JP2004305120A - Method for acclimatizing function of internal organ, tissue or cell constituting these - Google Patents

Method for acclimatizing function of internal organ, tissue or cell constituting these Download PDF

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JP2004305120A
JP2004305120A JP2003104266A JP2003104266A JP2004305120A JP 2004305120 A JP2004305120 A JP 2004305120A JP 2003104266 A JP2003104266 A JP 2003104266A JP 2003104266 A JP2003104266 A JP 2003104266A JP 2004305120 A JP2004305120 A JP 2004305120A
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tissue
organ
function
cell
cell constituting
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Japanese (ja)
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Tadashi Tokutomi
徳冨直史
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Kumamoto Technology and Industry Foundation
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Kumamoto Technology and Industry Foundation
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for keeping or recovering activity of internal organ or tissue in which an organism constantly generates biological electric signals and constitutional cells mutually receive these electric signals or a cell constituting these and to provide a functionally acclimatized internal organ or tissue and a cell constituting these as a specimen for evaluation of pharmacodynamic effects of medicines and toxicity test of medicines. <P>SOLUTION: The internal organ or the tissue in which the organism constantly generates biological electric signals and constituent these mutually receive electric signals or the cell constituting a cell is cultured under input of pulse electric signals of 0.5-50 Hz and 10 mV-10V or pulse magnetic signals of 0.5-50 Hz and 0.1 mT-10 mT. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【0001】
本発明は、生体での組織の移植方法に関する。本発明は特に、常時電気信号を発生し、構成細胞が互いに電気信号を受けている臓器、または組織、あるいはこれらを構成する細胞の機能馴化方法、好ましくは心筋組織の機能馴化方法に関する。
【0002】
【従来の技術】
再生医学の進歩により、胚幹細胞、造血幹細胞、間葉系幹細胞、更に成熟細胞組織中に僅かに存在する組織特有の幹細胞あるいは残存組織そのものを用いて、多くの組織が再生されるようになった。このことは、臓器移植のためのドナー不足という問題の解決に新たな可能性をもたらすものである。
【0003】
今後の課題として、(1)組織再生のための細胞の増殖、分化効率の向上と、(2)移植のための再生組織の適合性強化が挙げられる。
【0004】
これらのうち、課題(2)の再生組織の適合性の強化については、現在、一部の臓器、例えば、心筋、神経、骨格筋のようなその組織を構成する細胞自体が生体内で特有の電気活動を行って、臓器特有の電気的環境から常に細胞が電気的入力を受けている臓器、特に心臓を構成する心筋移植の試みは、充分に成功しているとは言いがたい。心筋に対する再生・移植に関しては、例えば、次のような特許文献が報告されている。
