JP2020006130A - Method for producing decellularized tissue - Google Patents
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Abstract
Description
本発明は、脱細胞化組織の製造方法に関する。 The present invention relates to a method for producing a decellularized tissue.
再生医療において、患者の欠損した器官を再生するための支持組織として、ヒトまたは異種哺乳動物の生体組織から、細胞質成分、細胞質ゾル成分、細胞骨格、細胞膜成分等の細胞成分が除去されている脱細胞化組織が再移植されている。脱細胞化組織は、エラスチン、コラーゲン（Ｉ型、ＩＶ型等）、ラミニン等の細胞外マトリックス成分を主成分とする。 In regenerative medicine, as a supporting tissue for regenerating a defective organ of a patient, a cellular tissue such as a cytoplasmic component, a cytosolic component, a cytoskeleton, or a cell membrane component is removed from a living tissue of a human or a heterologous mammal. Cellular tissue has been reimplanted. The decellularized tissue has as its main component an extracellular matrix component such as elastin, collagen (type I, type IV, etc.), laminin and the like.
特許文献１には、ポリエチレングリコール水溶液を用いて、生体組織の細胞を破壊する工程と、ＤＮａｓｅを用いて、細胞が破壊された生体組織に含まれるＤＮＡを分解させる工程と、ＰＢＳを用いて、ＤＮＡが分解した生体組織を洗浄する工程を含む脱細胞化組織の製造方法が開示されている（例えば、特許文献１参照）。 Patent Literature 1 discloses a process of destroying cells in a living tissue using an aqueous solution of polyethylene glycol, a process of decomposing DNA contained in the living tissue in which the cells have been destroyed by using DNase, and a process of using PBS. A method for producing a decellularized tissue including a step of washing a living tissue in which DNA has been degraded is disclosed (for example, see Patent Document 1).
しかしながら、上述したような技術では、得られた脱細胞化組織（支持組織）には、脂溶性成分が残存しているため、脱細胞化組織（支持組織）に細胞を接着させようとしても、脱細胞化組織（支持組織）に細胞が接着しにくいという問題があった。 However, in the technique as described above, since the obtained decellularized tissue (supporting tissue) has a fat-soluble component remaining, even if the cells are to be adhered to the decellularized tissue (supporting tissue), There is a problem that cells are hardly adhered to the decellularized tissue (supporting tissue).
本発明の一態様は、脱細胞化組織の細胞接着性を向上させることが可能な脱細胞化組織の製造方法を提供することを目的とする。 An object of one embodiment of the present invention is to provide a method for producing a decellularized tissue capable of improving the cell adhesion of the decellularized tissue.
本発明の一態様は、脱細胞化組織の製造方法において、細胞が破壊されている生体組織に含まれるＤＮＡを分解させる工程と、液化ガスを含む流体を用いて、該ＤＮＡが分解した生体組織を脱脂する工程を含む。 One aspect of the present invention is a method for producing a decellularized tissue, wherein the step of decomposing the DNA contained in the living tissue in which the cells have been destroyed, and the use of a fluid containing a liquefied gas, Degreasing.
本発明の一態様によれば、脱細胞化組織の細胞接着性を向上させることが可能な脱細胞化組織の製造方法を提供することができる。 According to one aspect of the present invention, it is possible to provide a method for producing a decellularized tissue capable of improving the cell adhesion of the decellularized tissue.
以下、本発明の実施形態を図面を参照して説明する。 Hereinafter, embodiments of the present invention will be described with reference to the drawings.
（脱細胞化組織の製造方法）
図１に、本実施形態の脱細胞化組織の製造方法の一例を示す。
(Method for producing decellularized tissue)
FIG. 1 shows an example of a method for producing a decellularized tissue according to the present embodiment.
脱細胞化組織の製造方法は、生体組織の細胞を破壊する工程（Ｓ０）と、細胞が破壊された生体組織に含まれるＤＮＡを分解する工程（Ｓ１）と、液化ガスを含む流体を用いて、ＤＮＡが分解された生体組織を脱脂する工程（Ｓ２）を含む。このため、ＤＮＡが分解した生体組織から脂溶性成分を十分に除去することができ、脱細胞化組織の細胞接着性を向上させることができる。 The method for producing a decellularized tissue includes a step of destroying cells in a living tissue (S0), a step of decomposing DNA contained in the living tissue in which the cells have been destroyed (S1), and using a fluid containing liquefied gas And delipidating the living tissue in which the DNA has been degraded (S2). Therefore, the fat-soluble component can be sufficiently removed from the living tissue in which the DNA has been degraded, and the cell adhesion of the decellularized tissue can be improved.
