JP5495486B2 - Cultured cartilage manufacturing method and cultured cartilage - Google Patents
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- 102000008186 Collagen Human genes 0.000 claims description 9
- 108010035532 Collagen Proteins 0.000 claims description 9
- 229920001436 collagen Polymers 0.000 claims description 9
- 108010010803 Gelatin Proteins 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
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- 235000019322 gelatine Nutrition 0.000 claims description 6
- 235000011852 gelatine desserts Nutrition 0.000 claims description 6
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- 238000010899 nucleation Methods 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 description 7
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- 238000012744 immunostaining Methods 0.000 description 3
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- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 230000022159 cartilage development Effects 0.000 description 2
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- 230000006698 induction Effects 0.000 description 2
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- 238000012404 In vitro experiment Methods 0.000 description 1
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- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 101150106167 SOX9 gene Proteins 0.000 description 1
- 102000056172 Transforming growth factor beta-3 Human genes 0.000 description 1
- 108090000097 Transforming growth factor beta-3 Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
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- 230000003848 cartilage regeneration Effects 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
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- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 1
- 229960003957 dexamethasone Drugs 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000001177 diphosphate Substances 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 1
- 235000011180 diphosphates Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
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- 238000009331 sowing Methods 0.000 description 1
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- 238000002054 transplantation Methods 0.000 description 1
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Description
本発明は、培養軟骨製造方法および培養軟骨に関するものである。 The present invention relates to a method for producing cultured cartilage and cultured cartilage.
従来、骨髄由来間葉系幹細胞から軟骨形成を誘導する方法が知られている（例えば、特許文献１参照。）。
しかし、骨髄液を採取するには侵襲性が高く、また微量の軟骨しか形成させることができないという不都合がある。
本発明は、骨髄由来間葉系幹細胞を用いた方法に比べて低侵襲で細胞ソースを獲得でき、多量の軟骨を形成できる培養軟骨製造方法および培養軟骨を提供する。
However, collecting bone marrow has the disadvantage that it is highly invasive and can only form trace amounts of cartilage.
The present invention provides a cultured cartilage production method and a cultured cartilage that can acquire a cell source with less invasiveness and can form a large amount of cartilage as compared with a method using bone marrow-derived mesenchymal stem cells.
上記目的を達成するために、本発明は以下の手段を提供する。
本発明は、臍帯血由来間葉系幹細胞を多孔質のスキャホールドに播種し、該スキャホールドに播種された前記臍帯血由来間葉系幹細胞を培養し、前記臍帯血由来間葉系幹細胞を培養する間、前記スキャホールドおよび臍帯血由来間葉系幹細胞に応力を与えて培養軟骨を形成する培養軟骨製造方法を提供する。
In order to achieve the above object, the present invention provides the following means.
The present invention seeds umbilical cord blood-derived mesenchymal stem cells in a porous scaffold, cultures the cord blood-derived mesenchymal stem cells seeded in the scaffold, and cultures the cord blood-derived mesenchymal stem cells Meanwhile, a method for producing cultured cartilage is provided in which stress is applied to the scaffold and umbilical cord blood-derived mesenchymal stem cells to form cultured cartilage.
上記発明においては、前記スキャホールドは、気孔率が７５−９０％になるような連続した気孔構造を持つ多孔質のコラーゲンまたはゼラチンからなることが好ましい。 In the above invention, the scaffold is preferably made of porous collagen or gelatin having a continuous pore structure with a porosity of 75-90%.
また、本発明は、臍帯血由来間葉系幹細胞を多孔質のスキャホールドに播種し、該スキャホールドに播種された前記臍帯血由来間葉系幹細胞を培養し、前記臍帯血由来間葉系幹細胞を培養する間、前記スキャホールドおよび臍帯血由来間葉系幹細胞に応力を与えて製造される培養軟骨を提供する。
上記発明においては、前記スキャホールドが、気孔率が７５−９０％になるような連続した気孔構造を持つ多孔質のコラーゲンまたはゼラチンからなっていてもよい。
Further, the present invention seeds umbilical cord blood-derived mesenchymal stem cells in a porous scaffold, cultures the umbilical cord blood-derived mesenchymal stem cells seeded in the scaffold, and the umbilical cord blood-derived mesenchymal stem cells A cultured cartilage produced by applying stress to the scaffold and umbilical cord blood-derived mesenchymal stem cells during culturing is provided.
In the above invention, the scaffold may be made of porous collagen or gelatin having a continuous pore structure with a porosity of 75 to 90%.
本発明によれば、骨髄由来間葉系幹細胞を用いた方法に比べて低侵襲で細胞ソースを獲得でき、多量の軟骨を形成できるという効果を奏する。 According to the present invention, the cell source can be acquired with less invasiveness compared to the method using bone marrow-derived mesenchymal stem cells, and a large amount of cartilage can be formed.