【0005】
【特許文献1】
特表平9−505471号公報
【特許文献2】
特表2001−502720号公報
【特許文献3】
特開2002−128676号公報
【特許文献4】
特表2002−535358号公報
【0006】
特許文献1は、心筋細胞または骨格筋芽細胞を哺乳動物の心筋組織に注入した心筋組織を移植体として用いるものであり、移植体の心機能への適応能力について不明な点が多く、必ずしも実験医学の域を出ているとは言えない。特許文献2は、心臓組織に粘膜下組織を培養する方法に関する発明である。特許文献3と特許文献4は、免疫抑制剤に関する発明である。
【0007】
臓器に対する組織の移植がうまくいかない原因は、主として免疫系による拒絶反応が挙げられている。他の臓器は免疫抑制剤によりかなりの成果が得られているが、心筋組織では、かなりの割合で定着せず、壊死や心臓が不整脈を起こすことがしばしば認められている。特許文献2の技術も組織移植で好結果が得られているものの、移植された心臓は自然収縮が遅い、あるいは心臓が細動する等のトラブルを生じている。
【0008】
発明者は、生体の定常的に生体電気信号を発生し、構成細胞が互いに電気信号を受ける臓器、または組織を構成する細胞、特に心筋細胞が培養中に次第に外部からの高頻度電気刺激に対する追従能力を失う現象に着目し、その対策について鋭意検討した結果、本発明に到達した。
【0009】
【発明が解決しようとする課題】
本発明は、生体の定常的に生体電気信号を発生し、構成細胞が互いに電気信号を受ける臓器、または組織、あるいはこれらを構成する細胞の活性を維持もしくは取り戻す方法を提供することを目的としている。本発明はまた、そのようにして機能馴化された臓器、または組織あるいは構成する細胞を医薬品の薬効評価および毒性試験のための検体として提供することを目的としている。
【0010】
【課題を解決するための手段】
すなわち、本発明は、 生体の定常的に生体電気信号を発生し、構成細胞が互いに電気信号を受ける臓器、または組織、あるいはこれらを構成する細胞を、0.5〜50Hz、10mV〜10Vのパルス電気信号、または、0.5〜50HZ、0.1mT〜10mTのパルス磁気信号入力下で活性化することを特徴とする臓器、または組織、あるいはこれらを構成する細胞の機能馴化方法である。
【0011】
本発明では臓器として、特に心臓を対象とする。
【0012】
本発明では組織として、特に心筋組織を対象とする。
【0013】
機能馴化は臓器、または組織を移植する前に行うことが好ましい。
【0014】
本発明はまた上記方法で機能馴化された組織、または細胞からなる医薬品の薬効評価および毒性試験用検体である。
【0015】
【発明の実施の形態】
本発明の臓器、または組織、あるいはこれらを構成する細胞の機能馴化方法につき詳しく説明する。
【0016】
本発明の機能馴化方法の対象とする生体とは、人間を始め、犬、ねこ、馬、豚、羊、マウス、ラット等の哺乳動物をはじめ、鳥類、爬虫類、両生類、魚類、細菌、ウイルス等の微生物、植物をも包含する概念である。これらのうちでは、神経及び心臓を有する動物、好ましくは哺乳類、特に好ましくは人間を対象とする。
【0017】
本発明で機能馴化方法の対象とされる臓器・組織としては、生体のあらゆる組織、臓器を包含する。これらのなかでは特に、生体内で興奮性の電気刺激が常在する臓器、組織または組織、例えば心臓、骨格筋、平滑筋、末梢神経、脳等の中枢神経、とりわけ心臓と末梢および中枢の神経組織を対象とする。
【0018】
本発明において移植のために機能馴化する組織としては、上記生体の組織、臓器のうち、常時臓器特有の生体電気信号に晒されている臓器の一部を形成する心筋、すなわち特殊心筋と固有心筋組織を特に対象とする。
【0019】
本発明で機能馴化するための臓器、組織、あるいは細胞は、生体から摘出・採取したものに限定されず、例えば、胚幹細胞(ES細胞)、造血幹細胞、間葉系幹細胞等の幹細胞から分化・培養して得た組織も、用いることができる。
【0020】
本発明で移植のために機能馴化する臓器、組織、または細胞は、生体、例えばMHCを人間のそれに適合させたトランスジェニックブタ等の哺乳類の臓器の一部から摘出した臓器、組織、または細胞でありうる。
【0021】
本発明で機能馴化するための組織は、例えば生体、例えば哺乳動物、例えば人間から採取した細胞をin vitroで増殖させて得た組織でありうる。
【0022】
本発明では、上記方法で得た組織を培養条件下で機能馴化を行なう。この機能馴化は、対象の臓器、組織または細胞を、培地を含有する容器に入れて行なう。
【0023】
なお、本発明における機能馴化とは、移植用に培養・増殖させたあるいは摘出した臓器、組織または細胞が、本来の臓器、組織と同様に生体の電気的興奮入力に適応できるように、一定期間電気的刺激雰囲気内に晒す操作をいう。なお、以後、本案件におけるこれらの操作を一括して「機能馴化」という。
【0024】
