JP2011505890A - 試料処理システムおよび方法 - Google Patents
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Abstract
Description
a)上記および本明細書で説明されるようなシステムを提供するステップと、b)試料の少なくとも第1の成分および第2の成分を分離するように、第1の処理ステップを行うステップと、およびc)試料の処理した成分に試料分離ステップを行うステップであって、分離は、処理した試料成分から標識および非標識成分を分離するステップを含む、ステップと、を含む、または包含する、方法に関する。処理ステップはまた、分離ステップ後に行うこともできる。
例えば、幹細胞移植と併せた、移植工学と、
臓器移植と、
急性骨髄性白血病や慢性骨髄性白血病を含む、白血病、および腎細胞癌、乳癌、黒色腫、膵臓癌等の固形腫瘍を含むが、それに限定されない癌治療と、
全身性エリテマトーデスまたは全身性強皮症、1型糖尿病、多発性硬化症等の、難治性自己免疫疾患の治療と、
エフェクター細胞を直接利用するステップを含むがそれに限定されない、細胞療法と、
感染性疾患の治療と、
心筋梗塞、肝臓障害、または神経変性疾患を含むが、それらに限定されない、組織再生と、
移植または自己免疫疾患を含むが、それらに限定されない、耐性誘導と、
を含むが、それらに限定されない。
上記で説明されるような基本操作によって製造される細胞生成物を、直接臨床使用のために構成することができることは、本発明の特異的かつ新規の特性である。当技術分野で公知の方法は、臨床要件に適応させるために、作り出された細胞生成物の手動後処理を必要とする。本発明のシステムにより、細胞生成物を即時利用のために直接処方することができる。処方ステップは、所望の体積または細胞濃度への調整、注射用液による処理液の交換、安定剤(自家血漿または血清、血清アルブミン、他のタンパク質、または合成ポリマー等)またはアジュバントの添加、後の貯蔵のためのDMSO等の凍結防止剤の補充、品質管理のための保持試料の引き抜き、注射用のバッグまたはシリンジの組み合わせへの送達を含む。最終処方のいくつかの成分、例えば、血漿、血小板、または血小板成分は、起源試料に由来してもよい。
ヒト臍帯血を、CliniMACS PBS/EDTA緩衝剤で希釈し、遠心分離によって規定の標識量に調整し、CliniMACS CD34またはCD133試薬を添加し、指定の標識時間にわたって細胞試料とともにインキュベートさせ、遠心分離機チャンバの中で洗浄することによってアクセス試薬を除去し、CD34またはCD133陽性細胞を、細胞懸濁から濃縮し、細胞培養培地とともに直接MACSカラムから溶出させ、細胞培養チャンバの中へ移転させる。培地補充物(ヒト血清アルブミン、サイトカイン)を、管類セットに取り付けられたバイアルから自動的に添加する。濃縮または単離幹細胞を、最大で3週間にわたって培養する。補充細胞培養培地を断続的に添加する。細胞培養培地を除去するように、拡張臍帯血細胞を遠心分離によって洗浄し、細胞をヒト血清アルブミンが補充された輸液(生理食塩水)中で再懸濁し、最終生成物容器(輸液バッグまたはシリンジ)に移転させる。
ヒト全血またはアフェレーシス収穫を、CliniMACS PBS/EDTA緩衝剤で希釈し、遠心分離によって規定の標識量に調整し、CliniMACS CD14試薬を添加し、指定の標識時間にわたって細胞試料とともにインキュベートさせ、遠心分離機チャンバの中で洗浄することによってアクセス試薬を除去し、CD14陽性単球を、細胞懸濁から濃縮し、細胞培養培地とともに直接MACSカラムから溶出させ、細胞培養チャンバの中へ移転させる。培地補充物(ヒト血清アルブミン、サイトカイン)を、管類セットに取り付けられたバイアルから自動的に添加する。濃縮または単離単球を、未熟単球由来樹状細胞に培養する。培地を遠心分離によって交換し、付加的な補充物を、管類セットに取り付けられたバイアルから自動的に添加する。細胞を培養し、成熟時に、抗原(組み換えタンパク質、ペプチド、細胞溶解物、DNA)を、管類セットに取り付けられたバイアルから自動的に添加する。単球由来樹状細胞を抗原処理のために培養する。細胞培養培地を、遠心分離によって除去し、細胞をヒト血清アルブミンが補充された輸液(生理食塩水)中で再懸濁し、最終生成物容器(輸液バッグまたはシリンジ)に移転させる。
