JP6523683B2 - 加工食用肉 - Google Patents
加工食用肉 Download PDFInfo
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- JP6523683B2 JP6523683B2 JP2014523009A JP2014523009A JP6523683B2 JP 6523683 B2 JP6523683 B2 JP 6523683B2 JP 2014523009 A JP2014523009 A JP 2014523009A JP 2014523009 A JP2014523009 A JP 2014523009A JP 6523683 B2 JP6523683 B2 JP 6523683B2
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Description
本出願は、2011年7月26日に出願された米国仮特許出願第61/511,948号の利益を主張するものであり、該仮特許は、その全体が参照により本明細書に組み込まれる。
従来の材料と方法を用いて作製される加工組織製品は、内部の細胞に栄養を与えるガスおよび栄養素の拡散可能距離が短いことから、サイズに限界がある。また、既存の手法は、加工製品を大量生産するための適切な速度と処理能力を提供できていない。その結果、肉製品の生産に使われる既存の組織工学方法では、訴求力のない薄いシートとペーストが、商業的に実現不能な規模でしか生産できない。
多くの自己接着細胞型を用いて、本明細書に記載の多細胞体、層、および加工肉を形成できる。いくつかの実施形態では、従来の肉製品に似るように加工肉製品を設計し、従来の肉製品に見られる細胞型に近似するように細胞型を選択する。さらなる実施形態では、加工肉製品、層、および多細胞体は、非ヒト筋細胞を含む。加工肉製品、層、および多細胞体は、非ヒト筋細胞、および/または内皮細胞、および/または脂肪細胞、および/または線維芽細胞を含む。
本明細書において、互い接着および/または密着している複数の非ヒト生細胞を含む多細胞体を開示する。また、本明細書において、互いに接着および/または密着している複数の生きた非ヒト筋細胞を含む複数の多細胞体を調製することと;支持基板上に隣接して複数の多細胞体を配置することと;この複数の多細胞体を融合させて、加工肉形成用の実質的に平坦な層を形成することと;を含む方法を開示する。いくつかの実施形態では、多細胞体は、互いに接着および/または密着している複数の細胞を含んでなり、この複数の細胞は、所望の三次元形状をなし、組織工学等の生体工学プロセス中の容易な操作および取扱いを可能にする粘弾性粘稠性および十分な完全性を有する。いくつかの実施形態では、十分な完全性とは、後続の取扱い中、多細胞体が、その物理形状(剛性ではなく、粘弾性粘稠性を有する形状)を保持し、かつ細胞の生命力を維持できることを意味する。
本明細書に開示される加工肉は、複数の層を含む。いくつかの実施形態では、1つの層は、互いに接着および/または密着している複数の非ヒト生細胞を含んでなる複数の多細胞体を含む。また、本明細書において、多細胞体を支持基板上に隣接して配置するステップと、多細胞体を融合させて、加工食用肉製品の形成用に実質的に平坦な層を形成するステップと、を含む方法を開示する。いくつかの実施形態では、本明細書に記載の手法を用いて各層をバイオプリントする。
いくつかの実施形態では、加工肉製品、加工層、および/または多細胞体は、1つ以上の栄養補助剤を含む。さらなる実施形態では、1つ以上の栄養補助剤は、ビタミン、ミネラル、繊維、脂肪酸、およびアミノ酸から選ばれる。いくつかの実施形態では、商業的訴求力(例えば外観、味覚、色、匂い等)を高めるため、加工肉製品、層、および/または多細胞体は、1つ以上の添加物を含む。さらなる実施形態では、加工肉製品、層、および/または多細胞体は、1つ以上の香味剤、1つ以上の着色剤、および/または1つ以上の着臭剤を含む。
本明細書において、加工食用肉の形成用に実質的に平坦な層を形成するための、支持基板上に隣接して配列される複数の多細胞層を、いくつかの実施形態で開示する。また、本明細書において、支持基板上に隣接して複数の多細胞体を並べて実質的に平坦な層を形成することと、単一の支持基板上に隣接して複数の層を配置することと、これらの層を融合させて加工肉を形成することと、を含む方法を、いくつかの実施形態で開示する。例えば、複数の異なる基板上に、上記のように複数の層を同時に形成し、多細胞体が十分に融合して除去可能になった時点で、それぞれの基板から層を除去し、複数の層を互いに積み重ねるか、または単一の基板上に重ねてよい。