本発明の機能馴化方法において用いることのできる培地としては、通常はNa、Mg、Ca等の金属イオンを含んだ生理食塩水に各種アミノ酸、例えばL−アルギニン, L−シスチン, L−グルタミン, L−ヒスチジン、及び各種ビタミン、例えば葉酸、パントテン酸、ニコチンアミド、ピリドキサール、リボフラビン等を含んだ培地、好ましくはEagleの MEM培地あるいはDulbecco&Smodified 培地としてバイオフルイド社から入手できる培地等を用いることができる。
【0025】
対象を機能馴化するための温度は、通常10℃から45℃、好ましくは30℃から37℃の範囲である。
【0026】
上記培地に従来から組織の再生において提案されている各種サイトカイン類、例えばインターロイキン類,ニューロトロピン類,血小板由来成長因子,上皮成長因子,線維芽細胞成長因子を1ng/mlから100ng/mlの範囲の量で配合することが好ましい。
【0027】
本発明の機能馴化方法では、機能馴化中に対象に対して電気信号を与える。電気信号はパルス電気信号もしくはパルス磁気信号であることが好ましい。機能馴化する際、電気信号は連続的に与えてもよいし、断続的に与えてもよい。
【0028】
対象に電気信号を与える方法としては、培地および/または機能馴化対象に電極を接触させて0.5〜50HZ、好ましくは1〜10HZ、10mV〜10V、好ましくは100mV〜1Vのパルス電気信号を与えることが必要である。
【0029】
本発明における機能馴化方法においては、電気的刺激は、最初は低電圧とし、徐々に強度を増加させるようにすることが好ましい。
【0030】
上記方法において与える電流は、パルス信号として流すことが必要である。このため、上記方法において電気流を与える装置は、一定の間隔で電流をパルス状に変化させうる、すなわちパルス電流を発生させうるファンクションジェネレーターと連結されていること好ましい。
【0031】
機能馴化すべき対象に電気信号を与える他の方法としては、図.1に示したように機能馴化すべき対象、好ましくは機能馴化すべき対象の入った容器を磁場、好ましくは磁束密度を変動させうる磁場におき、磁場の磁束密度を変化させることにより機能馴化すべき対象内部に渦電流を発生させる方法を挙げることもできる。
【0032】
上記の磁束密度を変動させうる磁場としては、例えば電磁石で知られているように電線をコイル状にまき、その中心部に移植すべき対象、好ましくは機能馴化すべき対象の入った容器を置き、コイルに電流、好ましくはパルス電流を与えてコイル内の磁束密度を変化させることにより機能馴化すべき対象内の渦電流の強度をパルス的に変化させる、すなわちパルス信号として与えることができる。この後者の方法は機能馴化すべき対象に電極等の異物を接触させることなく刺激が可能である点からより好ましい。
【0033】
このとき機能馴化すべき対象に与える磁気信号は0.5〜50HZ、好ましくは1〜10HZ、0.1mT〜10mT、好ましくは0.5mT〜5mT範囲のパルス信号とすることが必要である。
【0034】
【発明の効果】
本発明の機能馴化方法で興奮性入力に慣らされた臓器、または組織、あるいはこれらを構成する細胞は、生体電気信号に馴らされているため、例えば、移植後直ちに移植されて臓器において生体電気信号を受けても周囲組織と協調した興奮伝導を行うことができる。特に心筋組織の移植において、従来の方法で作成された移植体より極めて高い適応能を有しかつ、移植体が不整脈等の発生源となることを極力防止できる。また、本発明の機能馴化方法による処置を受けた組織、または細胞、特に心筋組織や心筋細胞は常に生理的条件に近い刺激追従能力を保持しているので、より信頼性の高い循環器作用薬の薬効評価および毒性試験用の検体として利用することができる。
【0035】
【実施例】
以下に、本発明の好適な実施例を示す。なお、下の実施例に示される材料、使用量、割合、手順等は、本発明の趣旨を逸脱しない限り適宜変更することができる。したがって、本発明の範囲は以下に示す具体例により限定的に解釈されるべきものではない。
【0036】
(機能馴化細胞の観察)
以下の実施例、比較例における機能馴化心筋の電気刺激に対する応答性の変化と磁気刺激による機能回復については、位相差顕微鏡に取り付けたデジタルム−ビ−カメラを用いて1例ごとに35秒間の動画デ−タを取得保存し、心拍数の測定と集計を行った。
【0037】
(磁気発生装置)
図1に示した磁気発生装置を用いた。
【0038】
(心筋組織の摘出/活性化)
BALB/cマウス(3〜4週令)より心臓を摘出し、カナマイシン(100mg/l)を含む無カルシウム・リン酸緩衝生理食塩水(PBS(−))にて2〜3回洗浄後、3mg/mlコラゲナーゼ(type1,Sigma)を含む同PBS(−)にて37℃,30min、ランゲンドルフ法による灌流処置を行った。
【0039】
【参考例1】
コラゲナーゼ処置した心臓切片を同PBS(−)にて2〜3回洗浄後、先端径をバーナー炎で小さくした(200〜400mm)パスツールピペットを用いて、ピペツテイングにより細胞を単離した。そのようにして作った細胞懸濁液を遠心分離し、沈降した細胞塊を、カルシウムを含む通常のPBSに懸濁し、適量をプレインキュベーション済みの培養用dishに2000〜3000/dishの細胞数で播いて温度37℃、湿度99%、CO濃度5%の条件下で培養を開始した。培養開始24h後、EAGLEs培地を交換し培養をつづけた。なお、プレインキュベーションは、10%ウシ胎児血清を含むEAGLEs培地(日本水産)1.5mlを、35mmカルチャーdish(FALCON,3801)に入れ、0.5〜1時間行った。
