JPH1176400A - Artificial organ - Google Patents
Artificial organInfo
- Publication number
- JPH1176400A JPH1176400A JP9261045A JP26104597A JPH1176400A JP H1176400 A JPH1176400 A JP H1176400A JP 9261045 A JP9261045 A JP 9261045A JP 26104597 A JP26104597 A JP 26104597A JP H1176400 A JPH1176400 A JP H1176400A
- Authority
- JP
- Japan
- Prior art keywords
- matrix
- plasma
- artificial organ
- living cells
- artificial
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
- A61M1/3621—Extra-corporeal blood circuits
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
- A61M1/3621—Extra-corporeal blood circuits
- A61M1/3623—Means for actively controlling temperature of blood
Landscapes
- Health & Medical Sciences (AREA)
- Heart & Thoracic Surgery (AREA)
- Vascular Medicine (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Anesthesiology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Cardiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- External Artificial Organs (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、肝細胞、膵細胞、
腎細胞等の生細胞を用いた人工臓器に関する。TECHNICAL FIELD The present invention relates to hepatocytes, pancreatic cells,
The present invention relates to an artificial organ using living cells such as kidney cells.
【０００２】[0002]
【従来の技術】従来より、肝細胞等の生細胞を、マトリ
ックスの中に分散させて保持した人工臓器が知られてい
る。公知のこの種の人工臓器は、マトリックス中に分散
された生細胞を中空繊維膜の内部に収納し、体液を循環
させて生細胞にその細胞本来の生理作用を行わせ、処理
後の体液を再び患者に戻すことにより、機能が低下又は
喪失された患者の臓器を補助又は代行するものである。
マトリックスとしては、コラーゲン、フィブロネクチン
のような細胞間物質や、ポリ−Ｎ−ｐ−ビニルベンジル
−Ｄ−ラクトアミド（ＰＶＬＡ）等の樹脂が知られてい
る。2. Description of the Related Art Conventionally, an artificial organ in which living cells such as hepatocytes are dispersed and held in a matrix has been known. Known artificial organs of this type contain living cells dispersed in a matrix inside a hollow fiber membrane, circulate a body fluid to allow the living cells to perform their original physiological action, and remove the treated body fluid. By returning to the patient again, the organ of the patient whose function has been reduced or lost is assisted or substituted.
Known matrices include intercellular substances such as collagen and fibronectin, and resins such as poly-Np-vinylbenzyl-D-lactoamide (PVLA).
【０００３】[0003]
【発明が解決しようとする課題】しかしながら、生細胞
を用いる従来の人工臓器では、生細胞の機能を高く維持
したまま、長期間に亘って生細胞を生かしておくことが
できず、現在までのところ、この種の人工臓器は実用化
されていない。However, in a conventional artificial organ using living cells, the living cells cannot be kept alive for a long period of time while maintaining the function of the living cells at a high level. However, this kind of artificial organ has not been put to practical use.
【０００４】従って、本発明の目的は、生細胞の機能を
高く維持したまま、長期間に亘って使用可能な、生細胞
を用いる人工臓器を提供することである。Accordingly, it is an object of the present invention to provide an artificial organ using living cells that can be used for a long period of time while maintaining the function of the living cells at a high level.
【０００５】[0005]
【課題を解決するための手段】本願発明者らは、鋭意研
究の結果、ラミニンをマトリックスとして用いることに
より、生細胞が自然な形で三次元的に保持され、その結
果、生細胞の機能を高く維持したまま、長期間に亘って
生細胞を生かしておくことができることを見出し本発明
を完成した。Means for Solving the Problems As a result of intensive studies, the present inventors have found that by using laminin as a matrix, living cells are three-dimensionally retained in a natural form, and as a result, the function of living cells is reduced. The present inventors have found that living cells can be kept alive for a long period of time while maintaining a high level, and completed the present invention.
【０００６】すなわち、本発明は、ラミニンを５０重量
％以上含むマトリックスと、該マトリックス中に分散さ
れた生細胞と、該マトリックス及び生細胞を収納し、外
部との液体の流出入が可能な収納体とを具備する人工臓
器を提供する。That is, the present invention provides a matrix containing laminin in an amount of 50% by weight or more, living cells dispersed in the matrix, the matrix and the living cells, and a liquid that can flow into and out of the liquid. An artificial organ having a body is provided.
