JP4753119B2 - 大量処理の生体外エレクトロポレーション法 - Google Patents
大量処理の生体外エレクトロポレーション法 Download PDFInfo
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Description
本明細書は、同時継続で2003年3月14日出願のウォルターズ及びキングの米国特許出願第60/454,360号、「大量処理の生体外エレクトロポレーション法」に基づく優先権を主張するものである。
式1:
式中、σは伝導度(ジーメンス/cm)、ギャップの単位はcm、電極面積の単位はcm2
更に、
式2:
及び、
式3:
1)全ての細胞は同じ電界強度及び電界方向に露出されないこと、
2)挿入する物質の密度、及び細胞密度が一定である保証がないこと、
3)均一なパルス電圧のみを印加できることである。
米国特許第6,010,613号に開示されるような様々な矩形パルス波形は使用できない。
であり、大きな処理能力は細胞の単層のため困難である。臨床用途は記載されていない。
上記に鑑みて、本発明の目的は、臨床目的及び治療目的に使用することができる大量処理の生体外エレクトロポレーション法を提供することであり、この方法では、生体外又は試験管内の全ての細胞がほぼ同一処理条件にかけられる。
a)小胞と外来物質との懸濁液を、電極を備えるチャンバ中の処理体積中に保持し、このチャンバは電極ギャップの2乗(cm2)をチャンバ体積(cm3)で割った商によって定義される形態係数を有し、この形態係数は0.1(cm−1)以下であり、この小胞と外来物質との懸濁液は培養液が0.01ミリジーメンスから1.0ミリジーメンスにわたる範囲の伝導度を有するように調節された培養液中にあり、この懸濁液は処理の際にチャンバ中に閉じ込められる工程(ステップ)と、
b)このチャンバ中に閉じ込められた懸濁液を1つ又は複数のパルス電界で処理する工程とを含み、
この懸濁液の処理体積は上記のa)及びb)に従って拡大縮小可能であり、また、この小胞のチャンバ中での処理時間は実質的に一定である、方法が提供される。
先に述べた重要な問題は、エレクトロポレーション用の培養液の伝導度である。この方法では、低伝導度の培養液を使用してこの培養液の全抵抗を小さく保ち、また、実質的に加熱をなくす。いかなる培養液伝導度でも使用できるわけではない。培養液のイオン含有量が減少するにつれて、細胞膜を横切って電荷(電圧)を構築するのに役立つ自由イオンの数が減少する。この効果は膜を充電するのに必要となる時間量を増やすことである。この方法は、エベルハードノイマン(Eberhard Neumann)、アーサーソワーズ(Arthur Sowers)、及びキャロルジョーダン(Carol Jordan)によって編集された、細胞生化学におけるエレクトロポレーション及び電気融合(Electroporation and Electrofusion in Cell Biology)、プレナムプレス(Plenum Press)、1989年の71頁の式によって記述される。10ミクロンの典型的な細胞直径を仮定すると、80μS/cmで充電時間は20マイクロ秒である。80μS/cmより下では、充電時間が長くなり過ぎ、また、細胞膜中の通路の形成が止まる。下の表6に培養液の抵抗を電極チャンバ体積及び伝導度の関数として示す。
参照文献: 細胞生化学におけるエレクトロポレーション及び電気融合(Electroporation and Electrofusion in Cell Biology)、エベルハードノイマン(Eberhard Neumann)、アーサーソワーズ(Arthur Sowers)、及びキャロルジョーダン(Carol Jordan)により編集、プレナムプレス(Plenum Press)、1989年
細胞直径 > 1ミリメートル
チャンバ体積 > 1ミリリットル
処理すべき材料の伝導度 > 1マイクロジーメンス/cm
処理すべき材料のチャンバ中の全抵抗 > 1オーム
チャンバの形態係数 < 0.1cm−1
である。
ソルビトール 280ミリモル
カルシウムアセテート 0.1ミリモル
マグネシウムアセテート 0.5ミリモル
Claims (41)
- 小胞内に外来物質を挿入するための外来物質で小胞を処理する方法において、
a)前記小胞と前記外来物質との懸濁液を、電極を含む少なくとも2ミリリットルのチャンバ体積を有するチャンバ中の処理体積中に保持する工程であって、
前記チャンバは電極ギャップの2乗(cm2)をチャンバ体積(cm3)で割った商によって定義される形態係数(cm−1)を有し、