【0040】
(機能馴化結果)
培養の時間経過とともに、活性化心筋の心拍数が、本来のマウスの心拍数(486〜738回/分、すなわち8.1〜12.3HZ)から低下し、さらに培養二週間目には電気刺激(強度3V,持続3msec)に対する刺激応答性、すなわち培養した心筋細胞の追従可能な刺激頻度が2〜5Hzにまで低下した。
【0041】
【実施例1】
(移植前機能馴化操作)
上記活性化二週間目の心筋細胞で構成された組織に対し、上記磁気刺激装置を用いて、6mTの磁気刺激を応答性の強さに応じて1〜10HZの頻度で3日間与えた。図2に培養中の心筋細胞の写真を示す。12例全ての刺激応答性が回復し、10HZの上記電気刺激に追従可能であった。
【0042】
【比較例1】
実施例1において、磁気刺激を与えないで実施例1と同様にして、二週間の心筋細胞の培養を行った。
【0043】
(刺激応答性の検討結果)
培養14日目の心筋組織の電気刺激(強度3V, 持続3msec)に対する追従可能な頻度は、検討した全例(11例)において僅か2〜5HZであり、10HZ以上の刺激に対する応答性の回復は全く見られなかった。
【図面の簡単な説明】
【図1】図1は本発明の馴化方法で用いられる装置の1例を示した概略図である。
【図2】図2は、本発明の馴化方法で培養中の心筋細胞の1例の写真である。
[0001]
The present invention relates to a method for implanting tissue in a living body. In particular, the present invention relates to a method for acclimating the function of an organ or a tissue which constantly generates an electric signal and receiving the electric signal to each other, or the cells constituting the organ or tissue, preferably the function of a myocardial tissue.
[0002]
[Prior art]
With the progress of regenerative medicine, many tissues have been regenerated using embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, and even stem cells peculiar to mature tissues or residual tissues themselves. . This opens up new possibilities for solving the problem of shortage of donors for organ transplantation.
[0003]
Future challenges include (1) improving cell proliferation and differentiation efficiency for tissue regeneration and (2) enhancing the suitability of regenerated tissue for transplantation.
[0004]
Among these, regarding the enhancement of the compatibility of the regenerated tissue of the problem (2), at present, some organs, for example, cells constituting the tissue, such as myocardium, nerve, and skeletal muscle, have specific cells in vivo. It is hard to say that attempts to transplant myocardium, which is an organ in which cells are constantly receiving electrical input from the electrical environment unique to the organ by performing electrical activity, particularly the heart, have been sufficiently successful. Regarding regeneration / transplantation of myocardium, for example, the following patent documents have been reported.