本明細書に記載の特性を持つ多細胞体は、様々な方法で作製できる。いくつかの実施形態では、複数の生細胞を含有し、または所望の細胞密度と粘度を有する細胞ペーストから、多細胞体を製作することができる。さらなる実施形態では、細胞ペーストを所望の形状にして、成熟(例えばインキュベーション)を通じて多細胞体を形成できる。特定の実施形態では、複数の生細胞を含む細胞ペーストを所望の形状(例えば円柱、球形等)に成形することにより、多細胞体を作製する。さらなる実施形態では、細胞ペーストを制御環境内でインキュベートして、細胞同士を接着および/または密着させることにより、多細胞体を形成する。別の特定の実施形態では、複数の生細胞を含む細胞ペーストを、細胞ペーストを三次元形状に保持するデバイス内で成形することにより、多細胞体を作製する。さらなる実施形態では、細胞ペーストを三次元形状に保持しながら、十分な時間をかけて制御環境内で細胞ペーストをインキュベートして、本明細書に記載のように、平面上で自身を支えるのに十分な密着力を持つ細胞体を作製する。
支持基板上に多細胞体を配置して所望の三次元構造物(例えば実質的に平坦な層)を作製する方法として、いくつか適切な方法がある。例えば、いくつかの実施形態では、複数の多細胞体を、互いに接触するように手作業で配置するか、ピペット、ノズル、もしくは針から押し出すことにより所定位置に堆積させるか、またはバイオプリンター等の自動機械により接触するように位置付ける。
支持基板上に複数の層を並べて加工肉を作製する方法として、いくつか適切な方法がある。例えば、いくつかの実施形態では、層が互いに接触するように手作業で配置し、または、バイオプリンター等の自動のコンピューター援用機械によりコンピュータースクリプトに従って層を所定位置に堆積させる。さらなる実施形態では、実質的に平坦な層を積み重ねて加工肉を形成する。
本明細書において、加工肉製品がいくつかの実施形態で開示される。また、本明細書において、支持基板上に隣接して並べられて、加工肉の形成用に実質的に平坦な層を形成する複数の多細胞体も、種々の実施形態で開示される。
2%アガロース溶液を調製するため、2gのUltrapure Low Melting Point(LMP)アガロースを100mLの超純水/緩衝液(1:1、体積/体積)に溶かした。緩衝液は、随意にPBS(1×ダルベッコリン酸緩衝食塩水)またはHBSS(1×ハンクス平衡塩類溶液)のどちらかである。温水(80℃超)の入ったビーカーにアガロース溶液を入れ、アガロースが完全に溶けるまでホットプレート上に保持した。温度が36℃超であり限り、アガロース溶液は液体のままである。36℃を下回ると相転移が発生し、粘度が上昇して、最終的にアガロースがゲルを形成する。
単離した直後のブタ大動脈平滑筋細胞(PASMC)を、10%ウシ胎仔血清(ハイクローンラボラトリーズ、ユタ州)、10%ブタ血清(インビトロジェン)、L−アスコルビン酸、硫酸銅、HEPES、L−プロリン、L−アラニン、L−グリシン、およびペニシリンGを含有する低グルコースDMEM中で増殖させた(上記栄養補助剤はすべてミズーリ州セントルイスのシグマから購入した)。0.5%ゼラチン(ブタ皮膚ゼラチン;シグマ)を塗布した皿(テクノプラスチックプロダクツ、ミズーリ州セントルイス)の上で細胞株を培養し、5%CO2を含有する加湿雰囲気中において37℃で維持した。PASMCを最高で7代目まで継代培養した後、多細胞体の形成に用いた。
細胞培養物をリン酸緩衝食塩水(PBS、インビトロジェン)で2回洗浄し、0.1%トリプシン(インビトロジェン)で10分間処理し、1500RPMで5分間、遠心分離した。細胞を4mLの細胞型特異的培養液で再懸濁させ、10mLの組織培養フラスコ(ベルコグラス、ニュージャージー州バインランド)内で、37℃にて、5%CO2存在下で、旋回シェーカー(ニューブランズウィックサイエンティフィック、ニュージャージー州エジソン)上で1時間インキュベートして接着を回復させ、3500RPMで遠心分離した。