本明細書及び特許請求の範囲において、脱脂とは、生体組織より脂溶性成分を除去することである。 In the present specification and claims, defatting refers to removing fat-soluble components from living tissue.
工程（Ｓ０）では、例えば、生体組織に細胞を破壊することが可能な液体を接触させる。 In the step (S0), for example, a liquid capable of destroying cells is brought into contact with a living tissue.
細胞を破壊することが可能な液体としては、特に限定されないが、界面活性剤、高圧水、液化ジメチルエーテル又は超臨界二酸化炭素を含む液体が好ましい。 The liquid that can destroy cells is not particularly limited, but a liquid containing a surfactant, high-pressure water, liquefied dimethyl ether, or supercritical carbon dioxide is preferable.
工程（Ｓ１）では、例えば、細胞が破壊されている生体組織に、ＤＮＡを分解することが可能な液体を接触させる。 In the step (S1), for example, a liquid capable of decomposing DNA is brought into contact with a living tissue in which cells are destroyed.
ＤＮＡを分解することが可能な液体としては、特に限定されないが、酵素又は界面活性剤を含む液体が好ましい。 The liquid capable of decomposing DNA is not particularly limited, but a liquid containing an enzyme or a surfactant is preferable.
酵素としては、特に限定されないが、ＤＮａｓｅ（例えば、ＤＮａｓｅＩ）、トリプシン等が挙げられる。 Examples of the enzyme include, but are not particularly limited to, DNase (for example, DNase I), trypsin, and the like.
界面活性剤としては、特に限定されないが、イオン系界面活性剤（例えば、ドデシル硫酸ナトリウム（ＳＤＳ））、非イオン系界面活性剤（例えば、ＴｒｉｔｏｎＸ−１００）等が挙げられる。 The surfactant is not particularly limited, and examples thereof include an ionic surfactant (for example, sodium dodecyl sulfate (SDS)) and a nonionic surfactant (for example, Triton X-100).
液体としては、生理的に適合すれば、特に限定されないが、生理食塩水、ＰＢＳ（リン酸緩衝化生理食塩水）等が挙げられ、二種以上を併用してもよい。これらの中でも、生理食塩水が好ましい。 The liquid is not particularly limited as long as it is physiologically compatible. Examples of the liquid include physiological saline and PBS (phosphate-buffered physiological saline). Two or more liquids may be used in combination. Of these, physiological saline is preferred.
細胞が破壊されている生体組織に、ＤＮＡを分解することが可能な液体を接触させる方法としては、特に限定されないが、細胞が破壊されている生体組織と、ＤＮＡを分解することが可能な液体を混合して撹拌する方法、細胞が破壊されている生体組織に、ＤＮＡを分解することが可能な液体を流通させる方法等が挙げられる。これらの中でも、ＤＮＡを分解することが可能な液体と細胞が破壊されている生体組織との接触の効率が高く、効率的にＤＮＡを分解することが可能な点で、細胞が破壊されている生体組織に、ＤＮＡを分解することが可能な液体を流通させる方法が好ましい。 The method for bringing a liquid capable of decomposing DNA into contact with a living tissue in which cells are destroyed is not particularly limited, and a living tissue in which cells are destroyed and a liquid capable of decomposing DNA are used. And a method in which a liquid capable of decomposing DNA is passed through living tissue in which cells are destroyed. Among these, cells are destroyed in that the efficiency of contact between a liquid capable of degrading DNA and a living tissue in which cells are destroyed is high, and the DNA can be degraded efficiently. A method of flowing a liquid capable of decomposing DNA through a living tissue is preferable.
なお、細胞が破壊されている生体組織と、ＤＮＡを分解することが可能な液体を混合して撹拌する場合、ＤＮＡを分解することが可能な液体を適宜交換してもよい。 When the living tissue in which the cells are destroyed and the liquid capable of decomposing DNA are mixed and stirred, the liquid capable of decomposing DNA may be appropriately replaced.