【０００７】[0007]
【発明の実施の形態】本発明の人工臓器に用いられる生
細胞は、体液に対して何らかの生理作用を行う細胞であ
り、好ましい例として肝細胞、膵細胞、腎細胞を挙げる
ことができるがこれらに限定されるものではない。生細
胞の由来は、特に限定されず、ヒト又はヒトの内臓と同
様な性能を発揮する動物であればよく、好ましい動物と
してブタ、ヒヒ、サル、ヒツジ、ウシ、イヌ等を挙げる
ことができる。また、これらの動物には遺伝子操作を施
された動物も含まれる。DESCRIPTION OF THE PREFERRED EMBODIMENTS Live cells used in the artificial organ of the present invention are cells that exert some physiological action on body fluids, and preferred examples include hepatocytes, pancreatic cells, and kidney cells. It is not limited to. The origin of the living cells is not particularly limited, and may be any animal that exhibits the same performance as humans or human internal organs, and preferred animals include pigs, baboons, monkeys, sheep, cows, dogs, and the like. These animals also include those that have been genetically engineered.
【０００８】生細胞を保持するマトリックスとしては、
ラミニンを５０重量％以上含むものが用いられる。マト
リックス中のラミニンの含量は５０重量％以上であり、
全量がラミニンであってもよい。ラミニンとコラーゲン
IVの混合物も好ましく、この場合、ラミニンとコラーゲ
ンIVの重量比は、好ましくは６：４〜８：２程度であ
る。ラミニンとコラーゲンIVの合計量は、マトリックス
全体の７０重量％以上が好ましく、さらには９０重量％
以上、特には９５重量％以上が好ましい。マトリックス
には、ラミニン及びコラーゲンIV以外の物質も本発明の
効果を有意に損なわない範囲で含まれていてもよい。こ
のような物質の例として従来からマトリックスとして用
いられているエンタクチン、プロテオグリカンやＰＶＬ
Ａのような樹脂並びにラミニンやコラーゲンIVに付随す
ることがある不純物等を例示することができる。これ
ら、ラミニン及びコラーゲンIV以外の物質の量は本発明
の効果を有意に損なわないであり、通常、３０重量％以
下、好ましくは１０重量％以下、さらに好ましくは５重
量％以下である。[0008] As a matrix for retaining living cells,
Those containing 50% by weight or more of laminin are used. The content of laminin in the matrix is 50% by weight or more;
The whole amount may be laminin. Laminin and collagen
Mixtures of IV are also preferred, in which case the weight ratio of laminin to collagen IV is preferably of the order of 6: 4 to 8: 2. The total amount of laminin and collagen IV is preferably 70% by weight or more of the whole matrix, and more preferably 90% by weight.
Above, especially 95% by weight or more is preferable. The matrix may contain substances other than laminin and collagen IV as long as the effects of the present invention are not significantly impaired. Examples of such substances are entactin, proteoglycan and PVL which have been conventionally used as a matrix.
Examples of the resin A include impurities that may accompany laminin and collagen IV. The amounts of these substances other than laminin and collagen IV do not significantly impair the effects of the present invention, and are usually 30% by weight or less, preferably 10% by weight or less, and more preferably 5% by weight or less.
【０００９】生細胞は、上記マトリックス中に分散状態
で保持される。上記マトリックスを用いることにより、
生細胞は生体内に近い自然な状態で三次元的に維持され
るので、細胞を高密度に充填しても細胞の機能を高く維
持したまま長期間に亘って細胞を生きたまま保つことが
できる。マトリックスと生細胞の混合物中における生細
胞の密度は、特に限定されないが、通常１０5 個／ｍｌ
〜１０9 個／ｍｌ程度であり、好ましくは、１０7 個／
ｍｌ〜１０8 個／ｍｌ程度である。また、生細胞の総数
は、生細胞の種類や、求める人工臓器の性能等に応じ適
宜選択されるが、例えば、肝細胞の場合、１０10ないし
４ｘ１０10程度が適当である。[0009] Living cells are maintained in a dispersed state in the matrix. By using the above matrix,
Since living cells are three-dimensionally maintained in a natural state close to the body, even if the cells are packed at a high density, it is possible to keep the cells alive for a long time while maintaining the function of the cells at a high level. it can. The density of the living cells in the mixture of the matrix and the living cells is not particularly limited, but is usually 10 5 cells / ml.
About 10 9 cells / ml, preferably 10 7 cells / ml.
ml~10 is about 8 / ml. The total number of living cells is appropriately selected according to the type of living cells and the performance of the artificial organ to be obtained. For example, in the case of hepatocytes, about 10 10 to 4 × 10 10 is appropriate.