前記形態係数は0.1(cm−1)以下であり、
前記小胞と前記外来物質との前記懸濁液は、0.01ミリジーメンスから1.0ミリジーメンスにわたる範囲の伝導度を有するように調節された培養液中にあり、
前記懸濁液は処理の際に前記チャンバ中に閉じ込められる、工程と、
b)前記チャンバ中に閉じ込められた前記懸濁液を1つ又は複数のパルス電界で処理する工程とを含み、
前記工程a)及びb)に従って、前記懸濁液の前記処理体積は拡大縮小可能であり、
前記チャンバ中の前記小胞の処理時間は実質的に均一である、外来物質で小胞を処理する方法。 - 前記チャンバは閉鎖チャンバである、請求項1に記載の方法。
- 前記チャンバは少なくとも2ミリリットルの処理能力を有する、請求項1に記載の方法。
- 前記チャンバ及びその含有物は無菌である、請求項1に記載の方法。
- 前記チャンバは前記懸濁液の導入及び排出用の入口及び出口を備える、請求項1に記載の方法。
- 前記電極は平行板電極である、請求項1に記載の方法。
- 前記電界は前記処理体積全体にわたって実質的に均一である、請求項1に記載の方法。
- 前記電界は、前記処理体積全体にわたって実質的に均一な、100ボルト/cmより大きく5,000ボルト/cmより小さな均一パルス電界を、前記平行板電極間に発生させるような矩形電圧パルス波形を含む、請求項1に記載の方法。
- 前記小胞は生体細胞であり、
前記培養液は生理的培養液であり、且つ50μS/cmと500μS/cmとの間の伝導度を有する、請求項1に記載の方法。 - 前記処理される生体細胞の数は少なくとも1000万個である、請求項1に記載の方法。
- 前記処理される生体細胞の数は少なくとも2000万個である、請求項1に記載の方法。
- 前記小胞は、外来物質での処理の後でドナーに戻すべき自己細胞である、請求項1に記載の方法。
- 前記小胞はドナー以外の受容菌に供与すべき同系細胞である、請求項1に記載の方法。
- 前記小胞は異系細胞である、請求項1に記載の方法。
- 前記小胞は人工リポソームである、請求項1に記載の方法。
- 前記パルス電界は、前記物質に対して100V/cm以上の電界強度を有する少なくとも3つの一連の非正弦波の電気パルスである電気パルスからのパルス電界であって、前記少なくとも3つの一連の非正弦波の電気パルスは、以下の特徴:(1)前記少なくとも3つのパルスのうち少なくとも2つはパルス振幅が互いに異なる;(2)前記少なくとも3つのパルスのうち少なくとも2つはパルス幅が異なる;(3)前記少なくとも3つのパルスのうちの第1の組の2つの第1パルス間隔は、前記少なくとも3つのパルスのうちの第2の組の2つの第2パルス間隔と異なる;のうちの1つ、2つ、又は3つを有する、請求項1に記載の方法。
- 処理中の温度上昇は極めて少ない、請求項1に記載の方法。
- 2ミリリットルから10ミリリットルにわたる範囲で拡大縮小可能な、請求項1に記載の方法。
- 連続的なバッチで実施される、請求項1に記載の方法。
- 前記外来物質は治療物質である、請求項1に記載の方法。
- 治療生成物が前記小胞の前記外来物質での前記処理から形成される、請求項1に記載の方法。
- 前記外来物質はポリヌクレオチドである、請求項1に記載の方法。
- 前記外来物質はDNA及びRNAからなる群から選択される、請求項1に記載の方法。
- 前記外来物質はポリペプチドである、請求項1に記載の方法。
- 前記外来物質はタンパク質である、請求項1に記載の方法。
- 前記外来物質は有機化合物である、請求項1に記載の方法。
- 前記外来物質は少なくとも8つの塩基対を含む、請求項1に記載の方法。
- 前記チャンバはチャンバ体積を有し、前記懸濁液は懸濁液体積を有し、前記懸濁液体積は前記チャンバ体積より大きく、
前記懸濁液体積の初めの部分が、前記チャンバ中に移動され、保持され、前記チャンバ中で処理され、且つ前記チャンバから外へ移動され、また
前記懸濁液体積の追加の部分が、前記チャンバ中に移動され、保持され、前記チャンバ中で処理され、且つ前記チャンバから外へ移動される、請求項1に記載の方法。 - 前記懸濁液体積の更なる部分が、連続的に前記チャンバ中に移動され、保持され、前記チャンバ中で処理され、且つ前記チャンバから外へ移動される、請求項1に記載の方法。
- 前記懸濁液体積の更なる部分が、前記懸濁液体積が枯渇するまで連続的に前記チャンバ中に移動され、保持され、前記チャンバ中で処理され、且つ前記チャンバから外へ移動される、請求項1に記載の方法。