細胞が破壊されている生体組織に、ＤＮＡを分解することが可能な液体を接触させる環境の温度は、４〜４０℃であることが好ましい。細胞が破壊されている生体組織に、ＤＮＡを分解することが可能な液体を接触させる温度が４℃以上であると、生体組織に含まれる細胞外マトリックスの氷結晶による損傷を抑制することができ、４０℃以下であると、生体組織に含まれるタンパク質の変性を抑制することができる。 The temperature of the environment in which a liquid capable of decomposing DNA is brought into contact with living tissue in which cells are destroyed is preferably 4 to 40 ° C. When the temperature at which the liquid capable of decomposing DNA is brought into contact with the living tissue in which the cells are destroyed is 4 ° C. or higher, damage due to ice crystals of the extracellular matrix contained in the living tissue can be suppressed. , 40 ° C. or lower, denaturation of proteins contained in living tissue can be suppressed.
工程（Ｓ２）では、例えば、工程（Ｓ１）により、ＤＮＡが分解された生体組織に、液化ガスを含む流体を接触させる。 In the step (S2), for example, a fluid containing a liquefied gas is brought into contact with the living tissue in which the DNA has been degraded in the step (S1).
本明細書及び特許請求の範囲において、液化ガスとは、常温常圧（０℃、１ａｔｍ（０．１０１３２５ＭＰａ）で気体である物質の液化物である。 In the present specification and claims, the liquefied gas is a liquefied substance that is a gas at normal temperature and normal pressure (0 ° C., 1 atm (0.101325 MPa)).
液化ガスとしては、生体組織を脱脂することが可能であれば、特に限定されないが、ジメチルエーテル、エチルメチルエーテル、ホルムアルデヒド、ケテン、アセトアルデヒド、プロパン、ブタン、液化石油ガス等が挙げられ、二種以上を併用してもよい。これらの中でも、比較的低温低圧で液化する点で、エチルメチルエーテル、ジメチルエーテルが好ましく、ジメチルエーテルが特に好ましい。 The liquefied gas is not particularly limited as long as it is capable of defatting a living tissue, and includes dimethyl ether, ethyl methyl ether, formaldehyde, ketene, acetaldehyde, propane, butane, liquefied petroleum gas, and the like. You may use together. Among these, ethyl methyl ether and dimethyl ether are preferable, and dimethyl ether is particularly preferable, in that liquefaction is performed at a relatively low temperature and low pressure.
ジメチルエーテルは、１〜４０℃、０．２〜５ＭＰａ程度で液化するため、装置のコストが安価となる。また、液化ジメチルエーテルは、常温常圧下で容易に気化することから、脱細胞化組織に残留しにくい。 Since dimethyl ether is liquefied at 1 to 40 ° C. and about 0.2 to 5 MPa, the cost of the apparatus is reduced. In addition, liquefied dimethyl ether is easily vaporized at normal temperature and normal pressure, and therefore hardly remains in the decellularized tissue.
工程（Ｓ２）は、液化ガスの液体状態を維持するため、気密状態の抽出槽内等の飽和蒸気圧以上の環境下で実施される。 The step (S2) is performed in an environment having a saturation vapor pressure or higher, such as in an airtight extraction tank, in order to maintain the liquid state of the liquefied gas.
ＤＮＡが分解された生体組織に、液化ガスを含む流体を接触させる方法としては、特に限定されないが、ＤＮＡが分解された生体組織を、液化ガスを含む流体に浸漬する方法等が挙げられる。 The method for bringing the fluid containing a liquefied gas into contact with the biological tissue in which the DNA has been decomposed is not particularly limited, and examples thereof include a method in which the biological tissue in which the DNA has been decomposed is immersed in a fluid containing the liquefied gas.
液化ガスを含む流体は、液化ガスとは異なる液体をさらに含んでいてもよい。 The fluid including the liquefied gas may further include a liquid different from the liquefied gas.
液化ガスとは異なる液体としては、生理的に適合すれば、特に限定されないが、生理食塩水、ＰＢＳ（リン酸緩衝化生理食塩水）等が挙げられ、二種以上を併用してもよい。これらの中でも、生理食塩水が好ましい。 The liquid different from the liquefied gas is not particularly limited as long as it is physiologically compatible, and examples thereof include physiological saline and PBS (phosphate buffered physiological saline). Two or more liquids may be used in combination. Of these, physiological saline is preferred.
