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JP4294316B2 - 胎盤幹細胞の回収方法 - Google Patents

胎盤幹細胞の回収方法 Download PDF

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JP4294316B2
JP4294316B2 JP2002548091A JP2002548091A JP4294316B2 JP 4294316 B2 JP4294316 B2 JP 4294316B2 JP 2002548091 A JP2002548091 A JP 2002548091A JP 2002548091 A JP2002548091 A JP 2002548091A JP 4294316 B2 JP4294316 B2 JP 4294316B2
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perfusate
placenta
perfusion
stem cells
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Description

先行仮出願の利益
本特許出願は、2000年12月6日に出願された同時係属中の先行米国特許仮出願第60/251,900号(発明の名称「胚性幹細胞の回収方法」、出願人と発明者は同一の名前で、Robert J. Hariri)の利益を請求するものである。
発明の背景
1. 発明の分野
本発明は、一般的には、幹細胞回収の分野、特に、胎盤からの胚様幹細胞及び他の多能幹細胞の回収に関する。これらの胚様幹細胞は分娩後に回収された胎盤に由来するものである。これらの胚様幹細胞は、胚性幹細胞の特徴を有するが、胚に由来するものではない。
2. 背景技術の説明
ヒトの幹細胞は、種々の成熟ヒト細胞系を生成することができる全能性又は多能性の前駆細胞である。この能力は器官及び組織の発達に必要な細胞の分化及び特殊化の基礎として役立つ。そのような幹細胞の移植における最近の成功は、疾患に起因する骨髄剥離、有毒な化学物質もしくは放射線への曝露後の骨髄を再構成及び／又は補充するための新しい臨床用ツールを提供してきた。幹細胞を、全てではないとしても多くの組織を再形成させ、生理学的及び解剖学的機能を回復させるのに用いることができることを証明するさらなる証拠が存在する。組織工学、遺伝子治療送達及び細胞治療薬における幹細胞の利用も急速に進歩している。
いくつかの理由から、十分なヒト幹細胞を取得することは問題の多いことであった。第1に、成体組織における正常に発生する幹細胞集団の単離は、技術的に困難であり、費用が高く、量が極く限られていた。第2に、胚又は流産児を含む胎児組織から得たこれらの細胞の入手は、多くの倫理的及び道徳的関心を提起してきた。ヒト胚及び胎児は独立した生命を形成するという広く行き渡った考えは、いかなる目的のためのそのような資源の使用に対しても一時的禁止を正当化してきた。独立した生命の尊厳を冒涜しない代替的な資源は、幹細胞の臨床的な使用におけるさらなる進歩にとって必須であろう。
臍帯血は、造血再構成における使用のために冷凍保存される造血多能前駆幹細胞の公知の供給源である。この目的のための臍帯血の使用は周知であり、広く用いられる治療手順になりつつある。臍帯血の回収に関する従来の技術は、胎盤から臍帯血を排液するために重力の助けを借りながら用いられる針又はカニューレの使用に基づく。通常、この針又はカニューレを臍静脈中に置き、胎盤を穏やかにマッサージして、胎盤からの臍帯血の排液を援助する。その後の排液された胎盤は利用できないものと考えられ、通常は廃棄されてきた。臍帯血からの幹細胞の入手の主要な限界は、得られる臍帯血の容量が不十分であることが多く、移植後の骨髄を再構成するには不十分な細胞数しか得られないことであった。
幹細胞は決定的に供給不足である。これらは悪性腫瘍、先天性代謝異常、異常ヘモグロビン症、及び免疫不全などの種々の障害の治療にとって重要である。より多くの胚性幹細胞の供給源を有することは極めて有利であろう。
従って、本発明の主な課題は、瀉血された胎盤から造血幹細胞を抽出し、回収する方法を提供することである。
また、本発明の課題は、排液された胎盤の抽出物から他の胚様及び／又は分化全能性の幹細胞を単離する方法を提供することである。
本発明のさらなる課題は、造血多能前駆幹細胞の最良の起源である臍帯静脈から幹細胞を回収する方法を提供することである。