【００１０】生細胞とマトリックスの混合物は、外部と
の液体の流出入が可能な収納体に収納される。収納体の
好ましい形態として中空繊維膜を例示することができ
る。すなわち、中空繊維膜の内側（すなわち、繊維の内
側の中空部分）又は外側に生細胞とマトリックスの混合
物を充填する。この場合、中空繊維膜の分画分子量は、
細胞が流出せず、かつ、体液の流出入が円滑に行われる
大きさであり、すなわち、数万〜１００万ダルトン程度
が好ましく、特に好ましくは約１０万ダルトン程度であ
る。また、中空繊維膜の内径は特に限定されないが２５
０〜３５０μｍ程度が好ましく、また、長さも特に限定
されないが通常１００〜３５０ｍｍ程度である。また、
中空繊維膜の材質は、体液に対して有害な作用を行うも
のでなければいずれのものであってもよく、例えばポリ
スルホン、ポリエステル等を挙げることができることが
できるがこれらに限定されるものではない。このような
中空繊維膜に生細胞／マトリックス混合物を充填したも
のを数百本ないし数千本束ね、液体流入口と液体流出口
とを具備するカートリッジに収納することが好ましい。
なお、上記の物理的特性を有する中空繊維膜を数百本な
いし数千本束ね、液体流入口と液体流出口とを具備する
カートリッジに収納したものは、人工透析装置として市
販されており、本発明では、このような市販の人工透析
装置を人工臓器の収納体として好ましく利用することが
できる。[0010] The mixture of living cells and the matrix is housed in a housing that allows liquid to flow in and out of the outside. A hollow fiber membrane can be exemplified as a preferred form of the housing. That is, the mixture of living cells and matrix is filled inside (ie, inside the hollow portion of the fiber) or outside the hollow fiber membrane. In this case, the molecular weight cutoff of the hollow fiber membrane is
The size is such that the cells do not flow out and the flow of the bodily fluid flows smoothly, that is, about tens of thousands to one million daltons is preferable, and particularly preferably about 100,000 daltons. The inner diameter of the hollow fiber membrane is not particularly limited, but may be 25 mm.
The length is preferably about 0 to 350 μm, and the length is not particularly limited, but is usually about 100 to 350 mm. Also,
The material of the hollow fiber membrane may be any material as long as it does not have a harmful effect on body fluids, and examples thereof include polysulfone and polyester, but are not limited thereto. . It is preferable to bundle hundreds or thousands of such hollow fiber membranes filled with a living cell / matrix mixture and store them in a cartridge having a liquid inlet and a liquid outlet.
A bundle of hundreds to thousands of hollow fiber membranes having the above-described physical properties and housed in a cartridge having a liquid inlet and a liquid outlet is commercially available as an artificial dialysis device. In the present invention, such a commercially available artificial dialysis device can be preferably used as a container for an artificial organ.
【００１１】なお、収納体は、上記のような中空繊維膜
に限定されるものではなく、例えば袋状の平膜や、通常
の容器のような形態であってもよく、要するに前記マト
リックス／生細胞混合物を収容して、これに体液を接触
させ、かつ接触後の体液を取出せる構造になっているも
のは全て本発明の範囲に含まれる。The container is not limited to the hollow fiber membrane as described above, but may be, for example, a bag-shaped flat membrane or an ordinary container. Any structure that accommodates the cell mixture, makes the body fluid come into contact with it, and can take out the body fluid after the contact is included in the scope of the present invention.