液化ガスとは異なる液体の添加量は、液化ガス中の溶解度以下となるようにすることが好ましい。これにより、液化ガスを含む流体を均一にすることができる。 It is preferable that the added amount of the liquid different from the liquefied gas be equal to or less than the solubility in the liquefied gas. Thereby, the fluid containing the liquefied gas can be made uniform.
ＤＮＡが分解された生体組織に、液化ガスを含む流体を接触させた後、常温常圧に戻すと、液化ガスは、気化するため、ＤＮＡが分解された生体組織から液化ガスが除去される。 When the fluid containing the liquefied gas is brought into contact with the living tissue in which the DNA has been decomposed, if the temperature is returned to normal temperature and normal pressure, the liquefied gas is vaporized, so that the liquefied gas is removed from the living tissue in which the DNA has been decomposed.
脱細胞化組織の製造方法は、脱脂された生体組織を洗浄する工程（Ｓ３）をさらに含んでいてもよい。 The method for producing a decellularized tissue may further include a step of washing the delipidated living tissue (S3).
本明細書及び特許請求の範囲において、洗浄とは、生体組織から水溶性成分を除去することである。 In the present specification and claims, washing refers to removing water-soluble components from living tissue.
工程（Ｓ３）では、例えば、工程（Ｓ２）により、脱脂された生体組織に、生体組織を洗浄することが可能な液体を接触させる。 In the step (S3), for example, a liquid capable of washing the living tissue is brought into contact with the delipidized living tissue in the step (S2).
生体組織を洗浄することが可能な液体としては、生理的に適合すれば、特に限定されないが、生理食塩水、ＰＢＳ（リン酸緩衝化生理食塩水）等が挙げられ、二種以上を併用してもよい。これらの中でも、生理食塩水が好ましい。 The liquid capable of washing the living tissue is not particularly limited as long as it is physiologically compatible, and examples thereof include physiological saline and PBS (phosphate buffered physiological saline). You may. Of these, physiological saline is preferred.
脱脂された生体組織に、生体組織を洗浄することが可能な液体を接触させる方法としては、特に限定されないが、脱脂された生体組織と、生体組織を洗浄することが可能な液体を混合して撹拌する方法、脱脂された生体組織に、生体組織を洗浄することが可能な液体を流通させる方法等が挙げられる。これらの中でも、効率的に洗浄することが可能な点で、脱脂された生体組織に、生体組織を洗浄することが可能な液体を流通させる方法が好ましい。 The method of contacting the defatted living tissue with a liquid capable of washing the living tissue is not particularly limited, and the defatted living tissue is mixed with a liquid capable of washing the living tissue. A method of stirring, a method of flowing a liquid capable of washing the living tissue through the delipidated living tissue, and the like can be given. Among these, a method of flowing a liquid capable of washing the living tissue through the delipidated living tissue is preferable in that the washing can be performed efficiently.
なお、脱脂された生体組織と、生体組織を洗浄することが可能な液体を混合して撹拌する場合、生体組織を洗浄することが可能な液体を適宜交換してもよい。 In addition, when mixing and stirring the delipidated living tissue and a liquid capable of washing the living tissue, the liquid capable of washing the living tissue may be appropriately replaced.
脱脂された生体組織に、生体組織を洗浄することが可能な液体を接触させる環境の温度は、４〜４０℃であることが好ましい。脱脂された生体組織に、生体組織を洗浄することが可能な液体を接触させる環境の温度が４℃以上であると、生体組織に含まれる細胞外マトリックスの氷結晶による損傷を抑制することができ、４０℃以下であると、生体組織に含まれるタンパク質の変性を抑制することができる。 The temperature of the environment where the delipidated living tissue is brought into contact with a liquid capable of washing the living tissue is preferably 4 to 40 ° C. When the temperature of the environment in which the liquid capable of washing the living tissue is brought into contact with the delipidized living tissue is 4 ° C. or higher, damage due to ice crystals of the extracellular matrix contained in the living tissue can be suppressed. , 40 ° C. or lower, denaturation of proteins contained in living tissue can be suppressed.
（生体組織の細胞破壊方法）
生体組織の細胞を破壊する際には、例えば、生体組織に、細胞を破壊することが可能な液体を接触させる。
(Method of destroying cells in living tissue)
When destroying cells of a living tissue, for example, a liquid capable of destroying the cells is brought into contact with the living tissue.
