JP4463124B2 - Method for reducing the amount of cells contained in living tissue and acellularized living tissue - Google Patents
Method for reducing the amount of cells contained in living tissue and acellularized living tissue Download PDFInfo
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- 239000012299 nitrogen atmosphere Substances 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 6
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- 210000003954 umbilical cord Anatomy 0.000 claims description 4
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- 238000010257 thawing Methods 0.000 claims description 3
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- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 1
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- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
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- VEZXCJBBBCKRPI-UHFFFAOYSA-N beta-propiolactone Chemical compound O=C1CCO1 VEZXCJBBBCKRPI-UHFFFAOYSA-N 0.000 description 1
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Description
本発明は、生体組織に含まれる細胞量を減少させる方法及び無細胞化された生体組織に関する。特に、本発明は、生体組織、例えば、角膜を無細胞化する方法及び無細胞化された角膜に関する。 The present invention relates to a method for reducing the amount of cells contained in a living tissue and a decellularized living tissue. In particular, the present invention relates to a method for decellularizing a living tissue, for example, the cornea and a decornized cornea.
角膜疾患における最終的な治療方法は角膜移植である。しかし、日本においては、移植に用いられる角膜の入手は必ずしも容易ではなく、移植待機患者も多いのが、実態である。そこで、考えられるのが異種移植である。しかしながら、異種移植では、拒絶反応、免疫反応も多く、現状では、異種移植は現実的解決策ではない。拒絶反応、免疫反応を軽減するためには、移植される組織内の細胞量を低減させ、好ましくは無細胞化することが必要である。 The final treatment method for corneal diseases is corneal transplantation. However, in Japan, it is not always easy to obtain a cornea used for transplantation, and there are many patients waiting for transplantation. Therefore, xenotransplantation can be considered. However, xenotransplantation has many rejection reactions and immune reactions, and at present, xenotransplantation is not a practical solution. In order to reduce the rejection reaction and the immune reaction, it is necessary to reduce the amount of cells in the transplanted tissue, and preferably to eliminate the cell.
組織の無細胞化については、特表2001-520542号公報(特許文献1)に記載がある。特許文献1に記載の組織の無細胞化方法は、「無細胞化された組織を生成する方法であって、i）生物学的組織を、該組織の細胞が溶解するような条件下で低張液と接触させること、そして、ii）（ｉ）の段階から得られる組織を、核酸が分解するような条件下でヌクレアーゼと接触させることを含み、該無細胞化された組織がそれによって生成される上記方法」である。
特許文献１に記載の組織の無細胞化方法は、主に心臓弁を対象として開発されたものである。それに対して本発明は、心臓弁も含む種々の生体組織を無細胞化の対象とし、特に、角膜の無細胞化を大きな目的としている。角膜は、無細胞化された後、眼内に移植されることになるが、角膜の代用として機能するためには、通常の角膜と同程度の透明性を保持する必要がある。 The tissue acellularization method described in Patent Document 1 has been developed mainly for heart valves. On the other hand, the present invention aims at decellularization of various biological tissues including heart valves, and particularly has a large object of decellularization of the cornea. The cornea is decellularized and then transplanted into the eye. In order to function as a substitute for the cornea, it is necessary to maintain the same degree of transparency as the normal cornea.
即ち、本発明は、角膜を無細胞化した場合、透明性を保持した無細胞化角膜が得られる方法、及び無細胞化された角膜を提供することを目的とする。 That is, an object of the present invention is to provide a method for obtaining a cell-free cornea having transparency when the cornea is cell-free, and a cell-free cornea.
さらに、本発明は、角膜以外の組織についても、角膜と同様に無細胞化し得る方法、及び無細胞化された角膜以外の組織を提供することも目的とする。角膜以外の組織についても、移植に際して、拒絶反応、免疫反応を低減することは強く要望されているからである。 Furthermore, another object of the present invention is to provide a method for decellularization of tissues other than the cornea as well as a non-corneal tissue other than the cornea. This is because there is a strong demand for reducing rejection and immune responses in transplantation of tissues other than the cornea.