【００１２】本発明の人工臓器は、上記マトリックスを
構成する物質（以下、「マトリックス物質」ということ
がある）の溶液と、生細胞の懸濁液を混合し、この混合
液を上記収納体に充填することにより製造することがで
きる。マトリックス物質の溶液の溶媒としては、マトリ
ックス物質を溶解することができ、生細胞に悪影響を与
えないものならばいずれのものでも用いることができ、
好ましい例として、培養液、生理食塩水、緩衝液等を挙
げることができる。また、マトリックス物質溶液中のマ
トリックス物質の濃度は特に限定されないが、０．１〜
１．５w/v ％程度が好ましく、特には約１w/v ％程度が
好ましい。一方、生細胞の懸濁液の溶媒としては、生理
食塩緩衝液等、生細胞の懸濁に常用されているものを用
いることができる。生細胞懸濁液中の細胞密度は、特に
限定されないが１０5 〜１０9 個／ｍｌ程度が好まし
く、さらに好ましくは、１０7 〜１０8 個／ｍｌ程度で
ある。マトリックス物質溶液と生細胞懸濁液との混合比
率は、上記した好ましい生細胞密度を達成するように選
択される。収納体が中空繊維膜である場合には、マトリ
ックス物質溶液と生細胞懸濁液との混合物を中空繊維の
中空部分に流し込み、３７℃で放置することにより、マ
トリックス物質がゲル化してゲル状のマトリックス中に
生細胞が分散したものが得られる。The artificial organ of the present invention is obtained by mixing a solution of a substance constituting the matrix (hereinafter, sometimes referred to as a “matrix substance”) with a suspension of living cells, and placing the mixture in the container. It can be manufactured by filling. As a solvent for the solution of the matrix substance, any substance can be used as long as it can dissolve the matrix substance and does not adversely affect living cells,
Preferred examples include culture solutions, physiological saline, buffers and the like. Further, the concentration of the matrix substance in the matrix substance solution is not particularly limited, but 0.1 to
It is preferably about 1.5 w / v%, particularly preferably about 1 w / v%. On the other hand, as a solvent for the suspension of living cells, a solvent commonly used for suspension of living cells, such as a physiological saline buffer, can be used. Cell density of viable cells in suspension are particularly preferred although about 105 to 109 cells / ml are not limited, and more preferably from 10 7 to 10 about 8 / ml. The mixing ratio between the matrix material solution and the viable cell suspension is chosen to achieve the preferred viable cell density described above. When the container is a hollow fiber membrane, the mixture of the matrix material solution and the living cell suspension is poured into the hollow portion of the hollow fiber and left at 37 ° C., whereby the matrix material gels to form a gel. A dispersion of living cells in a matrix is obtained.
【００１３】本発明の人工臓器の好ましい使用態様の１
例を図１に基づいて説明する。患者の大腿動脈からポン
プ１０により血液を抜き出してプラズマセパレーター１
２に導き、血球成分と血漿に分離する。ポンプ１０によ
る送液速度は６０〜１５０ｍｌ／分が好ましい。血漿成
分をポンプ１４により熱交換器１６に導く。ポンプ１４
の送液速度は３０ｍｌ／分程度が好ましい。熱交換器１
６は、血漿の温度をヒトの体温（３７℃）に維持するた
めに用いられる。血漿は次いで、熱交換器１６に隣接し
て設けられているオキシゲネーター１８に導かれ、ここ
で酸素を供給される。酸素の供給は、生細胞の生存にと
って有利であるが、必須的ではない。オキシゲネーター
１８を通った血漿は本発明の人工臓器２０に導かれ、マ
トリックス中に保持された生細胞と接触する。図示の人
工臓器２０は、多数の中空繊維膜の中空部分にマトリッ
クス／生細胞混合物を充填したものを、液体流入口２２
と液体流出口２４とを具備するカートリッジに収納した
ものである。マトリックス／生細胞混合物が中空繊維膜
に充填されている場合には、生細胞との接触を十分に確
保するために、血漿を中空繊維膜の内側の中空部分に導
き、中空繊維膜の側面にある多数の孔から流出してきた
処理後の血漿を液体流出口２４から送り出すようにする
ことが好ましい。液体流出口２４から送り出された処理
後の血漿は、プラズマセパレーター１２で分離された血
球成分と合流して患者の体内に戻される。なお、人工臓
器の使用態様は上記のものに限定されるものではなく、
例えば、体内に埋め込むタイプも可能である。One preferred embodiment of the use of the artificial organ of the present invention is as follows.
An example will be described with reference to FIG. Blood is drawn from the femoral artery of the patient by the pump 10 and the plasma separator 1
2 and separate into blood cell components and plasma. The liquid sending speed by the pump 10 is preferably 60 to 150 ml / min. The plasma component is led to the heat exchanger 16 by the pump 14. Pump 14
Is preferably about 30 ml / min. Heat exchanger 1
No. 6 is used to maintain the temperature of plasma at human body temperature (37 ° C). The plasma is then led to an oxygenator 18 located adjacent to the heat exchanger 16 where it is supplied with oxygen. The supply of oxygen is advantageous, but not essential, for the survival of living cells. The plasma that has passed through the oxygenator 18 is guided to the artificial organ 20 of the present invention, and comes into contact with living cells held in a matrix. The illustrated artificial organ 20 is obtained by filling a hollow portion of a large number of hollow fiber membranes with a matrix / viable cell mixture at a liquid inlet 22.