[0005]
[Patent Document 1]
Japanese Patent Publication No. 9-505471 [Patent Document 2]
Japanese Patent Publication No. 2001-502720 [Patent Document 3]
Japanese Patent Application Laid-Open No. 2002-128676 [Patent Document 4]
JP 2002-535358 A
Patent Literature 1 uses a myocardial tissue obtained by injecting cardiac muscle cells or skeletal myoblasts into a myocardial tissue of a mammal as a transplant, and there are many unclear points about the adaptability of the transplant to cardiac function. He is not out of medicine. Patent Document 2 is an invention relating to a method of culturing a submucosal tissue in a heart tissue. Patent Literature 3 and Patent Literature 4 are inventions relating to immunosuppressants.
[0007]
The cause of the failure of tissue transplantation into organs is mainly due to rejection by the immune system. Although other organs have achieved considerable success with immunosuppressive drugs, a significant proportion of myocardial tissue does not colonize and is often found to cause necrosis and cardiac arrhythmias. Although the technique of Patent Document 2 has also obtained good results by tissue transplantation, the transplanted heart has troubles such as slow natural contraction or fibrillation of the heart.
[0008]
The inventor of the present invention has proposed that a living body constantly generates a bioelectric signal, and that constituent cells receive an electric signal from each other, or that cells constituting a tissue, particularly cardiomyocytes, gradually follow external high-frequency electrical stimulation during culture. As a result of paying attention to the phenomenon of losing the ability and intensively studying countermeasures, the present invention has been reached.
[0009]
[Problems to be solved by the invention]
An object of the present invention is to provide a method for generating or constantly generating a bioelectric signal of a living body, and for maintaining or regaining the activity of an organ or tissue in which constituent cells receive an electric signal from each other, or the cells constituting these. . Another object of the present invention is to provide an organ, a tissue or a cell constituting the function-adapted organ as described above as a specimen for evaluating the efficacy of a drug and testing toxicity.
[0010]
[Means for Solving the Problems]
That is, the present invention provides an organ or a tissue in which a living body constantly generates a bioelectric signal, and constituent cells receive an electric signal with each other, or a cell constituting the same, by applying a pulse of 0.5 to 50 Hz, 10 mV to 10 V. This is a method for accommodating the function of an organ or a tissue, or a cell constituting the same, which is activated under the input of an electric signal or a pulsed magnetic signal of 0.5 to 50 HZ and 0.1 mT to 10 mT.
[0011]
In the present invention, a heart is particularly targeted as an organ.
[0012]
In the present invention, myocardial tissue is particularly targeted.
[0013]
The functional acclimation is preferably performed before transplanting the organ or tissue.
[0014]
The present invention is also a specimen for evaluating the efficacy and toxicity of a drug comprising a tissue or a cell that has been adapted to function by the above method.
[0015]
BEST MODE FOR CARRYING OUT THE INVENTION
The method for acclimating the function of an organ or a tissue or cells constituting the same according to the present invention will be described in detail.
[0016]
The living body to be subjected to the function acclimatization method of the present invention includes humans, dogs, cats, horses, pigs, sheep, mice, rats, and other mammals, birds, reptiles, amphibians, fish, bacteria, viruses, and the like. This concept includes microorganisms and plants. Among them, animals having nerves and hearts, preferably mammals, particularly preferably humans are targeted.
[0017]
The organs / tissues to be subjected to the function acclimation method in the present invention include all tissues and organs of a living body. Among these, in particular, organs, tissues or tissues in which excitatory electrical stimulation is resident in the living body, such as the central nervous system such as the heart, skeletal muscle, smooth muscle, peripheral nerve, and brain, particularly the heart and peripheral and central nerves Target organizations.
[0018]
In the present invention, the tissue to be adapted for transplantation includes, as the tissues of the living body, the myocardium forming a part of the organ that is constantly exposed to the bioelectric signal specific to the organ, that is, the special myocardium and the intrinsic myocardium. Especially for organizations.