本発明のさらなる課題は、排液された胎盤から、より高濃度のさらなる胚様幹細胞を取得することができる方法及び手段を提供することである。
本発明のさらなる課題は、限定されるものではないが、リンパ球及び幹細胞などの内因性細胞の増殖にとって良好な環境を提供するバイオリアクターとしての、単離され灌流された胎盤の利用方法を提供することである。
本発明のさらなる課題は、出産及び子宮からの胎盤の娩出からかなりの時間が経過した後に幹細胞を取得することができる方法及び手段を提供することである。
発明の概要
瀉血後のヒト胎盤の回収及び残存する血液の回収に際して、臍帯動脈及び臍帯静脈のいずれか又は双方を用いる灌流技術により、排液された胎盤から胚様幹細胞を抽出する方法が開発された。次いで、実質組織及び血管外空間内の残存する幹細胞を集めて空の微小循環(それら細胞は灌流により集合血管中に流れ込んで洗浄され得る)中に移動させる、ex vivoの天然のバイオリアクター環境を確立するような方法で、胎盤を処理する。
発明の詳細な説明
I. 胎盤を排液し抽出する方法
臍帯血の排液及び新鮮な胎盤の保存
この方法は、従来の臍帯血回収方法にかけられた新鮮な排液後のヒト胎盤を、その胎盤から実質的に全ての臍帯血を排液することにより、利用する必要がある。この胎盤を胚性幹細胞の好適な供給源とする場合、これを適切に保存し、排液することが重要である。一般的には、胎盤を凝固防止剤溶液中、5〜25℃(摂氏)の温度で、48時間を超えない期間で保存した後、臍帯血を回収するべきである。好適な凝固防止剤溶液は周知である。好ましい凝固防止剤溶液は、ヘパリン(1:1000溶液中、1％ w/w)の溶液を含む。一般的には、排液された胎盤を、胚様幹細胞を回収する前に36時間を超えない期間で保存するべきである。
細胞の抽出
好ましい胚様幹細胞の抽出方法は、0.9 N塩化ナトリウム溶液などの任意の好適な等張性水溶液に溶解した凝固防止剤などの好適な水溶液を用いた、排液された胎盤の灌流に基づく。灌流液用の凝固防止剤は、残存する臍帯血のクロットの形成を防止するのに十分な濃度のヘパリン又はワルファリンナトリウムを含んでもよい。一般的には、100〜1000ユニットのヘパリンを用いることができる。
抽出方法は、臍帯動脈及び臍帯静脈を最も高い地点に置くような様式で吊り下げた胎盤への重力流を用いた、臍帯動脈及び臍帯静脈のいずれか又は双方を通じた灌流液の通過に基づく。図1に示すように、臍帯動脈及び臍帯静脈を同時に、可撓性コネクタを介して灌流液の貯蔵器に接続されたピペットに接続しておき、灌流液を臍帯静脈及び臍帯動脈中に通過させた後、懐胎期間に母体の子宮に付着していた胎盤表面から好適な開口容器中に回収するのが好ましい。
回収技術は、臍帯血の排液後に残された細胞の回収をもたらす十分量の灌流液の使用に基づく。最初に灌流液を回収する場合、この液体は残存する赤血球により着色されているが、灌流液が胎盤を通過するに従って透明になる傾向があることが観察された。一般的には、胚様細胞を回収するには30〜100 mlの灌流液で十分であるが、観察される結果に応じてそれ以上でもそれ以下の量でも使用することができる。
II. 排液され灌流された胎盤のバイオリアクターとしての使用方法
排液された胎盤の灌流
上記で考察したように、臍帯基部(胎盤板中への挿入部分の4〜5 cm(センチメートル)以内)を採血及びクランプした後、胎盤を無菌条件下で回収し、胎盤の血液を回収し、その胎盤を滅菌断熱輸送デバイス(胎盤の温度を20〜28℃に維持する)中で、例えば、図2a〜eに示すように、クランプした胎盤を滅菌したジップロックプラスチックバッグに入れた後、それを断熱したStyrofoam容器又は減圧断熱容器に入れて、処理のために実験室に輸送する。
バイオリアクターとしての胎盤の使用
胎盤を滅菌したたらいにいれ、500 mLのリン酸緩衝生理食塩水で洗浄する。この洗浄液を廃棄する。図3に示すように、臍帯静脈に、灌流マニホールドに接続されている滅菌管に接続したテフロン又はプラスチックカニューレを挿入する。ついで、たらいを覆い、胎盤を2〜24時間の間、室温(20〜25℃)に維持する。その後、胎盤を定期的に、好ましくは4、8、12、及び24時間の時点で、一定容量の灌流液、好ましくは、100 mLの灌流液(1000 u/Lのヘパリン及び／もしくはEDTA及び／もしくはCPDA(クレアチンリン酸デキストロース)を補充した、又は補充しない滅菌生理食塩水)を用いて灌流する。反対側表面で胎盤から漏出する流出液を回収し、目的の幹細胞を単離するために処理する。胎盤を維持する条件及び灌流液の性質に変更を加えて、流出液の容量及び組成を調節することができる。