アフェレーシス収穫を、CliniMACS PBS/EDTA緩衝剤で希釈して、自動的にCD19を枯渇させ、後に、MACSカラムを介して、CD304(BDCA−4、CD1c、Neuropilin−1)またはCD1c(BDCA−1)について濃縮する。DCを、細胞培養培地とともに直接MACSカラムから溶出させ、細胞培養チャンバの中へ移転させる。培地補充物(ヒト血清アルブミン、サイトカイン、組み換えタンパク質、ペプチド、細胞溶解物、DNAのような活性化成分)を、管類セットに取り付けられたバイアルから自動的に添加し、最適な成熟および活性化を達成するように、細胞を24時間にわたって培養する。細胞培養培地を、遠心分離によって除去し、細胞をヒト血清アルブミンが随意で補充された輸液(生理食塩水)中で再懸濁し、最終生成物容器(輸液バッグまたはシリンジ)に移転させる。生成物容器は、さらなる使用のために、本発明の管類セットから除去する。
ヒト全血またはアフェレーシス収穫を洗浄し、細胞培養培地で希釈し、細胞培養チャンバの中へ移転させる。抗原(組み換えタンパク質、ペプチドプール、または腫瘍細胞溶解物)および培地補充物(ヒト血清アルブミン、サイトカイン)を、管類セットに取り付けられたバイアルから自動的に添加する。抗原特異的T細胞を再刺激するように、細胞および抗原を3〜16時間にわたって培養する。細胞懸濁を、遠心分離によって規定の標識量に調整し、CliniMACS IFN−ガンマCatchmatrix試薬を添加し、指定の標識時間にわたって細胞試料とともにインキュベートさせ、遠心分離機チャンバの中で洗浄することによってアクセス試薬を除去する。細胞を細胞培養容器に移転させ、サイトカインの放出のためにインキュベートする。細胞懸濁を、遠心分離によって規定の標識量に調整し、CliniMACS IFN−ガンマ濃縮試薬を添加し、指定の標識時間にわたって細胞試料とともにインキュベートさせ、遠心分離機チャンバの中で洗浄することによってアクセス試薬を除去し、抗原特異的細胞を、細胞懸濁から磁気的に濃縮し、補充輸液を使用するMACSカラムからの直接溶出によって、わずかな注射用量に濃縮し、最終生成物容器(輸液バッグまたはシリンジ)に移転させる。
ヒト全血またはアフェレーシス収穫を、CliniMACS PBS/EDTA緩衝剤で希釈し、遠心分離によって規定の標識量に調整し、CliniMACS CD3試薬を添加し、指定の標識時間にわたって細胞試料とともにインキュベートさせ、遠心分離機チャンバの中で洗浄することによってアクセス試薬を除去し、CD3陽性細胞を細胞懸濁から枯渇させる。CD3陰性標的細胞を標識量に調整し、CliniMACS CD56試薬を添加し、指定の標識時間にわたって細胞試料とともにインキュベートさせ、遠心分離機チャンバの中で洗浄することによってアクセス試薬を除去し、CD56陽性細胞を濃縮し、細胞培養培地とともに直接MACSカラムから溶出させ、細胞培養チャンバの中へ移転させる。培地補充物(ヒト血清アルブミン、IL−2および/またはIL−15等のサイトカイン)を、管類セットに取り付けられたバイアルから自動的に添加する。濃縮または単離NK細胞を、8〜48時間にわたって培養する。細胞培養培地を、遠心分離によって細胞生成物から除去し、細胞をヒト血清アルブミンが補充された輸液(生理食塩水)中で再懸濁し、最終生成物容器(輸液バッグまたはシリンジ)に移転させる。
