骨分化を特に促進できることを確認できた。 In addition, it was confirmed that the biological evaluation was x as in Example 2 including only one of heparin and bFGF and Example 4 not including both. Therefore, it was confirmed that cartilage differentiation can be particularly promoted by adding heparin and bFGF simultaneously as in Example 1 instead of adding only one of heparin and bFGF.
［例６〜例１９］
上述の表６に示した共通成分と、各実験例について表８に示した各成分とを混合した点以外は、例１と同様にして軟骨分化培養液を調製し、評価を行った。結果を表８に示す。
[Examples 6 to 19]
A cartilage differentiation culture solution was prepared and evaluated in the same manner as in Example 1 except that the common components shown in Table 6 and the components shown in Table 8 for each experimental example were mixed. The results are shown in Table 8.
例６〜例９については、ヘパリン、及びｂＦＧＦのいずれか一方または両方を含まない例２〜例５と比較すると軟骨分化を促進する効果は確認できたものの、例１と比較すると、軟骨分化を促進する効果が若干劣ることが確認された。 About Examples 6-9, although the effect which accelerates | stimulates cartilage differentiation was confirmed compared with Examples 2-5 which do not contain any one or both of heparin and bFGF, compared with Example 1, cartilage differentiation was It was confirmed that the promoting effect was slightly inferior.
また例１０〜例１９については、生物学的評価が例１と同様に〇となっており、例１と同様に軟骨分化を促進する効果を有することが確認できた。 Moreover, about Example 10-Example 19, biological evaluation is (circle) similarly to Example 1, and it has confirmed having the effect which accelerates | stimulates cartilage differentiation similarly to Example 1. FIG.
これらの結果から、ヘパリンの添加量は１Ｕｎｉｔ／ｍＬ以上１００００Ｕｎｉｔ／ｍＬ以下が好ましく、ｂＦＧＦの添加量は１ｎｇ／ｍＬ以上１００００ｎｇ／ｍＬ以下が好ましいことを確認できた。 From these results, it was confirmed that the addition amount of heparin is preferably 1 Unit / mL or more and 10,000 Unit / mL or less, and the addition amount of bFGF is preferably 1 ng / mL or more and 10,000 ng / mL or less.
［例２０〜例２２］
上述の表６に示した共通成分と、各実験例について表９に示した各成分とを混合した点以外は、例１と同様にして軟骨分化培養液を調製し、評価を行った。結果を表９に示す。
[Examples 20 to 22]
A cartilage differentiation culture solution was prepared and evaluated in the same manner as in Example 1 except that the common components shown in Table 6 and the components shown in Table 9 for each experimental example were mixed. The results are shown in Table 9.
例２０〜例２２については生物学的評価が△＋になっていることから従来技術と比較して、軟骨分化を促進する効果は確認できたものの、例１に比べると、その効果は若干劣ることが確認できた。これらの結果から、軟骨分化培養液は、ＴＧＦ−β３、インスリン、及びＩＴＳ＋も含有していることが好ましいことを確認できた。 In Examples 20 to 22, although the biological evaluation is Δ +, the effect of promoting cartilage differentiation was confirmed as compared with the prior art, but the effect was slightly inferior to Example 1. I was able to confirm. From these results, it was confirmed that the cartilage differentiation culture solution preferably also contains TGF-β3, insulin, and ITS +.
Claims (5)
ｂＦＧＦと、を含み、
前記ｂＦＧＦの含有量が、１ｎｇ／ｍＬ以上である、軟骨分化培養液。 With heparin,
and bFGF, only including,
A cartilage differentiation culture medium , wherein the content of bFGF is 1 ng / mL or more .
前記ｂＦＧＦの含有量が、１００００ｎｇ／ｍＬ以下である、請求項１に記載の軟骨分化培養液。 The heparin content is 1 Unit / mL or more and 10000 Unit / mL or less ,
The content of the bFGF is a 1 0000ng / mL or less, cartilage differentiation culture of claim 1.
ＴＧＦ−β３と、
インスリンと、
ＩＴＳ＋と、を含む、請求項１または２に記載の軟骨分化培養液。 further,
TGF-β3,
Insulin,
Including the ITS +, and cartilage differentiation culture solution according to claim 1 or 2.
前記インスリンの含有量が、１μｇ／ｍＬ以上１００００μｇ／ｍＬ以下であり、
前記ＩＴＳ＋の含有量が、０．１体積％以上１０体積％以下である、請求項３に記載の軟骨分化培養液。 The content of the TGF-.beta.3 is not more than 0.1 ng / mL or more 1000 ng / mL,
The content of the insulin is not more than 1 [mu] g / mL or more 10000 / mL,
The content of ITS + is 10 vol% or less than 0.1% by volume, cartilage differentiation culture of claim 3.
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