JP2004529938A - Organ chamber for blood-free metabolic support system - Google Patents

Abstract

A blood-free metabolic support system for maintaining an organ (40) or tissue at a near normal metabolic rate is disclosed. The system comprises an organ chamber (32) containing a container (33) and a support (36) adapted to limit movement of the organ (40) within the container (33) during perfusion and / or transfer. Is used. The organ chamber (32) further includes a conduit (43) for receiving venous outflow of the perfusion solution and preventing contact of the external surface of the organ (40) with the venous outflow. The conduit (47) for receiving the organ product allows for collection of the organ product resulting from the functional organ (40) during perfusion. The use of the organ chamber (32) provides the necessary configuration for long-term maintenance of the organ for transplantation, resuscitation and active repair of organs suspected of having undergone warm ischemic injury, and transport of the removed organ. New or continuous synthesis of the components is supported.

Description

【００４３】
【表１−１】

【００４４】
【表１−２】

【００４５】
【表１−３】

該灌流溶液は、酸化的代謝のための分子状酸素を器官に与える働きをする一つ以上の酸素運搬化合物（「酸素運搬体」）を含む。このような酸素運搬体は当業者らに良く知られており、ヘモグロビン、及びジアスピリン架橋ヘモグロビン（ＤＣＬＨｂ）、ｏ−ラフィノース架橋ヘモグロビン、及びグルタルアルデヒド架橋ヘモグロビンなどの架橋ポリヘモグロビンを含む安定化ヘモグロビン誘導体、及びポリオキシエチレン複合体（polyoxyethylene conjugates）（ＰＨＰ）、ＰＥＧ複合ヘモグロビンなどの複合ヘモグロビン（conjugated hemoglobin)、組換えヘモグロビン生産物、パーフルオロ化合物（ＰＦＣ）エマルジョン及び／又はパーフルオロ化合物超微粒気泡（集合的に「パーフルオロ化合物」と称する）を含むが、これらに限定されない。このような酸素運搬体の一つはパーフルブロン・エマルジョン（臭化ペルフルオロオクチル、ＰＦＯＢ）などのパーフルオロ化合物である。酸素運搬体として役立つと言われている他のパーフルオロ化合物・エマルジョンは、例えば米国特許第５，４０３，５７５号、４，８６８，３１８号、４，８６６，０９６号、４，８６５，８３６号、４，６８６，０２４号、４，５３４，９７８号、４，４４３，４８０号、４，４２３，０７７号、４，２５２，８２７号、４，１８７，２５２号、４，１８６，２５３号、４，１１０，４７４号、及び３，９６２，４３９号に記載されている。このような液状ＰＦＣエマルジョンには、臭化ペルフルオロオクチル、二臭化ペルフルオロオクチル（perfluorooctyl dibromide）、ブロモフルオロカーボン、ペルフルオロエーテル、フルオゾルＤＡ（商標）、Ｆ−４４Ｅ、１，２−ビスペルフルオロブチル−エチレン、Ｆ−４−メチル オクタヒドロキノール−イジジン（F-4-methyl octahydroquinol-idizine) 、９〜１２炭素ペルフルオロアミン、ペルフルオロデカリン、ペルフルオロインダン、ペルフルオロトリメチル・ビシクル［３，３，１］イナン、ペルフルオロメチル・アダマンテ（perfluoromethyl adamante) 、ペルフルオロジメチル・アダマンチン（perfluoromethyl adamantine) が含まれるが、これらに限定されない。このような酸素運搬体は、本発明の灌流溶液の調製の際に基礎溶液に添加し溶解される添加成分を容量で約０％から約５９％、又は灌流溶液全体の約０％から約２０％（ｖ／ｖ）を含む。
【００４６】
また、赤血球（ＲＢＣ）が酸素運搬体として、灌流される器官による代謝を支えるのに効果的な量で用いられる場合がある。一般的に、灌流溶液５００ｍｌにつき約５ｃｃのＲＢＣ（即ち、約１％）が効果的な量である。パーフルオロ化合物のエマルジョン又は複合ヘモグロビンと比較した場合、ＲＢＣは代謝している器官による酸素消費と同等の酸素濃度を供給する。これは一般的に０.１〜０.３ｃｃ／分／ｇｍの速度で発生する。加えて、灌流の２４から４８時間後にはＲＢＣがクレチネート（cretinate）されたという証拠はなかった。従ってこの範囲のＲＢＣ量は、血液に基づく灌流物と関連した器官への機構的損傷の問題を与えない。
【００４７】
ＲＢＣ（灌流液５００ｃｃにつき５ｃｃ）を、＞２００ｍｍＨｇのＰＯ₂が得られるまで循環している灌流液に添加した。安定した灌流圧、血管流速、及び利尿が得られた（表２参照）。
【００４８】
（外１）

（外２）

好ましい実施態様では、化学的に修飾されたヘモグロビン、例えばポリエチレングリコール修飾ヘモグロビンは、灌流液の酸素張力（oxygen tension）を灌流されている器官による代謝を支えるのに十分なレベルに維持するために用いてもよい。一般的に、灌流溶液５００ｍｌにつき約０.０１ｇから０.５ｇ未満が効果的な量である。化学的に修飾されたヘモグロビンは、組換えられたか又はヒト若しくは他の動物から単離されたかのいずれかで、酸素を除去し、例えば架橋結合又は重合（polymeraization）により、又はポリエチレングリコールやポリオキシエチレンなどの付加物の添加により化学的に改変されたヘモグロビンを指す。ポリエチレングリコール修飾ヘモグロビンは、ポリエチレングリコール（α−ヒドロ−ω−ヒドロキシポリ−（オキシ−１，２−タンジイル）と結合するように修飾されたヘモグロビンを指し、化学的に改変されたヘモグロビンの調製法は当分野で知られており、例えば米国特許第５，２３４，９０３号、５，３８６，０１４号、６，０５４，４２７号、５，９８５，８２５号、及び５，８１４，６０１号がある。その内容は参照により本明細書にその全体がインコーポレートされる。
【００４９】
本発明の灌流溶液は更に、生理学的に有効な量で血管拡張剤を含み、これは微細血管を適切に拡張するためばかりでなく平滑筋細胞弛緩を介して大血管を適切に拡張するための手段をも提供する。脈管構造の正常な透過性が維持されていることを保証するため、この血管拡張は内皮細胞に依存した形で制御される。本発明の灌流溶液で使用する血管拡張成分には、（ｉ）アセチルコリン、ドーパミン、ブラジキニン、及びアルギニンなどの内皮細胞が媒介する血管拡張のための基質、（ii）プロスタサイクリン（及び類似体、例えばカルバサイクリン）及びＭｇ⁺などの微細血管の血管拡張のための基質、及び（iii)アデノシン（及び類似体、例えばシクロヘキシルアデノシン）、及びカルシウム・チャンネル遮断により媒介される血管膨張に対するこれらを組み合わせ効果のためのベラパミルが含まれる。本発明が包含する他のカルシウム・チャンネル遮断薬は、フルナリジン、ニフェジピン、ＳＮＸ−１１、クロルプロマジン、及びジルチアゼムを含む。前述の血管拡張剤の使用は脈管構造が十分拡張し、同時にその完全性と正常な障壁機能を保持することを確実にする。この血管拡張剤は、本発明の灌流溶液を調製する際に基礎溶液に添加される添加成分の組み合わせを約１％から約５０容量％（ｗ／ｖ）で含む。
【００５０】
ＥＭＳ灌流部分システム
本発明は、好ましい実施態様を参照しながら説明される。このシステムの様々な構成要素は組み合わされる場合や、設計上の選択としてシステムに導入された別々の部品として提供される場合があることを理解すべきである。
【００５１】
図１は本発明の器官灌流循環経路の一つの実施態様の図である。殆どの場合、一つの灌流経路のみが必要となる。しかしながら、代謝的に維持されるべき器官が肝臓である場合は、二つの灌流回路が必要である。この二つの灌流経路は図２に示してあり、以下で更に詳細に説明する。このシステムを通る循環に利用できる灌流液は全て、ポンプ１２により循環経路１０を通って推進される。この灌流溶液の流れの方向は流路内に矢印で示してある。本発明の灌流液を循環させるのにどんなポンプを用いても良いが、脈動ポンプ、例えばモデル番号ＭＯＸ−１００（商標）（ウォーターズ・インストルメンツ，インク．，ロチェスター，ミネソタ州）が好ましい。
【００５２】
該灌流液は、器官４０に入る前に熱交換器１４を通過して２５℃〜３７℃の範囲内の温度となる。器官による最適な代謝にとって好ましい温度はこの範囲内にある。この熱交換器１４は、灌流経路１０内に配置されている温度センサー１８からの入力を受信する温度コントローラー１６で制御される。一つの実施態様では、この温度コントローラーは、温度を所望の範囲に保つために必要な場合には、灌流液の温度を感知する熱電対と、熱電対で活性化される熱交換器を含む一つのユニットである。別の実施態様では、上記温度は温水を灌流液貯蔵槽又は酸素供給器の周囲に温水を循環させるための水加熱装置により制御しうる。さらに別の実施態様では、器官室内に配置された温度センサーが灌流液の温度を監視しそして制御するのに用いられる。
【００５３】
酸素運搬体を含む該灌流液は、器官と接触する前に、中空糸酸素供給器若しくは膜酸素供給器２０、例えばサルネスから入手できるもののような小児用サイズの酸素供給器を介して酸素化し、大抵の場合は１００〜２４０ｍｍＨｇの範囲内の酸素分圧が得られる。しかしながら、肝臓の門脈系に灌流液を供給する灌流経路に配置される場合、この分圧は大抵８０〜１４０ｍｍＨｇの範囲内に維持される。好ましい実施態様では、この酸素供給器２０は熱交換器１４とｐＨセンサー２２の間の灌流溶液流路１０に配置される。この酸素供給器は独立したユニットであっても良いが、例えば熱交換器と組み合わせたり又は器官室の一部として、本システムの別の構成単位と組み合わせて一つのユニットを形成しても良い。
【００５４】
該灌流溶液のｐＨ及びＰａＣＯ₂は、ＣＯ₂による灌流液の自動間欠ガス発生で制御され、７.３０〜７.４５の範囲のｐＨと３０〜６０ｍｍＨｇのＣＯ₂分圧を有する循環間流液となる。最終的にこの灌流液は、器官室３２に入る直前に従来型の気泡トラップ３０で気泡が除去される。この灌流液は静脈廃液として器官から出て行き、大抵の灌流システムでは、器官を保持する器官室３２の小室の下に配置された廃液貯蔵槽３８に重力により直接排液される。好ましい実施態様では、この廃液は器官４０の静脈内に挿入された従来型の非外傷性のカニューレによるか、又は以下に更に詳しく説明するように静脈が導管４３と連結し続けるように静脈を支えるための装置４９によって静脈に接続される導管４３を用いて廃液貯蔵槽３８に向けられる。
【００５５】
器官室
用いられる器官室３２の仕様は維持されるべき器官に左右される。例えば、器官が肝臓の場合、器官室には二つ同時に作動する灌流流路を収容する二つ以上の入口がなくてはならない（図２）。器官が心臓の場合には、心臓の灌流液の入口の上部の灌流流路から上方に延びる開口した垂直な管が、鼓動している心臓による灌流液の押し出しに対抗するのに十分な圧力が維持されるように灌流液の柱を提供する。
【００５６】
しかしながら、一般的に器官室３２（図１〜４に示すとおり）は、維持すべき器官４０を収容するのに適したサイズの下部と上部を含み、覆い又は蓋３４を有するコンテナ３３を含む。室全体は医療用の品質で、無毒の、血を吸わない（non-leeching) 、殺菌可能な硬い素材でできている。一つの実施態様では、この器官室はステンレス鋼製で、１枚以上の透明若しくは半透明のパネルのような、室の内部を見るための手段を有している。灌流中に器官を観察できることは極めて望ましく、従って好ましい実施態様では、器官室全体が透明な硬い素材でできている。様々な熱可塑性物質が市販されている。例えば、ルサイト、又はＬＥＸＡＮ（登録商標）などのポリカーボネート素材である。本発明の器官室は、ふさわしい素材の組み合わせを含んでもよい。
【００５７】
本発明の器官室は、灌流の間器官の機械的制約を与えるコンテナ３３の中で器官４０を支えるための器官室コンテナ３３の上部に配置できる少なくとも一つの支持体３６を含む。これにより、例えば輸送中の器官の突発的な移動により器官又はその血管が障害を受けたり損傷するのを最小限に抑えられる。器官室３２は更に、灌流液が器官の外部表面及び器官室の内部表面に接触するのを避けて汚染の危険を最小限にするため、器官からの灌流液の静脈流出を温保存システムの貯蔵槽に直接流す一つ以上の灌流液入口４２、導管４３を含む。また、この器官室は器官４０から出る器官生産物を回収する導管４８を含む。任意選択的に、コンテナ３３の下部は灌流液貯蔵槽３８及び／又は灌流システムの他の構成要素を含む。
【００５８】
器官支持体３６は、器官がコンテナ内で移動するのを阻害するように適合される。器官支持体３６は、固形の素材若しくは半固形の素材から作りうる、又は器官を吊り下げたような状態でぶらさげるメッシュ状の布で作ってもよい。一つの実施態様では、この支持体は器官が空洞内に納まるように器官の一部の形状に形を合わせた空洞を持つ固形素材又は半固形素材である。好ましい実施態様では、この支持体は上に載せる器官の形に適合できるガス（不活性ガス）、流体（例えば、生理的食塩水又は他の適切な非毒性液体）、又はゲル（例えば、シリコン）を充填したパウチ又は袋などの弾性のある物質である。支持体の外部表面及び内部の素材は両者とも、不活性で生態適合性があり殺菌できる素材でなければならない。支持体は底面上、及び部分的に側面で器官と接触するが、灌流の間器官を観察できるように、器官室コンテナの側面又は覆いから見える部分を残している。器官の目視観察は水腫の徴候を早期に発見するため、適切に灌流されていない器官の場所又は器官の機能不全の他の徴候を発見するために必要である。
【００５９】
支持体は器官室内で器官を支え、重要なことに、室内での器官の移動を阻止し、それにより器官の損傷を防ぐ。室内で器官が滑ったり転がったりするのを防ぐことにより、特に器官の輸送中に主要な血管、例えば腎臓の腎静脈及び動脈が伸びるのも排除する。振動や衝撃による影響が減少する。さらに、このタイプの器官支持は、器官が身体内で隣接する組織に支えられそして隣接する組織と接触している正常な生理的環境により近い。さらに、成形性のある支持体により、器官の側に置くだけでなく、器官を様々な向き（生理学的なものも含む）に向けることができる。
【００６０】
図３は、器官室３２に器官４０の静脈４１から灌流液を器官室（図３に示したとおり）の一部であるか又は器官室とは別のものである（図１及び２）灌流液貯蔵槽３８に直接導く手段を有する灌流液出口４３がある実施態様を表す。静脈４１を出て行く灌流液が器官４０の側面を流れ落ち、器官室の下に配置された貯蔵槽に滴り落ちる現存する器官室とは異なり、本発明の器官室は、灌流液が器官の外部表面と接触するのを防止することにより、灌流液の汚染の可能性を実質的に減少させる。任意選択的に、この器官室は、図３及び図５に図示するように静脈支持体４８を更に含む。この実施態様のさらなる利点は、器官に隣接して配置された殺菌可能な先が分かれた小さな部品（図３及び図５に示す）のような静脈支持手段により、静脈にカニューレを挿入する必要性及びカニューレの挿入に関連する障害や損傷を避け得ることである。静脈支持体４８は静脈と器官の外壁との交差点に近い場所で静脈を支持し、静脈排液を器官から離して貯蔵槽３８に導く管の端に対して静脈の開口端を安全に保持することができる。
【００６１】
本発明の器官室は使い捨てのユニットであっても良く、又は殺菌して再使用できるステンレス鋼やポリカーボネートなどの適切な素材でできていても良い。一つの実施態様では、本発明の器官室は器官室を外部の温保存システムに取り外し可能なように接続するためのコネクターを含む。適切なコネクターは、いったん器官室をその中に設置すると流路の完全性を確実にするための、長い不活性な生物学的適合性のある管又はより複雑な機械装置を含むがこれらに限定されない。
【００６２】
任意選択的に、この器官室は酸素供給器、貯蔵槽、気泡トラップ及び流量計、これらに限定されないが、含む温灌流システムの様々なコンポーネントを含んでもよい。また、圧力、温度、ｐＨ、ＰａＯ₂、ＰａＣＯ₂、ブドウ糖などの代謝の指標、及びＮＯ（安定な終末生産物）ＰＣＮＡ又はＺＯ−１などの合成機能のマーカーを測定する手段が器官室に組み込まれる場合がある。更に本発明の器官室は、灌流液をサンプリングするための一つ以上の出入口、及び灌流液を再生利用するための一つ以上の出入口を含む。
【００６３】
図３は、器官室コンテナの下部に灌流液貯蔵槽を有する本発明の器官室の一実施態様を例示する。器官代謝を示す灌流液のパラメータ、例えばブドウ糖濃度、ＰａＯ₂、ＰａＣＯ₂などを監視するためのセンサーが、灌流液が器官に入る直前と器官から出るときの灌流液の特性を測定するために配置されている。器官の前後の灌流液の値の変化は器官代謝を示す。
【００６４】
ｐＨ及び温度などの灌流液のその他のパラメータのためのセンサーを器官室内に配置して、灌流の間、灌流液のパラメータを最適に制御させても良い。
【００６５】
大抵の保存システムでは、器官から出る静脈廃液は、支持手段３６内の穴３７のいずれかを通って重力により器官室３２の貯蔵槽３８に直接流れて回収される。本発明では、静脈流出は重力による流れによって回収されるが、静脈を例えばある長さの管４３に連結して、器官から離して運ばれる。一つの実施態様では、これは非外傷性のカニューレを用いて静脈にカニューレを挿入することで実施される。しかしながら、再移植の際の切除で利用できる静脈の長さは、カニューレ挿入の間に発生する何らかの損傷により減少する。従って、好ましい実施態様では、導管内で静脈４１を支持する静脈支持体４６は、静脈４１が器官４０の外壁と交差する点で器官に隣接した状態で静脈の下に配置される。図６及び図７に示されるように、この支持体の上部は静脈及び導管をこの２つの合流点で支持するのに十分なだけ広い。これにより、静脈４１の結紮された端を導管に、例えばある長さの管４３に、カニューレ挿入する必要なく、例えば管４３の端を静脈４１の上部で単に滑らせるだけで接続することが可能になる。または、静脈は部分的に開いた導管内に落ち着かせてもよい。静脈の更なる損傷はこれにより回避される。
【００６６】
器官生産物の回収
本発明の器官室は、器官生産物を灌流液と分けて回収するか、或いは器官生産物を灌流液の一部として再循環させることができる。器官生産物を回収する手段４８はバルブ及び／又は出入口を含み、それによって灌流中に器官が生産した器官生産物の全て又は一部を試料として採取しその器官にとっての正常な器官機能を示すパラメータについて検定することができる。
【００６７】
本発明の器官室は、器官生産物を灌流液から分けて任意選択的に回収する手段を更に含む。例えば、灌流された腎臓により生産される尿は、尿管から器官生産物流路へ出て行く。この器官生産物経路内に配置されるのは、開いたときに器官生産物経路を静脈灌流液経路と流体が流れるように連結させるバルブである。このバルブが閉じた位置にあれば、器官生産物は灌流液から迂回され、試料採取用又は回収用出入口により該システムから除去されうる。この器官の機能的特性はこの回収した器官生産物の化学的パラメータ、生産物の生産速度、又は消失速度を決定するために添加したトレーサー分子の濃度を評価することにより求めることができる。或いは、器官生産物は灌流液容量、容積モル浸透圧濃度及び化学的完全性が保持できるよう、灌流液に再混合される。このようにして、器官代謝、灌流及び機能が測定でき、保存される器官の状態が先を見越して評価できる。
【００６８】
該廃液は、ミリポア社から入手できるような０.２２ミクロンの孔径を持つフィルター４４を通して濾過し、破砕物及び細菌性汚染物を除去する。回収した廃水はこのように、空気との接触による灌流液の汚染を最小にするという別の利点ももたらす。いったん灌流溶液が廃液貯蔵槽３８に回収されると、この廃液は灌流液交換又は灌流透析部分システム５０内で透析するかのいずれかで再生され、該システムを再循環する。
【００６９】
本発明の器官室は更に、灌流溶液のパラメータを監視し制御するための適切なマイクロプロセッサ及びセンサーを含む。
【００７０】
灌流再生
当発明のシステムにおける灌流溶液の構成物の器官による連続的な代謝利用のため、灌流液は補給するか再生しなければ遂には使い果たすであろう。一つの実施態様では、代謝を支えるのに必要な成分は灌流液交換、即ち、貯蔵槽内の装置４６により消耗した灌流液の一部を除去し、新鮮な灌流液で置き換えることにより補給できる。
【００７１】
灌流交換
一つの実施態様では、灌流液は容量を調節できる二つのポンプ４７及び４９を用いて交換される。ポンプ４７は消耗した灌流液を抽出するための貯蔵槽内の装置４６に接続され、ポンプ４９は酸素供給器２０のすぐ前の灌流液経路１０に配置される。消耗した灌流溶液のポンプ４７による除去の直ぐ後にポンプ４９によりこのシステムに等量の新鮮な灌流液を導入する。この方法の交換は連続的又は断続的にできる。交換の速度は、温度及び／又は灌流される器官の代謝速度によって決まる。
【００７２】
新鮮な灌流液を酸素供給器２０およびｐＨ制御システムのすぐ前にある灌流経路に導入することにより、灌流液のＰａＯ₂及びｐＨは一定に保たれる。このように代謝の副産物は除去され、アシドーシスを進行させずに器官による進行中の代謝を維持するため、新鮮な溶液が投与される。灌流液の量は一定のままであり、容積モル浸透圧濃度も一定のままである。
【００７３】
灌流液透析
しかしながら、代謝速度に追いつくためには器官の代謝を維持するのに必要な栄養分を灌流液交換で補充するのが不適切な場合もある。従って別の実施態様では、灌流液は単独で又は灌流液交換と組み合わせて連続的に透析により再生される。
【００７４】
本発明の透析部分システムでは、代謝の継続に必要な栄養分及び化学エネルギー基質は一つ以上の透析ユニットにより灌流液中に再導入される。このようなユニットは、一つで又はいくつか繋いで、灌流液から異化代謝産物を除去する透析に類似した方法で、補給対象の灌流液中に栄養分を供給することができる。
【００７５】
該ＥＭＳの灌流透析部分システムは、図１に示すように、灌流流路１０と流体が流れるように連結している廃液貯蔵層５０及び一つ以上の栄養分貯蔵槽Ｄ１、Ｄ２、Ｄ３、Ｄｎを含む。栄養分貯蔵槽Ｄ１、Ｄ２、Ｄ３、Ｄｎは分子量カットオフが１，０００〜８０，０００ダルトンの範囲で変わる半透水性膜Ｍ１、Ｍ２、Ｍ３、Ｍｎを経て廃液貯蔵槽５０と繋がっている。これらの栄養分貯蔵槽には灌流溶液を再生するのに必要な濃縮した量の構成成分が入れてあり、これらは廃液貯蔵槽５０の灌流液中に拡散させられる。
【００７６】
一つの実施態様では、二つ以上で使用する場合には、灌流液の流れはまず最大サイズの分子量を持つ栄養素と接し、それに続いて接する栄養分の分子量が小さくなるよう、栄養分貯蔵槽を分子量が大きいものから順に配置する。これにより、最初の透析ユニットで失われたかもしれない如何なる構成成分も、灌流液透析システムを出る前に、次のユニットを通ることにより確実に補給される。このシステムは、廃液貯蔵槽と栄養分貯蔵槽の間の平衡が維持されるように灌流液溶液の容量を調節する装置を更に含む。
【００７７】
監視システム及び制御システム
灌流溶液監視部分システムは、灌流溶液の様々なパラメータを監視するための標準的な構成要素とハードウェアを用いて複数の機能を実行する。一つの実施態様では、熱交換器に連結された熱電対又は温度センサー１８で温度を検知し制御する。例えば気泡トラップ３０の構成部品として、灌流溶液流路に配置された一つ以上の圧力計３１は、灌流液に対する血管抵抗及び呼吸ガスの部分圧を監視するのに用いられる。流量計５２、例えば、トラソニック社から入手可能なものなどの、灌流液を運搬する管の上に留めてあるセンサー５４が付いた流速を検知するための超音波装置が、灌流液の流速を監視するのに用いられる。好ましい実施態様では、この流量計は気泡トラップと器官室の動脈入口の間の流路に配置される。灌流液の容積モル浸透圧濃度は従来の氷点降下法を用いて測定する。または、容積モル浸透圧濃度は灌流溶液の主成分の濃度を評価するインライン検出手段を用いて監視しても良い。灌流液の試料は気泡トラップ３０又はこの測定のために灌流液流路に配置された他の何らかの手段により除去してもよい。
【００７８】
別の実施態様では、灌流溶液のパラメータを監視及び制御は器官室内に組み込まれたセンサー及びマイクロプロセッサを用いて行われる。これらは全て、通常の技術者の技術範囲内にある。制御部分システムの構成要素は、設計上の選択の問題としてシステムに組み込まれる別個の部品として組み合わされ又は提供されてもよい。
【００７９】
ｐＨと呼吸ガスの維持
灌流溶液は、１００〜２５０ｍｍＨｇのＯ₂の部分圧（ＰａＯ₂）を維持するため、１００％の酸素２８を膜若しくは中空糸の酸素供給器２０を介して導入することにより連続的に酸素が供給される。本発明のこのシステムは、灌流液のｐＨを７.３２と７.３８の間に、そして灌流液中のＣＯ₂レベルを３０〜６０ｍｍＨｇの部分圧に維持する新規なメカニズムを含む。灌流液のｐＨレベル及びＣＯ₂レベルの調節は、ＣＯ₂での灌流液の制御された断続的なガス発生により達成される。このシステムは、例えば灌流経路１０にあるｐＨ電極又はセンサー２２でｐＨを連続的に監視する。このｐＨセンサー２２は、ｐＨの厳格な制御を維持するために必要なＣＯ₂２６ガス発生を調節するためのコントローラー２４及びソレノイド２５に機能しうるように接続される。
【００８０】
一つの実施態様では、例えばハンナ・インストルメント製のモデルＨＩ８７１１などの一つ以上のｐＨメーター２１が、灌流流路１０に配置されたセンサー又は電極２２により循環している灌流液のｐＨを監視する。ブレオニクス社製のデータロジャーなどのインライン・コントローラー２４は、コントローラー２４に接続されたｐＨメーター２１を介してｐＨセンサー２２からの入力を受信し、ソレノイド２５を用いてＣＯ₂２６を灌流溶液中に放出するようバルブ接点２３を操作する。ｐＨが７.３５を超え、それによりコントローラー２４の設定値を超えると、ガス発生システムがバルブ接点２３及びソレノイド２５により稼動して、ｐＨが再び７．３５に達するまでＣＯ₂２６が灌流溶液中に注入され、その点でこのシステムは動作を停止し、このガスは停止する。重炭酸塩緩衝液システムを有する灌流溶液と組み合わせて用いる場合、このようにしてＣＯ₂を用いて灌流溶液の間欠的なガス発生を行なうことは、呼吸系による血液ｐＨの厳格な生理的制御を模倣する際に特に有効である。灌流溶液の他のパラメータと同様に、ｐＨ及び呼吸ガスはこの用途で当業者に知られているセンサー及びマイクロプロセッサーを用いて監視し制御しうる。
【００８１】
ＥＭＳ内での器官の固定
本発明に従って、いったん器官が摘出されると、カニューレを通して主要な動脈血液供給中に十分な量の灌流溶液をゆっくりと導入して、特定の器官の廃液から血液がなくなるまでその器官を灌流する。器官を洗い流すのに十分な灌流溶液の量が特定の器官のタイプ及びサイズ、並びにその器官が血流を失った時間に左右されるということは、当業者らに理解されるであろう。例えば、１〜３時間の間に血流を失ったヒトの腎臓を流すのには、灌流溶液は２００〜６００ｍｌで十分であろう。このようにして血管内に蓄積された全ての阻血血液及びアシドーシス産物は除去される。さらにｐＨが復元され、新鮮な基質が供給されて、嫌気的代謝及び細胞の完全性及び機能の維持に必要な他の細胞経路がサポートされる。
【００８２】
洗い流した後、器官は例えば器官室内に設置できる支持体の上に器官を置くことで器官室の内部空間内に固定され、器官の移動防止が図られる。例えばこの支持体は、特に輸送中に器官が器官室内を動き回らないよう、流体、ガス、又はゲルを充填した袋などの器官の外部表面の形になじむ弾力性のある支持体であっても良く、また器官の外部表面に概ね形を合わせた空洞を含む固体又は半固体の支持体であっても良い。器官は次いでカニューレを挿入した動脈を器官室の入口孔と繋ぐ短い管により灌流溶液に接続される。本発明で使用する管はすべて不活性で殺菌可能な素材、例えば、シリコンやＴｙｇｏｎ（登録商標）製の管であることが望ましい。
【００８３】
器官は、灌流溶液の温度を２５℃から３７℃の範囲に維持しながら灌流する。同時に、該システムは灌流液のＰａＯ₂、ＰａＣＯ₂、及びｐＨをガス発生部分システムで調節し、流速、血管圧及び容積モル浸透圧濃度を監視する。
【００８４】
灌流期間中、器官の機能の完全性も監視される。様々な生理学的パラメータ及び代謝パラメーが機能完全性の指標として使用できる。例えば、表１に示したような既知の処方で作る灌流液を使用すると、代謝機能の一定の側面の測定を容易にすることができる。時間に対して、器官を通って循環する間に灌流液内のブドウ糖濃度が失われる割合は、器官機能の一つの尺度となる。選択された時点に関して、ブドウ糖利用の正常レベルの範囲より低いブドウ糖濃度は器官の損傷又は代謝の阻害の指標になり得る。器官機能の尺度の別の一例は、灌流液が器官内を循環している間の酸素消費である。不適切な酸素消費、即ち、正常範囲に比べて低い酸素消費は、組織の低酸素症を表す場合がある。器官機能の尺度のまた別の一例は、標準的な灌流圧を使用して器官を通って循環している灌流液の血管流速である。正常範囲に比べて低い流速は、血管収縮、水腫、血管内皮細胞の膨張、及び損傷を受けた器官における血管完全性の喪失を表しうる。
【００８５】
これらの器官機能の特徴は、ＥＭＳ温保存システムの灌流経路に配置されたセンサーによって容易に監視される。センサーの最適な配置は、器官室自体の中で、保存溶液が器官に入る直前で且つ器官を出て行く溶液の静脈流出の近くである。センサーを器官のすぐ前及びすぐ後に配置すると、どんな差異も器官自体に帰することができるという点で、テストの精度が上がる。センサーを他の配置にすることも可能ではあるが、センサーを器官に対して遠くに配置するとエラーの可能性がはいってくる。これは、ブドウ糖などの代謝マーカーでは大した問題ではないが、酸素に対しては大きな懸念となる。
【００８６】
本発明のシステムはまた、器官の機能的完全性の他の指標を評価するための二つめの監視用部分システムをも備える。例えば、灌流期間中に器官から器官生産物を抽出する器官室の導管は、器官の産出物を追跡し、灌流された器官の生産物の物理的若しくは化学的パラメータを正常な器官機能を表す範囲と比較して評価することで、器官機能の監視を可能にする。当業者は、保存対象の器官に応じてどの特性を監視するかを簡単に決定することができる。例えば器官が腎臓であれば、利尿速度、尿浸透圧、比重、クレアチニン・クリアランスなどを含むパラメータを計測することにより腎臓生理学の個々の側面だけでなく腎機能全体が測定できる。監視部分システムの性質は灌流される器官に依存し、胆汁酵素、心臓酵素、心電計の動きなどを測定する手段を含むことがある。
【００８７】
本発明を実施するため、器官生産物の化学的若しくは物理的パラメータについての正常な値及び範囲、及び器官の正常な機能を表す他の計測値は、生理学及び臨床化学上の医療分野の当業者らに知られる教科書で参照される値のプラスマイナス２０％である。「臨床化学のＣＲＣハンドブック」マリオワーナー編，ＣＲＣプレス、スチュアート・アイラ・フォックス著「ヒト生理学」第６版，ウィリアム・シー・ブラウン、パブリッシャーを参照。
【００８８】
様々な器官及び組織の器官培養を支える場合の本発明の有効性を評価した。本発明を使用し、イヌの腎臓、ウシの腎臓、ラットの心臓、ヒトの胎盤及びウシの肢で効果を立証した。
【実施例１】
【００８９】
イヌの腎臓を摘出し、腎動脈にカニューレを挿入し、本発明のプロセス及びシステムを用いて溶液を適用した。全ての血液を腎臓から除去し、これらの腎臓を溶液で灌流した。該腎臓をＥＭＳ器官培養技術で支えて３２℃で３日間維持した。同様に、ＥＭＳ器官培養の期間中、生理学的ｐＨ、容積モル浸透圧濃度、圧力、流速及び酸素消費を維持した。該腎臓は、器官培養の間、無傷のままであり、代謝し続けた。腎臓内で進行する代謝は、機能の継続という結果を得るのに十分であった。即ち、該腎臓は器官培養の期間中、尿を生産し続けたのである。無傷の全腎臓の培養の結果を表２に列挙する。このＥＭＳ器官培養の期間中、どのパラメータ範疇においても代謝又は機能の低下はなかった。同様に、進行した水腫もなく、組織学的な評価の後に観察された壊死もなかった。
【００９０】
【表２】