ヒト全血またはアフェレーシス収穫を、CliniMACS PBS/EDTA緩衝剤で希釈し、遠心分離によって規定の標識量に調整し、CliniMACS CD3試薬を添加し、指定の標識時間にわたって細胞試料とともにインキュベートさせ、遠心分離機チャンバの中で洗浄することによってアクセス試薬を除去し、CD3陽性細胞を細胞懸濁から枯渇させる。CD3陰性標的細胞を標識量に調整し、CliniMACS CD56試薬を添加し、指定の標識時間にわたって細胞試料とともにインキュベートさせ、遠心分離機チャンバの中で洗浄することによってアクセス試薬を除去し、CD56陽性細胞を濃縮し、細胞培養培地とともに直接MACSカラムから溶出させ、細胞培養チャンバの中へ移転させる。培地補充物(ヒト血清アルブミン、サイトカイン)を、管類セットに取り付けられたバイアルから自動的に添加する。CD2およびCD335(NKp46)、またはCD314(NKG2D)およびCD335(NKp46)に対する抗体が搭載された細胞拡張ビーズを、バイアルから細胞培養区画に移転させる。濃縮または単離NK細胞を、1週間にわたって培養し、第7日より開始して、補充細胞培養培地を1:1の比率で3日ごとに添加し、細胞を第14〜21日まで培養する。
ヒト全血またはアフェレーシス収穫を、CliniMACS PBS/EDTA緩衝剤で希釈し、遠心分離によって規定の標識量に調整し、CliniMACS CD4試薬を添加し、指定の標識時間にわたって細胞試料とともにインキュベートさせ、遠心分離機チャンバの中で洗浄することによってアクセス試薬を除去し、CD4陽性細胞を濃縮し、細胞培養培地とともに直接MACSカラムから溶出させ、細胞培養チャンバの中へ移転させる。培地補充物(ヒト血清アルブミン、サイトカイン)を、管類セットに取り付けられたバイアルから自動的に添加する。CD2、CD3、およびCD28、または代替としてCD3およびCD28に対する抗体が搭載された細胞拡張ビーズを、バイアルから細胞培養区画に移転させる。単離Tヘルパー細胞を培養し、第3日より開始して、補充細胞培養培地を1:1の比率で2日ごとに添加し、細胞を第14日まで培養する。細胞拡張ビーズを除去するように、拡張T細胞をMACSカラムの上方に通す。細胞培養培地を、遠心分離によって細胞生成物から除去し、細胞をヒト血清アルブミンが補充された輸液(生理食塩水)中で再懸濁し、最終生成物容器(輸液バッグまたはシリンジ)に移転させる。生成物容器は、さらなる使用のために、本発明の管類セットから除去する。
CD133抗原は、幹細胞マーカーであり、一部の未成熟CD34+細胞上、循環内皮前駆細胞上、およびCD34−幹細胞サブセット上で特異的に発現される(De Wynterら,1998)。CliniMACS CD133システムを使用する、濃縮または単離CD133陽性細胞は、臍帯血からの造血前駆細胞の生体外拡張のため、ならびに自家(Pasinoら,2000)および同種(Koehlら,2002)移植で使用されている。
CD14抗原は、LPS受容体複合体に属し、単球は、抗原を強く発現する。CD14選択単球は、ヒト単球由来樹状細胞の後続の生成のために使用することができる(MoDCs;Campbellら、2005)。樹状細胞には、固形腫瘍、血液学的悪性腫瘍、ウイルス感染症、および自己免疫疾患を含む、種々の疾患に対する細胞ワクチンとしての大きな可能性がある。
末梢血において、CD304(BDCA−4、Neuropilin−1)を、ヒト形質細胞様樹状細胞(PDC)上で発現させる。PDCは、最も強力なI型インターフェロン産生細胞である(Dzionekら、2002)。CD304は、(MoDCについて)培養期間なしで、末梢血から樹状細胞を直接濃縮または単離するために使用することができる。
CD4は、T細胞による、MHCクラスII抗原と関連した外来抗原の認識におけるアクセサリー分子である。Tヘルパー細胞、より低い程度で、単球および樹状細胞は、CD4を発現する。
MHCクラスI分子に対するコレセプターである、CD8抗原は、細胞障害性T細胞上で、およびかすかに一部のNK細胞上で発現される。
Ficoll−PaqueTMは、骨髄、末梢血、および臍帯血からの単核細胞の単離のための滅菌密度媒体である。それは、Ficoll PM400(高度に分岐した高質量の親水性多糖)、ジアトリゾ酸ナトリウム、およびエデト酸カルシウム二ナトリウムEDTAから成る、密度1.077g/mlの水溶液である。赤血球および顆粒球が、1.077g/ml以上の密度を有する一方で、リンパ球および単球は、より低い密度を有する。ヒト血液がFicoll−Paqueの最上部で層状になると、遠心力下でリンパ球および単球を赤血球および顆粒球から分離することができる。