［請求項１］
摘出組織である生体組織を無酸素状態に置くことで、前記生体組織に含まれる細胞量を減少させることを含む、細胞量を減少させた生体組織の作成方法。
［請求項２］
無酸素状態が、窒素雰囲気である請求項１に記載の方法。
［請求項３］
生体組織が哺乳類の組織である、請求項１または２に記載の方法。
［請求項４］
生体組織が角膜、心臓弁、腱、靭帯、動脈、静脈、横隔膜、心膜、臍帯、筋膜、硬膜、鼓膜、またはそれらの一部である請求項１〜３のいずれか１項に記載の方法。
［請求項５］
生体組織に含まれる細胞の95%以上を消滅させる請求項１〜４のいずれか１項に記載の方法。
［請求項６］
摘出組織である角膜を無酸素状態に置くことで、前記角膜を無細胞化することを含む、無細胞化した角膜の作成方法。
［請求項７］
角膜が哺乳類の角膜である、請求項６に記載の方法。
［請求項８］
摘出組織は、凍結保存組織を解凍したものである請求項１〜７のいずれか１項に記載の方法。
［請求項９］
無酸素状態に置く時間を１時間〜１４日の範囲とする、請求項１〜８のいずれか１項に記載の方法。
［請求項１０］無酸素状態に置く温度を−１５０〜４０℃の範囲とする、請求項１〜９のいずれか１項に記載の方法。
[Claim 1]
A method for producing a biological tissue with a reduced cell amount, comprising reducing the amount of cells contained in the biological tissue by placing the biological tissue, which is an extracted tissue, in an anoxic state.
[Claim 2]
The method according to claim 1, wherein the oxygen-free state is a nitrogen atmosphere.
[Claim 3]
The method according to claim 1 or 2, wherein the biological tissue is a mammalian tissue.
[Claim 4]
The biological tissue is cornea, heart valve, tendon, ligament, artery, vein, diaphragm, pericardium, umbilical cord, fascia, dura mater, tympanic membrane, or a part thereof. the method of.
[Claim 5]
The method according to any one of claims 1 to 4, wherein 95% or more of cells contained in the living tissue are eliminated.
[Claim 6]
A method for producing a decellularized cornea, comprising decellularizing the cornea by placing the cornea as an excised tissue in an anoxic state.
[Claim 7]
The method of claim 6, wherein the cornea is a mammalian cornea.
[Claim 8]
The method according to any one of claims 1 to 7, wherein the excised tissue is obtained by thawing a cryopreserved tissue.
[Claim 9]
The method according to any one of claims 1 to 8, wherein the time for placing in anoxic state is in the range of 1 hour to 14 days.
[10] The method according to any one of [1] to [9], wherein the temperature at which the oxygen-free state is placed is in the range of -150 to 40 ° C.
本発明によれば、生体組織を簡単な操作により、無細胞化し得る。無細胞化により組織の免疫抗原性が低下する。特に、生体組織が角膜である場合、その透明性を損なうことなく、無細胞化し得るという利点がある。 According to the present invention, a living tissue can be acellularized by a simple operation. Acellularization reduces the immunogenicity of the tissue. In particular, when the biological tissue is a cornea, there is an advantage that it can be acellular without impairing its transparency.
本発明は、生体組織を無酸素状態に置くことで、前記生体組織に含まれる細胞量を減少させる方法に関する。無酸素状態とは、酸素を実質的に含まない状態であり、好ましくは窒素雰囲気である。生体組織の細胞量減少に悪影響を及ぼさない程度の不純物としての酸素を含む状態は、「酸素を実質的に含まない状態」に含まれる。 The present invention relates to a method for reducing the amount of cells contained in a living tissue by placing the living tissue in an anoxic state. The oxygen-free state is a state that substantially does not contain oxygen, and is preferably a nitrogen atmosphere. The state containing oxygen as an impurity that does not adversely affect the decrease in the amount of cells in the living tissue is included in the “state substantially free of oxygen”.
窒素雰囲気を形成する場合、市販の窒素ガスをそのまま使用することができ、また、適宜、脱酸素した後の窒素ガスを使用することもできる。具体的には、生体組織を密閉容器中に置き、容器中の空間を窒素ガスで置換した後、容器の密閉状態を維持する。 When forming a nitrogen atmosphere, commercially available nitrogen gas can be used as it is, and nitrogen gas after deoxidizing can also be used suitably. Specifically, the living tissue is placed in a sealed container, and the space in the container is replaced with nitrogen gas, and then the sealed state of the container is maintained.
生体組織を無酸素状態に置く時間及び温度は、生体組織の種類や細胞量減少の程度を考慮して適宜決定される。無酸素状態に置く時間としては、例えば、１時間〜１４日の範囲とすることができる。また、無酸素状態に置く温度は、例えば、−１５０〜４０℃の範囲とすることができる。 The time and temperature at which the living tissue is kept in an oxygen-free state are appropriately determined in consideration of the type of living tissue and the degree of cell mass reduction. As time to leave in an oxygen-free state, it can be set as the range of 1 hour-14 days, for example. Moreover, the temperature put in an oxygen-free state can be made into the range of -150-40 degreeC, for example.