細胞を破壊することが可能な液体としては、特に限定されないが、液化ガスを含む液体、界面活性剤を含む液体等が挙げられる。これらの中でも、脱細胞化組織に、実質的に損傷がなく、液化ガスが残留しにくくなる点で、液化ガスを含む液体が好ましい。 The liquid capable of destroying cells is not particularly limited, but includes a liquid containing a liquefied gas, a liquid containing a surfactant, and the like. Among them, a liquid containing a liquefied gas is preferable, since the decellularized tissue is not substantially damaged and the liquefied gas hardly remains.
ここで、液化ガスを含む液体は、細胞膜成分を溶解するため、生体組織の細胞を破壊することができる。 Here, the liquid containing the liquefied gas dissolves cell membrane components, and thus can destroy cells of a living tissue.
液化ガスとしては、生体組織の細胞を破壊することが可能であれば、特に限定されないが、ジメチルエーテル、エチルメチルエーテル、ホルムアルデヒド、ケテン、アセトアルデヒド、プロパン、ブタン、液化石油ガス等が挙げられ、二種以上を併用してもよい。これらの中でも、比較的低温低圧で液化する点で、エチルメチルエーテル、ジメチルエーテルが好ましく、ジメチルエーテルが特に好ましい。 The liquefied gas is not particularly limited as long as it can destroy cells of a living tissue, and includes dimethyl ether, ethyl methyl ether, formaldehyde, ketene, acetaldehyde, propane, butane, liquefied petroleum gas, and the like. The above may be used in combination. Among these, ethyl methyl ether and dimethyl ether are preferable, and dimethyl ether is particularly preferable, in that liquefaction is performed at a relatively low temperature and low pressure.
ジメチルエーテルは、１〜４０℃、０．２〜５ＭＰａ程度で液化するため、装置のコストが安価となる。また、液化ジメチルエーテルは、常温常圧下で容易に気化することから、脱細胞化組織に残留しにくい。 Since dimethyl ether is liquefied at 1 to 40 ° C. and about 0.2 to 5 MPa, the cost of the apparatus is reduced. In addition, liquefied dimethyl ether is easily vaporized at normal temperature and normal pressure, and therefore hardly remains in the decellularized tissue.
液化ガスを含む液体を生体組織に接触させる場合は、液化ガスの液体状態を維持するため、気密状態の抽出槽内等の飽和蒸気圧以上の環境下で実施される。 When a liquid containing a liquefied gas is brought into contact with a living tissue, the liquid is kept in an airtight state in an extraction tank or the like under an environment of a saturated vapor pressure or higher in order to maintain the liquid state of the liquefied gas.
細胞を破壊することが可能な液体を生体組織に接触させる方法としては、特に限定されないが、細胞を破壊することが可能な液体に生体組織を浸漬する方法等が挙げられる。 The method for bringing a liquid capable of destroying cells into contact with a living tissue is not particularly limited, and examples thereof include a method of immersing a living tissue in a liquid capable of destroying cells.
液化ガスを含む液体は、液化ガスとは異なる液体をさらに含んでいてもよい。 The liquid containing a liquefied gas may further contain a liquid different from the liquefied gas.
液化ガスとは異なる液体としては、特に限定されないが、エタノール、水、生理食塩水、ＰＢＳ（リン酸緩衝化生理食塩水）等が挙げられ、二種以上を併用してもよい。 The liquid different from the liquefied gas is not particularly limited, but includes ethanol, water, physiological saline, PBS (phosphate buffered physiological saline) and the like, and two or more kinds may be used in combination.
液化ガスとは異なる液体の添加量は、液化ガス中の溶解度以下となるようにすることが好ましい。これにより、液化ガスを含む液体を均一にすることができる。 It is preferable that the added amount of the liquid different from the liquefied gas be equal to or less than the solubility in the liquefied gas. Thereby, the liquid containing the liquefied gas can be made uniform.
液化ガスの温度は、１〜４０℃であることが好ましく、１０〜３０℃であることがさらに好ましい。 The temperature of the liquefied gas is preferably 1 to 40C, more preferably 10 to 30C.