【実施例２】
【００９１】
種による差異を評価するため、ウシの腎臓もテストした。ウシの腎臓は、実施例１に記載したのと同じ技術を用いて、ＥＭＳ器官培養の中に３日間置いた。イヌの腎臓を使用したときの結果と同様に、ウシの腎臓はＥＭＳ器官培養で、進行中の代謝や得られる機能を損失することなく、３日間無傷で維持できた。イヌの腎臓で得られた結果と同様に、ウシの腎臓は器官培養の期間中、安定した灌流圧、流速及び水腫の欠如を示した。組織学的評価に基づき、このウシの腎臓は正常であり、腎臓構造の全構成要素が優れた状態に保たれているように見えた。
【実施例３】
【００９２】
ラット（複数）から心臓を摘出し、その大動脈にカニューレを挿入した。これらの心臓を本発明のＥＭＳ器官培養内に置いて２４時間維持した。ラットの心臓を用いたこのテストの結果を表３に列挙する。
【００９３】
【表３】

ＥＭＳ器官培養期間の後、これらの心臓が生命を維持するのに十分な完全性を持つか否かを評価するため、複数の心臓のうちの三つを移植した。この三つの心臓は全て、補助なしに自発的に鼓動し、生命を維持することができた。
【実施例４】
【００９４】
臍帯がまだ付いたままのヒトの胎盤を摘出した。臍帯の血管にカニューレを挿入し、本発明の溶液でその血液を臍帯から洗い出した。該器官の血液を洗い流し、血管内部を該溶液で満たした直後に、該器官をＥＭＳ器官培養の中に置いた。該器官はＥＭＳ器官培養技術を用いて約２２時間、無傷で維持された。この器官培養期間中、胎盤を通る溶液の流速は９２ｃｃ／分で最大血圧は８０ｍｍＨｇであった。ＥＭＳ器官培養の終わりに、この無傷の胎盤を組織学的に評価した。次いで臍帯と胎盤自体の両方から単離細胞を採取した。その結果を表４に示す。
【００９５】
【表４】

２２時間のＥＭＳ器官培養後に器官全体から生存能力のある細胞を採取することができた。臍帯血管及び無傷の胎盤の双方からコラゲナーゼ消化により組織を単離した。無傷の器官から単離された細胞は、培養フラスコの表面に付着する能力、及び標準的な組織培養で複製する能力を保持していた。この単離された細胞は標準的な組織培養で複製を続け、遂には３回継代した。従って、この無傷の器官はＥＭＳ器官培養での維持が成功し、そのため代謝的に活性であったのである。器官培養期間後に器官全体から単離された細胞のその後の生存能力と機能は、この解釈を支持する。
【実施例５】
【００９６】
無傷のウシの肢を入手した。大腿動脈にカニューレを挿入し、この無傷の肢からその血液を洗い出した。本発明の溶液を用いて血管内部を満たし、該肢をＥＭＳ器官培養で２日間維持した。器官培養の完了時点で、該肢を組織学的に評価した。これらの評価の結果から、該肢の構成要素は、壊死を観察することなく、完全に保存されたことが示された。２日のＥＭＳ器官培養後の無傷の肢から単離した細胞は、同様に生存能力があった。この無傷の肢から単離した細胞は標準的な細胞培養でフラスコの表面に付着した。これらの細胞は増殖し続け、３回継代された。器官全体で得られた結果と同様に、無傷の肢などの解剖による切除部分も本発明のＥＭＳ器官培養技術で維持することができた。
【実施例６】
【００９７】
本発明のＥＭＳ器官培養技術を用いて、長さが約２〜２.５フィートに達するヒトの臍帯を維持した。このヒトの臍帯はＥＭＳ器官培養内で少なくとも７日間無傷で維持された。該臍帯の血管は一方の端にカニューレを挿入した。もう一方の端で、血管と２本の動脈をｙコネクターに繋がったシリコン管で接続し閉回路を形成した。ＥＭＳ器官培養の完了時点で、該臍帯血管の内腔表面をコラゲナーゼで消化した。単離された内皮細胞は生存能力があり、標準的な組織培養でフラスコ表面に付着した。単離された細胞は組織培養で内皮細胞の正常な特性を示した。即ち、単離された細胞は付着し、複製し、抗血友病因子抗原を発現した。これらの結果は、本発明のＥＭＳ器官培養技術が２〜２.５フィートの長さのヒトの組織をＥＭＳ器官培養で７日間、組織内の細胞の生存能力を損なうことなく、無傷で維持するのに使用できることを実証する。これらの研究は、ＥＭＳ器官培養技術が無傷の器官又は解剖による大きな切除部分を長期間維持する上で有効であることを証明する。
【００９８】
本発明のＥＭＳ全器官培養技術はイン・ビボ適用にも使用できる。特定の器官は、器官に栄養分を供給する主動脈源の摘出、及び静脈流出を同時に摘出・回収することにより摘出できる。器官培養技術のイン・ビボ適用は、イナベーション（enervation）系及びリンパ系の生理学的な維持を含む。標的化された器官又は解剖による切除部分に栄養分を供給する動脈にカニューレを挿入し、器官廃液を受け取る静脈も同様にカニューレを挿入し、血液を組織内から流し出す。血管内腔を本発明の溶液で再び満たす。器官又は解剖による切除部分はイン・ビボで維持されるが、生理学的システムからは「切断」されており、ＥＭＳ器官培養技術により維持される。
【実施例７】
【００９９】
イヌの腎臓は、腎の動脈と静脈にカニューレを挿入し、腎臓から血液を洗い流し、本発明の溶液で再び満たすことにより単離した。この腎臓は、ＥＭＳ器官培養技術を用いて３７℃で８時間そのままで維持した。この分離の間、該腎臓は酸素消費及びブドウ糖消費により測定すると、イン・ビボＥＭＳ器官培養で代謝を続けた。同様に、代謝は継続的な利尿が生ずる程に十分なレベルのものであった。該腎臓は、残りの静脈系から切り離されＥＭＳ器官培養技術で維持されている間、尿を製造し続け接続された膀胱を満たし続けた。ＥＭＳ器官培養が終わるとカニューレを除去し、該器官を血液で再灌流した。該腎臓は正常に機能し続けた。１０頭のイヌ全てが、腎臓をＥＭＳ器官培養後に正常な血清の化学的性質を示した。
【実施例８】
【０１００】
摘出された器官又は局所化された組織の灌流は、全身毒性を減少させながら高用量化学療法を実施するのに極めて適している。最近いくつかの癌研究所における、化学療法剤を送達するのに単離器官の灌流を行なおうとする試みは、標的外の組織の損傷を惹起する危険性が高かった。標的の器官は、本発明の器官培養技術を用いれば、如何なる損傷をも惹起することなく維持できる。化学療法剤を送達するために器官培養技術を用いる利点は、通常の全身性副作用を減少させ又は排除さえしながら同時に遙かに高用量を送達するという機会である。
【０１０１】
薬物送達技術としてのＥＭＳ器官培養の有効性は、移植が不可能なヒトの腎臓で確立された。この腎臓は、腎細胞癌と同定された腎腫瘍のため移植不可能であった。このヒトの腎臓は、ＥＭＳ灌流の間、安定した灌流圧、血管流速及び酸化的代謝を示した。同様に、ヒトの腎臓はまた、酸素及びブドウ糖を消費し、尿を生産し、そしてクレアチニンを浄化した。腎臓の生検は、組織学的評価のためＥＭＳ全器官培養灌流の前後の両方で行った。１８時間のＥＭＳ器官培養後に観察された組織学的変化はなかった。従って、ＥＭＳ器官培養は、腎細胞癌を持つ腎臓を、損傷を惹起することなく維持し、それにより標的化された薬物療法のための送達システムを提供できる。
【実施例９】
【０１０２】
心臓発作や脳梗塞などの閉塞性疾病に続く阻血性発作は、ＥＭＳ器官培養技術の別のイン・ビボ適用を表す。最近開発された血栓溶解剤は、脳梗塞や心臓発作を患う患者に大きな展望を与える。このような療法の有効性は、まだ閉塞現象後の短い時間内に薬物を投与できるかどうかにかかっている。閉塞箇所の下流の組織が血液を奪われ、その結果阻血性損傷が起きる。ＥＭＳ器官培養技術は、閉塞箇所から下流の組織を再灌流しそして維持するのに使用でき、それにより血栓溶解療法を実施する機会の時間枠を拡大する。
【０１０３】
大きな損傷後の蘇生で主に問題となるのは、適切な栄養素の送達と容量交換である。事後の敗血症を伴う出血及び多器官不全は、外科集中治療室での死亡の約６０％の原因となる。本発明のＥＭＳ器官培養技術は、ショックからの蘇生においてイン・ビボで組織の完全性と機能を維持するために使用できる。正常血液量は生理的温度の本発明の無細胞溶液で回復でき、これにより、生命に関わる失血をしている患者での二次的な再灌流傷害の進行は最小限になる。ＥＭＳ器官培養技術は摘出した器官を維持する。又は患者の全体を維持するのに用いてもよい。
【実施例１０】
【０１０４】
本発明の灌流溶液に適切な増殖因子及びホルモンを加えると、損傷を受けた器官における細胞修復工程が支えられれる。６０〜１２０分の温阻血を経験している腎臓を、本発明のＥＭＳ全器官培養技術で維持した。本発明の器官培養技術で６〜８時間器官培養した後、ヘンレ係蹄のシック・アセンディング・リム（thick ascending limb）の遠位尿細管で有糸分裂像が観察された。見掛けの損傷のない腎臓内の領域、即ち、近位尿細管および糸球体では修復プロセスの証拠はなかった。これらの研究は、損傷を受けた組織の修復及び再生におけるＥＭＳ器官培養技術の実質的な可能性を実証する。
【０１０５】
死後２時間で取り出した腎臓は、次のようにＥＭＳ灌流の間、蘇生及び修復がなされた。最初の灌流圧及び血管流速は極めて異常であり、平均灌流圧が２４／２０ｍｍＨｇ、平均血管流速が４ｃｃ／分／ｍｇであった。同様に、最初の酸素消費も損なわれ、平均値は０.０８ｃｃ／分／ｇｍであった。これらの器官の最初の査定は、一次機能なし（ＰＮＦ）又は生存能力なしと予測した。
【０１０６】
本明細書に記載のプロセス及び溶液を用いて８時間ＥＭＳ灌流する間に、この腎臓は修復された。灌流圧及び血管流速は正常になり、酸素消費は平均値０.２２ｃｃ／分／ｇｍまで増え、尿流量が元に戻った。これらのパラメータの正常化は、ＰＮＦから軽度の急性腎尿細血管壊死を伴う生存可能な器官に結果を変化させるのに十分な程この腎臓が蘇生し、回復したことを示す。これらの腎臓のうちの二つは８時間のＥＭＳ灌流後の生存可能性を測定し、その後移植した。三つ目の腎臓は１８時間のＥＭＳ灌流の後、再移植した。下の表５に挙げた結果は器官の生存可能性を示すだけでなく、死後２時間の温阻血からの蘇生及び損傷の修復におけるＥＭＳ技術の役割をも証明する。
【０１０７】
【表５】