And a liquid outlet 24. When the matrix / viable cell mixture is filled in the hollow fiber membrane, the plasma is led to the hollow portion inside the hollow fiber membrane to ensure sufficient contact with the living cells, It is preferable that the processed plasma flowing out from a number of holes is sent out from the liquid outlet 24. The processed plasma sent out from the liquid outlet 24 is combined with the blood cell component separated by the plasma separator 12 and returned to the patient's body. In addition, the use form of the artificial organ is not limited to the above,
For example, a type that can be implanted in the body is also possible.
【００１４】本発明の人工臓器は、当該臓器の機能が低
下し又は喪失された患者の当該臓器の補助又は代行を行
うために利用することができる。例えば、生細胞が肝細
胞の場合、本発明の人工臓器を用いることにより、劇症
肝炎や術後肝不全のような重篤な肝疾患患者の生命を維
持することができる。The artificial organ of the present invention can be used for assisting or substituting for the organ of a patient whose function has been reduced or lost. For example, when living cells are hepatocytes, the use of the artificial organ of the present invention can maintain the lives of patients with severe liver diseases such as fulminant hepatitis and postoperative liver failure.
【００１５】[0015]
【実施例】以下、本発明を実施例に基づきより具体的に
説明する。もっとも、本発明は、下記実施例に限定され
るものではない。DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below more specifically based on embodiments. However, the present invention is not limited to the following examples.
【００１６】実施例１ (1) 人工肝臓の作製 体重２０ｋｇのブタ（雄、２０週齢）を畜殺し、肝臓を
摘出した。脱血後、コラーゲナーゼ処理を行い、肝細胞
を分散した。これを遠心して、緩衝液にて数回洗浄し
た。ラミニン、コラーゲンIV混合液（２：１）１．３％
（w/v ）１００ｍｌを洗浄した肝細胞懸濁液（溶媒：培
養液）１００ｍｌと混合し、ホモゲナイズした。該混合
物（２００ｍｌ）中の肝細胞の数は２×１０10個であっ
た。この混合物を市販の人工透析装置の中空繊維内に加
圧しながら封入した。この市販の人工透析装置は、分画
分子量１０万ダルトン、内径２６０μｍ、肉厚７５μ
ｍ、長さ１８０ｍｍのポリスルホン中空繊維膜２５０本
を直径５ｃｍ、長さ２０ｃｍのカートリッジに収納した
ものである。 Example 1 (1) Preparation of Artificial Liver A pig (male, 20 weeks old) weighing 20 kg was killed and the liver was removed. After blood removal, the cells were treated with collagenase to disperse hepatocytes. This was centrifuged and washed several times with buffer. Laminin, collagen IV mixed solution (2: 1) 1.3%
(W / v) 100 ml was mixed with 100 ml of the washed hepatocyte suspension (solvent: culture solution) and homogenized. The number of hepatocytes in the mixture (200 ml) was 2 × 10 10 . This mixture was sealed under pressure into the hollow fibers of a commercially available artificial dialysis machine. This commercially available artificial dialysis machine has a molecular weight cut off of 100,000 daltons, an inner diameter of 260 μm, and a wall thickness of 75 μm.
A 250-m long polysulfone hollow fiber membrane having a length of 180 mm was housed in a cartridge having a diameter of 5 cm and a length of 20 cm.
【００１７】一方、比較のため、マトリックス物質のみ
を変えて上記と同じ方法により、市販のプラスチック
（ポリウレタン）（比較例１）、コラーゲンＩ（比較例
２）、ＰＶＬＡ（比較例３）をマトリックス物質とする
人工肝臓を作製した。On the other hand, for comparison, a commercially available plastic (polyurethane) (Comparative Example 1), collagen I (Comparative Example 2), and PVLA (Comparative Example 3) were prepared in the same manner as described above except that only the matrix material was changed. Was prepared.
【００１８】(2) 人工肝臓の性能評価 体重２５ｋｇのブタ（雄、２７週齢）を開腹し、肝動脈
を結索、門脈と下大静脈を縫合した。これを肝不全モデ
ルとして使用した。このブタの大腿動脈を図１に示す体
外血液循環装置（人工臓器として、実施例１又は比較例
１、２若しくは３の人工肝臓を具備する）に連結し、図
１に基づいて先に説明した方法により、血液の処理を行
った。(2) Performance Evaluation of Artificial Liver A pig (male, 27 weeks old) weighing 25 kg was laparotomized, the hepatic artery was ligated, and the portal vein and the inferior vena cava were sutured. This was used as a liver failure model. The femoral artery of this pig was connected to the extracorporeal blood circulation apparatus shown in FIG. 1 (including an artificial liver of Example 1 or Comparative Examples 1, 2 or 3 as an artificial organ), and was described above with reference to FIG. Blood was processed according to the method.