[0019]
The organs, tissues, or cells for functional acclimation in the present invention are not limited to those extracted and collected from a living body, and for example, differentiated from stem cells such as embryonic stem cells (ES cells), hematopoietic stem cells, mesenchymal stem cells, and the like. A tissue obtained by culturing can also be used.
[0020]
An organ, tissue, or cell that is adapted for transplantation in the present invention is an organ, tissue, or cell isolated from a living body, for example, a part of a mammalian organ such as a transgenic pig whose MHC has been adapted to that of a human. It is possible.
[0021]
The tissue for functional adaptation in the present invention can be, for example, a tissue obtained by growing cells collected from a living body, for example, a mammal, for example, a human, in vitro.
[0022]
In the present invention, the tissue obtained by the above method is subjected to functional conditioning under culture conditions. This functional adaptation is performed by placing the target organ, tissue or cell in a container containing a medium.
[0023]
The function acclimation in the present invention means that an organ, tissue or cell cultured or proliferated or removed for transplantation can adapt to an electrical excitation input of a living body for a certain period of time in the same manner as the original organ or tissue. This refers to the operation of exposing to an electrically stimulated atmosphere. Hereinafter, these operations in this case will be collectively referred to as “functional acclimation”.
[0024]
As the medium that can be used in the method for acclimating the function of the present invention, usually, various amino acids such as L-arginine, L-cystine, L-glutamine, L-glutamine, and the like are added to physiological saline containing metal ions such as Na, Mg, and Ca. -A medium containing histidine and various vitamins, for example, folic acid, pantothenic acid, nicotinamide, pyridoxal, riboflavin, etc., preferably Eagle's MEM medium or a medium available from Biofluid as Dulbecco & Modified medium can be used.
[0025]
The temperature for adapting the subject to function is usually in the range of 10 ° C to 45 ° C, preferably 30 ° C to 37 ° C.
[0026]
Various cytokines conventionally proposed for tissue regeneration, such as interleukins, neurotropins, platelet-derived growth factor, epidermal growth factor, and fibroblast growth factor, in the above medium in the range of 1 ng / ml to 100 ng / ml. Is preferably blended in an amount of
[0027]
In the function acclimatization method of the present invention, an electric signal is given to an object during the function acclimation. Preferably, the electrical signal is a pulsed electrical signal or a pulsed magnetic signal. When the function is adapted, the electric signal may be given continuously or intermittently.
[0028]
As a method of giving an electric signal to a subject, an electrode is brought into contact with a culture medium and / or a function-conditioned subject to give a pulse electric signal of 0.5 to 50 HZ, preferably 1 to 10 HZ, 10 mV to 10 V, preferably 100 mV to 1 V. It is necessary.
[0029]
In the function acclimatization method of the present invention, it is preferable that the electric stimulus is initially set to a low voltage and the intensity is gradually increased.
[0030]
The current applied in the above method needs to flow as a pulse signal. For this reason, the device for applying an electric current in the above method is preferably connected to a function generator capable of changing the current in a pulsed manner at regular intervals, that is, generating a pulsed current.
[0031]
Figure 2 shows another method of giving an electric signal to the object to be adapted. As shown in FIG. 1, the object to be functionalized, preferably the container containing the object to be functionalized is placed in a magnetic field, preferably a magnetic field capable of changing the magnetic flux density, and the function is adapted by changing the magnetic flux density of the magnetic field. A method of generating an eddy current inside a target object may be used.
[0032]
As the magnetic field capable of varying the magnetic flux density, for example, an electric wire is wound in a coil shape as is known by an electromagnet, and a container containing an object to be transplanted, preferably an object to be functionalized, is placed at the center thereof. By applying a current, preferably a pulse current, to the coil to change the magnetic flux density in the coil, the intensity of the eddy current in the object to be functionalized can be changed in a pulse manner, that is, given as a pulse signal. This latter method is more preferable in that the stimulation can be performed without bringing a foreign object such as an electrode into contact with the object to be adapted.