本発明の遠心分離チャンバは、細胞培養フラスコまたはバッグと同様に、細胞の培養に使用することができる。3.2E5/mlのヒト細胞株K562を、10%ウシ胎仔血清が補充されたRPMI1640細胞培養培地の30mlの体積中で、遠心分離チャンバに塗布した。チャンバは、5%CO2のCO2インキュベータの中に入れた。24、48、および70時間後に、細胞計数および生存評価のために、内容物のアリコートをチャンバから除去した。播種した細胞は、80%、95%、および95%生存率で、4.1E5/ml、6.4E5/ml、および9.2E5/mlの生存細胞に拡大した。
CD133+細胞の分離手順全体は、以下の作業を伴う、いくつかのステップを含む。
1.緩衝剤を入れることによる管類セットの準備
2.遠心分離チャンバへの骨髄試料の移転
3.遠心分離チャンバの中での骨髄試料の調製
4.CD133+細胞の磁気分離
5.最終体積低減
1.緩衝剤を入れることによる管類セットの準備
第1のステップでは、PEB(2mmol/1EDTA、0.5%HASが補充されたリン酸緩衝生理食塩水)を入れて、管類セットを準備した。
2.遠心分離チャンバへの骨髄試料の移転
管類セットの準備後、骨髄試料を試料バッグ312から遠心分離チャンバ332に移転させた。
3.遠心分離チャンバの中での骨髄試料の調製。遠心分離チャンバの中での試料調製は、上清がチャンバから除去される、いくつかのステップを含む。上清除去中、細胞を含まない上清のみ、または血小板洗浄の場合は血小板を伴う上清が、移転されるべきである。白血球(WBC)、したがってCD133+細胞が、上清とともに除去されることを回避することが目標である。試料調製は、以下のステップを含む。
−血漿の生成
−血小板の低減
−CD133 Microbeads(Miltenyi Biotec, Germany)によるインキュベーション
−非結合CD133 Microbeadsの除去
試料調製過程の始めに生成される血漿は、以下の2つの機能を果たす。
1.単球のFc受容体を遮断し、単球に付着するマイクロビーズに結合されたCD133抗体のFc部を妨害する、遮断試薬としての役割を果たすこと。これは、単球による免疫磁気分離過程の生成物の低減した純度につながる。
2.最終細胞生成物が懸濁される、緩衝液に対する補充物としての役割を果たすこと。これは、使用した緩衝剤単独よりも、細胞生成物に対する生理的環境を提供する。血漿の生成および収集のために、赤血球(RBC)、白血球(WBC)、および血小板としての材料の細胞成分の大部分がペレット化されるまで、試料が遠心分離される。細胞を含まない上清は、貯留部としての役割を果たす容器(303)に移転される。
血漿生成がチャンバの中の全上清を除去すると、70mlの残留量がチャンバの中にある。懸濁を希釈するように150mlのPEBを添加し、220mlの合計量をもたらす。次いで、ドラムが632rpm/分の減速率で1000rpmまで減速される前に、RBCおよびWBCをペレット化するために、ドラムを2000rpmまで加速し、沈殿を100秒間行う。この沈殿時間内に、チャンバの減速が開始すると、血小板のほんの一部しかペレットに達しない。次いで、全上清を6ml/分の速度で除去する。試料および除去した上清の分析は、50.8%の実験的判定血小板除去を示した。残りの血小板は、フィルタ(310)を使用して除去する。
CD133マイクロビーズを、隔壁を有するガラスバイアル中で提供する。無菌状態を確保するために、0.2μmの細孔径(306)のフィルタは、バイアルに接続された管類セットの分岐に統合される。バイアルは、通気式バイアルアダプタ(305)によって管類セットに接続される。試薬がチャンバに送出されると、ドラムの前の管類セットの部品内の圧力が監視される。バイアルが空になると、空気がフィルタへと移動する。フィルタが試薬によって湿潤されると、空気がフィルタの中へ移動する時に、細孔は依然として液体で充填されている。毛細管力により、空気はフィルタの膜を通過することができない。ポンプが稼働し続けると、圧力降下が圧力センサ(329)によって検出され、ソフトウェアがポンプを止める。このように、残留試薬を除去する洗浄ステップと組み合わされて、試薬がチャンバ中の細胞懸濁に自動的に移転される。