本発明の一実施形態に係る培養軟骨製造方法および培養軟骨について、図１を参照して以下に説明する。
本実施形態に係る培養軟骨製造方法は、臍帯血由来間葉系幹細胞を多孔質のスキャホールドに播種するステップＳ１と、該スキャホールドに播種された前記臍帯血由来間葉系幹細胞を、スキャホールドおよび臍帯血由来間葉系幹細胞に応力を与えて培養するステップＳ２とを備えている。
スキャホールドは、気孔率が７５−９０％になるような連続した気孔構造を持つ多孔質のコラーゲンまたはゼラチンであることが好ましい。
A cultured cartilage manufacturing method and cultured cartilage according to an embodiment of the present invention will be described below with reference to FIG.
The cultured cartilage production method according to the present embodiment includes a step S1 of seeding umbilical cord blood-derived mesenchymal stem cells in a porous scaffold, and the umbilical cord blood-derived mesenchymal stem cells seeded in the scaffold are scaffolded. And step S2 of applying stress to the umbilical cord blood-derived mesenchymal stem cells and culturing them.
The scaffold is preferably porous collagen or gelatin having a continuous pore structure with a porosity of 75-90%.
本実施形態に係る軟骨製造方法によれば、骨髄由来間葉系幹細胞を使用することなく、臍帯血由来間葉系幹細胞を使用するので、患者に対する侵襲が少なくて済み、患者にかかる負担を軽減することができるという利点がある。
したがって、低侵襲で細胞ソースを獲得でき、多量の軟骨を形成することができるという利点がある。
According to the cartilage production method according to the present embodiment, since cord blood-derived mesenchymal stem cells are used without using bone marrow-derived mesenchymal stem cells, less invasiveness to the patient is achieved, and the burden on the patient is reduced. There is an advantage that you can.
Therefore, there is an advantage that a cell source can be acquired with minimal invasiveness and a large amount of cartilage can be formed.
次に、本実施形態に係る軟骨製造方法および培養軟骨の一実施例について説明する。
（実施例１）Ｉｎ ｖｉｔｒｏでの実験
１５mlのポリプロピレン製チューブに臍帯血由来間葉系幹細胞を２×１０５個加え、遠心分離によりペレットを形成させ、１０ｎｇ／ｍＬのＴＧＦ−β３および５００ｎｇ／ｍＬのＢＭＰ−２を含む軟骨誘導培地［ＤＭＥＭ（ｈｉｇｈ ｇｌｕｃｏｓｅ）、５０ｍｇ／ｍＬのＩＴＳ＋Ｐｒｅｍｉｘ、１０−７Ｍのデキサメサゾン、５０μｇ／ｍＬのアスコルビン酸二リン酸、４０μｇ／ｍＬのプロリン、１００μｇ／ｍＬのピルビン酸］で培養した。
Next, an example of the cartilage manufacturing method and cultured cartilage according to this embodiment will be described.
(Example 1) In vitro experiment 2 × 10 5 umbilical cord blood-derived mesenchymal stem cells were added to a 15 ml polypropylene tube, and a pellet was formed by centrifugation. 10 ng / mL TGF-β3 and 500 ng / mL Cartilage induction medium containing BMP-2 [DMEM (high glucose), 50 mg / mL ITS + Premix, 10 −7 M dexamethasone, 50 μg / mL ascorbic acid diphosphate, 40 μg / mL proline, 100 μg / mL pyruvin Acid].
その結果、誘導に伴い（１〜６週間）、ペレットの大きさと透明度が増大し、軟骨関連遺伝子のＳｏｘ９、コラーゲンタイプ２Ａ１、アグリカン、コラーゲンタイプ１０Ａ１を発現した。培養３週目のペレットのパラフィン切片を作製し、トルイジンブルー染色した結果、赤紫色（メタクロマジー）に染色された。 As a result, with the induction (1 to 6 weeks), the size and transparency of the pellet increased, and the cartilage-related genes Sox9, collagen type 2A1, aggrecan, and collagen type 10A1 were expressed. As a result of preparing a paraffin section of the pellet at the third week of culture and staining it with toluidine blue, the pellet was stained purple (metachromy).
また、免疫染色により軟骨細胞マーカーのタイプＩＩコラーゲンも陽性反応を示した。これらの結果から、臍帯血由来間葉系幹細胞は軟骨細胞に分化できることが明らかになった。さらに、他の組織由来の間葉系幹細胞である骨髄由来間葉系幹細胞から誘導した軟骨様ペレットに比べ、その大きさが著しく増大していた。このことから、軟骨再生における間葉系幹細胞ソースとして臍帯血由来間葉系幹細胞は有力なものであることが実証された。 The chondrocyte marker type II collagen also showed a positive reaction by immunostaining. These results revealed that cord blood-derived mesenchymal stem cells can differentiate into chondrocytes. Furthermore, the size was remarkably increased compared to the cartilage-like pellet derived from bone marrow-derived mesenchymal stem cells, which are mesenchymal stem cells derived from other tissues. This demonstrates that umbilical cord blood-derived mesenchymal stem cells are a powerful source of mesenchymal stem cells in cartilage regeneration.