[0033]
At this time, it is necessary that the magnetic signal given to the object to be functionalized be a pulse signal in the range of 0.5 to 50 HZ, preferably 1 to 10 HZ, 0.1 mT to 10 mT, preferably 0.5 mT to 5 mT.
[0034]
【The invention's effect】
Since the organ or tissue accustomed to the excitatory input in the function acclimatization method of the present invention, or the cells constituting these, are accustomed to the bioelectric signal, for example, they are transplanted immediately after transplantation and the bioelectric signal in the organ is obtained. Even in response to this, excitement conduction can be performed in cooperation with surrounding tissues. In particular, in transplantation of myocardial tissue, it has a much higher adaptability than a transplant prepared by a conventional method, and can prevent the transplant from becoming a source of arrhythmia or the like as much as possible. In addition, the tissue or cells treated by the method for acclimating the function of the present invention, particularly myocardial tissue and myocardial cells, always maintain a stimulus tracking ability close to physiological conditions, so that a more reliable cardiovascular agent It can be used as a specimen for the evaluation of the efficacy and toxicity test of.
[0035]
【Example】
Hereinafter, preferred embodiments of the present invention will be described. The materials, amounts, ratios, procedures, and the like shown in the following examples can be appropriately changed without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples described below.
[0036]
(Observation of functionally adapted cells)
In the following Examples and Comparative Examples, changes in responsiveness to electrical stimulation of functionally conditioned myocardium and recovery of function by magnetic stimulation were measured using a digital movie camera attached to a phase-contrast microscope for 35 seconds for each case. The video data was acquired and saved, and the heart rate was measured and counted.
[0037]
(Magnetic generator)
The magnetic generator shown in FIG. 1 was used.
[0038]
(Removal / activation of myocardial tissue)
The heart was excised from a BALB / c mouse (3-4 weeks old), washed with calcium-free / phosphate-buffered saline (PBS (-)) containing kanamycin (100 mg / l) 2-3 times, and then 3 mg. Perfusion treatment was performed by the Langendorff method in the same PBS (-) containing 1 / ml collagenase (type 1, Sigma) at 37 ° C. for 30 minutes.
[0039]
[Reference Example 1]
After the collagenase-treated heart section was washed with the same PBS (-) two or three times, cells were isolated by pipetting using a Pasteur pipette whose tip diameter was reduced by burner flame (200 to 400 mm). The cell suspension thus prepared is centrifuged, and the sedimented cell mass is suspended in normal PBS containing calcium, and an appropriate amount is added to a pre-incubated culture dish at a cell count of 2000 to 3000 / dish. The seeding was performed, and the culture was started under the conditions of a temperature of 37 ° C., a humidity of 99%, and a CO 2 concentration of 5%. 24 hours after the start of the culture, the EAGLEs medium was replaced and the culture was continued. The preincubation was performed for 0.5 to 1 hour by placing 1.5 ml of EAGLEs medium (Nippon Suisan) containing 10% fetal bovine serum in a 35 mm culture dish (FALCON, 3801).
[0040]
(Functional acclimation results)
With the lapse of time in the culture, the heart rate of the activated myocardium decreased from the original heart rate (486-738 beats / minute, ie, 8.1-12.3 HZ) of the mouse, and the electrical stimulation was performed in the second week of the culture. (Intensity 3 V, continuous 3 msec), i.e., the stimulus frequency that can be followed by cultured cardiomyocytes decreased to 2 to 5 Hz.
[0041]
Embodiment 1
(Function adaptation operation before transplantation)
To the tissue composed of the myocardial cells two weeks after the activation, 6 mT magnetic stimulation was applied at a frequency of 1 to 10 HZ for 3 days using the magnetic stimulator according to the strength of the response. FIG. 2 shows a photograph of cardiomyocytes during culture. The stimulus responsiveness of all 12 cases recovered, and it was possible to follow the electrical stimulation at 10 HZ.