細胞量減少させる生体組織は、特に制限はなく、摘出組織であっても、凍結保存組織を解凍したものであってもよい。凍結保存した組織は無細胞化に先立って解凍するが、これは凍結保護物質が除去されることによってそれによる毒性が回避されるような条件下で行うことが適当である。好適な解凍条件は当該分野でよく知られている。 There are no particular limitations on the biological tissue that reduces the amount of cells, and it may be an excised tissue or a thawed cryopreserved tissue. The cryopreserved tissue is thawed prior to decellularization, which is suitably performed under conditions such that the cryoprotectant is removed thereby avoiding toxicity. Suitable thawing conditions are well known in the art.
生体組織は、例えば、哺乳類の組織であることができる。本方法への使用に好適な生体には、ヒトまたは動物への移植に好適なものがある。組織はヒトまたは非ヒト（例えばウシ、ブタ、または非ヒト霊長類）に由来してもよい。さらに、生体組織は、角膜、心臓弁、腱、靭帯、動脈、静脈、横隔膜、心膜、臍帯、筋膜、硬膜、鼓膜、またはそれらの一部であることができる。 The biological tissue can be, for example, a mammalian tissue. Suitable living organisms for use in the present methods include those suitable for transplantation into humans or animals. The tissue may be from a human or non-human (eg, a cow, pig, or non-human primate). Furthermore, the biological tissue can be the cornea, heart valve, tendon, ligament, artery, vein, diaphragm, pericardium, umbilical cord, fascia, dura mater, tympanic membrane, or part thereof.
生体組織に含まれる細胞量の減少の程度は、本発明の方法で処理した組織の用途に応じて適宜決定することができる。本発明の方法で処理した組織を、移植に使用する場合には、70%以上、好ましくは80%以上、より好ましくは90%以上、さらに好ましくは95%以上、もっとも好ましくはほぼ100%細胞を消滅させる。例えば、生体組織が角膜であって、移植用の角膜のは場合、角膜を無酸素状態に置くことで、前記角膜を無細胞化する、即ち、100%細胞を消滅させることが好ましい。 The degree of decrease in the amount of cells contained in the living tissue can be appropriately determined according to the use of the tissue treated by the method of the present invention. When the tissue treated by the method of the present invention is used for transplantation, 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, and most preferably almost 100% cells. Extinguish. For example, when the living tissue is the cornea and the cornea for transplantation, it is preferable to make the cornea acellular by placing the cornea in an anoxic state, that is, to eliminate 100% of the cells.
さらに本発明は、本発明の方法で無細胞化された生体組織に関する。この生体組織は哺乳類の組織であることができ、さらに、生体組織は、角膜、心臓弁、腱、靭帯、動脈、静脈、横隔膜、心膜、臍帯、筋膜、硬膜、鼓膜、またはそれらの一部であることができる。特に、本発明においては、生体組織が角膜または角膜の一部であることが好ましい。 Furthermore, this invention relates to the biological tissue made acellular by the method of this invention. The biological tissue can be mammalian tissue, and further, the biological tissue can be cornea, heart valve, tendon, ligament, artery, vein, diaphragm, pericardium, umbilical cord, fascia, dura mater, tympanic membrane, or those Can be part. In particular, in the present invention, the living tissue is preferably the cornea or a part of the cornea.
無細胞化した組織を最終的に種々の滅菌法のいずれかを使用して滅菌することができる。例えば、ガンマ線照射、エチレンオキシド、過酢酸、β−プロピオラクトン、ポビドンヨード、またはＵＶ照射を、感光性物質の存在下または非存在下で組織に施与してもよい。効果的な最終滅菌のための好適な条件は当該分野でよく知られている。 Acellularized tissue can ultimately be sterilized using any of a variety of sterilization methods. For example, gamma radiation, ethylene oxide, peracetic acid, β-propiolactone, povidone iodine, or UV radiation may be applied to the tissue in the presence or absence of a photosensitive material. Suitable conditions for effective terminal sterilization are well known in the art.
無細胞化した組織は固定することもでき、固定は、グルタルアルデヒド固定を含む、当該分野で知られる方法を使用して行うことができる。しかしながら、非固定化組織も使用できる。非固定化組織に種々の薬剤のいずれかを含浸させることができ、それらの薬剤には、ｉｎ ｖｉｖｏにおいて無細胞化した組織の移植の際に再細胞化を刺激するものがある。そのような薬剤の例には、成長因子、グリコサミノグリカンのような接着因子、そしてフィブロネクチン、ラミニン、ビトロネクチンなどのような可溶性細胞外マトリクスグリコプロテインがある。使用できる他の薬剤には血液相溶性（hemocompatability）、血栓モジュレーター、および抗菌性を増加するものがある。好適な含浸方法は当該分野で知られている。組織が心臓弁の場合、標準的な方法を用いて生物学的または非生物学的ステントと共に製作することができる。 Acellularized tissue can also be fixed, and fixation can be performed using methods known in the art, including glutaraldehyde fixation. However, non-immobilized tissue can also be used. Non-immobilized tissue can be impregnated with any of a variety of agents, some of which stimulate recellularization upon transplantation of acellularized tissue in vivo. Examples of such agents include growth factors, adhesion factors such as glycosaminoglycans, and soluble extracellular matrix glycoproteins such as fibronectin, laminin, vitronectin and the like. Other drugs that can be used include those that increase hemocompatability, thrombus modulators, and antibacterial properties. Suitable impregnation methods are known in the art. If the tissue is a heart valve, it can be fabricated with biological or non-biological stents using standard methods.