液化ガスの圧力は、０．２〜５ＭＰａであることが好ましく、０．３〜０．７ＭＰａの範囲内であることがさらに好ましい。 The pressure of the liquefied gas is preferably from 0.2 to 5 MPa, and more preferably from 0.3 to 0.7 MPa.
液化ガスを含む液体を生体組織に接触させた後、常温常圧に戻すと、液化ガスは、気化するため、液化ガスを簡便に除去することができる。 When the liquid containing the liquefied gas is brought into contact with the living tissue and then returned to normal temperature and normal pressure, the liquefied gas is vaporized, so that the liquefied gas can be easily removed.
なお、液化ガスを含む液体を生体組織に接触させる工程を複数回繰り返してもよい。 The step of bringing the liquid containing the liquefied gas into contact with the living tissue may be repeated a plurality of times.
（生体組織）
生体組織としては、細胞壁を有する植物、菌類、古細菌、真正細菌、もしくは細胞壁を有しない動物のいずれかから得られる組織のことを意味する。特に限定されないが、葉、枝、樹木、花弁、茎、根、果肉、果皮及び種子や、ヒトまたは異種哺乳動物由来の皮膚、血管、心臓弁膜、角膜、羊膜、硬膜等を含む軟組織またはその一部、心臓、腎臓、肝臓、膵臓、脳等を含む臓器またはその一部等、骨、軟骨、腱またはその一部等が挙げられる。
(Living tissue)
Biological tissue means a tissue obtained from any of plants, fungi, archaea, eubacteria, or animals without a cell wall having a cell wall. Although not particularly limited, leaves, branches, trees, petals, stems, roots, pulp, pericarp, and seeds, and human or xenogeneic mammal-derived skin, blood vessels, heart valves, cornea, amniotic membrane, dura mater, and the like, or soft tissues thereof A part thereof includes an organ including a heart, a kidney, a liver, a pancreas, a brain, or a part thereof, and a bone, a cartilage, a tendon, a part thereof, and the like.
以下に、本発明の実施例を説明するが、本発明は、実施例に限定されない。 Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to the embodiments.
［実施例１］
（細胞が破壊されたラットの由来の脳組織のＤＮＡ分解、脱脂及び洗浄）
細胞が破壊されたラット由来の脳組織を、ＤＮａｓｅＩ（ロシュ・ダイアグノスティックス社製）、ＭｇＣｌ２（和光純薬工業社製）を含む生理食塩水に入れた後、振とうさせ、細胞が破壊されたラット由来の脳組織に含まれるＤＮＡを分解した。次に、ＤＮＡが分解されたラット由来の脳組織を、液化ジメチルエーテルに接触させ、脱脂した。次に、脱脂されたブタ由来の脳組織を、生理食塩水に入れた後、振とうさせ、洗浄した。その結果、ラット由来の脳組織の脱細胞化組織を得た。
[Example 1]
(DNA degradation, defatting and washing of brain tissue derived from a rat whose cells have been destroyed)
The rat-derived brain tissue whose cells have been destroyed is placed in a physiological saline solution containing DNase I (Roche Diagnostics) and MgCl 2 (Wako Pure Chemical Industries), and shaken. DNA contained in the brain tissue from the disrupted rat was degraded. Next, the rat-derived brain tissue from which the DNA was degraded was brought into contact with liquefied dimethyl ether and defatted. Next, the defatted pig-derived brain tissue was placed in physiological saline, shaken, and washed. As a result, a decellularized tissue of rat brain tissue was obtained.
［比較例１］
ＤＮＡが分解されたラット由来の脳組織を脱脂する際に、液化ジメチルエーテルの代わりに、エタノールを用いた以外は、実施例１と同様にして、ラット由来の脳組織の脱細胞化組織を得た。
[Comparative Example 1]
A decellularized tissue of rat-derived brain tissue was obtained in the same manner as in Example 1 except that ethanol was used instead of liquefied dimethyl ether when the DNA-degraded rat-derived brain tissue was delipidated. .
次に、脱細胞化組織の細胞接着性を評価した。 Next, the cell adhesion of the decellularized tissue was evaluated.