- 少なくとも2ミリリットルのチャンバ体積、前記チャンバ内に含まれる一対のエレクトロポレーション電極、及び電極ギャップの2乗(cm2)をチャンバ体積(cm3)で割った商によって定義される0.1(cm−1)以下の形態係数(cm−1)を有するチャンバと;
前記エレクトロポレーション電極間の前記チャンバ中に含まれる、懸濁液中に小胞を持つエレクトロポレーション培養液であって、50μS/cmと500μS/cmとの間の伝導度を有する培養液と;
前記エレクトロポレーション電極に電気的に接続されたパルス電圧源と;
内部でエレクトロポレーション処理するための、物質を前記チャンバに添加する手段、及び前記チャンバから処理済物質を排出する手段と;
を備える、エレクトロポレーション装置。 - 封止チャンバを提供するための、前記チャンバに接続された封止手段を更に備える、請求項31に記載の装置。
- 前記封止手段は大量のエラストマ材を含む、請求項32に記載の装置。
- 前記封止チャンバは前記チャンバ内部が無菌である、請求項32に記載の装置。
- 前記封止チャンバは、液体が前記チャンバ内に移動するとき空気を排気する排気手段を備える、請求項32に記載の装置。
- 前記排気手段は前記チャンバの壁面中にフィルタ部材を備える、請求項35に記載の装置。
- 前記排気手段は前記チャンバと流体連通した排気小室を備える、請求項35に記載の装置。
- 前記チャンバはチャンバ入口とチャンバ出口を備える、請求項31に記載の装置。
- 前記チャンバ内に導入する前に前記小胞担持エレクトロポレーション培養液を収容するための、前記チャンバ入口に流体連通した第1リザーバーと;
処理済み小胞担持培養液を前記チャンバから流し出すためにチャンバフラッシング材料を収容するための、前記チャンバ入口に流体連通した第2リザーバーと;
前記チャンバから流し出された処理済み小胞担持培養液を受け入れるための、前記チャンバ出口に流体連通した第3リザーバーと;
を更に備える、請求項31に記載の装置。 - 前記第1リザーバー、前記第2リザーバー、及び前記第3リザーバーは可撓性バッグからなっている、請求項39に記載の装置。
- 前記チャンバ入口と前記第1リザーバーと前記第2リザーバーとの間に接続された入口バルブと;
前記チャンバ出口と前記第3リザーバーとの間に接続された出口バルブと;
を更に備える、請求項39又は40に記載の装置。
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PCT/US2004/005237 WO2004083379A2 (en) | 2003-03-14 | 2004-03-15 | Large volume ex vivo electroporation method |
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US20010007153A1 (en) * | 1997-06-16 | 2001-07-05 | Jennifer June Brown | Solid chimeric organs, animal models having same, process for preparing same, non-tumorigenic immortalized human cell lines, susceptible cells and cytopathic mammalian viruses |
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EP1620537A4 (en) | 2007-07-04 |
WO2004083379A2 (en) | 2004-09-30 |
JP2006520254A (ja) | 2006-09-07 |
WO2004083379A3 (en) | 2006-03-02 |
EP1620537A2 (en) | 2006-02-01 |
US20060108229A1 (en) | 2006-05-25 |
CN1826408A (zh) | 2006-08-30 |
CN1826408B (zh) | 2011-01-26 |
EP1620537B1 (en) | 2012-10-24 |
US9982251B2 (en) | 2018-05-29 |
CA2519065C (en) | 2014-06-17 |
CA2519065A1 (en) | 2004-09-30 |
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