本発明の方法により作成した無細胞化組織は、哺乳類、特にヒトにおいて欠陥のある組織の代替として使用する、即ち、移植することができる。移植の方法は、既に知られている。 The acellular tissue produced by the method of the present invention can be used as a substitute for, or transplanted to, a defective tissue in mammals, particularly humans. The method of transplantation is already known.
以下に本発明を実施例によりさらに詳細に説明する。 Hereinafter, the present invention will be described in more detail with reference to examples.
実施例１（角膜）
ブタ角膜は市場に流通する眼球より得た。角膜を眼球から外した後、無菌リン酸緩衝液もしくは生理食塩水で洗浄し、密栓できる容器に移した。その後、その容器に窒素を注入し、１００％窒素雰囲気にした。この状態で７日放置し、その後、角膜の状態を観察した。放置前と放置後の角膜断面の写真を図１に示す。放置前の角膜には、顆粒状の細胞が多数観察されるのに対して、放置後の角膜には、顆粒状の細胞は全く観察されず、無細胞化されていることがわかる。
Example 1 (cornea)
Porcine cornea was obtained from commercially available eyeballs. After removing the cornea from the eyeball, the cornea was washed with sterile phosphate buffer or physiological saline, and transferred to a container that could be sealed. Thereafter, nitrogen was injected into the container to make a 100% nitrogen atmosphere. This state was left for 7 days, and then the state of the cornea was observed. A photograph of the corneal cross section before and after standing is shown in FIG. It can be seen that many granular cells are observed in the cornea before being left standing, whereas no granular cells are observed in the cornea after being left standing, and the cells are decellularized.
上記で得られた無細胞化された角膜を家兎の眼に移植した。移植後４カ月後の眼の写真を図２に示す。移植後４カ月後においても、透明な状態が保たれており、無細胞化により、拒絶反応が抑制されていることがわかる。 The acellularized cornea obtained above was transplanted into the eye of a rabbit. A photograph of the eye 4 months after transplantation is shown in FIG. Even after 4 months from the transplantation, the transparent state is maintained, and it can be seen that the rejection reaction is suppressed by decellularization.
実施例２（皮膚）
マウスから得た皮膚を実施例１と同様に、１００％窒素雰囲気にして７日放置した。放置前と放置後の皮膚の断面を観察した結果を図３に示す。放置前の皮膚には、顆粒状の細胞が多数観察されるのに対して、放置後の皮膚には、顆粒状の細胞は全く観察されず、無細胞化されていることがわかる。
Example 2 (Skin)
Skin obtained from mice was left in a 100% nitrogen atmosphere for 7 days in the same manner as in Example 1. The result of observing the cross section of the skin before and after leaving is shown in FIG. It can be seen that many granular cells are observed in the skin before standing, whereas no granular cells are observed in the skin after standing, and the cells are acellularized.
実施例３（心筋）
マウスから得た心筋を実施例１と同様に、１００％窒素雰囲気にして７日放置した。放置前と放置後の心筋の断面を観察した結果を図４に示す。放置前の心筋には、顆粒状の細胞が多数観察されるのに対して、放置後の心筋には、顆粒状の細胞は全く観察されず、無細胞化されていることがわかる。
Example 3 (myocardial)
The myocardium obtained from the mouse was left in a 100% nitrogen atmosphere for 7 days in the same manner as in Example 1. The result of observing the cross section of the myocardium before and after being left is shown in FIG. It can be seen that many granular cells are observed in the myocardium before being left standing, whereas no granular cells are observed in the myocardium after being left standing, and the cells are decellularized.
本発明によれば、移植に適した、免疫抗原性が低下した組織を容易に作成することができ、移植用組織の製造に活用できる。 ADVANTAGE OF THE INVENTION According to this invention, the structure | tissue which the immuno-antigenicity suitable for a transplant can be created easily, and it can utilize for manufacture of the tissue for a transplant.
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