[0042]
[Comparative Example 1]
In Example 1, cardiomyocytes were cultured for two weeks in the same manner as in Example 1 without applying magnetic stimulation.
[0043]
(Study response results)
The frequency with which the myocardial tissue on the 14th day of culture can be followed for electrical stimulation (intensity 3 V, sustained 3 msec) is only 2 to 5 HZ in all the examined cases (11 cases), and the responsiveness to the stimulation of 10 HZ or more is recovered. I didn't see it at all.
[Brief description of the drawings]
FIG. 1 is a schematic diagram showing an example of an apparatus used in a conditioning method of the present invention.
FIG. 2 is a photograph of one example of a cardiomyocyte being cultured by the adaptation method of the present invention.

Claims (5)

生体の定常的に生体電気信号を発生し、構成細胞が互いに電気信号を受ける臓器、または組織、あるいはこれらを構成する細胞を、0.5〜50HZ、10mV〜10Vのパルス電気信号、または、0.5〜50HZ、0.1mT〜10mTのパルス磁気信号入力下で培養することを特徴とする臓器、または組織、あるいはこれらを構成する細胞の機能馴化方法。An organ or a tissue that constantly generates a bioelectric signal of a living body and the constituent cells receive an electric signal from each other, or a cell constituting the organ or tissue, is subjected to a pulse electric signal of 0.5 to 50 HZ, 10 mV to 10 V, or 0 A method for acclimating a function of an organ or a tissue, or a cell constituting the organ or tissue, which is cultured under a pulse magnetic signal input of 0.5 to 50 HZ and 0.1 mT to 10 mT. 臓器が心臓であることを特徴とする請求項1記載の臓器、または組織、あるいはこれらを構成する細胞の機能馴化方法。2. The method according to claim 1, wherein the organ is a heart. 組織が、心筋であることを特徴とする請求項1記載の臓器、または組織、あるいはこれらを構成する細胞の機能馴化方法。2. The method according to claim 1, wherein the tissue is a myocardium. 機能馴化は、臓器、または組織を移植する前に行うことを特徴とする請求項1〜3記載の臓器、または組織、あるいはこれらを構成する細胞の機能馴化方法。The method for acclimating a function of an organ or a tissue or cells constituting the organ or the tissue according to claim 1, wherein the function acclimation is performed before transplanting the organ or the tissue. 請求項1〜4の方法で機能馴化された組織、または細胞からなる薬効評価および毒性試験用検体。A sample for evaluating the efficacy and toxicity of a tissue or a cell adapted to function according to the method of claims 1 to 4.
JP2003104266A 2003-04-08 2003-04-08 Method for acclimatizing function of internal organ, tissue or cell constituting these Pending JP2004305120A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005071057A1 (en) * 2004-01-22 2005-08-04 Japan Science And Technology Agency Magnetic or electric field stimulating device and method for promoting, restraining, or obstructing growth and function of living cell or living tissue using the magnetic or electric field stimulating device
WO2017204111A1 (en) * 2016-05-26 2017-11-30 株式会社Ifg Apparatus and method for non-contact electrical stimulation of cells in liquid culture medium

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005071057A1 (en) * 2004-01-22 2005-08-04 Japan Science And Technology Agency Magnetic or electric field stimulating device and method for promoting, restraining, or obstructing growth and function of living cell or living tissue using the magnetic or electric field stimulating device
WO2017204111A1 (en) * 2016-05-26 2017-11-30 株式会社Ifg Apparatus and method for non-contact electrical stimulation of cells in liquid culture medium
JP6258567B1 (en) * 2016-05-26 2018-01-10 株式会社Ifg Non-contact electrical stimulation apparatus and non-contact electrical stimulation method for cells in culture medium
CN109153957A (en) * 2016-05-26 2019-01-04 株式会社Ifg The non-contact electrical stimulation device and non-contact electrical stimulation method of cell in culture solution
CN109153957B (en) * 2016-05-26 2021-07-06 株式会社Ifg Non-contact electrical stimulation device and non-contact electrical stimulation method for cells in culture solution

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