ＥＭＳ技術は、現在臨床的移植における修復不可能な損傷であると考えられている阻血傷害から器官を蘇生させ修復することができる。ＥＭＳ灌流の期間は、修復の程度に直接相関し、腎臓を長く灌流させればそれだけ良く修復する。８時間のＥＭＳ灌流では、移植後第４日目でピーク値の血清クレアチニンを持ち移植可能になり、第１２日と１３日目で正常化する程にＰＮＦの腎臓を十分に修復した。１８時間のＥＭＳ灌流では更なる修復が見られ、血清クレアチニン値がわずかに上昇し、２日でピークとなり、移植後３日目で正常値になった。これは再移植前に８時間灌流した腎臓よりも１週間早い。
【実施例１１】
【０１０８】
ＥＭＳ器官培養技術の、肝臓をほぼ生理学的な温度で保存する効果、及び死後６０分の温阻血（ＷＩ）後に肝機能を蘇生する能力を検討した。酸素消費、及び急性の損傷を表す標準的な肝臓酵素を含む、肝臓の生存能力及び代謝機能を表す選択されたパラメータの測定を行った。代謝機能の評価には、胆汁生産の測定及び胆汁中の酵素とビリルビンの濃度の評価が含まれた。
【０１０９】
図２に記載の肝臓灌流システムは、動脈及び静脈血管系の双方を支える必要があるため、腎臓システム及び肝臓システムとは異なる。本発明で記載するシステムは、より低い血管流速（＜２５０ｃｃ／分）で肝臓動脈を経て高いＰａＯ₂（１００〜２４０ｍｍＨｇ）を送達し、同時に門脈系を経て低いＰａＯ₂（＜１４０ｍｍＨｇ）とそれに対応する高い血管流速（＞２４０ｃｃ／分）を供給する点で独特である。正味の効果は、胆管系が保護され、灌流中の胆汁生産が最大になるよう、より生理学的な灌流を供給することである。
【０１１０】
図２に示したように、廃液貯蔵槽３８は、ポンプ６４で灌流液を肝臓の門脈６６に運搬する二つ目の灌流経路６０に排出させる二つ目の廃液排出口６２を有する。腎臓と同様に、該ＥＭＳシステムは灌流液の温度、灌流圧、血管流速、容積モル浸透圧濃度、ｐＨ、ＰａＯ₂、ＰａＣＯ₂、栄養分送達および老廃副産物の除去を制御する。これは二つの流路で灌流液をそれぞれ別に監視、制御することにより、又は肝臓動脈を供給する灌流経路のみを調節することにより行われる。従って、一つの実施態様では、門脈系用の流路はそれ自体の温度制御器、酸素供給器及び呼吸ガス制御器、ｐＨ制御器、気泡トラップ、及び流速、血管抵抗、ＯｓＭ、ＰａＯ₂、及びＰａＣＯ₂を検知する手段を更に含む。
【０１１１】
肝動脈流路と同様に、門脈流路の灌流温度は２５℃〜３７℃の範囲で維持される。一つ以上のインライン酸素供給器と酸素運搬体が、肝動脈と門脈供給について上で述べた酸素張力を与える。一つ以上のｐＨ電極、制御器及びソレノイドのシステムがＣＯ₂の流入を制御し、両方の流路で灌流液のｐＨを７.３０〜７.４５の範囲に維持する。
【０１１２】
他の器官と同様に、長期間の肝臓の保存は、灌流液交換、透析、又はその組み合わせにより灌流液中の枯渇した栄養分を補充することにより、そして蓄積した代謝老廃物を透析で除去することにより行われる。肝臓に特有の合成的特性は、タンパク質合成と新たな細胞増殖を生み出すことにより再生の機会を与える。
【０１１３】
死後３０分（ｎ＝５）又は６０分（ｎ＝５）の温阻血（ＷＩ）のいずれかの後、子ウシの肝臓を摘出した。肝上の大静脈を横隔膜で切り離した。左上側から始め、リンパ組織、胃動脈及び総胆管を切って肝臓を解剖した。門脈と肝動脈を切り離し、それぞれ１２ｍｍと５ｍｍのカニューレでカニューレ挿入した。この肝臓を約４時間、３４℃で灌流した。灌流の間、ビリルビン、ＡＬＴ、ＡＳＴ、ＡＬＰ及び血液ガス（blood gases）を測定するため、灌流液の試料を２時間と４時間の時点で採取した。水腫又は変色はなく、ＥＭＳ器官培養技術の肝臓修復能力を証明した。
【０１１４】
死後３０分のＷＩを受けた肝臓は、ＥＭＳで蘇生させた後、全て高い酸素消費速度を示した。ＷＩ傷害が６０分に及んでも、酸素消費速度には有意義な違いは検出されなかった。死後３０分のＷＩに付された肝臓は、代謝の回復後、３１ｍｍＨｇの平均動脈血圧（ＭＡＰ）（肝動脈と門脈を合わせて）とこれに対応する３２０ｃｃ／分の合併流速を示した。血管抵抗は０.１であった。ＷＩに曝される時間が死後６０分に増えると、ＭＡＰは２０ｍｍＨｇまで下がり、流速は平均４２０ｃｃ／分まで増加した。この結果得られた血管抵抗は０.０６であった。
【０１１５】
標準的な肝機能スクリーニングを実施して、ＡＬＴ、ＡＳＴ、アルカリホスファターゼ（ＡＬＰ）及び総ビリルビン／直接型ビリルビンをテストした。ＥＭＳ器官培養技術で蘇生させた後、死後３０分のＷＩで傷害を受けた肝臓は、２時間で一つの肝臓を除き、テストした両方の時点でＡＬＴ、ＡＬＰ及びビリルビンの正常値を示した（表４）。同様に、ＷＩ傷害が死後６０分に増加した場合、２時間及び４時間のＥＭＳ灌流後のＡＬＴ、ＡＬＰ及びビリルビンの値は正常であった。
【０１１６】
胆汁生産
死後３０分のＷＩの後にＥＭＳで蘇生させた肝臓は、温灌流の期間中胆汁を生産した。胆汁流速の変化を観察すると、１.５〜６ｃｃ／時の範囲であった。胆汁の色は暗緑色または緑黄色であった。摘出時点で胆嚢から取り出した胆汁から成る正常な対照は、３１５ｍｇ／ｄＬの総ビリルビン量、１８ＩＵ／ＬのＡＬＰを示し、ＡＳＴ及びＡＬＴについては陰性であった。死後３０分のＷＩで傷害を受けた肝臓は広範囲のビリルビン濃度、１.１〜４１８ｍｇ／ｄＬを持つ胆汁を生産した（表５）。しかしながら、平均値は正常な胆汁のビリルビン濃度に近かった。上述のＥＭＳ灌流液での肝臓酵素の濃度について観察された結果と同様に、胆汁ＡＳＴレベルは３０分のＷＩを受けた肝臓を２から４時間灌流する間に半減し、一方６０分のＷＩで損傷を受けた肝臓では胆汁ＡＳＴレベルは一定のままであった。ＡＬＴは３０分のＷＩ後に２時間灌流した肝臓のうち２つから得た胆汁で見出された。死後６０分のＷＩを受けた肝臓で生産された胆汁は、２時間と４時間のＥＭＳ灌流の両方でＡＬＴをも含んでいた。同様に、回復した肝機能は、胆汁生産の継続を支えるのに十分であった。
【０１１７】
これらの結果は、ＥＭＳ器官培養技術が実質的な死後のＷＩ傷害後の肝機能を蘇生させ修復させる能力を与えることを示唆する。３０分及び６０分のＷＩを受けた肝臓では、代謝はＥＭＳ灌流でほぼ生理学的な酸素消費を復旧するのに十分回復した。肝機能のテストの結果は肝臓が６０分程度のＷＩ損傷から蘇生できるという更なる証拠を示す。
【０１１８】
冷阻血損傷を予防するためのＥＭＳ技術の使用
冷虚血に曝す前のＥＭＳ灌流は組織を保護し、同種移植片の冷保存期間の延長を可能にする。現在、同種移植片は日常的に冷保存で２４時間しか臨床的に保存されない。冷保存の期間を延長すると、高率で機能の遅延をもたらし、多くの場合、腎臓が機能を回復できるまで透析により患者を支えることが必要である。冷阻血に曝す前に無傷の器官を無細胞の灌流に適合させることで、冷損傷から細胞を保護することができる。
【０１１９】
従って、本発明はまた、組織、移植片又は移植用の器官を貯蔵する方法であって、血液及び血液産物を除去するために組織、移植片又は器官を血液を含まない緩衝化生理学的溶液で洗い流す工程、組織、移植片又は器官への損傷を阻害するのに十分な時間ほぼ正常な代謝速度で組織、移植片又は器官を維持できる、本明細書に記載したものなどの温保存システム中で組織、移植片又は器官を灌流する工程、及びこの器官を４℃〜８℃で貯蔵する工程を含む方法を提供する。損傷を抑制するのに十分な時間とは、摘出された器官が灌流パラメータ、例えば流速や血管抵抗を正常化する機会を得た時間である。一般的に、３０分から２４時間の灌流期間で十分である。
【０１２０】
本発明の器官室は器官がコンテナ内で動くのを抑えるので、これは器官の輸送にも特に適している。器官は上で述べたように貯蔵のために調製できる。続いて、温保存システムから器官室を単に取り出し、出荷用に冷蔵又は冷包装することにより器官は輸送される。必要に応じて、移植場所に到着した際、器官を含む器官室を互換性のある温灌流システムに再固定でき、移植者に移植される前にこの器官を温灌流できる。更に、温灌流の期間を利用して移植前に該器官の機能的特性を監視することができる。
【０１２１】
温保存システムのコンポーネントが該器官室に組み込まれている実施態様では、適切な電源を用いて器官の灌流を輸送中に続けることができる。
【０１２２】
イヌの腎臓の対照グループ（ｎ＝３）を摘出し、ＶｉａＳｐａｎ（商標）ですぐに冷洗し、次いで２重に袋に入れ、氷の中に静的に包装して４８時間保存する。テストグループの腎臓は切除後、冷保存の前にＥＭＳ灌流に置いた。このグループ（ｎ＝２）では、切除した腎臓はＥＭＳ灌流溶液で血液を洗い出し、３２℃〜３４℃でＥＭＳ灌流に６時間置いた。ＥＭＳ灌流期間の後、該腎臓を４℃の２重の袋で約２００ｃｃのＶｉａＳｐａｎ（商標）で再度洗い流し、静的に包装して氷の中で４８時間保存した。
【０１２３】
冷蔵した腎臓を再移植する直前に、反対側の腎臓を腎摘出した。テスト腎臓と対照腎臓は次いで、腎動脈と大動脈の間と腎動脈から大静脈の間に作った末端−側面吻合を用いて自家移植した。移植後、該イヌを縫合し回復させた。毎朝、該イヌの前足から採血し、臨床経過を測定するため化学検査を実施した。
【０１２４】
結果
冷蔵前のＥＭＳ灌流は、静的な冷蔵４８時間後に正常な機能が回復するまでの時間が半減するという、より良い結果をもたらした。対照グループの腎臓をゆっくりと再灌流したが、手術台上(on the table)では尿は生産されなかった。ごく少量の尿が移植後の第一夜の間に生産された。対照グループのイヌは、可逆的な急性尿細管壊死（ＡＴＮ）の期間を経験した。移植後２４時間の血清クレアチニン値は、全て正常値を上回っており、それぞれ５.３、２.６、及び２.８ｍｇ／ｄＬであった。血清クレアチニン値は上昇し続け、頂点に達し、その後徐々に降下し、それぞれ７、９、及び１２日に正常化した。
【０１２５】
テストグループでは、腎臓は再灌流の数分以内に手術台上で尿を生産した。これらの腎臓は移植後の期間中、尿を生産し続けた。移植後２４時間の血清の化学は上昇し、血清クレアチニン値は２.２及び２.５ｍｇ／ｄＬであった。対照のイヌとは対照的に、上昇した血清化学が頂点に達したのは移植後２日目であった。血清クレアチニン値はそれぞれ５日と６日に正常化した。安楽死直後、該腎臓は巨視的に正常であった。組織学的研究もまた、正常な腎病理学を明らかにした。
【０１２６】
再移植前のＥＭＳ灌流は、従って、温灌流されたテスト腎臓はより軽度で期間がより短いＡＴＮを経験するという点で、冷阻血に誘発される損傷に対する保護を与える。ＥＭＳ灌流されたテストグループの腎臓はまた、ＥＭＳ灌流を受けていない対照グループの腎臓と比べると、移植後２４時間後の血清クレアチニン値がより低かった。ＥＭＳ灌流された腎臓はまた、すぐに冷蔵された対照グループの腎臓よりもより早く（５.５日対９.３日）血清クレアチニン値が正常化した。
【０１２７】
標的化された薬物の送達システムとしてのＥＭＳの使用
ＥＭＳ灌流は、器官、解剖による切除部分及び又は組織に標的化された方法で薬物を送達するのに用いることができる。さらに、どの手順においても、ＥＭＳで支えられた進行中の代謝及び機能を監視することができ、薬物の影響及びそれに対応する如何なる細胞の応答をも定量化するメカニズム提供することができる。例えば、熱ショックタンパク質の高レベル調節を惹起することで知られる化合物、ヘムオキシゲナーゼ−１を、本発明の技術に従って灌流されている摘出腎臓に投与した。該ＥＭＳ技術はエクス・ビボ灌流の間の腎臓の代謝と機能を適切に支え、６時間以内にヘムオキシゲナーゼ−１（ＨＯ−１）酵素の発現を増加させた。
【０１２８】
テストグループ（ｎ＝３）のイヌの腎臓を摘出し、カニューレ挿入し、ＥＭＳ灌流溶液で血液を洗い流し、コバルト・プロトポルフィリン（ＣｏＰＰ）を５、２５又は５０ｕＭのいずれかを灌流液に添加して３２℃〜３４℃でＥＭＳ灌流に６時間置いた。代謝と機能の評価は、ＨＯ−１誘導期間の間のエクス・ビボの腎機能の定量化から成るものであった。ＥＭＳ技術を使用することで、腎臓はほぼ生理学的な温度での灌流により代謝的に活性状態に維持された。約７０〜９０ｇｍの重さの腎臓が、脳死とそれに付随する僅か１５分の温阻血の後に得られた。これらの腎臓を、次いで３２℃から３４℃のＥＭＳ灌流に移した。該腎臓は、酸化的代謝、血管動力学及び器官機能について評価した。器官パラメータは、機能の評価の前に１時間安定化させた。動脈系及び静脈系からの試料を灌流中に生産された尿とともに採取した。動脈及び静脈の試料についてのＰＯ₂測定にはラジオメータＡＢＬ５を用いた。
【０１２９】
酸素消費は、
ｍｌ／分／ｇｍ＝（ＰＯ₂動脈−ＰＯ₂静脈）×（流速）
として計算し、
血管抵抗は、
血管抵抗＝平均動脈圧／平均流速
として計算した。
【０１３０】
（外３）

クレアチニンをトレーサーとして灌流液に添加した。３２℃〜３４℃でのＥＭＳ灌流の間に作られた尿を採取してテストした。クレアチニン濃度をＩＤＥＸＸヴェットラブ化学アナライザー上で測定し、糸球体濾過速度（ＧＦＲ）を
ＧＦＲ＝ （尿中クレアチニン）×（尿流量）／（灌流液中クレアチニン）×（時間）
として計算した。
【０１３１】
これらの腎臓のエクス・ビボの酸化的代謝は、三つ全てのＣｏＰＰ濃度と匹敵しており、６時間のＥＭＳ灌流の間中安定したままであった。回復した酸化的代謝は、酸素消費で測定したとき、０.１７から０.２３ｃｃ／分／ｇｍの範囲であった。しかしながら、この酸素消費は２５μＭのＣｏＰＰで灌流した腎臓では全ての時点で最高であった。灌流圧、血管流速及び血管抵抗もまた三つのＣｏＰＰ濃度グループで匹敵していた。従って、ＥＭＳ灌流中のＣｏＰＰの処置は血管動力学又は細胞酸化的代謝に悪影響を与えなかった。しかしながら、ＣｏＰＰ誘導の濃度の増加に従い、尿流量、クレアチニン清浄化、及びタンパク尿の発生における差異が観察された。
【０１３２】
器官機能に対するＣｏＰＰ濃度の効果
ＣｏＰＰ濃度が増加するに従い、尿流量は抑制された。ＣｏＰＰ濃度が５から２５μＭに増加した場合は尿流量は６６％だけ低下し、ＣｏＰＰが５０μＭに増加した場合は９０％を越えて低下した。糸球体濾過速度（ＧＦＲ）は、ＧＦＲの計算が尿流量と尿中クレアチニン濃度の両方に依存するため、それに応じて低かった。ＣｏＰＰ濃度を５μＭから２５μＭに増やすとＧＦＲが７１％低下した。同様に、５０μＭのＣｏＰＰ濃度はＧＦＲの９１％抑制をもたらした。ＣｏＰＰの濃度の増加はタンパク尿で示されるような「漏出性内皮（leaky endothelium）」の発生とも関連付けられた。
【０１３３】
（外４）