［脱細胞化組織の細胞接着性］
実施例１及び比較例１のラット由来の脳組織の脱細胞化組織と、ラット由来の脳組織に、神経細胞を播種し、７日経過した後、神経細胞の存在位置において蛍光を発する蛍光色素を導入したサンプルの断面を、光学顕微鏡を用いて観察した。
[Cell adhesion of decellularized tissue]
Fluorescent dye that emits fluorescence at the position where the nerve cells are present after 7 days have passed since neurons were seeded on the decellularized tissue of the rat-derived brain tissue and the rat-derived brain tissue of Example 1 and Comparative Example 1. Was observed using an optical microscope.
図３に、脱細胞化組織の細胞接着性の評価結果を示す。なお、図３の左側がラット由来の脳組織の脱細胞化組織であり、右側がラット由来の脳組織である。 FIG. 3 shows the results of evaluating the cell adhesion of the decellularized tissue. The left side of FIG. 3 is a decellularized tissue of rat-derived brain tissue, and the right side is rat-derived brain tissue.
図３から、実施例１のラット由来の脳組織の脱細胞化組織は、比較例１のラット由来の脳組織の脱細胞化組織に比べて、蛍光を発する領域（図中、点線部）が大きいことから、液化ジメチルエーテルを用いて、ＤＮＡが分解されたラット由来の脳組織を脱脂することで、脱細胞化組織の細胞接着性が向上していることがわかる。 From FIG. 3, the decellularized tissue of the rat-derived brain tissue of Example 1 has a fluorescent region (dotted line in the figure) as compared to the decellularized tissue of the rat-derived brain tissue of Comparative Example 1. The large size indicates that the cell adhesion of the decellularized tissue is improved by delipidating rat-derived brain tissue from which DNA has been degraded using liquefied dimethyl ether.
［実施例２］
（ブタ由来の大動脈組織の細胞破壊）
図２に示す細胞破壊装置を用いて、ブタ由来の大動脈組織５５の細胞を破壊した。
[Example 2]
(Cell destruction of pig-derived aortic tissue)
The cells of the aortic tissue 55 derived from pigs were destroyed using the cell disrupting apparatus shown in FIG.
具体的には、抽出槽５４に、ブタ由来の大動脈組織５５を仕込んだ。次に、分離槽６０を予めジメチルエーテルで置換し、バルブ５２、５３、５６、５８、５９を閉状態とした。次に、背圧弁５７の設定圧力を０．７ＭＰａとし、バルブ５２、５３、５６、５８を開状態とし、ポンプ５１を用いて、液化ジメチルエーテルを流通させ、液化ジメチルエーテルで抽出槽５４が満たされたところで、バルブ５３、５６を閉状態とし、ブタ由来の大動脈組織５５を液化ジメチルエーテルに浸漬し、リン脂質等の細胞膜成分を抽出した。次に、バルブ５３、５６を開状態とし、液化ジメチルエーテルの流量を１ｍＬ／ｍｉｎに調整して、抽出液を分離槽６０で回収した。次に、バルブ５８を閉状態とし、分離槽６０を装置から取り外し、ドラフト内で大気圧として、液化ジメチルエーテルを揮発させた。 Specifically, swine-derived aortic tissue 55 was charged into the extraction tank 54. Next, the separation tank 60 was replaced with dimethyl ether in advance, and the valves 52, 53, 56, 58, and 59 were closed. Next, the set pressure of the back pressure valve 57 was set to 0.7 MPa, the valves 52, 53, 56, and 58 were opened, and liquefied dimethyl ether was passed using the pump 51, and the extraction tank 54 was filled with liquefied dimethyl ether. By the way, the valves 53 and 56 were closed, and the aortic tissue 55 derived from pig was immersed in liquefied dimethyl ether to extract cell membrane components such as phospholipids. Next, the valves 53 and 56 were opened, the flow rate of liquefied dimethyl ether was adjusted to 1 mL / min, and the extract was collected in the separation tank 60. Next, the valve 58 was closed, the separation tank 60 was removed from the apparatus, and the liquefied dimethyl ether was volatilized at atmospheric pressure in the fume hood.
上記の操作により、液化ジメチルエーテル６０ｍＬとブタ由来の大動脈組織５５を接触させた後、バルブ５３を閉状態とし、バルブ５６、５８、５９を開状態とし、抽出槽５４内の圧力を大気圧とし、抽出槽５４内の液化ジメチルエーテルを揮発させて排気した。その結果、細胞が破壊されたブタ由来の大動脈組織を得た。 By the above operation, after contacting 60 mL of liquefied dimethyl ether with the aortic tissue 55 derived from pig, the valve 53 is closed, the valves 56, 58, and 59 are opened, and the pressure in the extraction tank 54 is set to atmospheric pressure. The liquefied dimethyl ether in the extraction tank 54 was volatilized and exhausted. As a result, swine-derived aortic tissue in which cells were destroyed was obtained.