【００１９】血液処理開始後１日、４日、９日に人工肝
臓中の肝細胞の一部をサンプリングし、細胞中のアルブ
ミン及びβ−アクチンのｍＲＮＡ量を常法であるノーザ
ン分析により定量し、アルブミンｍＲＮＡ／β−アクチ
ンｍＲＮＡの比率を計算した。結果を図２に示す。On days 1, 4 and 9 after the start of the blood treatment, a part of the hepatocytes in the artificial liver was sampled, and the mRNA levels of albumin and β-actin in the cells were quantified by a conventional Northern analysis. The ratio of albumin mRNA / β-actin mRNA was calculated. The results are shown in FIG.
【００２０】β−アクチンｍＲＮＡは平面培養で最も高
い値を示すことが知られており、これを分母としてアル
ブミンｍＲＮＡ量を分子とする比率をとることにより、
肝細胞の活力の指標とすることができる。図２から明ら
かなように、本発明の人工肝臓を用いた場合には、血液
処理開始後９日目においても、肝細胞は高い活力を維持
しているのに対し、本発明で規定されるマトリックス以
外のマトリックスを用いた比較例１〜３の人工肝臓で
は、肝細胞の活力が血液処理開始後１日から既に低く、
９日後にはほとんど機能していないことがわかる。It is known that β-actin mRNA shows the highest value in a plate culture, and by using this as a denominator, the ratio of the amount of albumin mRNA to the molecule is obtained.
It can be used as an indicator of hepatocyte vitality. As is clear from FIG. 2, when the artificial liver of the present invention was used, hepatocytes maintained a high vitality even on the ninth day after the start of blood treatment, but were defined by the present invention. In the artificial liver of Comparative Examples 1 to 3 using a matrix other than the matrix, the vitality of hepatocytes was already low from 1 day after the start of blood treatment,
It turns out that it hardly functions after 9 days.
【００２１】実施例２、比較例４〜７ (1) 人工肝臓の作製 肝細胞がラット由来であり、マトリックス物質溶液と肝
細胞懸濁液の混合物の量が２５ｍｌである（従って、肝
細胞の総数は２．２５×１０9 個）であることを除き、
実施例１と同様の方法により人工肝臓を調製した（実施
例２）。また、比較のため、マトリックス物質としてＰ
ＶＬＡ（比較例４）、コラーゲンゲルサンドイッチ（肝
細胞をコラーゲンゲルでサンドイッチ状に挟んだもの）
（比較例５）、コラーゲンゲル（比較例６）、コラーゲ
ンで市販のプラスチック（ポリウレタン）をコーティン
グしたもの（比較例７）を用いて同様に人工肝臓を作製
した。 Example 2, Comparative Examples 4 to 7 (1) Preparation of Artificial Liver Hepatocytes were derived from rats, and the amount of the mixture of the matrix substance solution and the hepatocyte suspension was 25 ml (thus, Except that the total number is 2.25 × 10 9 )
An artificial liver was prepared in the same manner as in Example 1 (Example 2). For comparison, P is used as a matrix material.
VLA (Comparative Example 4), collagen gel sandwich (hepatocytes sandwiched between collagen gels)
An artificial liver was prepared in the same manner using (Comparative Example 5), collagen gel (Comparative Example 6), and a product coated with a commercially available plastic (polyurethane) with collagen (Comparative Example 7).
【００２２】(2) 人工肝臓の性能評価 作製された人工肝臓を、実施例１と同じ体外血液循環シ
ステムに組み入れ、実施例１と同様な肝不全モデルブタ
を用いて血液処理を行った。血液処理開始１日〜２２日
後に、処理後の血漿中のアルブミン量（すなわち、肝細
胞による分泌アルブミン量）を、市販の血清アルブミン
抗体を用いた免疫測定法により定量した。結果を図３に
示す。(2) Performance Evaluation of Artificial Liver The prepared artificial liver was incorporated into the same extracorporeal blood circulation system as in Example 1, and blood treatment was performed using the same hepatic failure model pig as in Example 1. One to 22 days after the start of the blood treatment, the amount of albumin in the plasma after the treatment (that is, the amount of albumin secreted by hepatocytes) was quantified by an immunoassay using a commercially available serum albumin antibody. The results are shown in FIG.