（外５）

ＥＭＳ灌流は、ＣｏＰＰに対する細胞が応答してヘムオキシゲナーゼ−１の発現を増加させるのに十分な程、進行中の代謝と機能を効果的に支えた。更に、ＥＭＳ灌流の間、ＣｏＰＰの投与は灌流の特性又は細胞代謝に悪影響を与えなかった。しかしながら、器官機能は、ＣｏＰＰ濃度が増加するにつれ投与量に依存する形で、尿流量の低下、糸球体濾過速度の低下及びタンパク尿の発生という面で、影響を受けることが見出された。従って、薬物に誘発される毒性はＥＭＳ灌流を通した投与の間に検知できる。治療用量で、ＥＭＳ灌流はＣｏＰＰによるＨＯ−１発現の誘発を支えるのに効果的であった。
【０１３４】
遺伝子治療でのＥＭＳ技術の使用
ＥＭＳ技術の関連のある使用は、標的化された遺伝子の送達システムとしてである。遺伝子治療の長期にわたる利益は、所望の遺伝子をある特定の場所に送達するという問題によって妨げられている。器官培養技術は、所望の場所に遺伝子を送達する一つの機構を提供する。遺伝子治療は、外因性遺伝子を移植に先立って導入できるので、移植された器官に関しては特に魅力的である。医薬品が遺伝子治療ベクターである場合は、その構築物は内因性タンパク質又は外因性タンパク質を機能的にコードしてもよく、これらは次いで移植後に標的中で発現することができる。このような遺伝子導入は標的組織によるさまざまなタンパク質の発現を可能にする。
【０１３５】
灌流システムは、標的の器官が適切な血液循環システムを有する、例えば腎臓、肝臓、心臓などである限り、数々の器官への遺伝子導入と医薬品投与に特に適している。灌流システムを経由する遺伝子送達の最も明白な利点は、有効性の向上、遺伝子導入の標的の特異性、及びごく少量のベクター物質の使用である。更に、体外の灌流システムは被験者の全身循環に大量の外来性遺伝子物質を投与することの危険性を減少する。これは免疫適格性の個体には特に重要である。
【０１３６】
低体温灌流を用いて無傷の腎臓に遺伝子産物を送達しようとする最近の試みは、標的細胞におけるトランスフェクション効率及び遺伝子発現の双方の低減という結果に終わった（ツァイグラー ＳＴ、カービー ＪＤ、キュリエル ＤＴ、ディーセルム ＡＧ、トンプソン ＪＡ：Transpl 61:812，1996）。より重要なことに、この遺伝子導入は近位尿細管上皮細胞に局在化することが見出された。血管壁に沿っての透過性の変化は、低体温を含む様々な要因によって仲介されることが知られている。透過性が改変された結果、通常は静電荷によって血管壁で撃退される粒子又は粒子サイズが基層への透過性を増加させるという障壁機能の損傷である。
【０１３７】
長時間器官保存への本発明の貢献に加え、本発明のＥＭＳ技術は、生体外又は正常の場所のいずれかで、標的化された遺伝子の導入を達成するために用いることができる。該ＥＭＳは、トランスフェクション期間の前、間及び後に、トランスフェクトされる器官又は組織を代謝的に活性な状態に維持する。標的外の組織と接触することなく、長期間に渡ってほぼ生理学的な条件の下で標的化された組織と直接作用する能力は、ウイルス性ベクターの必要性の排除を含む代替的トランスフェクションの手順を使用する機会を提供する。例えば、ＥＭＳ器官培養灌流中に、より高い組み込み率を容易にするために、リポソーム複合体又はウイルス性ベクター及び血管内皮細胞特異的プロモーターを、治療用ＤＮＡをほぼ生理学的な温度で導入するために、灌流システムに導入することができる。
【０１３８】
ＥＭＳの間に代謝を保存するもう一つの利点は、代謝を抑制し細胞膜電位を変える低体温を使用するのとは対照的に、トランスフェクトの前に器官又は組織の機能状態を確定することの実現可能性である。これは器官が心拍のあるドナー以外のドナーから得られたものであり、何らかの阻血傷害を受けたかもしれない場合には特に重要である。更に、トランスフェクションの間に器官の代謝を監視することができ、無傷の器官の場合には最終的な器官の機能、即ち利尿などを定量化できる。細胞膜は正常な流体状態にあるので、正常な障壁機能が維持され、治療用の遺伝子（単数又は複数）を血管細胞に直接標的化する能力を持つ。
【０１３９】
ＥＭＳ灌流が腎臓内で血管壁の完全性と正常な障壁機能を保存する能力は、血管細胞に限定したトランスフェクションを達成する機構を提供する。器官内の血管細胞に治療用遺伝子（単数又は複数）を標的化させる能力はいくつかの重要な利点をもたらす。例えば、レポーター遺伝子産物は直接血流と接触し、それにより効果的な遺伝子治療送達システムを提供する。血管内皮は器官内で免疫性の接触面となるため、遺伝子治療は移植疾患及び自己免疫疾患の両方で、器官特異的な免疫調節療法の機会を与える。
【０１４０】
更なる利点は、ＥＭＳが無細胞であること、そしてＥＭＳが初期の免疫応答を誘発することなく、治療用遺伝子（単数又は複数）の核への最適なトランスフェクションのための環境を提供することである。従って、該ＥＭＳは、正常の場所または生体外で適用される場合、標的遺伝子送達用の高効率の送達ビークルを表し、そして代謝と機能を同時に監視することを可能にする。
【０１４１】
本発明の方法に従って、目的の遺伝子を現在知られているビークルの宿主を介して標的の組織に送達しうる。外因性ＤＮＡと標的組織のゲノムの間の相同組換えによる遺伝子治療は、ゲノム内の特定の遺伝子座の修飾という結果をもたらす。トランスフェクションのいくつかの方法には、遺伝子、リポソーム、プラスミドＤＮＡなどを含むウイルスベクターによる送達が含まれる。更に、本発明の方法によるエピソームＤＮＡの送達が実施可能であり、ゲノムとは無関係の標的遺伝子ベクターのエピソーム反復に起因する送達遺伝子のゲノム組み込みの必要性を回避する。
【０１４２】
器官又は組織について、本発明の標的送達方法を実施する際は、標的の組織を含む器官を生体から摘出し、温ＥＭＳ溶液で血液を洗い流し、ＥＭＳ灌流の上に置く。該灌流は機能の基準線が確立するのに十分な時間行う。外因性分子、例えばウイルス性ベクターを灌流内に注入し、そして薬学的に有効な量の外因性分子を送達するのに十分な時間灌流を継続する。器官の機能は、該器官を受容体に戻す前に、送達後の一定期間監視する。
【実施例１２】
【０１４３】
緑色蛍光タンパク質（ＧＦＰ）遺伝子をコードする複製障害性のアデノウイルス（Adeno CMV5-GFP）を用いて、３４℃でエクス・ビボ灌流する間、無傷のウシ腎臓への遺伝子導入を支えるＥＭＳ灌流システムの有効性を評価した。ウシの腎臓は温ＥＭＳ溶液で血液を洗い流し、ＥＭＳ灌流に置いた。この灌流は約６０分実施し、機能の基準線を確立した。ウイルスベクター（１×１０⁹ＰＦＵ）をシステム中に注入し、灌流を２時間続けた。ＥＭＳ灌流中のウイルスベクターの投与とその結果の感染力は器官代謝又は器官機能に悪影響を与えなかった。
【０１４４】
対照腎臓は、ビアスパンをベースとした冷灌流液を用いて洗浄し４℃で灌流した腎臓から成る。代謝及び機能は４℃では阻害されるため、対照腎臓の代謝または機能に対するウイルスベクターの如何なる効果も測定できなかった。
【０１４５】
遺伝子導入に続き、これらの腎臓は、ウイルス粒子を循環させるあらゆる残留物を除去するため、腎動脈を経由して十分に洗い流した。次いで脈間構造の内腔をコラゲナーゼ溶液で満たして各腎臓内の血管細胞を単離し、これらの細胞を形態学的に確認した。消化され単離された血管内皮細胞を腎臓から洗い流し、これらを洗浄し１×１０⁶／ｍｌの濃度で一次培養中に置いた。緑色蛍光タンパク質をコードするトランスフェクトされた遺伝子の発現は表７に記載する。
【０１４６】
該ＥＭＳ灌流は感染率の上昇をもたらし、ＧＦＰを発現する培養では１８時間までに血管細胞の４０％が感染したのに対し、冷灌流された対照の腎臓では１７％であった。組織培養で４８時間で、ＥＭＳ灌流した腎臓から単離された内皮細胞は細胞の６０％でＧＦＰの強い発現を示した。対照的に、トランスフェクションが冷温で実施された対照の腎臓では、発現レベルの上昇は検出されなかった。
【０１４７】
コラゲナーゼ消化により血管内皮細胞を単離した後、実質性の細胞をＥＭＳ灌流された腎臓から分離した。分離された実質細胞は、組織培養で２４時間及び４８時間の時点ではＧＦＰ発現が陰性であることが見出された。これらの結果は、ＥＭＳ灌流の間にＧＦＰをコードする遺伝子の感染力及び導入が腎臓の血管内部の血管細胞に限定されたことを示唆する。従って、ＥＭＳで灌流されたテスト腎臓ではより良いトランスフェクション率とより高いレポーター遺伝子発現が両方の時点で観察され、トランスフェクションの証拠は血管細胞でしか見つからなかった。
【０１４８】
【表６】

【０１４９】
【表７】

本発明のＥＭＳ温保存技術は、損傷を負わせることなく器官を保存し、（エクス・ビボ器官灌流後の）器官の再移植又は（正常場所灌流後の）再灌流の際、直ちに正常機能を得るのに十分なレベルで代謝を支える。これらの理由で、ＥＭＳ灌流は治療薬、例えばヌクレオチドの送達が望まれる遺伝子治療における治療薬の送達に特に良く適している。更に、本発明のＥＭＳ温保存技術は現在の方法論より優れた遺伝子送達／トランスフェクションシステムを提供する。
【０１５０】
例えば、トリグヴァソンらに対する米国特許第５，８７１，４６４号は医薬品送達用の灌流装置及び方法を開示し、正常場所及びエクス・ビボの両方で成功した遺伝子トランスフェクションを記載している。しかしながら、この結果は過剰な利尿（灌流の最初の１時間で８００ｃｃ）で証明された実質的な腎臓損傷が発生したことを示唆する。また、利尿は２時間を越えて１，２００ｃｃ／時まで劇的に増加している。これは該腎臓が灌流中に損傷を受けたことの別の示唆である。更に、正常場所での灌流は２時間に及ばず、その後の時点では尿生産の記載はなく、エクス・ビボ灌流の場合には該腎臓は再移植されなかった。従って処置後の腎機能は記載されていない。
【０１５１】
トリグヴァソンに記載されたクレブス・リンガー液を用いた摘出腎臓の灌流が、再移植すると機能しない損傷器官を生ずる結果となることが証明された。当業者は摘出した腎臓がほぼ生理学的な温度で灌流された場合には数時間生存可能であるに過ぎないことを認めるはずである。
【０１５２】
以前に報告された研究を超えるＥＭＳ灌流の改良は、器官がほぼ生理学的な温度に維持され、損傷を与えられずに、器官代謝が支えられ、エクス・ビボの灌流後の再移植又は正常場所での灌流後の再灌流された場合、すぐに正常機能が得られるというものである。更に、ＥＭＳ灌流は数日間行うことができ、損傷を与えずに効果的な遺伝子トランスフェクションを可能にするだけでなく、ＥＭＳは遺伝子産物を完全に発現するのに十分長く器官を支えることができる。ほぼ生理学的な代謝で数日間器官を支える能力は、再移植又は再灌流の前にトランスフェクトされた遺伝子によりコードされるタンパク質産物を合成するという独特の能力を示し、これは既存の技術では不可能だったものである。
【０１５３】
次の例は、遺伝子送達及び遺伝子導入の間の器官の完全性を保つというＥＭＳ技術の優れた能力を証明する。対になった腎臓を子ウシから摘出した。この切除された腎臓から血液を洗い出し、対のうちの一方をＥＭＳ灌流上に置き、他方をトリグヴァソンにより記載されたシステムと溶液を用いた灌流の上に置いた。灌流の間、酸化的代謝は毎分毎グラム消費されるＯ₂、利尿と対応する器官機能により酸素消費を計算して評価した。器官の機能は、その正常な障壁機能を保持し、血管内部に大きな分子を保持する糸球体の能力として測定した。ＥＭＳ灌流の場合、器官機能はコロイド、血清アルブミンのタンパク質漏出なしに尿を生産する能力、及びトリグヴァソン特許がコロイドを含まないため、血管内腔に赤血球を保持する能力、即ち赤血球について陰性の尿を生産する能力である。
【０１５４】
（外６）

米国特許第５，８７１，４６４号に記載された方法に基づく灌流、即ち、インライン酸素供給器を持つガンブロ機器上で、１７％のヘマトクリットを得るために赤血球を補給されたクレブス・リンガー液を用いた灌流は、器官の水腫、マノメーターで測定したとき灌流圧の上昇、低い酸素消費、及び灌流液と同等のヘマトクリットを含む尿を生ずる障壁機能の低下という結果をもたらす。組織学は器官損傷の進行を支える更なるデータを示した。対照的に、ＥＭＳ灌流は水腫を防ぎ、安定した灌流圧と酸素消費を維持し、タンパク質を濾過した尿生産をもたらす。組織学はＥＭＳ灌流中の腎臓の完全性が保存されたことを確証した。
【０１５５】
（外７）

長期間保存
ＥＭＳ技術を用いて、器官をエクス・ビボで長期間、代謝的に活発な状態で且つほぼ生理学的な温度で保存することができる。ＥＭＳ技術を用いて、１０個のイヌの腎臓をエクス・ビボで２４時間、４８時間及び７２時間保存した。エクス・ビボ灌流の期間中、灌流圧、血管流速、血管抵抗、酸素消費、ブドウ糖消費、及び利尿は安定したままであった。更に、これらの腎臓は組織学的な評価で正常であるように見えた。灌流期間中の様々な時点でＥＭＳ灌流溶液を交換することにより、適切な代謝サポートが達成された。これらの器官のうち三つが、その後の生存可能性と機能を測定するために再移植された。
【０１５６】
（外８）