得られたペレットを300μm径(サッターインスツルメント、カリフォルニア州)または500μm径(ドラモンドサイエンティフックカンパニー、ペンシルベニア州ブルーモール)のキャピラリーマイクロピペットに移し、37℃、5%CO2存在下で15分間インキュベートした。球形の多細胞体の場合、円柱状に押し出して均等断片にカットし、旋回シェーカー上で一晩かけて自然に丸めた。この手順により、マイクロピペットの直径に応じた、所定のサイズと細胞数を持つ一定の球形体が得られた。円柱形の多細胞体の場合、機械により円柱状に押し出し、バイオプリンターを用いて個別に調製された非粘着性のTeflon(登録商標)またはアガロースの型枠に流し込んだ。型枠内で一晩かけて成熟した細胞円柱体は、堆積できるほど十分に密着していた。
実施例3に記載の通りに、円柱形の多細胞体を調製する。この多細胞体は異種細胞性であり、実施例2のPASMCと、ブタ冠動脈内皮細胞(PCAEC、ジェンランティス、カリフォルニア州サンディエゴ、製品番号PP30005)で構成される。多細胞体中の内皮細胞に対する筋細胞の比率は、約6:1である。多細胞体の断面径は300μmであり、長さは2cm、3cm、4cm、または5cmである。多細胞体を成熟させ、複数のカートリッジ(内径300μmのマイクロピペット)に入れてパッケージ化し、このカートリッジをバイオプリンターに挿入する。
Claims (26)
- 加工肉製品であって、
ボリュームのある本体を含み;
本体は複数の20〜80層に積層された平面層を含み、層は少なくとも部分的に融合しており、各平面層は複数の少なくとも部分的に融合した、非ヒト筋細胞を含む多細胞体を含み;
本体はいかなる血管も支持基板も含まず、肉製品の中間領域内の平面層では、加工肉の外側の層より前に細胞死が起こり、さらに、加工肉製品は食用で摂食するためのものである、前記加工肉製品。 - 本体のボリュームが10cm3より大きい、請求項1に記載の加工肉製品。
- 本体が複数の40〜60層に積層された平面層を含む、請求項1に記載の加工肉製品。
- 各平面層は、複数の少なくとも部分的に融合した、非ヒト筋細胞と非ヒト内皮細胞を含む多細胞体を含む、請求項1に記載の加工肉製品。
- 本体の積層された平面層が完全に融合した、請求項1に記載の加工肉製品。
- 筋細胞が、哺乳類、鳥類、爬虫類、魚類、甲殻類、軟体動物または頭足類由来である、請求項1に記載の加工肉製品。
- 前記筋細胞が骨格筋細胞である、請求項1に記載の加工肉製品。
- 前記筋細胞が心筋細胞である、請求項1に記載の加工肉製品。
- 前記筋細胞が平滑筋細胞である、請求項1に記載の加工肉製品。
- 前記筋細胞が整列している、請求項1に記載の加工肉製品。
- 複数の50層より多くの層に積層された層を含むボリュームを含み、前記ボリュームはいかなる血管も支持基板も含まず、各層は複数の少なくとも部分的に融合した、非ヒト筋細胞を含む多細胞体を含み、各層における多細胞体は互いに隣接して配置され;
前記各層は、栄養素を拡散させて、培養中に前記非ヒト筋細胞の維持と増殖が充分行われるように調整した厚さを有し、
加工肉は食用で摂食するためものである、加工肉。 - ボリュームが10cm3より大きい、請求項11に記載の加工肉。
- 前記各層の厚さが、約100μm〜約1000μmである、請求項11に記載の加工肉。
- 食用加工肉製品の形成方法であって、
複数の多細胞体を平面に隣接して配置することにより複数の平面層を形成する工程、ここで、各多細胞体は複数の密着した非ヒト筋細胞を含み;
少なくとも各層内の複数の多細胞体が融合し始めるまで各平面層を培養する工程;
50層より多くの層の少なくとも部分的に融合した層を積層して、加工肉の層状ボリュームを形成する工程;および
少なくとも積層が融合し始めるまでボリュームを培養する工程を含み、
前記ボリュームはいかなる血管も支持基板も含まない、方法。 - 少なくとも細胞が互いに密着するまで複数の非ヒト筋細胞と非ヒト内皮細胞を培養することにより、複数の多細胞体を調製する工程をさらに含む、請求項14に記載の方法。
- 積層には、平面基板上の層の上に平面層を連続的に積層することが含まれ、培養後に加工肉成分を前記平面基板から分離することをさらに含む、請求項14に記載の方法。
- 加工肉ボリュームを冷凍する工程をさらに含む、請求項14に記載の方法。
- 積層には、約100層より多くの層を積層することが含まれる、請求項14に記載の方法。
- 加工肉の形成方法であって、
互いに密着した複数の非ヒト筋細胞を含む複数の多細胞体を調製する工程;