【００２３】アルブミンの分泌は肝細胞の重要な機能で
ある。本発明の人工肝臓では、肝細胞のアルブミン分泌
量が血液処理開始１６日以降も高く維持されているのに
対し、比較例の人工肝臓では、血液処理開始１６日以降
は、最も高い比較例５のものでも本発明の半分以下であ
る。よって、本発明によれば、肝細胞の能力を高く維持
したまま、肝細胞を長期間に亘って維持できることがわ
かる。Albumin secretion is an important function of hepatocytes. In the artificial liver of the present invention, the amount of albumin secretion of hepatocytes is maintained high after 16 days from the start of the blood treatment, whereas in the artificial liver of the comparative example, after the 16 days from the start of the blood treatment, Comparative Example 5 was the highest. Is less than half of the present invention. Therefore, according to the present invention, it can be seen that hepatocytes can be maintained for a long period of time while maintaining the ability of the hepatocytes at a high level.
【００２４】[0024]
【発明の効果】本発明によれば、生細胞を用いる人工臓
器において、ラミニンを主成分とするマトリックスを用
いることにより、生細胞が自然な形で三次元的に保持さ
れ、その結果、生細胞の機能を高く維持したまま、長期
間に亘って生細胞を生かしておくことができる。According to the present invention, a living cell is three-dimensionally retained in a natural form by using a matrix containing laminin as a main component in an artificial organ using living cells. The living cells can be kept alive for a long period of time while maintaining the function of the cell at a high level.
【図１】本発明の人工臓器の好ましい使用態様の１例を
示す模式図である。FIG. 1 is a schematic view showing an example of a preferred use mode of the artificial organ of the present invention.
【図２】本発明及び比較例の人工肝臓における、血液処
理開始後の日数と、肝細胞中にアルブミンｍＲＮＡ／β
−アクチンｍＲＮＡ比の関係を示す図である。FIG. 2 shows the number of days after the start of blood treatment in the artificial livers of the present invention and the comparative example, and albumin mRNA / β in hepatocytes.
It is a figure which shows the relationship of actin mRNA ratio.
【図３】本発明及び比較例の人工肝臓における、血液処
理開始後の日数と、肝細胞により分泌されたアルブミン
量の関係を示す図である。FIG. 3 is a graph showing the relationship between the number of days after the start of blood treatment and the amount of albumin secreted by hepatocytes in the artificial livers of the present invention and a comparative example.
１０ ポンプ １２ プラズマセパレーター １４ ポンプ １６ 熱交換器 １８ オキシゲネーター ２０ 人工臓器 ２２ 人工臓器の液体流入口 ２４ 人工臓器の液体流出口 DESCRIPTION OF SYMBOLS 10 Pump 12 Plasma separator 14 Pump 16 Heat exchanger 18 Oxygenator 20 Artificial organ 22 Liquid inlet of artificial organ 24 Liquid outlet of artificial organ
Claims (6)
クスと、該マトリックス中に分散された生細胞と、該マ
トリックス及び生細胞を収納し、外部との液体の流出入
が可能な収納体とを具備する人工臓器。1. A matrix comprising 50% by weight or more of laminin, living cells dispersed in the matrix, and a storage body which stores the matrix and the living cells and allows liquid to flow in and out of the outside. Artificial organs.
ンIVを含む請求項１記載の人工臓器。2. The artificial organ according to claim 1, wherein the matrix contains laminin and collagen IV.
である請求項１又は２記載の人工臓器。3. The artificial organ according to claim 1, wherein the living cells are hepatocytes, pancreatic cells, or kidney cells.
載の人工臓器。4. The artificial organ according to claim 3, wherein the living cells are hepatocytes.
マトリックス及び生細胞は、該中空繊維膜中又は膜外に
充填される請求項１ないし４のいずれか１項に記載の人
工臓器。5. The artificial organ according to claim 1, wherein the container is a hollow fiber membrane, and the matrix and the living cells are filled in or outside the hollow fiber membrane. .