ＥＭＳ技術で２４時間を越えて灌流した該腎臓のうち三つを再移植した。この三つの腎臓は全て再灌流すると直ちに良い膨張を示し、再灌流して数分以内で尿を生産した。この三つの腎臓は全て直ぐに正常な機能を示し、終了時間までに膀胱がいっぱいになった。この３匹のイヌは全て、長期間ＥＭＳ灌流された腎臓のみで生存し、正常な血清化学値及び尿分析値示した。
【０１５７】
輸送用の器官を調製するためのＥＭＳシステムの使用
器官の維持と修復に加え、本発明は移植場所まで輸送するための器官の保存と器官の調製に役立つ方法を提供する。移植用の組織、移植片及び器官の貯蔵法は上に記載されている。同様に、本発明の器官室と温保存システムは移植場所への輸送するための器官を調製するためにも有利である。基本的に、器官は器官室に固定され、器官の損傷を修復し冷蔵又は出荷から生じうる更なる損傷から保護するために温保存システムで灌流する。この方法は（ａ）組織、移植片又は器官を血液を含まない緩衝化生理学的溶液で洗浄して血液及び血液産物を除去する工程、（ｂ）組織、移植片又は器官を、器官、組織又は移植片を支えることができる支持体を持ち、輸送中の器官、組織又は組織片が器官室内で動くのを阻止する器官室内で灌流する工程であって、該器官室が組織、移植片又は器官を保護するのに十分な期間ほぼ正常な代謝速度で組織、移植片又は器官を維持できる温保存システムの一部である工程、（ｃ）組織、移植片又は器官を冷灌流溶液を、例えば、４〜８℃の範囲の温度で灌流する工程、（ｄ）器官を含む器官室全体を温保存システムから取り出し、該器官を含む器官室を冷包装する工程、及び（ｅ）移植場所へ器官を含む器官室を輸送する工程を含む。
【図面の簡単な説明】
【０１５８】
【図１】図１は、灌流液を循環させる閉ループ灌流部分システム及びそれを再処理する透析部分システムを有する血液を含まない代謝サポートシステムの実施態様を示す。
【図２】図２は、灌流交換部分システム及び肝臓の保存に適する二つの灌流経路を有する実施態様を示す。
【図３】図３は本発明の器官室の実施態様の一つの透視図である。
【図４】図４は本発明の器官室であって、該器官質がさらに貯蔵庫を含むものの実施態様の一つの透視図ある。
【図５】図５は本発明の器官室の一つの実施態様の断面図である。
【図６】図６は器官の血管を支持し、灌流液を器官から直接貯蔵庫に導く導管と接触して血管をしっかりと保持する血管支持体の断面図である。
【図７】図７は血管支持体の透視図である。【Technical field】
[0001]
The present invention relates to metabolic support systems including solutions, methods and devices for maintaining organs for transplantation under near physiological conditions. More specifically, the present invention relates to organ repair and / or long-term maintenance of the organ chamber and the organ for transplantation, prediction of organ function after transplantation, preparation for temporary refrigerated storage of the organ. And the use of the organ chamber in supporting the overall function required for transport of the organ intended for implantation.
[Background Art]
[0002]
Transplant organs continue to be extremely scarce. Currently, the main limiting factor in clinical transplantation is the persistent shortage of organs. For example, kidney transplants rely heavily on the availability of organs recovered from dead donors with heartbeat. However, there are large sources of transplant organs that have not yet been utilized. That is, the cadaver in which the heartbeat is stopped. The cadaver with cardiac arrest has died from the injury and is the victim of an accident with a short survival time following trauma. In addition, a cadaver with a cardiac arrest results in the inability of the family to emotionally decide to stop the life support device and provide the organ of a deceased person. In such a situation, the organ is not used because once the heart stops beating, the circulating blood supply is insufficient (warm ischemia) and an injury cascade is created.
[0003]
Slightly functionally damaged organs due to warm ischemia are unable to tolerate further damage mediated by hypothermic conditions currently used to preserve organs for transplantation. Under these conditions, the lipid bilayer undergoes a phase change and becomes a gel with significant loss of fluidity. The completely frozen lipid in the cell membrane is O_TwoCancels the use of tension. The result of its metabolism is glycolysis, which is similar to a state of hypoxia. Below 18 ° C., hypothermia inhibits renal tubular and renal glomerular activity, and at 4 ° C., oxygen utilization has been described to be about 5% of that at normal body temperature.
[0004]
Hypothermic preservation may also cause edema following vasospasm in organs. Organs stored at hypothermia may experience swelling of renal glomerular endothelial cells and loss of vascular integrity associated with tubular necrosis. This is a phenomenon caused by the adopted hypothermia condition. Hypothermia can also inhibit Na / K dependent ATPase, resulting in a loss of cell volume regulating ability. Loss of volume regulation is what causes cell swelling and damage. An adequate supply of oxygen does not actively reduce the amount of this swelling. This is because the cell membrane freezes completely, preventing effective use of oxygen. Without proper oxygen delivery, anoxia leads to smaller blood vessel collapse after several hours of perfusion. Lack of oxygen and subsequent depletion of ATP storage means that anaerobic glycolysis is a major energy source under traditional storage conditions. Subsequent loss of nucleosides is probably a critical factor in the inability of tissues that have undergone warm and long term cold ischemia to regenerate ATP after restoring blood supply. Lack of adequate oxygen supply resulted in mechanical reliance on hypothermia for organ preservation. In the case of warm ischemia, circulatory arrest results in an oxygen deprivation in the absence of molecular oxygen for oxidative phosphorylation. Lack of molecular oxygen leads to NADH accumulation and depletion of ATP stores in mitochondria.
[0005]
Thus, ischemia (whether warm or cold) is a injury cascade of events that can be characterized as prelethal and lethal phases. The pre-lethal phase causes three detrimental effects: hypoxia, malnutrition, and failure to remove toxic metabolic waste. Depletion of molecular oxygen due to lack of circulating blood. The resulting hypoxia triggers a deficiency in energy storage, such as a deficiency in ATP storage in mitochondria. ATP deficiency results in cellular changes including edema, loss of normal cell integrity, and loss of cell membrane polarity. This cellular change triggers the accumulation of metabolic waste, activation of proteases, and a lethal phase of ischemia leading to cell death.
[0006]
Perfusion solutions, which represent the current state of the art in hypothermic organ preservation and provide optimal organ preservation in hypothermic conditions, are metabolites that promote organ function after transplantation, hypothermia-induced tissue edema And antioxidants, membrane stabilizers, colloids, ions, and salts (see Non-Patent Documents 1 and 2). This perfusate formulation is designed to preserve organs due to hypothermia-induced metabolic decline. While this minimizes the edema and vasospasm normally encountered during hypothermic storage, it does not provide for substantially expanded donor pool utilization.
[0007]
This is due to the fact that organs or tissues damaged by warm ischemia cannot tolerate further damage mediated by hypothermia. Even after 30 minutes of ischemia, organ function after transplantation can be compromised. For example, using organs from a heartbeat cadaver (no damage), the immediate nonfunction rate is estimated to be 25%. Within 30 minutes of warm ischemia, the immediate failure rate rises to about 60%. Thus, 60% of kidneys from cadaver with arrest are not functioning immediately due to pre-lethal ischemic injury. In addition, irreversible ischemic damage and injury is believed to result in organ blood loss within just a few hours (see US Pat. Unless new organ sources can be developed, the number of transplant procedures will not change. Also, the donor pool cannot be substantially expanded due to the lack of available processes / systems to repair pre-lethal ischemic damage in organs or tissues damaged by warm ischemia.
[0008]
Recently, efforts have been focused on preventing ischemic damage by intervening a solution immediately after cessation of blood flow. For example, the protective solutions disclosed in U.S. Pat. No. 4,415,556 may be used during surgery or on organs to be transplanted to prevent ischemic damage to the organ. This protective solution is used as a perfusate to promote aerobic metabolism during perfusion of the organ. U.S. Pat. No. 5,395,314 discloses hypothermic preservation solutions (about 8 DEG C. to less) which are designed to lower organ metabolism, supply oxygen and inhibit free radical damage after disrupting the blood supply. (10 ° C.) through the brain to resuscitate the brain.
[0009]
While such methods and storage solutions are beneficial in preventing ischemic damage in organs, their beneficial effects have to be discounted due to practical and functional limitations. First, in order for such methods and solutions to be effective in preventing ischemic damage, they must be applied immediately (within minutes) after disruption of the blood supply. Logistical constraints severely limit the use of such methods and solutions, such as when the accident victim becomes an organ donor. For example, in an accident scene or an ambulance where an ischemic injury cascade starts, it is impractical to use them. Second, irreversible ischemic damage and injury is likely to result in loss of blood flow to the organ within minutes (eg, brain) or within just a few hours (heart, kidney). Organs or tissues that are slightly but functionally damaged by warm ischemia are unable to tolerate further damage mediated by hypothermic preservation before transplantation or restoration of blood flow after transplantation. One reason is that restoring blood circulation after ischemia-reperfusion can paradoxically result in further tissue damage (see Non-Patent Document 3). During reperfusion, re-oxygenation of the ischemic injured tissue results in further tissue damage due to the formation of oxygen free radicals, loss of free radical scavengers, and release of chemoattractants.
[Patent Document 1]
U.S. Patent No. 5,395,314 to Kratz et al.
[Non-patent document 1]
South Aard et al., Transpl. 49: 251, 1990.
[Non-patent document 2]
South Ard, Transpl. Proc. 21: 1195, 1989.
[Non-Patent Document 3]
McCord et al., N Engl J Med 312: 159-163, 1985.
DISCLOSURE OF THE INVENTION
[Problems to be solved by the invention]
[0010]
Accordingly, there is a need for a system that includes a storage solution that is useful for initial organ irrigation and useful as a perfusate for in situ or ex vivo storage of transplanted organs. This system employs warm preservation techniques to minimize, effectively repair, and sustain organs at near-normal metabolic rates, due to warm ischemia. The portability and automation of this system is especially important in situations where this system is used to initiate organ preservation in vivo before or shortly after life support is stopped or externally after an accident in which cardiac arrest has occurred. It is.
[0011]
Summary of the Invention
In one aspect, the invention relates to blood for maintaining a section of anatomy in an organ, tissue, or anatomy outside the body's circulatory system or at least separated from the circulatory system and in a near-normal metabolic state. A metabolic support system that does not contain The system comprises an organ chamber holding the organ having means for collecting the product of the organ produced during perfusion, one or more for circulating and regenerating a warm perfusion solution capable of maintaining the organ in a substantially normal metabolic state. Perfusion fluid delivery system including a perfusion fluid path, a controlled gas generating subsystem for adjusting the pH of the respiratory gas and perfusion fluid, a temperature controller for controlling the temperature of the perfusion fluid, and a variety of perfusion fluids It includes a monitoring subsystem that monitors various parameters.
[0012]
In a related aspect, the present invention relates to a monitoring subsystem in which monitoring of various parameters of the perfusion solution is controlled by a microprocessor. Such a system is arranged in a flow path of a microprocessor and a perfusion solution, and works in conjunction with the microprocessor to control the temperature, pH, pressure, flow rate, PaO of the perfusion solution._Two, PaCO_TwoAnd a sensor that senses at least one of osmolarity and provides the sensed information to a microprocessor.
[0013]
In another aspect, the invention relates to an organ chamber for use in a blood-free metabolic support system for preserving an organ, comprising: a container; at least one support positionable within the container for supporting the organ within the container. Pertains to an organ chamber including a support member, the support being designed to restrain movement of the organ within the organ chamber. In one embodiment, the support of the present invention is a resilient support that conforms to the outer surface portion of the organ, such as a gas bag, a liquid bag, or a bag filled with gel. In another embodiment, this is a rigid support that includes a cavity contoured to fit the outer surface portion of the organ. The organ can be heart, liver, kidney, pancreas, lung or other tissue from an adult or pediatric donor.
[0014]
The organ chamber includes a conduit for directing the venous outflow of the perfusion solution circulating through the organ from the organ to a reservoir, a conduit for separating and recovering the organ product from the perfusion fluid, a flow rate of the perfusion solution, pH, PaO_Two, PaCO_TwoTemperature, vascular pressure, and oxygen consumption, glucose consumption, consumption of at least one of the citric acid cycle components, CO2_TwoIt further includes at least one sensor monitoring at least one parameter of the perfusate selected from metabolic indicators such as production.
[0015]
In yet another aspect, the present invention relates to an organ chamber that further comprises at least one component of a warm storage system, such as a storage tank, a heat exchanger, an oxygenator, and / or a pump. Alternatively, the organ chamber of the present invention includes a connector for removably connecting the organ chamber to an external warm storage system.
[0016]
In yet another aspect, the invention relates to a method of storing an organ, comprising placing the organ in the container on a resilient support designed to prevent movement of the organ within the container. This organ is then connected to a warm preservation system, such as the metabolic support system of the present invention, and is perfused with a warm preservation solution that can maintain the organ at near normal metabolic rates.
[0017]
In another related aspect, the invention provides for securing and maintaining an organ in a warm storage system including an organ chamber, such as the blood-free metabolic support system of the invention, and monitoring the functional integrity of the organ. A method for maintaining an organ or tissue for transplantation, comprising the steps of:
[0018]
In another aspect, the invention relates to the use of the organ chamber of the invention in combination with a warm perfusion system that supports the continuation of de novo synthesis sufficient for an active repair process to occur.
[0019]
In another aspect, the invention relates to a method for securing an organ to an organ chamber, such as those described herein, wherein the organ is maintained at a substantially normal metabolic rate for a time sufficient to prevent damage to the organ. Perfusing an organ in a first warm preservation system including an organ chamber, such as a blood-free metabolic support system described herein, and disconnecting the organ chamber from the warm preservation system and shipping the organ chamber including the organ And a method of transporting an organ, including a step of refrigeration or refrigerated packaging for transportation. If necessary, the organ chamber containing the organ should be secondarily placed upon arrival at the transplant site so that the organ can be warm-perfused and the integrity of the function of the organ monitored before transplantation to the recipient. Can be fixed to the warm storage system.
[0020]
In yet another related aspect, the invention provides a method of storing an organ comprising perfusing an organ with a warm preservation solution and directing the venous outflow of the preservation solution from the organ so that the preservation solution does not touch the exterior surface of the organ. About.
[0021]
In yet another aspect, the invention relates to a warm preservation solution employed in a blood-free metabolic support system that includes hemoglobin modified with polyethylene glycol. The hemoglobin may be of human, animal or recombinant origin.
[0022]
DETAILED DESCRIPTION OF THE INVENTION
Numerous perfusion devices have been described elsewhere. For example, U.S. Patent Nos. 5,856,081, 5,716,378, 5,362,622, 5,356,771, 5,326,706, 4,186,565 and 3,995. , 444. However, none can employ warm storage solutions to support and control the ongoing metabolism of the organ and the functions performed by the organ intended for transplantation. To do so, the organ's natural activities must be maintained and controlled with a metabolic support system (EMS), such as those described herein. The EMS perfusion system of the present invention delivers a warm perfusion solution containing all the components needed to repair the organ as needed and to support oxidative metabolism by the organ. The perfusion system also reprocesses the perfusion solution to ensure a continuous supply of nutrients and chemical energy substrates and to remove metabolic by-products. In addition, the EMS is temperature, vascular pressure, perfusion flow rate, OsM, pH, PaO_Two, PaCO_TwoMonitor and control various perfusion parameters, including nutrient delivery and waste removal.
[0023]
In the following description, certain conventions are used in using the terms. The term “excised part by organ, tissue or anatomy” refers to the resected viable part and all parts of the body maintained by EMS etc. of the present invention, such as kidney, heart, liver, lung, small intestine, pancreas , Brain, eyes, skin, limbs, or intact organs, including but not limited to a quadrant. The term "organ product" refers to any substance formed as a result of the organ's secretory function, usually a fluid, such as bile from the liver, urine from the kidney, but a mechanistic function such as kidney filtration or cardiac pumping. Including.
[0024]
The terms "stock solution," "perfusion solution," and "perfusate" are used interchangeably and provide a means of restoring the integrity and function of cells in an organ that may have undergone ischemic injury before or during removal. Refers to a blood-free, buffered physiological solution that allows an organ or tissue to be maintained at a near-normal metabolic rate. The term "blood-free" is intended to exclude substantially any perfusate containing whole blood or its individual components. However, the perfusion solution of the present invention may contain minimal amounts of whole blood or blood components, such as red blood cells, serum or plasma.
[0025]
The terms "nearly normal rate of metabolism" and "nearly normal rate of metabolism" can be said to be normal function for a particular organ having functional properties of the organ as described in US Pat. No. 5,699,793. It is defined as about 70-100% of the normal metabolic rate of a particular organ as determined by measuring and assessing if it is within the range. Examples of functional properties include cardiac electrical activity as measured by electrocardiogram, physical and chemical parameters of organ products, such as oxygen consumption and glucose utilization as determined from perfusate concentrations, pancreatic enzymes, cardiac enzymes, creatinine It includes, but is not limited to, clearance and filtration functions, specific gravity of urine, and the like.
[0026]
The term "therapeutic agent" refers to any molecule used to cause a functional, synthetic, metabolic, immunogenic, or genomic change in an organ or tissue. These include viral vectors, liposomes, episomes, naked DNA, either sense or antisense molecules, RNA, chemotherapeutics, biologics such as cyokines and chemnokines, It is not limited to these.
[0027]
The process of the present invention includes the step of removing an organ, tissue or anatomical site from the rest of the physiological system by removing or blocking an arterial source of blood that feeds the desired tissue or tissues. Similarly, venous outflow from an organ or anatomical resection is blocked and venous waste is recovered. If the tissue is completely dissociated from the body, the nerve enervation of the tissue and the lymph glands will also be dissociated. The organ or tissue is then flushed through the arterial system with a solution of the present invention at about 25-37 ° C. to remove blood and blood products. The tissue is then placed in the blood-free metabolic support system of the invention and perfused with the solution of the invention, while various parameters of the solution are monitored by the system to maintain proper organ and tissue metabolism. Adjust as needed. For example, collecting organ products, such as urine and bile, and assessing whether the physical and chemical parameters of the organ products are within ranges for normal function for that particular organ, Is also monitored. The invention may thus optionally include a sub-system for assessing the functional status of the organ. Thus, organ function is monitored using in-line sensing means and in-line test methodology.
[0028]
Perfusing the excised organ or dissected portion with a solution at approximately physiological temperatures of about 25 ° C. to 37 ° C. according to the present invention performs various functions. This perfusion maintains the cellular environment at physiological pH, maintaining near normal oxygenation, temperature, and osmolarity. This perfusion maintains the normal barrier function of the tissue to macromolecules, which results in a stable perfusion pressure and a stable vasculature flow rate. The perfusion appropriately dilates and fills the vasculature, delivers appropriate nutrients to maintain near normal metabolic levels in the excised organ or anatomical resection, and supplies high energy compounds to deliver artificial energy. Supports aerobic metabolism that has been blocked. This perfusion supports ongoing oxidative metabolism with auxiliary substrates, which may include but are not limited to glucose, pyruvate, uridine 5-triphosphate (UTP). This ongoing oxidative metabolism is further supported by maintaining the storage of adenine compounds. The citrate cycle and electron transport system are supported by providing metabolic support and delivery of appropriate substrates to maintain function in isolated organs and tissues. The ongoing metabolism supported by the methods and solutions of the present invention provides the appropriate metabolites and nutrients and maintains tissue integrity with tight cell function and normal membrane polarity.
[0029]
The methods and systems of the present invention allow for the removal of blood in organs or anatomical resections and refilling of blood vessels and pericellular spaces with the solutions of the present invention. In addition, this system has a pH, PaO_TwoMaintain temperature, osmolarity and hydrostatic pressure to deliver the appropriate substrate to support the metabolism required for cell integrity. The ongoing metabolism provided by the combination of this method, solution and system is the ongoing functioning of the resected part by a particular organ or dissection during the time that the tissue (s) is removed from the body or circulatory system. It is a level enough to support
[0030]
By way of illustration, but not of limitation, this solution is at a systolic pressure appropriate to the tissue (s) maintained by the EMS organ culture technique of the present invention, at a near normal flow rate of that particular organ or tissue. Perfuse until reaching. By way of illustration, and not by way of limitation, a human kidney can be perfused with a solution of systolic pressure <80 mmHg, flow rate> 80 cc / min. The pH is adjusted to CO 2 via an oxygenator._TwoOr O_TwoIs maintained in the physiological range by injection. Suitable oxygen is provided to the organ by including an oxygen transport compound as a component of the solution.
[0031]
The methods, solutions and systems of the present invention provide the oxygen supply, metabolic nutrients, colloid osmotic pressure, pH, perfusion pressure, temperature and flow rates necessary to support appropriate organ metabolism in their respective approximate physiological ranges. I do. As defined above, a near-normal metabolic rate is about 70-100% of a normal metabolic rate. Further, the methods, solutions and systems of the present invention support levels of metabolism during periods of EMS organ culture that support sufficient oxidative metabolism to produce a normal functional product of the organ or dissected resection.
[0032]
Thus, the blood-free metabolic support (EMS) technology of the present invention provides a number of advantages over conventional cold storage methods. (1) The organ for transplantation can be maintained in a metabolically active state for a long time before transplantation, during which time it can assess its functional integrity and assess its likelihood of functioning after transplantation it can. (2) Organs previously thought to be unsuitable for transplantation because of too long warm ischemia can be revived and actively recovered. (3) Performing EMS perfusion before exposure to cold ischemia with conventional cold preservation protocols protects the tissue and prolongs the duration of cold preservation of allografts compared to preservation immediately after organ harvest. Will be able to (4) EMS perfusion provides a means of targeted delivery of therapeutics, for example, in chemotherapy or gene therapy. (5) Stimulate cells inside an organ or tissue and actively synthesize de novo compounds.
[0033]
Perfusion solution
Organ preservation and perfusate solutions are known in the art as including buffered physiological solutions, such as saline solution, to which various defined additional ingredients can be added, and basal solutions consisting of basal media, such as cell cultures. I have. The perfusion solution of the present invention includes a buffer system that maintains a sufficient amount of amino acids, ions, saline, serum proteins, carbohydrates, and pH at physiological levels to support protein synthesis by metabolizing organs. Use a good base solution. In addition, the perfusion solution of the present invention is designed to support the nutritional and metabolic needs of the vascular endothelium within the graft, thereby increasing the integrity of the vasculature, and subsequently the normal permeability of the organ. maintain.
[0034]
The buffered basal medium can be any of the commercially available saline or cell culture media (eg, Hank's BSS, Earl's BSS, Ham's F12, DMEM, Iskov's MEM, M199, RPMI 1640, RSM-210). . One embodiment of the perfusion solution of the present invention employs a bicarbonate buffer system. The bicarbonate buffer works in conjunction with the respiratory gas control sub-system of the EMS system and automatically adjusts the pH of the perfusion solution to a narrow range from 7.0 to 7.6, more preferably from 7.30 to 7.45. To maintain. This approximates the respiratory control of blood pH by the lungs.
[0035]
This basal medium contains a number of additional components, including but not limited to essential and non-essential amino acids, growth factors, vasodilators, vitamins, and chemical energy substrates, to support oxidative metabolism by organs or tissues. It is added in a physiologically effective amount.
[0036]
The amino acids to be included in the perfusion solution of the present invention include the basic 20 kinds of amino acids, and may be D- or L-amino acids or a combination thereof, and include citrulline, ornithine, homocysteine, homoserine, β-alanine Or a modified amino acid such as amino-caproic acid, or a combination thereof.
[0037]
Chemical energy substrates added to this perfusion solution include pyruvate, glucose, ATP, AMP, coenzyme A, flavin adenine dinucleotide (FAD), thiamine pyrophosphate hydrochloride (cocarboxylase), β-nicotinamide adenine dinucleotide ( DPN), β-nicotinamide adenine dinucleotide phosphate (TPN), uridine 5 ′ triphosphate (UTP) hydrochloride. These chemical energy substrates contain from about 0.01% to about 90% by volume of the combination of additional components added to the basal solution during the preparation of the perfusion solution of the present invention.
[0038]
The basal medium is also supplemented with nucleic acids for repair and synthesis of DNA including 2'deoxyadenosine, 2'deoxyguanosine, 2'deoxycytidine, adenosine, thymidine, guanosine, cytidine and uridine. The solution of the present invention comprises insulin, thyroid stimulating hormone (TSH) and platelet-derived growth factor (PDGF), fibroblast growth factors (FGF-1, FGF-2), insulin-like GFI and II, epithelial GF (epithelial GF). ), Epidermal GF, brain-derived FGF, somatomedins A1, A2, B and C, nerve growth factor (NGF), vascular endothelial growth factor (VEGF), heparin binding growth factor (HBGF), endothelial cell growth factor Growth factors (GF) such as (ECGF), transforming growth factor (TGF), glucocorticoids and urogastone may also be included. Also included are cytokines such as IL-1, colony stimulating factor (CSF), and erythropoietin.
[0039]
The perfusion solution of the present invention contains mucopolysaccharides such as serum albumin and / or chondroitin sulfate B, heparin, petastarch, hetastarch, and a plasma expander as colloid sources, and contains linoleic acid, arachidonic acid, linolenic acid, Also includes lipids such as icosapentaenoic acid, docosahexaenoic acid, and oil. In addition, attachment factors, antioxidants, vasodilators, and impermeable materials may be included in the perfusion solutions of the present invention.
[0040]
The high osmolality of the present invention is used for initial organ lavage and is used to maintain organs for extended periods in EMS systems using warm preservation techniques (18-35 ° C.) that do not cause excessive hypothermia. Used as perfusate. This solution is designed to support the nutritional, synthetic, and metabolic needs of the vascular endothelium within the graft, thereby maintaining vascular integrity and subsequent normal organ permeability . Some of the components of the solution of the present invention are similar to components of other known tissue culture media and other known perfusion solutions for organ transplantation with excessive hypothermia. The solution of the invention enables the stimulation and amplification of microvascular and macrovascular endothelial cells in particular, supports the integrity of the vascular endothelium in the graft, and supports normal permeability and metabolism without excessive hypothermia Designed. This solution's enhanced ability to serve as a preservation solution for organs for transplantation using warm preservation techniques, such as serum albumin as a source of protein and colloid, vasodilators to ensure proper swelling of the vasculature, survival Only as a source of trace elements that enable capacity and cell function, pyruvate and adenosine for oxidative phosphorylation support, transferrin as adsorbent, insulin and sugar for metabolic support, and impermeable substances Glutathione, which also removes toxic free radicals, cyclodextrin as a source of impermeable material, scavengers and potentiators of cell-adsorbing and growth factors, high Mg concentration to support microvascular metabolism, growth Mucopolysaccharide mainly containing chondroitin sulfate and heparin sulfate for enhancing action of factor and hemostasis, Fine colloid, seems to be attributed to the supplemented with ENDO GRO (TM) as a source of impermeable material and growth promoters. As a result, it has been found that the preservation solution of the present invention can preserve organs without causing excessive hypothermia, and the common problems that occur with cold preservation are edema, vasospasm, depletion of ATP storage, cessation of ion pump, It does not show glycolysis and the formation of harmful free radical intermediates induced by low temperatures. The preservation solutions of the present invention provide more effective preservation, thereby offering the potential for an expanded donor pool, ie, the use of dead cardiac arrest donors.
[0041]
One of skill in the art will recognize that other compounds can be replaced with functionally equivalent compounds with similar results. For purposes of explanation and not limitation, Table 1 lists the components of one embodiment of the perfusion solution of the present invention.
[0042]
[Table 1]

[0043]
[Table 1-1]

[0044]
[Table 1-2]

[0045]
[Table 1-3]

The perfusion solution contains one or more oxygen-carrying compounds ("oxygen carriers") that serve to provide molecular oxygen to the organs for oxidative metabolism. Such oxygen carriers are well known to those skilled in the art and include hemoglobin and stabilized hemoglobin derivatives including cross-linked polyhemoglobin, such as dispirin cross-linked hemoglobin (DCLHb), o-raffinose cross-linked hemoglobin, and glutaraldehyde cross-linked hemoglobin. And conjugated hemoglobin, such as polyoxyethylene conjugates (PHP), PEG conjugated hemoglobin, recombinant hemoglobin products, perfluoro compound (PFC) emulsions and / or perfluoro compound ultrafine bubbles ( (Collectively referred to as "perfluoro compounds"). One such oxygen carrier is a perfluoro compound such as perflubron emulsion (perfluorooctyl bromide, PFOB). Other perfluoro compound emulsions which are said to serve as oxygen carriers are described, for example, in U.S. Patent Nos. 5,403,575, 4,868,318, 4,866,096, 4,865,836. 4,686,024, 4,534,978, 4,443,480, 4,423,077, 4,252,827, 4,187,252, 4,186,253, Nos. 4,110,474 and 3,962,439. Such liquid PFC emulsions include perfluorooctyl bromide, perfluorooctyl dibromide, bromofluorocarbon, perfluoroether, Fluorsol DA ™, F-44E, 1,2-bisperfluorobutyl-ethylene, F-4-methyloctahydroquinol-idizine, 9-12 carbon perfluoroamine, perfluorodecalin, perfluoroindane, perfluorotrimethyl vehicle [3,3,1] inane, perfluoromethyl Adamantes (perfluoromethyl adamante) and perfluoromethyl adamantine include but are not limited to. Such an oxygen carrier provides about 0% to about 59% by volume of added components that are added and dissolved in the base solution during the preparation of the perfusion solution of the present invention, or about 0% to about 20% of the total perfusion solution. % (V / v).
[0046]
Red blood cells (RBCs) may also be used as oxygen carriers in amounts effective to support metabolism by the perfused organ. Generally, about 5 cc of RBC per 500 ml of perfusion solution (ie, about 1%) is an effective amount. When compared to perfluoro compound emulsions or complex hemoglobin, RBCs provide an oxygen concentration equivalent to oxygen consumption by metabolizing organs. This generally occurs at a rate of 0.1 to 0.3 cc / min / gm. In addition, there was no evidence that RBC was cretinate 24 to 48 hours after perfusion. Thus, RBC levels in this range do not present the problem of mechanical damage to organs associated with blood-based perfusates.
[0047]
RBC (5 cc per 500 cc of perfusate) with PO> 200 mmHg_TwoWas added to the circulating perfusate until was obtained. Stable perfusion pressure, vascular flow rate, and diuresis were obtained (see Table 2).
[0048]
(Outside 1)

(Outside 2)