平面支持基板上に隣接して一つより多くの多細胞体を置く工程;
前記多細胞体を融合させて第一の層を形成する工程;
第一の層上に20〜80の追加の平面層を置く工程であって、前記各層は、栄養素を拡散させて、培養中に前記非ヒト筋細胞の維持と増殖が充分行われるように調整した厚さを有する工程;
前記積層された平面層を培養して、互いに部分的に融合させる一方でボリュームの内部領域の平面層が死滅して加工肉のボリュームを形成する工程;を含み、
前記ボリュームはいかなる血管も支持基板も含まず、
加工肉は食用である、方法。 - 前記加工肉ボリュームを冷凍する工程をさらに含む、請求項19に記載の方法。
- 複数の多細胞体を調製する工程には、互いに密着した複数の非ヒト筋細胞を含む複数の細長い多細胞体を調製する工程と、互いに密着した複数の非ヒト筋細胞を含む複数の実質的に球状の多細胞体を調製する工程が含まれ;さらに、一つより多くの多細胞体を置く工程には、平面支持基板上に隣接して、一つより多くの細長い多細胞体と一つより多くの実質的に球状の多細胞体を置く工程が含まれる、請求項19に記載の方法。
- 前記細長い多細胞体の長さは、約1mm〜約10cmの範囲である、請求項21に記載の方法。
- 複数の多細胞体を調製する工程には、互いに密着した複数の非ヒト筋細胞を含む複数の実質的に球状の多細胞体を調製する工程が含まれ;さらに一つより多くの多細胞体を置く工程には、支持基板上に隣接して、一つより多くの実質的に球状の多細胞体を置く工程が含まれる、請求項19に記載の方法。
- 平面支持基板は液体および栄養素を透過可能であり、細胞培地を多細胞体の表面全てに接触させる、請求項19に記載の方法。
- 前記多細胞体が、栄養素を拡散することで培養中に前記非ヒト筋細胞と非ヒト内皮細胞の維持と増殖が充分行われるように調整した直径を持つ、請求項19に記載の方法。
- 前記多細胞体の直径は、約100μm〜約500μmである、請求項19に記載の方法。
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JP2014523009A Active JP6523683B2 (ja) | 2011-07-26 | 2012-07-26 | 加工食用肉 |
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2016
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JP2014521336A (ja) | 2014-08-28 |
PL2736357T3 (pl) | 2019-02-28 |
CN103747693A (zh) | 2014-04-23 |
KR20140050064A (ko) | 2014-04-28 |
ES2685638T9 (es) | 2019-02-04 |
AU2018214029A1 (en) | 2018-08-23 |
AU2016204474B2 (en) | 2018-08-23 |
ES2685638T3 (es) | 2018-10-10 |
US11707077B2 (en) | 2023-07-25 |
AU2016204474A1 (en) | 2016-07-21 |
CA2842837A1 (en) | 2013-01-31 |
EP2736357B1 (en) | 2018-05-02 |
CN103747693B (zh) | 2017-08-01 |
EP2736357A4 (en) | 2015-05-06 |
US20130029008A1 (en) | 2013-01-31 |
EP2736357A2 (en) | 2014-06-04 |
WO2013016547A3 (en) | 2013-05-10 |
WO2013016547A2 (en) | 2013-01-31 |
US20140093618A1 (en) | 2014-04-03 |
AU2012286817A1 (en) | 2014-02-13 |
US8703216B2 (en) | 2014-04-22 |
EP2736357B9 (en) | 2019-01-09 |
PL2736357T4 (pl) | 2019-02-28 |
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