1.ドラムを300rpmの速度まで加速する。これは、液体をチャンバの壁に移動させる。
2.遠心分離を10秒間実行する。
3.その後、ドラムを止める。液体が戻り、水平に整合した表面を有する。
4.チャンバが50秒間停止し続ける。
5.30分のインキュベーション時間が経過するまで、ステップ1−4を反復する。
標識化およびインキュベーション後に非結合試薬を除去するために、ビーズ洗浄を行う。洗浄過程では、以下のステップが3回反復される。
1.PEB懸濁を希釈するために遠心分離機に移転させ、230mlの合計量をもたらす。
2.RBCおよびWBCの沈殿を2000rpmで140秒間実行する。
3.チャンバの速度を2000rpmから1000rpmに低減する。
4.上清を20ml/分の速度で完全に除去する。ビーズ洗浄過程は、3つの洗浄ステップから成り、非結合試薬の少なくとも97.2%が除去される。
ビーズ洗浄過程後、磁気分離カラム(CliniMACSカラム、Miltenyi Biotec GmbH,Germany)を使用して、CD133+細胞を分離する。したがって、5ml/分の負荷速度で、細胞懸濁を分離カラム上に移転させる。磁気標識細胞を磁化カラムの中に保持し、カラムをPEBで洗浄することによって、非標識細胞から分離する。保持したCD133+細胞を、カラムから磁界を除去することによって溶出させる。CD133+細胞を管類セットのループの中へ送出し、分離カラム上に再搭載する。この過程をもう一度反復し、3度目の再搭載過程後に、最終的にCD133+細胞を20mlの溶出緩衝剤中で溶出させる(表3および図26を参照)。溶出速度は、600ml/分である。
心臓幹細胞療法のために、CD133+細胞は、最大限でも6mlの少ない最終量で入手可能となるべきである。通常、CD133+細胞は、最終的に20ml溶出緩衝剤中でCliniMACSカラムから溶出させられる。最終量の低減のために、3つの方法が可能である。単離または濃縮CD133+細胞の最終量は、以下によって低減することができる。
2.磁気分離後の濾過、または
3.AutoMACSカラム(Miltenyi Biotec GmbH,Germany)の使用
20mlの溶出緩衝剤中での溶出の代わりに、CD133+細胞を、6mlの溶出緩衝剤中でCliniMACSカラムから溶出させることができる。溶出速度は、600ml/分である。この方法を、骨髄からのCD133+細胞の濃縮によって試験した。濃縮細胞をFACS分析によって判定した(表4および図27を参照)。
20mlの溶出緩衝剤中でCliniMACS分離カラムからの溶出後、CD133+細胞を、4ml/分の速度でフィルタ(Pall IV−5、0.2μm、またはRoweFil 24、1.2μm)上に移転させることができる。その後、細胞をフィルタから2mlで溶出させる。この方法を、骨髄からのCD133+細胞の濃縮によって試験した。濃縮細胞をFACS分析によって判定した(表5および図28を参照)。
CliniMACSカラムからの溶出後、CD133+細胞を、4ml/分の速度でautoMACSカラム上に搭載することができる。その後、CD133+細胞をautoMACSカラムから4mlで溶出させる。この方法を、骨髄からのCD133+細胞の濃縮によって試験した。濃縮細胞をFACS分析によって判定した(表6および図29を参照)。
Claims (15)
- a)試料処理ユニットであって、
該試料処理ユニットは、少なくとも1つの試料チャンバを有する回転容器に連結される、入力ポートおよび出力ポートを備え、
該試料処理ユニットは、
試料に第1の処理ステップを提供することと、
該チャンバ内に堆積された試料に遠心力を適用し、該堆積試料の少なくとも第1の成分および第2の成分を分離するように、該容器を回転させることと
を実行するように構成される、試料処理ユニットと、
b)該試料処理ユニットの該出力ポートに連結される、試料分離ユニットであって、
該試料分離ユニットは、