（細胞が破壊されたブタ由来の大動脈組織のＤＮＡ分解、脱脂及び洗浄）
細胞が破壊されたブタ由来の大動脈組織を、０．２ｍｇ／ｍＬのＤＮａｓｅＩ（ロシュ・ダイアグノスティックス社製）、０．０５ＭのＭｇＣｌ２（和光純薬工業社製）を含む生理食塩水に入れた後、振とうさせ、細胞が破壊されたブタ由来の大動脈組織に含まれるＤＮＡを分解させた。次に、ＤＮＡが分解したブタ由来の大動脈組織を、液化ジメチルエーテルに接触させ、脱脂した。次に、脱脂されたブタ由来の大動脈組織を、生理食塩水に入れた後、振とうさせ、洗浄した。その結果、ブタ由来の大動脈組織の脱細胞化組織を得た。
(DNA degradation, delipidation and washing of aortic tissue derived from swine from which cells were destroyed)
The swine-derived aortic tissue from which cells have been destroyed is placed in physiological saline containing 0.2 mg / mL of DNase I (manufactured by Roche Diagnostics) and 0.05 M of MgCl 2 (manufactured by Wako Pure Chemical Industries). After that, the cells were shaken to decompose DNA contained in the aortic tissue derived from pigs whose cells were destroyed. Next, the pig-derived aortic tissue from which the DNA had been degraded was brought into contact with liquefied dimethyl ether and defatted. Next, the defatted swine-derived aortic tissue was placed in physiological saline, shaken, and washed. As a result, a decellularized tissue of an aortic tissue derived from pig was obtained.
［比較例２］
ＤＮＡが分解されたラット由来の脳組織を脱脂する際に、液化ジメチルエーテルの代わりに、エタノールを用いた以外は、実施例２と同様にして、ブタ由来の大動脈組織の脱細胞化組織を得た。
[Comparative Example 2]
A decellularized tissue of swine-derived aortic tissue was obtained in the same manner as in Example 2 except that ethanol was used instead of liquefied dimethyl ether when delipidating rat-derived brain tissue in which DNA was degraded. .
［実施例３〜６］
生体組織として、ブタ由来の大動脈組織の代わりに、ブタ由来の海綿骨組織、ブタ由来の皮膚組織、ブタ由来の骨組織、ブタ由来の軟骨組織を用いた点以外は、実施例２と同様にして、脱細胞化組織を得た。
[Examples 3 to 6]
In the same manner as in Example 2 except that a porcine-derived cancellous bone tissue, a porcine-derived skin tissue, a porcine-derived bone tissue, and a porcine-derived cartilage tissue were used as living tissues instead of the porcine-derived aortic tissue. Thus, a decellularized tissue was obtained.
［比較例３〜６］
生体組織として、ブタ由来の大動脈組織の代わりに、ブタ由来の海綿骨組織、ブタ由来の皮膚組織、ブタ由来の骨組織、ブタ由来の軟骨組織を用いた点以外は、比較例２と同様にして、脱細胞化組織を得た。
[Comparative Examples 3 to 6]
The same procedure as in Comparative Example 2 was carried out except that porcine-derived trabecular bone tissue, porcine-derived skin tissue, porcine-derived bone tissue, and porcine-derived cartilage tissue were used as living tissues instead of porcine-derived aortic tissue. Thus, a decellularized tissue was obtained.
実施例２〜６及び比較例２〜６の脱細胞化組織の細胞接着性を評価したところ、いずれの組織においても、液化ジメチルエーテルを用いて、ＤＮＡが分解されたブタ由来の生体組織を脱脂することで、脱細胞化組織の細胞接着性が向上していることがわかった。 When the cell adhesion of the decellularized tissues of Examples 2 to 6 and Comparative Examples 2 to 6 was evaluated, in any of the tissues, liquefied dimethyl ether was used to delipidate DNA-degraded swine-derived living tissue. Thus, it was found that the cell adhesion of the decellularized tissue was improved.
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