In a preferred embodiment, chemically modified hemoglobin, such as polyethylene glycol modified hemoglobin, is used to maintain the oxygen tension of the perfusate at a level sufficient to support metabolism by the perfused organ. You may. Generally, an effective amount is from about 0.01 g to less than 0.5 g per 500 ml of perfusion solution. Chemically modified hemoglobin can be deoxygenated, either recombinant or isolated from humans or other animals, for example, by cross-linking or polymerization, or by polyethylene glycol or polyoxyethylene. Refers to hemoglobin chemically modified by the addition of an adduct such as Polyethylene glycol-modified hemoglobin refers to hemoglobin modified to bind to polyethylene glycol (α-hydro-ω-hydroxypoly- (oxy-1,2-tandiyl)), and a method for preparing chemically modified hemoglobin is as follows. Known in the art, for example, U.S. Patent Nos. 5,234,903, 5,386,014, 6,054,427, 5,985,825, and 5,814,601. The contents of which are incorporated herein by reference in their entirety.
[0049]
The perfusion solution of the present invention further comprises a vasodilator in a physiologically effective amount, which not only properly dilates microvessels but also dilates large vessels via smooth muscle cell relaxation. Means are also provided. This vasodilation is controlled in an endothelial cell dependent manner to ensure that the normal permeability of the vasculature is maintained. The vasodilator components used in the perfusion solution of the present invention include (i) substrates for endothelial cell-mediated vasodilation such as acetylcholine, dopamine, bradykinin, and arginine; (ii) prostacyclin (and analogs such as Carbacycline) and Mg⁺And (iii) verapamil for their combined effects on vasodilation mediated by calcium channel blockade, and (iii) adenosine (and analogs such as cyclohexyl adenosine). . Other calcium channel blockers encompassed by the present invention include flunarizine, nifedipine, SNX-11, chlorpromazine, and diltiazem. The use of the aforementioned vasodilators ensures that the vasculature is sufficiently dilated while at the same time retaining its integrity and normal barrier function. The vasodilator comprises from about 1% to about 50% by volume (w / v) of a combination of additional components added to the basal solution when preparing the perfusion solution of the present invention.
[0050]
EMS perfusion subsystem
The invention is described with reference to preferred embodiments. It should be understood that the various components of the system may be combined or provided as separate parts introduced into the system as a design choice.
[0051]
FIG. 1 is a diagram of one embodiment of the organ perfusion circuit of the present invention. In most cases, only one perfusion path is needed. However, if the organ to be metabolically maintained is the liver, two perfusion circuits are required. These two perfusion paths are shown in FIG. 2 and are described in more detail below. All of the perfusate available for circulation through the system is propelled through circulation path 10 by pump 12. The direction of this perfusion solution flow is indicated by arrows in the flow path. Although any pump may be used to circulate the perfusate of the present invention, a pulsatile pump, such as Model No. MOX-100 ™ (Waters Instruments, Inc., Rochester, MN) is preferred.
[0052]
The perfusate passes through the heat exchanger 14 before entering the organ 40 to a temperature in the range of 25C to 37C. Preferred temperatures for optimal metabolism by the organ are within this range. The heat exchanger 14 is controlled by a temperature controller 16 that receives an input from a temperature sensor 18 disposed in the perfusion path 10. In one embodiment, the temperature controller includes a thermocouple that senses the temperature of the perfusate, as needed to maintain the temperature in the desired range, and a thermocouple-activated heat exchanger. One unit. In another embodiment, the temperature may be controlled by a water heater for circulating the hot water around the perfusate reservoir or oxygenator. In yet another embodiment, a temperature sensor located in the organ chamber is used to monitor and control the temperature of the perfusate.
[0053]
The perfusate containing the oxygen carrier is oxygenated via a hollow fiber oxygenator or a membrane oxygenator 20, such as that available from Sarnes, prior to contacting the organ, through a child-sized oxygenator, In most cases, an oxygen partial pressure in the range of 100-240 mmHg is obtained. However, when placed in a perfusion pathway that supplies perfusate to the portal system of the liver, this partial pressure is usually maintained in the range of 80-140 mmHg. In a preferred embodiment, the oxygenator 20 is located in the perfusion solution flow path 10 between the heat exchanger 14 and the pH sensor 22. The oxygenator may be a separate unit, but may be combined with other components of the system to form one unit, for example, in combination with a heat exchanger or as part of an organ chamber.
[0054]
PH and PaCO of the perfusion solution_TwoIs CO_TwoControlled by automatic intermittent gas generation of the perfusate with pH ranging from 7.30 to 7.45 and CO of 30 to 60 mmHg._TwoIt becomes a circulation liquid having a partial pressure. Finally, the perfusate is degassed by a conventional air bubble trap 30 just before entering the organ chamber 32. This perfusate exits the organ as a venous drainage and in most perfusion systems drains directly by gravity into a waste reservoir 38 located below the compartment of the organ chamber 32 that holds the organ. In a preferred embodiment, the drainage is by a conventional atraumatic cannula inserted into the vein of the organ 40 or by supporting the vein such that the vein remains connected to the conduit 43 as described in more detail below. To the waste storage tank 38 by means of a conduit 43 connected to the vein by means of an apparatus 49.
[0055]
Organ chamber
The specifications of the organ chamber 32 used will depend on the organ to be maintained. For example, if the organ is a liver, the organ chamber must have two or more inlets to accommodate two simultaneously operating perfusion channels (FIG. 2). If the organ is the heart, an open vertical tube extending upward from the perfusion channel above the perfusion fluid inlet of the heart will provide sufficient pressure to resist the ejection of perfusion fluid by the beating heart. Provide a column of perfusate to be maintained.
[0056]
However, in general, the organ chamber 32 (as shown in FIGS. 1-4) includes a container 33 having a cover or lid 34, including a lower and upper portion sized to accommodate the organ 40 to be maintained. The entire room is made of medical-grade, non-toxic, non-leeching, sterilizable, hard material. In one embodiment, the organ chamber is made of stainless steel and has means for viewing the interior of the chamber, such as one or more transparent or translucent panels. It is highly desirable to be able to observe the organ during perfusion, so in a preferred embodiment the entire organ chamber is made of a transparent, rigid material. Various thermoplastics are commercially available. For example, it is a polycarbonate material such as Lucite or LEXAN (registered trademark). The organ chamber of the present invention may include a suitable combination of materials.
[0057]
The organ chamber of the present invention includes at least one support 36 that can be placed on top of the organ chamber container 33 for supporting an organ 40 within the container 33 that provides mechanical constraints for the organ during perfusion. This minimizes damage or damage to the organ or its blood vessels, for example, due to sudden movement of the organ during transport. The organ chamber 32 also provides for a venous outflow of perfusate from the organ to prevent the perfusion fluid from contacting the exterior surfaces of the organ and the interior surfaces of the organ chamber to minimize the risk of contamination. It includes one or more perfusate inlets 42, conduits 43, which flow directly into the bath. The organ chamber also includes a conduit 48 for collecting organ products exiting the organ 40. Optionally, the lower portion of container 33 includes a perfusate reservoir 38 and / or other components of the perfusion system.
[0058]
Organ support 36 is adapted to inhibit movement of the organ within the container. The organ support 36 may be made of a solid or semi-solid material, or may be made of a mesh-like cloth that hangs the organ in a suspended state. In one embodiment, the support is a solid or semi-solid material having a cavity shaped to a portion of the organ so that the organ fits within the cavity. In a preferred embodiment, the support is a gas (inert gas), fluid (eg, saline or other suitable non-toxic liquid), or gel (eg, silicon) that can conform to the shape of the organ to be overlaid. An elastic material such as a pouch or bag filled with. Both the outer surface and the inner material of the support must be inert, biocompatible and sterilizable. The support contacts the organ on the bottom surface, and partially on the side, leaving a portion visible from the side or covering of the organ chamber container so that the organ can be observed during perfusion. Visual observation of the organ is necessary to detect early signs of edema and to find the location of the organ that is not properly perfused or other signs of organ dysfunction.
[0059]
The support supports the organ within the organ chamber and, importantly, prevents movement of the organ within the chamber, thereby preventing organ damage. Preventing organs from slipping and rolling in the room also eliminates the stretching of major blood vessels, such as the renal veins and arteries of the kidney, especially during organ transport. The effects of vibration and shock are reduced. Moreover, this type of organ support is closer to the normal physiological environment in which the organ is supported by and in contact with adjacent tissues in the body. In addition, the formable support allows the organ to be oriented in a variety of orientations (including physiological ones) as well as resting on the side of the organ.
[0060]
FIG. 3 shows a perfusion from the vein 41 of the organ 40 into the organ chamber 32, which is part of the organ chamber (as shown in FIG. 3) or separate from the organ chamber (FIGS. 1 and 2). 1 represents an embodiment in which there is a perfusate outlet 43 with means for directing it to a fluid reservoir 38. Unlike existing organ chambers, in which the perfusate exiting the vein 41 flows down the side of the organ 40 and drip into a reservoir located below the organ chamber, the organ chamber of the present invention is characterized in that the perfusate is external to the organ. Preventing contact with the surface substantially reduces the potential for contamination of the perfusate. Optionally, the organ chamber further comprises a vein support 48 as illustrated in FIGS. A further advantage of this embodiment is that the need for cannulating the vein with vein support means, such as a small sterile, pointed piece (shown in FIGS. 3 and 5) placed adjacent to the organ. And obstruction and damage associated with cannulation. The vein support 48 supports the vein near the intersection of the vein and the outer wall of the organ and securely holds the open end of the vein against the end of the tube that directs venous drainage away from the organ and into the reservoir 38. be able to.
[0061]
The organ chamber of the present invention may be a disposable unit or may be made of a suitable material such as stainless steel or polycarbonate that can be sterilized and reused. In one embodiment, the organ chamber of the present invention includes a connector for removably connecting the organ chamber to an external warm storage system. Suitable connectors include, but are not limited to, long inert biocompatible tubing or more complex mechanical devices to ensure the integrity of the flow path once the organ chamber is placed therein. Not done.
[0062]
Optionally, the organ chamber may include various components of a warm perfusion system, including, but not limited to, an oxygenator, a reservoir, a bubble trap, and a flow meter. Pressure, temperature, pH, PaO_Two, PaCO_TwoMeans for measuring metabolic indicators such as glucose, and markers of synthetic function such as NO (stable end product) PCNA or ZO-1 may be incorporated into the organ chamber. Further, the organ chamber of the present invention includes one or more ports for sampling perfusate and one or more ports for recycling perfusate.
[0063]
FIG. 3 illustrates one embodiment of the organ chamber of the present invention having a perfusate reservoir below the organ chamber container. Perfusate parameters that indicate organ metabolism, such as glucose concentration, PaO_Two, PaCO_TwoSensors for monitoring such as are located to measure the properties of the perfusate just before it enters the organ and as it exits the organ. Changes in perfusate values before and after the organ indicate organ metabolism.
[0064]
Sensors for other parameters of the perfusate, such as pH and temperature, may be located in the organ chamber to optimally control the parameters of the perfusate during perfusion.
[0065]
In most storage systems, venous waste fluid exiting the organ flows through one of the holes 37 in the support means 36 and flows directly by gravity to a reservoir 38 in the organ chamber 32 and is collected. In the present invention, the venous outflow is collected by gravity flow, but the vein is transported away from the organ, for example, by connecting it to a length of tubing 43. In one embodiment, this is performed by cannulating the vein with an atraumatic cannula. However, the length of vein available for resection during reimplantation is reduced due to any damage that occurs during cannulation. Thus, in a preferred embodiment, the vein support 46 supporting the vein 41 in the conduit is positioned below the vein adjacent to the organ at the point where the vein 41 intersects the outer wall of the organ 40. As shown in FIGS. 6 and 7, the top of the support is wide enough to support the veins and conduits at the two junctions. This makes it possible to connect the ligated end of the vein 41 to a conduit, for example a tube of a certain length, without having to cannulate it, for example by simply sliding the end of the tube 43 over the top of the vein 41 become. Alternatively, the vein may settle into a partially open conduit. Further damage of the veins is thereby avoided.
[0066]
Collection of organ products
The organ chamber of the present invention can recover the organ product separately from the perfusate, or recycle the organ product as part of the perfusate. Means 48 for collecting the organ product may include valves and / or ports, thereby sampling all or a portion of the organ product produced by the organ during perfusion and parameters indicative of normal organ function for that organ. Can be tested for
[0067]
The organ compartment of the present invention further comprises means for optionally collecting and separating the organ product from the perfusate. For example, urine produced by a perfused kidney exits the ureter into an organ product flow path. Disposed within the organ product pathway is a valve that, when opened, couples the organ product pathway to the venous perfusate fluid path. When the valve is in the closed position, organ product is diverted from the perfusate and can be removed from the system by a sampling or collection port. The functional properties of the organ can be determined by assessing the chemical parameters of the recovered organ product, the concentration of the tracer molecule added to determine the rate of production, or the rate of disappearance of the product. Alternatively, the organ product is re-mixed with the perfusate to maintain perfusate volume, osmolality and chemical integrity. In this way, organ metabolism, perfusion and function can be measured, and the state of the preserved organ can be proactively evaluated.
[0068]
The effluent is filtered through a filter 44 having a pore size of 0.22 microns, such as that available from Millipore, to remove debris and bacterial contaminants. The recovered wastewater thus also offers the further advantage of minimizing contamination of the perfusate by contact with air. Once the perfusate is collected in the waste reservoir 38, the waste is regenerated either by perfusate exchange or by dialysis in the perfusion dialysis sub-system 50 and recirculates the system.
[0069]
The organ chamber of the present invention further includes suitable microprocessors and sensors for monitoring and controlling perfusion solution parameters.
[0070]
Perfusion regeneration
Due to the continuous metabolic utilization of the components of the perfusion solution by the organs in the system of the present invention, the perfusion will eventually be exhausted unless replenished or regenerated. In one embodiment, the components required to support metabolism can be replenished by perfusion exchange, i.e., removing some of the depleted perfusion by the device 46 in the reservoir and replacing it with fresh perfusion.
[0071]
Perfusion exchange
In one embodiment, the perfusate is exchanged using two pumps 47 and 49 with adjustable volumes. A pump 47 is connected to a device 46 in the storage tank for extracting spent perfusate, and a pump 49 is located in the perfusate path 10 immediately before the oxygenator 20. Immediately after removal of spent perfusion solution by pump 47, pump 49 introduces an equal volume of fresh perfusion solution into the system. The exchange of this method can be continuous or intermittent. The rate of exchange depends on the temperature and / or metabolic rate of the perfused organ.
[0072]
By introducing fresh perfusate into the perfusion path just before the oxygenator 20 and the pH control system, the perfusate PaO_TwoAnd the pH is kept constant. In this way, by-products of metabolism are removed and fresh solutions are administered to maintain the ongoing metabolism by the organ without developing acidosis. The volume of perfusate remains constant and the osmolarity remains constant.
[0073]
Perfusate dialysis
However, in order to keep up with the metabolic rate, it may be inappropriate to replenish the nutrients necessary to maintain organ metabolism with perfusate exchange. Thus, in another embodiment, the perfusate is regenerated by continuous dialysis, alone or in combination with perfusate exchange.
[0074]
In the dialysis sub-system of the present invention, the nutrients and chemical energy substrates required for continuation of metabolism are reintroduced into the perfusate by one or more dialysis units. Such units, one or more in tandem, can provide nutrients in the perfusate to be replenished in a manner similar to dialysis, which removes catabolic metabolites from the perfusate.
[0075]
As shown in FIG. 1, the perfusion dialysis sub-system of the EMS includes a waste liquid storage layer 50 and one or more nutrient storage tanks D1, D2, D3, and Dn that are connected to the perfusion channel 10 so that the fluid flows. Including. The nutrient storage tanks D1, D2, D3, and Dn are connected to the waste liquid storage tank 50 via semipermeable membranes M1, M2, M3, and Mn whose molecular weight cutoff varies in the range of 1,000 to 80,000 daltons. These nutrient reservoirs contain concentrated amounts of the components required to regenerate the perfusion solution, which are diffused into the perfusate of the waste reservoir 50.
[0076]
In one embodiment, when used in more than one, the perfusate stream is first contacted with the nutrient with the largest molecular weight and then the nutrient reservoir is reduced in molecular weight such that the molecular weight of the nutrient contacted is reduced. Arrange them in descending order. This ensures that any components that may have been lost in the first dialysis unit are replenished by passing through the next unit before leaving the perfusate dialysis system. The system further includes a device for adjusting the volume of the perfusate solution such that an equilibrium is maintained between the waste reservoir and the nutrient reservoir.
[0077]
Monitoring and control systems
The perfusion solution monitoring subsystem performs several functions using standard components and hardware to monitor various parameters of the perfusion solution. In one embodiment, the temperature is sensed and controlled by a thermocouple or temperature sensor 18 coupled to the heat exchanger. For example, as a component of the bubble trap 30, one or more pressure gauges 31 located in the perfusion solution flow path are used to monitor the vascular resistance to the perfusion fluid and the partial pressure of the respiratory gas. An ultrasonic device for sensing the flow rate with a sensor 54 affixed to a tube carrying the perfusate, such as that available from Trasonic, Inc. Used to monitor. In a preferred embodiment, the flow meter is located in the flow path between the bubble trap and the artery entrance of the organ chamber. The osmolarity of the perfusate is measured using a conventional freezing point depression method. Alternatively, the osmolarity may be monitored using in-line detection means to evaluate the concentration of the main component of the perfusion solution. The perfusate sample may be removed by bubble trap 30 or some other means located in the perfusate flow path for this measurement.
[0078]
In another embodiment, monitoring and controlling the parameters of the perfusion solution is performed using sensors and a microprocessor integrated within the organ chamber. These are all within the skill of the ordinary engineer. The components of the control subsystem may be combined or provided as separate components that are incorporated into the system as a matter of design choice.
[0079]
Maintain pH and breathing gas
The perfusion solution is 100-250 mmHg O_TwoPartial pressure (PaO_Two2), oxygen is supplied continuously by introducing 100% oxygen 28 through a membrane or hollow fiber oxygenator 20. This system of the present invention provides for the pH of the perfusate to be between 7.32 and 7.38 and the CO_TwoIncludes a novel mechanism for maintaining levels at a partial pressure of 30-60 mmHg. Perfusate pH level and CO_TwoAdjustment of the level_TwoAchieved by controlled intermittent gassing of the perfusate at The system continuously monitors the pH with, for example, a pH electrode or sensor 22 in the perfusion path 10. This pH sensor 22 provides the CO2 needed to maintain tight control of pH._Two26 is operatively connected to a controller 24 and a solenoid 25 for regulating gas generation.
[0080]
In one embodiment, one or more pH meters 21, such as, for example, model HI8711 from Hanna Instruments, monitor the pH of the circulating perfusate by sensors or electrodes 22 located in the perfusion channel 10. . An in-line controller 24 such as a dataron jar manufactured by Bleonics receives an input from the pH sensor 22 via a pH meter 21 connected to the controller 24, and uses a solenoid 25 to control the CO._TwoOperate valve contact 23 to release 26 into the perfusion solution. When the pH exceeds 7.35 and thereby exceeds the set value of the controller 24, the gas generating system is activated by the valve contact 23 and the solenoid 25 until the pH reaches 7.35 again._Two26 is injected into the perfusion solution, at which point the system stops operating and the gas stops. When used in combination with a perfusion solution having a bicarbonate buffer system,_TwoPerforming intermittent gassing of the perfusion solution using is particularly effective in mimicking the strict physiological control of blood pH by the respiratory system. As with other parameters of the perfusion solution, pH and respiratory gases can be monitored and controlled using sensors and microprocessors known to those skilled in the art for this application.
[0081]
Organ fixation in EMS
In accordance with the present invention, once an organ is removed, a sufficient amount of perfusion solution is slowly introduced into the main arterial blood supply through the cannula to perfuse that organ until the waste fluid of that particular organ is free of blood. It will be appreciated by those skilled in the art that the amount of perfusion solution sufficient to flush the organ will depend on the type and size of the particular organ and the time at which that organ lost blood flow. For example, 200-600 ml of perfusion solution will be sufficient to flush a human kidney that has lost blood flow for 1-3 hours. In this way, all the ischemic blood and acidosis products accumulated in the blood vessels are removed. In addition, pH is restored and fresh substrate is provided to support anaerobic metabolism and other cellular pathways necessary for maintaining cellular integrity and function.
[0082]
After flushing, the organ is fixed in the internal space of the organ chamber by placing the organ on a support that can be placed in the organ chamber, for example, to prevent movement of the organ. For example, the support may be a resilient support that conforms to the outer surface of the organ, such as a bag filled with fluid, gas, or gel, so that the organ does not move around the organ chamber during transport. It may be a solid or semi-solid support that includes cavities generally conforming to the external surface of the organ. The organ is then connected to the perfusion solution by a short tube connecting the cannulated artery to the entry hole of the organ chamber. It is desirable that the tubes used in the present invention are all tubes made of an inert and sterilizable material, for example, silicon or Tygon (registered trademark).
[0083]
The organ is perfused while maintaining the temperature of the perfusion solution in the range of 25 ° C to 37 ° C. At the same time, the system is_Two, PaCO_Two, And pH are adjusted with the gas generating subsystem and the flow rate, vascular pressure and osmolarity are monitored.
[0084]
During the perfusion period, the integrity of the function of the organ is also monitored. Various physiological and metabolic parameters can be used as indicators of functional integrity. For example, the use of a perfusate made of a known formulation as shown in Table 1 can facilitate the measurement of certain aspects of metabolic function. The rate of loss of glucose concentration in the perfusate during circulation through the organ over time is a measure of organ function. For selected time points, glucose concentrations below the normal range of glucose utilization may be indicative of organ damage or metabolic inhibition. Another example of a measure of organ function is oxygen consumption while perfusate is circulating in the organ. Inappropriate oxygen consumption, ie, low oxygen consumption compared to the normal range, may be indicative of tissue hypoxia. Another example of a measure of organ function is the vascular flow rate of perfusate circulating through the organ using standard perfusion pressure. Low flow rates compared to the normal range may indicate vasoconstriction, edema, swelling of vascular endothelial cells, and loss of vascular integrity in the damaged organ.
[0085]
The features of these organ functions are easily monitored by sensors located in the perfusion path of the EMS warm storage system. The optimal placement of the sensor is in the organ chamber itself, just before the storage solution enters the organ and near the venous outflow of the solution leaving the organ. Placing the sensor immediately before and immediately after the organ increases the accuracy of the test in that any differences can be attributed to the organ itself. Other arrangements of the sensor are possible, but placing the sensor far from the organs introduces the possibility of error. This is not a major problem for metabolic markers such as glucose, but is a major concern for oxygen.
[0086]
The system of the invention also comprises a second monitoring subsystem for evaluating other indicators of the functional integrity of the organ. For example, organ chamber conduits that extract organ products from organs during the perfusion period can track the output of the organ and determine the physical or chemical parameters of the perfused organ product to represent normal organ function. Evaluation in comparison to allows for monitoring of organ function. One skilled in the art can easily determine which properties to monitor depending on the organ to be preserved. For example, if the organ is a kidney, by measuring parameters including diuretic rate, urine osmotic pressure, specific gravity, creatinine clearance, etc., not only individual aspects of renal physiology but also overall renal function can be measured. The nature of the monitoring subsystem depends on the organ to be perfused and may include means for measuring bile enzymes, cardiac enzymes, electrocardiograph movements, and the like.
[0087]
In order to practice the present invention, normal values and ranges for chemical or physical parameters of organ products, and other measurements representative of the normal functioning of the organ, will be apparent to those skilled in the physiological and clinical chemistry medical arts. ± 20% of the value referenced in textbooks known to them. See CRC Handbook of Clinical Chemistry, edited by Mario Warner, CRC Press, "Human Physiology", 6th edition, by Stuart Ira Fox, William C. Brown, publisher.
[0088]
The effectiveness of the present invention in supporting organ culture of various organs and tissues was evaluated. The present invention has been used to demonstrate efficacy in dog kidney, bovine kidney, rat heart, human placenta and bovine limb.
Embodiment 1
[0089]
The dog kidney was removed, the renal artery was cannulated, and the solution was applied using the process and system of the present invention. All blood was removed from the kidneys and these kidneys were perfused with the solution. The kidneys were maintained at 32 ° C. for 3 days supported by EMS organ culture technique. Similarly, physiological pH, osmolality, pressure, flow rate and oxygen consumption were maintained during EMS organ culture. The kidney remained intact during organ culture and continued to metabolize. The metabolism that progresses in the kidney was sufficient to result in continued function. That is, the kidney continued to produce urine during organ culture. The results of intact whole kidney cultures are listed in Table 2. During this EMS organ culture, there was no decrease in metabolism or function in any of the parameter categories. Similarly, there was no advanced edema and no necrosis observed after histological evaluation.
[0090]
[Table 2]