分離カラムホルダと、
ポンプと、
流体回路および該ホルダ内に配置された分離カラムを通る流量を少なくとも部分的に制御するように構成される、複数の弁と
を備え、
該分離カラムは、該カラムを通して流された試料の標識および非標識成分を分離するように構成される、試料分離ユニットと
を備える、システム。 - 前記回転容器は、該回転容器内の前記堆積試料の前記少なくとも第1の成分および前記第2の成分の分離の進展を検出する手段を備える、請求項1に記載のシステム。
- 前記分離の進展を検出する前記手段は、
光源からの光が、分離されている前記試料の少なくとも一部を、少なくとも部分的に貫通することと、
該試料の少なくとも一部を通過する光を、光検出器によって検出することと
が可能であるように位置する、請求項1または2に記載のシステム。 - 前記分離の進展を検出する前記手段は、前記回転容器の回転軸に対して本質的に垂直に、該回転容器に位置する、請求項1〜3のうちのいずれか一項に記載のシステム。
- 前記分離の進展を検出する前記手段は、前記回転容器に配置され、該手段は、該回転容器の前記回転軸に隣接する領域から該回転容器の周囲に隣接する領域まで本質的に及ぶ、請求項1〜4のうちのいずれか一項に記載のシステム。
- 前記回転容器の中の前記分離の進展を検出する前記手段は、窓、プリズム、または鏡である、請求項1〜5のうちのいずれか一項に記載のシステム。
- 前記プリズムまたは鏡は、前記回転容器の蓋に形成されるチャネルを覆うように配置され、該チャネルは、遠心分離中に前記試料の少なくとも一部がそれに流入することができるように構成される、請求項1〜6のうちのいずれか一項に記載のシステム。
- 前記回転容器は、細胞を培養するために使用可能であるように構成される、請求項1〜7のうちのいずれか一項に記載のシステム。
- 前記回転容器は、その上で細胞を増殖させるための少なくとも1つの層を備える、請求項1〜8のうちのいずれか一項に記載のシステム。
- 前記回転容器は、使い捨てであるか、および/または、滅菌することができる、請求項1〜9のうちのいずれか一項に記載のシステム。
- 前記回転容器は、
プラスチック、ポリスチロール(PS)、ポリスチレン、ポリ塩化ビニル、ポリカーボネート、ガラス、ポリアクリレート、ポリアクリルアミド、ポリメチルメタクリルレート(PMMA)、ポリエチレンテレフタレート(PET)、ポリテトラフルオロエチレン(PTFE)、熱可塑性ポリウレタン(TPU)、シリコーン、ポリエチレン(PE)、コラーゲン、キチン、アルギン酸塩、ヒアルロン酸誘導体、ポリラクチド(PLA)、ポリグリコリド(PGA)、およびそれらの共重合体、ポリスチロール(PS)、ポリスチレン、ポリカーボネート、ポリアクリレート、セラミック、ハイドロキシアパタイト(HA)およびリン酸カルシウムのようなガラス材料、および上記の材料のうちの1つ以上を備える組成物から成る群より選択される、材料を備えるか、または材料で作ることができる、請求項1〜10のうちのいずれか一項に記載のシステム。 - 請求項1〜11に記載のシステムを提供することと、
試料の少なくとも第1の成分および第2の成分を分離するように、処理ステップを実行することと、
該試料の処理された成分に試料分離ステップを実行することであって、該分離は、該処理された試料成分から標識および非標識成分を分離することを含む、ことと
を含む、方法。 - 具体的には前記試料の層の形成、pH値の変化、および/または温度の変化を検出することによって、前記分離の進展を検出することをさらに含む、請求項12に記載の方法。
- 前記試料は、生体試料であり、具体的には、血液、骨髄、細胞、細胞成分、白血球分離生成物、腫瘍またはその断片、組織、微生物、細菌、真菌、またはウイルスである、請求項12または13に記載の方法。
- 具体的にはコンピュータ上で実行されると、具体的には請求項12〜14のうちのいずれか一項に記載の方法を行うために、請求項1〜11のうちのいずれか一項に記載のシステムを制御するコンピュータプログラム。
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