ートリッジに複数本の前記中空繊維膜が収納されている
請求項５記載の人工臓器。6. The artificial organ according to claim 5, wherein a plurality of the hollow fiber membranes are housed in a cartridge having a liquid inlet and a liquid outlet.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9261045A JPH1176400A (en) | 1997-09-09 | 1997-09-09 | Artificial organ |
| PCT/JP1998/004030 WO1999012589A1 (en) | 1997-09-09 | 1998-09-09 | Artificial organ |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9261045A JPH1176400A (en) | 1997-09-09 | 1997-09-09 | Artificial organ |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2007242013A Division JP4304223B2 (en) | 2007-09-19 | 2007-09-19 | Artificial organ |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH1176400A true JPH1176400A (en) | 1999-03-23 |
Family
ID=17356299
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP9261045A Pending JPH1176400A (en) | 1997-09-09 | 1997-09-09 | Artificial organ |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JPH1176400A (en) |
| WO (1) | WO1999012589A1 (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003503022A (en) * | 1999-06-21 | 2003-01-28 | ザ・ジェネラル・ホスピタル・コーポレイション | Cell culture system and method for organ assist device |
| JPWO2004085606A1 (en) * | 2003-03-24 | 2006-06-29 | 独立行政法人国立環境研究所 | Cell culture substrate and solid phase preparation of cell adhesion protein or peptide |
| JP2012522511A (en) * | 2009-03-31 | 2012-09-27 | リージェンツ オブ ザ ユニバーシティ オブ ミネソタ | Methods for decellularizing and recellularizing organs and tissues |
| US10220056B2 (en) | 2005-08-26 | 2019-03-05 | Miromatrix Medical, Inc. | Decellularization and recellularization of solid organs |
| US10233420B2 (en) | 2010-09-01 | 2019-03-19 | Regents Of The University Of Minnesota | Methods of recellularizing a tissue or organ for improved transplantability |
| US11278643B2 (en) | 2016-09-06 | 2022-03-22 | Mayo Foundation For Medical Education And Research | Use of resected liver serum for whole liver-engineering |
| US11452797B2 (en) | 2013-03-15 | 2022-09-27 | Miromatrix Medical Inc. | Use of perfusion decellularized liver for islet cell recellularization |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102210889A (en) * | 2010-04-02 | 2011-10-12 | 瑞德肝脏疾病研究(上海)有限公司 | In-vitro biomaterial for artificial liver dialysis and preparation method thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6417653A (en) * | 1987-07-13 | 1989-01-20 | Otsuka Pharma Co Ltd | Laminate type hybrid artificial liver apparatus |
| JPH0669487B2 (en) * | 1991-02-15 | 1994-09-07 | 東京大学長 | Method for promoting and controlling biological cell growth and functional differentiation |
-
1997
- 1997-09-09 JP JP9261045A patent/JPH1176400A/en active Pending
-
1998
- 1998-09-09 WO PCT/JP1998/004030 patent/WO1999012589A1/en active Application Filing
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003503022A (en) * | 1999-06-21 | 2003-01-28 | ザ・ジェネラル・ホスピタル・コーポレイション | Cell culture system and method for organ assist device |
| JPWO2004085606A1 (en) * | 2003-03-24 | 2006-06-29 | 独立行政法人国立環境研究所 | Cell culture substrate and solid phase preparation of cell adhesion protein or peptide |
| JP4555773B2 (en) * | 2003-03-24 | 2010-10-06 | 独立行政法人国立環境研究所 | Cell culture substrate and solid phase preparation of cell adhesion protein or peptide |
| US10441609B2 (en) | 2005-08-26 | 2019-10-15 | Miromatrix Medical Inc. | Decellularization and recellularization of solid organs |
| US10220056B2 (en) | 2005-08-26 | 2019-03-05 | Miromatrix Medical, Inc. | Decellularization and recellularization of solid organs |
| JP2016039903A (en) * | 2009-03-31 | 2016-03-24 | リージェンツ オブ ザ ユニバーシティ オブ ミネソタ | Decellularization and recellularization of organs and tissues |
| JP2017195900A (en) * | 2009-03-31 | 2017-11-02 | リージェンツ オブ ザ ユニバーシティ オブ ミネソタ | Decellularization and recellularization of organs and tissues |
| JP2019134723A (en) * | 2009-03-31 | 2019-08-15 | ミロマトリックス メディカル インコーポレイテッド | Decellularization and recellularization of organs and tissues |
| JP2012522511A (en) * | 2009-03-31 | 2012-09-27 | リージェンツ オブ ザ ユニバーシティ オブ ミネソタ | Methods for decellularizing and recellularizing organs and tissues |
| US10233420B2 (en) | 2010-09-01 | 2019-03-19 | Regents Of The University Of Minnesota | Methods of recellularizing a tissue or organ for improved transplantability |
| US11414644B2 (en) | 2010-09-01 | 2022-08-16 | Regents Of The University Of Minnesota | Methods of recellularizing a tissue or organ for improved transplantability |
| US11452797B2 (en) | 2013-03-15 | 2022-09-27 | Miromatrix Medical Inc. | Use of perfusion decellularized liver for islet cell recellularization |
| US11278643B2 (en) | 2016-09-06 | 2022-03-22 | Mayo Foundation For Medical Education And Research | Use of resected liver serum for whole liver-engineering |
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