Embodiment 2
[0091]
Bovine kidney was also tested to assess species differences. Bovine kidneys were placed in EMS organ cultures for 3 days using the same technique as described in Example 1. Similar to the results when using canine kidney, bovine kidney could be maintained intact in EMS organ culture for 3 days without loss of ongoing metabolism or available function. Similar to the results obtained with canine kidney, bovine kidney showed stable perfusion pressure, flow rate and lack of edema during organ culture. Based on histological evaluation, the bovine kidney was normal and all components of the kidney structure appeared to be in excellent condition.
Embodiment 3
[0092]
Hearts were removed from the rats and the aorta was cannulated. These hearts were placed in the EMS organ culture of the present invention and maintained for 24 hours. The results of this test using rat hearts are listed in Table 3.
[0093]
[Table 3]

After the EMS organ culture period, three of the hearts were transplanted to assess whether these hearts were of sufficient integrity to support life. All three hearts spontaneously beat and support life without assistance.
Embodiment 4
[0094]
The human placenta with the umbilical cord still attached was removed. Umbilical cord blood vessels were cannulated and the blood was washed from the umbilical cord with the solution of the present invention. The organ was placed in EMS organ culture immediately after the organ blood was flushed and the vessel interior was filled with the solution. The organ was maintained intact for about 22 hours using EMS organ culture techniques. During this organ culture period, the flow rate of the solution through the placenta was 92 cc / min and the systolic blood pressure was 80 mmHg. At the end of the EMS organ culture, the intact placenta was evaluated histologically. The isolated cells were then harvested from both the umbilical cord and the placenta itself. Table 4 shows the results.
[0095]
[Table 4]

Viable cells could be harvested from the whole organ after EMS organ culture for 22 hours. Tissue was isolated from both umbilical cord blood vessels and intact placenta by collagenase digestion. Cells isolated from intact organs retained the ability to attach to the surface of the culture flask and to replicate in standard tissue culture. The isolated cells continued to replicate in standard tissue culture and were finally passaged three times. Thus, this intact organ was successfully maintained in EMS organ culture and was therefore metabolically active. The subsequent viability and function of cells isolated from whole organs after the organ culture period supports this interpretation.
Embodiment 5
[0096]
Intact bovine limbs were obtained. The femoral artery was cannulated and the blood was flushed from the intact limb. The interior of the vessel was filled with the solution of the invention and the limb was maintained in EMS organ culture for 2 days. Upon completion of organ culture, the limb was evaluated histologically. The results of these evaluations indicated that the components of the limb were completely preserved without observing necrosis. Cells isolated from intact limbs after 2 days of EMS organ culture were similarly viable. Cells isolated from this intact limb adhered to the surface of the flask in standard cell culture. These cells continued to grow and were passaged three times. Similar to the results obtained for whole organs, dissected excisions such as intact limbs could be maintained with the EMS organ culture technique of the present invention.
Embodiment 6
[0097]
Using the EMS organ culture technique of the present invention, human umbilical cords reaching approximately 2-2.5 feet in length were maintained. The human umbilical cord was maintained intact in EMS organ culture for at least 7 days. The umbilical cord blood vessel was cannulated at one end. At the other end, the blood vessel and the two arteries were connected by a silicon tube connected to the y connector to form a closed circuit. Upon completion of EMS organ culture, the luminal surface of the umbilical cord blood vessels was digested with collagenase. The isolated endothelial cells were viable and adhered to the flask surface in standard tissue culture. The isolated cells exhibited the normal characteristics of endothelial cells in tissue culture. That is, the isolated cells attached, replicated, and expressed the anti-hemophilic factor antigen. These results indicate that the EMS organ culture technique of the present invention maintains 2-2.5 ft long human tissue in EMS organ culture for 7 days without harming the viability of the cells in the tissue without damage. Demonstrate that it can be used for These studies demonstrate that the EMS organ culture technique is effective in maintaining long-term intact organs or large dissections by dissection.
[0098]
The EMS whole organ culture technique of the present invention can also be used for in vivo applications. Specific organs can be removed by simultaneously removing and retrieving the main arterial source that supplies nutrients to the organ and venous outflow. In vivo applications of organ culture techniques include the physiological maintenance of the enervation and lymphatic systems. The artery supplying nutrients to the targeted organ or anatomical resection is cannulated, and the vein receiving the organ drainage is also cannulated to drain blood from the tissue. The vessel lumen is refilled with the solution of the invention. The organ or dissection excision is maintained in vivo, but is "cut" from the physiological system and is maintained by EMS organ culture techniques.
Embodiment 7
[0099]
Canine kidney was isolated by cannulating the renal arteries and veins, flushing the blood from the kidney, and refilling with a solution of the invention. The kidneys were kept intact at 37 ° C. for 8 hours using the EMS organ culture technique. During this separation, the kidney continued to metabolize in vivo EMS organ culture as measured by oxygen consumption and glucose consumption. Similarly, metabolism was at a level sufficient to cause continuous diuresis. The kidney continued to produce urine and fill the connected bladder while disconnected from the rest of the venous system and maintained with EMS organ culture techniques. At the end of the EMS organ culture, the cannula was removed and the organ was reperfused with blood. The kidney continued to function normally. All ten dogs showed normal serum chemistry after EMS organ culture of the kidneys.
Embodiment 8
[0100]
Perfusion of an isolated organ or localized tissue is highly suitable for performing high-dose chemotherapy with reduced systemic toxicity. Recent attempts at perfusion of isolated organs to deliver chemotherapeutic agents in some cancer laboratories have been at increased risk of causing non-target tissue damage. The target organ can be maintained without causing any damage using the organ culture techniques of the present invention. An advantage of using organ culture techniques to deliver chemotherapeutic agents is the opportunity to deliver much higher doses at the same time while reducing or even eliminating the usual systemic side effects.
[0101]
The efficacy of EMS organ culture as a drug delivery technique has been established in non-transplantable human kidneys. The kidney was non-transplantable due to a renal tumor identified as renal cell carcinoma. The human kidney exhibited stable perfusion pressure, vascular flow rate and oxidative metabolism during EMS perfusion. Similarly, the human kidney also consumed oxygen and glucose, produced urine, and purified creatinine. Kidney biopsies were performed both before and after EMS whole organ culture perfusion for histological evaluation. No histological changes were observed after 18 hours of EMS organ culture. Thus, EMS organ cultures can maintain kidneys with renal cell carcinoma without causing damage, thereby providing a delivery system for targeted drug therapy.
Embodiment 9
[0102]
Ischemic stroke following an occlusive disease such as a heart attack or cerebral infarction represents another in vivo application of EMS organ culture technology. Recently developed thrombolytic agents have great prospects for patients suffering from stroke or heart attack. The effectiveness of such therapies still depends on the ability to administer the drug within a short time after the obstruction event. Tissue downstream of the occlusion is deprived of blood, resulting in ischemic damage. The EMS organ culture technique can be used to reperfuse and maintain tissue downstream from the occlusion, thereby extending the time frame of opportunity for performing thrombolytic therapy.
[0103]
The main problem in resuscitation after a major injury is the delivery of appropriate nutrients and volume exchange. Bleeding and multi-organ failure with post-septic sepsis account for approximately 60% of deaths in surgical intensive care units. The EMS organ culture technology of the present invention can be used to maintain tissue integrity and function in vivo during resuscitation from shock. Normal blood volume can be restored with the cell-free solution of the present invention at physiological temperatures, thereby minimizing the development of secondary reperfusion injury in patients with life-threatening blood loss. The EMS organ culture technique maintains the removed organ. Or it may be used to maintain the whole patient.
Embodiment 10
[0104]
The addition of appropriate growth factors and hormones to the perfusion solution of the present invention supports the cell repair process in the damaged organ. Kidneys experiencing 60-120 minutes of warm ischemia were maintained with the EMS whole organ culture technique of the present invention. After 6-8 hours of organ culture with the organ culture technique of the present invention, mitotic images were observed in the distal tubule of the thick ascending limb of Henle's loop. There was no evidence of a repair process in areas of the kidney without apparent damage, ie, proximal tubules and glomeruli. These studies demonstrate the substantial potential of EMS organ culture techniques in repairing and regenerating damaged tissue.
[0105]
Kidneys removed 2 hours after death were resuscitated and repaired during EMS perfusion as follows. Initial perfusion pressure and vascular flow rate were extremely abnormal with an average perfusion pressure of 24/20 mmHg and an average vascular flow rate of 4 cc / min / mg. Similarly, initial oxygen consumption was also impaired, with an average value of 0.08 cc / min / gm. Initial assessment of these organs predicted no primary function (PNF) or no viability.
[0106]
The kidney was repaired during 8 hours of EMS perfusion using the processes and solutions described herein. Perfusion pressure and vascular flow rates were normal, oxygen consumption increased to an average of 0.22 cc / min / gm, and urine flow returned. Normalization of these parameters indicates that the kidney was resuscitated and recovered sufficiently to change the outcome from PNF to a viable organ with mild acute renal tubular necrosis. Two of these kidneys were measured for viability after 8 hours of EMS perfusion and then transplanted. The third kidney was reimplanted after 18 hours of EMS perfusion. The results listed in Table 5 below not only indicate organ viability, but also demonstrate the role of EMS technology in resuscitation from warm ischemia 2 hours after death and repair of injury.
[0107]
[Table 5]

EMS technology can resuscitate and repair organs from ischemic injuries, which are now considered irreparable damage in clinical transplantation. The duration of EMS perfusion directly correlates to the degree of repair, and the longer the kidney is perfused, the better it repairs. After 8 hours of EMS perfusion, transplantation was possible on day 4 after transplantation with peak serum creatinine, and the PNF kidney was sufficiently repaired to normalize on days 12 and 13. Further repair was seen at 18 h EMS perfusion, with a slight increase in serum creatinine, peaking at 2 days and normal at 3 days after transplantation. This is one week earlier than a kidney that was perfused for 8 hours before reimplantation.
Embodiment 11
[0108]
The effect of the EMS organ culture technique on preserving the liver at near physiological temperatures and the ability to resuscitate liver function after warm ischemia (WI) 60 minutes after death was examined. Measurements of selected parameters representing liver viability and metabolic function were made, including oxygen consumption and standard liver enzymes representing acute injury. Evaluation of metabolic function included measurement of bile production and evaluation of enzyme and bilirubin concentrations in bile.
[0109]
The liver perfusion system described in FIG. 2 differs from the kidney and liver systems because it needs to support both the arterial and venous vasculature. The system described in the present invention provides high PaO via hepatic arteries at lower vascular flow rates (<250 cc / min)._Two(100-240 mmHg) while simultaneously delivering low PaO via the portal system._Two(<140 mmHg) and the corresponding high blood flow velocity (> 240 cc / min). The net effect is to provide a more physiological perfusion so that the biliary system is protected and bile production during perfusion is maximized.
[0110]
As shown in FIG. 2, the waste liquid storage tank 38 has a second waste liquid outlet 62 for discharging a perfusate to a second perfusion path 60 for transporting the perfusate to the portal vein 66 of the liver by the pump 64. Like the kidney, the EMS system provides perfusion fluid temperature, perfusion pressure, vascular flow rate, osmolarity, pH, PaO_Two, PaCO_TwoControls nutrient delivery and removal of waste by-products. This can be done by separately monitoring and controlling the perfusate in the two channels, or by adjusting only the perfusion path that supplies the hepatic artery. Thus, in one embodiment, the flow path for the portal system has its own temperature controller, oxygenator and respiratory gas controller, pH controller, bubble trap, and flow rate, vascular resistance, OsM, PaO._Two, And PaCO_TwoAnd means for detecting
[0111]
As with the hepatic artery flow path, the perfusion temperature of the portal vein flow path is maintained in the range of 25-37 ° C. One or more inline oxygenators and oxygen carriers provide the oxygen tension described above for hepatic artery and portal vein supply. The system of one or more pH electrodes, controllers and solenoids is CO_TwoAnd maintain the pH of the perfusate in both channels between 7.30 and 7.45.
[0112]
As with other organs, long-term liver preservation involves replenishing depleted nutrients in the perfusate by perfusate exchange, dialysis, or a combination thereof, and dialysis of accumulated metabolic waste. Is performed by The synthetic properties unique to the liver provide opportunities for regeneration by creating protein synthesis and new cell growth.
[0113]
After either 30 minutes (n = 5) or 60 minutes (n = 5) warm ischemia (WI) post-mortem, the calf liver was removed. The vena cava on the liver was dissected at the diaphragm. Beginning from the upper left, the liver was dissected with lymph tissue, gastric artery and common bile duct cut off. The portal vein and hepatic artery were cut off and cannulated with 12 mm and 5 mm cannulas, respectively. The liver was perfused for about 4 hours at 34 ° C. During perfusion, samples of perfusate were taken at 2 and 4 hours for measurement of bilirubin, ALT, AST, ALP and blood gases. No edema or discoloration demonstrated the ability of the EMS organ culture technique to repair liver.
[0114]
Livers receiving WI 30 minutes post-mortem all exhibited high rates of oxygen consumption after resuscitation with EMS. No significant difference in oxygen consumption rate was detected over 60 minutes of WI injury. Livers subjected to WI 30 min after death showed a mean arterial blood pressure (MAP) of 31 mmHg (combined hepatic artery and portal vein) with a corresponding combined flow rate of 320 cc / min after metabolic recovery. The vascular resistance was 0.1. As the time of exposure to WI increased to 60 minutes after death, MAP dropped to 20 mmHg and flow rates increased to an average of 420 cc / min. The resulting vascular resistance was 0.06.
[0115]
Standard liver function screens were performed to test ALT, AST, alkaline phosphatase (ALP) and total / direct bilirubin. After resuscitation with the EMS organ culture technique, livers injured at WI 30 min after death, except for one liver at 2 h, showed normal levels of ALT, ALP and bilirubin at both time points tested ( Table 4). Similarly, when WI injury increased at 60 minutes post-mortem, ALT, ALP and bilirubin values were normal after 2 h and 4 h EMS perfusion.
[0116]
Bile production
Livers resuscitated with EMS after 30 minutes post-mortem WI produced bile during warm perfusion. The change in bile flow rate was in the range of 1.5 to 6 cc / hour. Bile color was dark green or green-yellow. A normal control consisting of bile removed from the gallbladder at the time of removal showed a total bilirubin level of 315 mg / dL, an ALP of 18 IU / L, and was negative for AST and ALT. Livers injured 30 minutes after death at WI produced bile with a wide range of bilirubin concentrations, 1.1-418 mg / dL (Table 5). However, the mean values were close to normal bile bilirubin concentrations. Similar to the results observed for the concentration of liver enzymes in the EMS perfusate described above, bile AST levels were halved during 2 to 4 hours of perfusion of livers that had received 30 minutes WI, while at 60 minutes WI Bile AST levels remained constant in the damaged liver. ALT was found in bile from two of the livers perfused for 2 hours after 30 minutes WI. Bile produced in livers that had undergone WI 60 minutes post-mortem also contained ALT at both 2 and 4 hours of EMS perfusion. Similarly, restored liver function was sufficient to support continued bile production.
[0117]
These results suggest that the EMS organ culture technique provides the ability to resuscitate and repair liver function after a substantial postmortem WI injury. In livers that received WI for 30 and 60 minutes, metabolism was restored sufficiently to restore nearly physiological oxygen consumption with EMS perfusion. Liver function tests provide additional evidence that the liver can be resuscitated from WI injury for as little as 60 minutes.
[0118]
Use of EMS technology to prevent cold ischemic injury
EMS perfusion prior to exposure to cold ischemia protects the tissue and allows for extended allograft cold storage. Currently, allografts are routinely stored cold for 24 hours only in cold storage. Extended periods of cold storage result in a high rate of functional delay, often requiring dialysis support of the patient until the kidneys can recover function. Adapting intact organs to cell-free perfusion prior to exposure to cold ischemia can protect cells from cold injury.
[0119]
Accordingly, the present invention is also a method of storing a tissue, graft or organ for transplantation, wherein the tissue, graft or organ is treated with a buffered physiological solution without blood to remove blood and blood products. Flushing, in a warm storage system such as those described herein, capable of maintaining the tissue, graft or organ at a near normal metabolic rate for a time sufficient to inhibit damage to the tissue, graft or organ A method comprising perfusing a tissue, graft or organ, and storing the organ at 4-8 ° C. The time sufficient to suppress injury is the time during which the removed organ has had the opportunity to normalize perfusion parameters, such as flow velocity and vascular resistance. Generally, a perfusion period of 30 minutes to 24 hours is sufficient.
[0120]
This is particularly suitable for organ transport, as the organ chamber of the present invention restrains the organ from moving within the container. Organs can be prepared for storage as described above. The organ is then transported by simply removing the organ chamber from the warm storage system and refrigerated or packaged for shipping. If necessary, upon arrival at the implantation site, the organ chamber containing the organ can be re-secured to a compatible warm perfusion system, and the organ can be warm perfused before being transplanted into the recipient. In addition, the duration of warm perfusion can be used to monitor the functional properties of the organ prior to transplant.
[0121]
In embodiments where components of the warm preservation system are incorporated into the organ chamber, perfusion of the organ can be continued during transport using a suitable power source.
[0122]
A control group of dog kidneys (n = 3) is excised, immediately cold-washed with ViaSpan ™, then double bagged, statically packaged in ice and stored for 48 hours. Test group kidneys were placed in EMS perfusion after resection and before cold storage. In this group (n = 2), resected kidneys were flushed with EMS perfusion solution and placed in EMS perfusion at 32 ° -34 ° C. for 6 hours. After the EMS perfusion period, the kidneys were again rinsed in double bags at 4 ° C. with about 200 cc of ViaSpan ™, statically packaged and stored in ice for 48 hours.
[0123]
Immediately before re-implanting the refrigerated kidney, the contralateral kidney was removed. Test and control kidneys were then autografted using end-to-side anastomoses made between the renal arteries and the aorta and between the renal arteries and the vena cava. After implantation, the dog was sutured and allowed to recover. Every morning, blood was collected from the dog's forepaws and a chemical test was performed to determine the clinical course.
[0124]
result
EMS perfusion before refrigeration resulted in better results, with the time to normal function being restored after 48 hours of static refrigeration being halved. The kidneys of the control group were slowly reperfused, but no urine was produced on the table. Very little urine was produced during the first night after transplantation. Dogs in the control group experienced a period of reversible acute tubular necrosis (ATN). Serum creatinine levels 24 hours after transplantation were all above normal, 5.3, 2.6, and 2.8 mg / dL, respectively. Serum creatinine levels continued to rise, peaked, and then declined gradually, normalizing on days 7, 9, and 12, respectively.
[0125]
In the test group, the kidney produced urine on the operating table within minutes of reperfusion. These kidneys continued to produce urine during the post-transplant period. Serum chemistry increased 24 hours after transplantation, with serum creatinine values of 2.2 and 2.5 mg / dL. In contrast to control dogs, elevated serum chemistry peaked 2 days after transplantation. Serum creatinine levels normalized on days 5 and 6, respectively. Immediately after euthanasia, the kidney was macroscopically normal. Histological studies also revealed normal renal pathology.
[0126]
EMS perfusion before reimplantation thus provides protection against cold ischemia-induced damage in that the warm perfused test kidney experiences a milder and shorter duration ATN. The kidneys of the EMS-perfused test group also had lower serum creatinine levels 24 hours after transplantation when compared to the kidneys of the control group that did not receive EMS perfusion. The EMS-perfused kidney also immediately normalized serum creatinine levels earlier (5.5 vs. 9.3 days) than the refrigerated control group kidneys.
[0127]
Use of EMS as a targeted drug delivery system
EMS perfusion can be used to deliver drugs in a targeted manner to organs, anatomical resections and / or tissues. In addition, any procedure can monitor the ongoing metabolism and function supported by EMS and provide a mechanism to quantify the effects of the drug and any corresponding cellular responses. For example, heme oxygenase-1, a compound known to elicit high-level regulation of heat shock proteins, was administered to isolated kidneys that had been perfused according to the techniques of the present invention. The EMS technique adequately supported renal metabolism and function during ex vivo perfusion and increased heme oxygenase-1 (HO-1) enzyme expression within 6 hours.
[0128]
Dogs from the test group (n = 3) were enucleated, cannulated, flushed with EMS perfusion solution, and cobalt protoporphyrin (CoPP) was added to the perfusate at either 5, 25 or 50 uM. Placed in EMS perfusion at 32 ° C. to 34 ° C. for 6 hours. Metabolism and function assessment consisted of quantification of ex vivo renal function during the HO-1 induction period. Using EMS technology, the kidney was maintained metabolically active by perfusion at near physiological temperature. Kidneys weighing about 70-90 gm were obtained after brain death and the concomitant only 15 minutes of warm ischemia. The kidneys were then transferred to EMS perfusion at 32 ° C to 34 ° C. The kidneys were evaluated for oxidative metabolism, vascular kinetics and organ function. Organ parameters were allowed to stabilize for 1 hour before evaluation of function. Samples from the arterial and venous systems were collected along with urine produced during perfusion. PO for arterial and venous samples_TwoRadiometer ABL5 was used for the measurement.
[0129]
Oxygen consumption is
ml / min / gm = (PO_TwoArtery-PO_TwoVein) x (flow rate)
Calculated as
Vascular resistance,
Vascular resistance = mean arterial pressure / mean flow velocity
Calculated as
[0130]
(Outside 3)