（実施例２）Ｉｎ ｖｉｖｏでの実験
直径５ｍｍ、高さ３ｍｍからなるコラーゲンスポンジに臍帯血由来間葉幹系細胞を１×１０６個包埋し、軟骨分化誘導培地で２週間培養した。
その後、細胞を含むコラーゲンスポンジを５週齢のヌードマウス皮下に移植した。
(Example 2) In vivo experiment 1 × 10 6 cord blood-derived mesenchymal stem cells were embedded in a collagen sponge having a diameter of 5 mm and a height of 3 mm, and cultured in a cartilage differentiation-inducing medium for 2 weeks.
Thereafter, a collagen sponge containing cells was transplanted subcutaneously into 5-week-old nude mice.
その結果、図２（ａ）に示されるように、移植後４週目で白く堅い組織が形成された。その組織のパラフィン切片を作製し、図２（ｂ）に示されるようにトルイジンブルー染色したところ、図２（ｃ）に示されるようにメタクロマジーに染色された円形の軟骨様細胞がみられた。また、軟骨細胞マーカーのタイプＩＩコラーゲンに対する抗体により免疫染色を行った結果、図２（ｄ）に示されるように、円形の軟骨様細胞の周囲に陽性反応が示された。
これらの結果から、臍帯血由来間葉系幹細胞はヌードラット関節下環境において軟骨形成することが明らかになった。
As a result, as shown in FIG. 2 (a), a white and hard tissue was formed 4 weeks after transplantation. A paraffin section of the tissue was prepared and stained with toluidine blue as shown in FIG. 2 (b). As shown in FIG. 2 (c), circular chondrocyte-like cells stained with metachrome were observed. Further, as a result of immunostaining with an antibody against chondrocyte marker type II collagen, a positive reaction was shown around circular chondroid cells as shown in FIG. 2 (d).
These results revealed that umbilical cord blood-derived mesenchymal stem cells form cartilage in the nude rat subarticular environment.
（実施例３）任意の軟骨組織の製造、培養中のスキャホールドへの刺激
実施例２における臍帯血由来間葉系幹細胞の培養中、培養液交換時または１日おきにスキャホールド上に１ｇのウエイトを置いて６０Ｇで１分間の遠心力を与えた。
その結果、コラーゲンのスキャホールドは、細胞が均質に分布した移植しやすい平板状の形状に変化した。さらに、ウエイトを置かないものに比べて軟骨形成能優位であった。
なお、上記実施形態においてはスキャホールドとしては、コラーゲンを用いてきたが、これに代えてゼラチンでも同様の培養軟骨が形成される。
(Example 3) Manufacture of arbitrary cartilage tissue, stimulation of scaffold during culture During culture of umbilical cord blood-derived mesenchymal stem cells in Example 2, 1 g of scaffold was exchanged at the time of culture medium exchange or every other day A weight was applied and a centrifugal force of 1 minute was applied at 60 G.
As a result, the collagen scaffold was changed to a flat plate shape in which cells were uniformly distributed and easily transplanted. Furthermore, the cartilage formation ability was superior to those without weight.
In the above embodiment, collagen has been used as the scaffold. However, similar cultured cartilage can be formed with gelatin instead.
Ｓ１ 播種ステップ
Ｓ２ 培養ステップ
S1 sowing step S2 culture step
Claims (2)
該スキャホールドに播種された前記臍帯血由来間葉系幹細胞を培養し、
前記臍帯血由来間葉系幹細胞を培養する間、前記スキャホールドおよび臍帯血由来間葉系幹細胞に応力を与えて培養軟骨を形成し、
前記スキャホールドは、気孔率が７５−９０％になるような連続した気孔構造を持つ多孔質のコラーゲンまたはゼラチンからなる培養軟骨製造方法。 Seeding cord blood-derived mesenchymal stem cells in a porous scaffold,
Culturing the umbilical cord blood-derived mesenchymal stem cells seeded in the scaffold,
While culturing the cord blood-derived mesenchymal stem cells, stress is applied to the scaffold and cord blood-derived mesenchymal stem cells to form cultured cartilage ,
The scaffold is a porous collagen or gelatin Tona Ru cultured cartilage production method with continuous pore structure as the porosity becomes 75-90%.
該スキャホールドに播種された前記臍帯血由来間葉系幹細胞を培養し、
前記臍帯血由来間葉系幹細胞を培養する間、前記スキャホールドおよび臍帯血由来間葉系幹細胞に応力を与えて製造され、
前記スキャホールドは、気孔率が７５−９０％になるような連続した気孔構造を持つ多孔質のコラーゲンまたはゼラチンからなる培養軟骨。 Seeding cord blood-derived mesenchymal stem cells in a porous scaffold,
Culturing the umbilical cord blood-derived mesenchymal stem cells seeded in the scaffold,
While culturing the umbilical cord blood-derived mesenchymal stem cells, the scaffold and umbilical cord blood-derived mesenchymal stem cells are produced by applying stress .
The scaffold is a porous collagen or gelatin Tona Ru cultured cartilage with a continuous pore structure as the porosity becomes 75-90%.
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