Creatinine was added to the perfusate as a tracer. Urine made during EMS perfusion at 32 ° C. to 34 ° C. was collected and tested. Creatinine concentration was measured on an IDEXX Vetlab chemical analyzer and the glomerular filtration rate (GFR) was measured.
GFR = (creatinine in urine) × (urine flow rate) / (creatinine in perfusate) × (time)
Calculated as
[0131]
The ex vivo oxidative metabolism of these kidneys was comparable to all three CoPP concentrations and remained stable during 6 hours of EMS perfusion. The restored oxidative metabolism ranged from 0.17 to 0.23 cc / min / gm as measured by oxygen consumption. However, this oxygen consumption was highest at all time points in kidneys perfused with 25 μM CoPP. Perfusion pressure, vascular flow rate and vascular resistance were also comparable in the three CoPP concentration groups. Thus, treatment of CoPP during EMS perfusion did not adversely affect vascular kinetics or cell oxidative metabolism. However, with increasing CoPP-induced concentrations, differences in urine flow, creatinine clearance, and the development of proteinuria were observed.
[0132]
Effect of CoPP concentration on organ function
Urine flow was suppressed as CoPP concentration increased. Urinary flow decreased by 66% when CoPP concentration was increased from 5 to 25 μM, and by more than 90% when CoPP was increased to 50 μM. The glomerular filtration rate (GFR) was correspondingly low because the calculation of GFR was dependent on both urine flow and urinary creatinine concentration. Increasing CoPP concentration from 5 μM to 25 μM reduced GFR by 71%. Similarly, a CoPP concentration of 50 μM resulted in 91% inhibition of GFR. Increased levels of CoPP have also been associated with the development of "leaky endothelium" as shown by proteinuria.
[0133]
(Outside 4)

(Outside 5)

EMS perfusion effectively supported ongoing metabolism and functioning enough for cells to respond to CoPP and increase heme oxygenase-1 expression. Furthermore, during EMS perfusion, administration of CoPP did not adversely affect perfusion properties or cell metabolism. However, organ function was found to be affected in a dose dependent manner as CoPP concentration increased, in terms of decreased urine flow, decreased glomerular filtration rate, and the development of proteinuria. Thus, drug-induced toxicity can be detected during administration through EMS perfusion. At therapeutic doses, EMS perfusion was effective in supporting the induction of HO-1 expression by CoPP.
[0134]
Use of EMS technology in gene therapy
A relevant use of EMS technology is as a targeted gene delivery system. The long-term benefits of gene therapy have been hampered by the problem of delivering the desired gene to a particular location. Organ culture technology provides one mechanism for delivering genes to a desired location. Gene therapy is particularly attractive for transplanted organs because exogenous genes can be introduced prior to transplantation. If the medicament is a gene therapy vector, the construct may functionally encode an endogenous or exogenous protein, which can then be expressed in the target after transplantation. Such gene transfer allows the expression of various proteins by the target tissue.
[0135]
The perfusion system is particularly suitable for gene transfer to a number of organs and drug administration, as long as the target organ has a suitable blood circulation system, for example, kidney, liver, heart and the like. The most obvious advantages of gene delivery via a perfusion system are increased efficacy, specificity of the target for gene transfer, and the use of very little vector material. In addition, extracorporeal perfusion systems reduce the risk of administering large amounts of exogenous genetic material to the subject's systemic circulation. This is particularly important for immunocompetent individuals.
[0136]
Recent attempts to deliver gene products to intact kidneys using hypothermic perfusion have resulted in reduced transfection efficiency and gene expression in target cells (Zeigler ST, Kirby JD, Curiel DT, Dieselm AG, Thompson JA: Transpl 61: 812, 1996). More importantly, this gene transfer was found to be localized to proximal tubular epithelial cells. Changes in permeability along the vessel wall are known to be mediated by various factors, including hypothermia. Altered permeability is the impairment of the barrier function that particles or particle size that are repelled at the vessel wall, usually by electrostatic charge, increase permeability to the substrate.
[0137]
In addition to the present contribution to long-term organ preservation, the EMS technology of the present invention can be used to achieve targeted gene transfer, either in vitro or in a normal location. The EMS maintains the transfected organ or tissue metabolically active before, during and after the transfection period. The ability to work directly with targeted tissue under near physiological conditions for extended periods of time without contacting non-targeted tissues is a potential alternative to transfection, including the elimination of the need for viral vectors. Provide an opportunity to use the procedure. For example, to facilitate higher integration rates during EMS organ culture perfusion, liposome complexes or viral vectors and vascular endothelial cell-specific promoters can be used to introduce therapeutic DNA at near physiological temperatures. Can be introduced into the perfusion system.
[0138]
Another advantage of preserving metabolism during EMS is that establishing the functional state of an organ or tissue prior to transfection, as opposed to using hypothermia to suppress metabolism and alter cell membrane potential. Feasibility. This is particularly important if the organ was obtained from a donor other than a heartbeating donor and may have suffered any ischemic injury. In addition, organ metabolism can be monitored during transfection, and in the case of intact organs, the ultimate organ function, ie, diuresis, etc., can be quantified. Since the cell membrane is in normal fluid state, normal barrier function is maintained and it has the ability to target the therapeutic gene (s) directly to vascular cells.
[0139]
The ability of EMS perfusion to preserve vascular wall integrity and normal barrier function in the kidney provides a mechanism to achieve vascular cell-specific transfection. The ability to target the therapeutic gene (s) to vascular cells in an organ offers several important advantages. For example, the reporter gene product is in direct contact with the bloodstream, thereby providing an effective gene therapy delivery system. Because vascular endothelium provides an immune interface within an organ, gene therapy offers the opportunity for organ-specific immunomodulatory therapies in both transplant and autoimmune diseases.
[0140]
A further advantage is that EMS is cell-free and that EMS provides an environment for optimal transfection of the therapeutic gene (s) into the nucleus without eliciting an early immune response. It is. Thus, the EMS represents a highly efficient delivery vehicle for targeted gene delivery when applied in a normal location or ex vivo, and allows for simultaneous monitoring of metabolism and function.
[0141]
According to the methods of the present invention, the gene of interest may be delivered to the target tissue via a currently known vehicle host. Gene therapy by homologous recombination between exogenous DNA and the genome of the target tissue results in the modification of specific loci within the genome. Some methods of transfection include delivery by viral vectors containing genes, liposomes, plasmid DNA, and the like. Furthermore, delivery of episomal DNA by the method of the present invention is feasible, avoiding the need for genomic integration of the delivered gene due to episomal repetition of the target gene vector independent of the genome.
[0142]
In practicing the target delivery method of the present invention for an organ or tissue, the organ containing the target tissue is removed from the living body, the blood is washed away with a warm EMS solution, and placed on EMS perfusion. The perfusion is performed for a time sufficient to establish a baseline of function. An exogenous molecule, such as a viral vector, is injected into the perfusion and the perfusion is continued for a time sufficient to deliver a pharmaceutically effective amount of the exogenous molecule. The function of the organ is monitored for a period of time after delivery, before returning the organ to the receptor.
Embodiment 12
[0143]
An replication-defective adenovirus encoding the green fluorescent protein (GFP) gene (Adeno CMV5-GFP) is used to create an EMS perfusion system that supports gene transfer into intact bovine kidney during ex vivo perfusion at 34 ° C. The efficacy was evaluated. Bovine kidneys were flushed with warm EMS solution and placed in EMS perfusion. This perfusion was performed for about 60 minutes to establish a baseline of function. Viral vector (1 × 10⁹(PFU) was injected into the system and perfusion continued for 2 hours. Administration of the viral vector and the resulting infectivity during EMS perfusion did not adversely affect organ metabolism or function.
[0144]
The control kidney consisted of a kidney washed and perfused at 4 ° C. with a cold perfusate based on Viaspan. Because metabolism and function were inhibited at 4 ° C., no effect of the viral vector on control kidney metabolism or function could be determined.
[0145]
Following gene transfer, these kidneys were flushed through the renal artery to remove any residue that circulated the virus particles. The lumen of the vasculature was then filled with collagenase solution to isolate vascular cells in each kidney and these cells were morphologically confirmed. The digested and isolated vascular endothelial cells are washed out of the kidney, they are washed and 1 × 10⁶/ Ml in the primary culture. The expression of the transfected gene encoding green fluorescent protein is described in Table 7.
[0146]
The EMS perfusion resulted in an increased infection rate, with cultures expressing GFP infecting 40% of the vascular cells by 18 hours, compared to 17% in cold-perfused control kidneys. At 48 hours in tissue culture, endothelial cells isolated from EMS-perfused kidneys showed strong expression of GFP in 60% of the cells. In contrast, no elevated expression levels were detected in control kidneys where transfection was performed cold.
[0147]
After isolating vascular endothelial cells by collagenase digestion, parenchymal cells were separated from EMS-perfused kidneys. The separated parenchymal cells were found to be negative for GFP expression at 24 and 48 hours in tissue culture. These results suggest that during EMS perfusion, the infectivity and transduction of the gene encoding GFP was limited to vascular cells inside the blood vessels of the kidney. Thus, better transfection rates and higher reporter gene expression were observed at both time points in test kidneys perfused with EMS, and evidence of transfection was found only in vascular cells.
[0148]
[Table 6]

[0149]
[Table 7]

The EMS warm preservation technique of the present invention preserves organs without damaging them and immediately restores normal function upon organ retransplantation (after ex vivo organ perfusion) or reperfusion (after normal place perfusion). Supports metabolism at a level sufficient to obtain. For these reasons, EMS perfusion is particularly well-suited for delivery of therapeutics, for example, in gene therapy where delivery of nucleotides is desired. Further, the EMS warm storage technology of the present invention provides a gene delivery / transfection system that is superior to current methodologies.
[0150]
For example, U.S. Pat. No. 5,871,464 to Trigvason et al. Discloses a perfusion apparatus and method for drug delivery, and describes successful gene transfection both in situ and ex vivo. However, this result suggests that substantial diuresis (800 cc in the first hour of perfusion), which has been documented by excessive diuresis, has occurred. Also, diuresis has increased dramatically to over 1,200 cc / hr over 2 hours. This is another indication that the kidney was damaged during perfusion. In addition, perfusion at normal sites was less than 2 hours, at which time no urine production was described, and in the case of ex vivo perfusion, the kidney was not reimplanted. Thus, renal function after treatment is not described.
[0151]
Perfusion of the isolated kidney with Krebs-Ringer solution as described by Trigvason proved to result in a non-functional injured organ upon reimplantation. One skilled in the art will recognize that the isolated kidney can only survive for a few hours if it is perfused at near physiological temperatures.
[0152]
Improvements in EMS perfusion over previously reported studies may indicate that organs are maintained at near physiological temperature, are not damaged, organ metabolism is supported, and ex vivo perfusion re-implantation or normal sites Normal function is obtained immediately after reperfusion after perfusion with. In addition, EMS perfusion can be performed for several days, not only allowing efficient gene transfection without damage, but EMS can support organs long enough to fully express gene products . The ability to sustain the organ for several days with near physiological metabolism indicates the unique ability to synthesize the protein product encoded by the transfected gene prior to reimplantation or reperfusion, which is not possible with existing technologies. It was possible.
[0153]
The following example demonstrates the superior ability of EMS technology to preserve organ integrity during gene delivery and transfection. The paired kidneys were removed from the calves. Blood was washed from the excised kidney and one of the pairs was placed on EMS perfusion and the other was placed on perfusion with the system and solution described by Trigvason. During perfusion, oxidative metabolism is consumed by gram per minute O_TwoOxygen consumption was calculated and evaluated by diuresis and corresponding organ function. Organ function was measured as the ability of the glomerulus to retain its normal barrier function and retain large molecules inside the blood vessels. In the case of EMS perfusion, organ function is colloid, the ability to produce urine without protein leakage of serum albumin, and the ability of Trigvason to retain red blood cells in the lumen of blood vessels because it does not contain colloids, i.e., urine negative for red blood cells. The ability to produce.
[0154]
(Outside 6)

Perfusion based on the method described in U.S. Pat. No. 5,871,464, i.e., using a Krebs-Ringer solution supplemented with red blood cells to obtain a 17% hematocrit on a Gambro apparatus with an in-line oxygenator. Perfusion results in edema of the organ, increased perfusion pressure as measured by a manometer, low oxygen consumption, and reduced barrier function to produce urine containing hematocrit equivalent to perfusate. Histology showed further data supporting the progression of organ damage. In contrast, EMS perfusion prevents edema, maintains stable perfusion pressure and oxygen consumption, and results in protein-filtered urine production. Histology confirmed that kidney integrity during EMS perfusion was preserved.
[0155]
(Outside 7)

Long-term storage
Using EMS technology, organs can be stored ex vivo for extended periods of time in a metabolically active state and at near physiological temperatures. Using EMS technology, 10 dog kidneys were stored ex vivo for 24, 48 and 72 hours. During ex vivo perfusion, perfusion pressure, vascular flow rate, vascular resistance, oxygen consumption, glucose consumption, and diuresis remained stable. In addition, these kidneys appeared normal on histological evaluation. Proper metabolic support was achieved by changing the EMS perfusion solution at various times during the perfusion period. Three of these organs were reimplanted to determine their subsequent viability and function.
[0156]
(Outside 8)

Three of the kidneys perfused for more than 24 hours with the EMS technique were reimplanted. All three kidneys showed good swelling immediately upon reperfusion, producing urine within minutes of reperfusion. All three kidneys were functioning immediately and the bladder was full by the end time. All three dogs survived only on long-term EMS-perfused kidneys and showed normal serum chemistry and urine analysis.
[0157]
Use of an EMS system to prepare organs for transport
In addition to organ maintenance and repair, the present invention provides methods that aid in organ preservation and organ preparation for transport to the transplant site. Methods for storing tissues, grafts and organs for transplantation are described above. Similarly, the organ chamber and warm storage system of the present invention are advantageous for preparing organs for transport to an implantation site. Basically, the organ is fixed in the organ chamber and perfused with a warm storage system to repair the organ damage and protect it from further damage that may result from refrigeration or shipping. The method comprises the steps of (a) washing the tissue, graft or organ with a buffered physiological solution that does not contain blood to remove blood and blood products, and (b) replacing the tissue, graft or organ with the organ, tissue or organ. Perfusion in an organ chamber having a support capable of supporting the graft and preventing movement of the organ, tissue or tissue piece during transport in the organ chamber, wherein the organ chamber is a tissue, graft or organ A step that is part of a warm preservation system that can maintain the tissue, graft or organ at a near normal metabolic rate for a period sufficient to protect the tissue, graft or organ with a cold perfusion solution, e.g. Perfusing at a temperature in the range of 4-8 ° C., (d) removing the entire organ chamber, including the organ, from the warm storage system and cold packaging the organ chamber, including the organ, and (e) transferring the organ to the transplant site. Transporting the containing organ chamber.
[Brief description of the drawings]
[0158]
FIG. 1 shows an embodiment of a blood-free metabolic support system with a closed loop perfusion subsystem that circulates perfusate and a dialysis subsystem that reprocesses it.
FIG. 2 shows an embodiment with a perfusion exchange subsystem and two perfusion pathways suitable for liver preservation.
FIG. 3 is a perspective view of one embodiment of the organ chamber of the present invention.
FIG. 4 is a perspective view of one embodiment of the organ chamber of the present invention, wherein the organ quality further comprises a reservoir.
FIG. 5 is a cross-sectional view of one embodiment of the organ chamber of the present invention.
FIG. 6 is a cross-sectional view of a vascular support that supports the organ's blood vessels and contacts the conduits that direct perfusate from the organ to the reservoir to securely hold the blood vessels.
FIG. 7 is a perspective view of a vascular support.

Claims (40)

An organ chamber (32) for use in a system for storing an organ (40), said chamber comprising a container (33);
At least one resilient support (36) that can be disposed within the container (33) for supporting the organ (40) within the container (33);
To prevent the organ (40) from moving within the container (33) when the organ (40) is placed on a resilient support, the support (36) is placed on the outer surface of the organ (40). An organ chamber that fits the part. The organ chamber (32) of claim 1, wherein the support (36) comprises a fluid-filled bag. The organ chamber (32) of claim 1, wherein the support (36) comprises a bag filled with gel. The organ chamber (32) according to claim 1, wherein the support (36) comprises a bag filled with gas. An organ chamber (32) according to claim 1, wherein the support (36) is adapted to the kidney of the organ (40). The organ chamber (32) according to claim 1, wherein the support (36) is adapted to the heart of the organ (40). The organ chamber (32) according to claim 1, wherein the support (36) is adapted to the liver of the organ (40). The organ chamber (32) according to claim 1, wherein the support (36) is adapted to the pancreas of the organ (40). A conduit (43) connectable to the organ (40) for receiving the venous outflow of the preservation solution from the organ (40) and preventing the outflow from contacting the external surface of the organ (40); Organ chamber (32) according to claim 1. A conduit (43) connectable to the organ (40) for receiving the venous outflow of the preservation solution from the organ (40) and preventing the outflow from contacting the internal surface of the organ chamber (32); An organ chamber (32) according to claim 1, comprising: The organ chamber (32) of claim 10, further comprising a reservoir (38) for receiving venous outflow from the conduit (43). The organ chamber (32) according to claim 1, further comprising a conduit (47) connectable to the organ (40) for receiving the product of the organ. The organ chamber (32) according to claim 1, wherein the chamber (32) is connectable to a warm storage system. The organ chamber (32) of claim 1, further comprising at least one of a storage tank (38), a heat exchanger (14), an oxygenator (20), and a pump (12). The organ chamber (32) of claim 1, further comprising at least one sensor for monitoring at least one parameter of the circulating perfusion solution. The parameter is the flow rate, pH, PaO _2, PaCO _2, the temperature are those selected from the vascular pressure, and metabolic indicators, claim 15 organ chamber described (32). The metabolic indicator is oxygen consumption, glucose consumption, at least one of the consumption of citric acid circuit components, and CO ₂ is selected from the production, according to claim 16 organ chamber described (32). An organ chamber (32) used in a system for storing an organ (40),
Container (33),
A support (36), mountable within the container (33), for supporting the organ (40) within the container (33), adapted to prevent movement of the organ (40) within the container (33). Said support (36), and
A conduit (43) for delivering venous outflow to prevent contact with the external surface of the organ (40);
Organ chamber containing. 19. The organ chamber (32) of claim 18, further comprising a perfusion conduit (42) for delivering a warm preservation solution to the organ (40). 19. The organ chamber (32) according to claim 18, further comprising a conduit (47) for separately collecting organ products from the storage solution. 19. The organ chamber (32) of claim 18, further comprising a connector for removably connecting the organ chamber to a warm storage system. The organ chamber (32) according to claim 18, further comprising at least one of a storage tank (38), a heat exchanger (14), an oxygenator (20), and a pump (12). 20. The organ chamber (32) of claim 19, further comprising at least one sensor for monitoring at least one of the parameters of the storage solution. The parameter is the flow rate, pH, PaO _2, PaCO _2, temperature, vessel pressure, and is selected from metabolic indicator, claim 23 organ chamber described (32). The metabolic indicator is oxygen consumption, glucose consumption, at least one of the consumption of citric acid circuit component, and is selected from CO ₂ production, according to claim 24 organ chamber described (32). 19. The organ chamber (32) of claim 18, wherein the support (36) comprises a rigid material including a cavity shaped to accommodate the contours of an adult-sized kidney. 19. The organ chamber (32) of claim 18, wherein the support (36) comprises a rigid material including a cavity shaped to accommodate the contour of a child-sized kidney. 20. The organ chamber (32) according to claim 19, wherein the container (33) further comprises a reservoir (38) for receiving a perfusion solution circulated in the organ (40). A method for preserving an organ (40),
Placing the organ on a support (36) adapted to prevent movement of the organ (40);
A method comprising perfusing an organ (40) with a warm storage solution. 30. The method of claim 29, further comprising monitoring a functional property of the organ (40). A method of preserving an organ, comprising the steps of perfusing an organ with a warm preservation solution, and directing venous outflow of the preservation solution away from the organ so as to prevent the preservation solution from contacting the external surfaces of the organ. A method of transporting an organ (40),
Perfusing the organ (40) in the organ chamber (32) of a warm preservation system capable of maintaining the organ (40) at a substantially normal metabolic rate for a time sufficient to inhibit damage to the organ (40);
Removing the organ chamber (32) from the storage system;
Cooling the organ (40); and
A method comprising the step of transporting an organ (40). The organ chamber (32)
Container (33),
A support (36) mountable within the container (33) for supporting the organ (40) within the container (33), the support (36) being adapted to prevent movement of the organ (40) within the container (33). Support (36), and
Conduit (43) for delivering venous outflow and preventing the outflow from contacting the external surface of organ (40)
33. The method of claim 32, comprising: 33. The method of claim 32, further comprising relocating the organ chamber (32) containing the organ (40) to the warm storage system, and perfusing the organ (40). A storage solution for use in a warm storage system, comprising an oxygen carrier selected from cross-linked polyhemoglobin, complex hemoglobin, recombinant hemoglobin and a perfluoro compound. 36. The storage solution according to claim 35, wherein said complex hemoglobin is polyethylene glycol complex hemoglobin. 37. The storage solution of claim 36, wherein said polyethylene glycol modified hemoglobin is human hemoglobin or non-human hemoglobin. 38. The storage solution of claim 37, wherein said non-human hemoglobin is bovine hemoglobin. 37. The warm preservation solution of claim 36, wherein said non-human hemoglobin is equine hemoglobin. The warm storage solution according to claim 35, wherein the polyethylene glycol-modified hemoglobin is a recombinant hemoglobin.

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