次いで、例えば、密度勾配遠心、磁気細胞分離又はその他の許容し得る方法などの当業者には公知の技術を用いて、幹細胞を流出液から単離し、例えば、図4に示されるスキームに従って分別する。
この方法の変法では、胎盤中の細胞を刺激して、免疫グロブリンもしくはその他の分子などの生物活性分子を産生させるか、又は、例えば、エリスロポエチンの投与により、増殖させることができる。また、例えば、アデノウイルスもしくはレトロウイルスベクターなどのウイルスベクターを用いて、又はDNAのリポソームもしくは化学物質を介する取込みなどの機械的手段を用いて、未だバイオリアクター中に入ったままの採取前に、又は採取の時点で、この細胞を遺伝子操作することもできる。
その手順は以下のとおりである：
1. 胎盤を完全に瀉血し、全ての接着性の凝固夾雑物及び非接着性の細胞夾雑物を除去する。
2. 凝固防止剤を含むもしくは含まない、及び／又は抗菌剤を含むもしくは含まない灌流溶液(例えば、生理食塩水)を用いて胎盤を培養し、灌流する。
3. 浸出させた灌流液及び循環させた灌流液の双方を、滅菌容器中に回収する。
4. 例えば、限定されるものではないが、密度勾配遠心、磁気細胞分離、アフィニティー細胞分離又は別の接着技術などの当業者には公知の技術を用いることにより、回収された灌流液から細胞型を単離する。
本発明の一実施形態において、胎盤を、灌流溶液を用いて48時間インキュベートすることにより、限定されるものではないが、リンパ球及び種々の多能性及び／又は全能性幹細胞などの内因性細胞のためのバイオリアクターとして胎盤を用いる。
別の実施形態において、胎盤を処理して全ての内因性細胞を除去し、灌流された胎盤の環境に外来細胞を導入し、増殖させる。特定の実施形態においては、灌流された胎盤に、電磁放射線、UV、X線、γ線又はβ線を照射して、全ての残存する内因性生細胞を撲滅する。目的の外来細胞を、照射された胎盤バイオリアクター中で増殖させた後、導入する。
栄養素及び増殖因子を灌流溶液に導入することにより、外因性細胞を増殖させる。血清及び他の増殖因子を、増殖用灌流溶液又は培地に添加する。増殖因子は通常はタンパク質であり、限定されるものではないが、サイトカイン、リンホカイン、インターフェロン、コロニー刺激因子(CSF)、インターフェロン、ケモカイン、及びインターロイキンなどが挙げられる。他の増殖因子としては、リガンド、幹細胞因子、トロンボポエチン(Tpo)、インターロイキン、及び顆粒球コロニー刺激因子(G-CSF)などの組換えヒト造血増殖因子が挙げられる。灌流溶液中に導入された増殖因子は、未分化の幹細胞又は分化した造血細胞の増殖を刺激し、限定されるものではないが、免疫グロブリン、ホルモン、又は以前に記載された他の増殖因子などの生物活性分子の産生を刺激することができる。
本発明は以下の実施例を参照することによりさらに理解されるであろう。
実施例1：排液された胎盤の灌流
20 ml(ミリリットル)のリン酸緩衝生理食塩水(PBS)を灌流液に加え、10 ml分を回収し、3000 rpm(毎分回転数)で25分間遠心分離する。流出物を4個のチューブに分割し、氷浴中に置く。1％ウシ胎仔血清(FCS)溶液を含むPBSを2.5 ml加え、チューブを遠心分離する(140分 x 10 g(重力加速度))。ペレットを5 mlの1％ FCSに再懸濁し、2個のチューブを合わせて１つに混合する。総リンパ球量と総単球量を加算し、これに細胞懸濁液総容量を乗じることにより、総単核球量を算出する。
実施例2：胎盤の灌流及びインキュベーションにより得られた細胞の分析
Figure 0004294316
ＰＰ試料はFicoll処理後のもの。
Ficoll処理後のＰＰの合計細胞数は5.3×108であり、処理前のＣＢの数は6.3×108である。Lym％はリンパ球のパーセントである。MID％はミッドレンジ白血球のパーセントである。GRA％は顆粒球のパーセントである。
例えば、Auto Macs (Miltenyi)などの磁気細胞分離法を用いることにより、細胞の単離を達成する。好ましくは、CD34+細胞の単離を最初に実施する。
材料及び方法
民間臍帯血銀行プログラムに登録した妊娠中の母親から胎盤のドナーを募集し、臍帯血回収後の瀉血した胎盤の研究目的での使用を許諾するインフォームド・コンセントを得た。これらのドナーはまた、冷凍保存用の臍帯血標本の通常の処理から作製された盲検データの使用も許諾した。これにより、回収された臍帯血と以下に記載するこの実験方法を用いて回収された流出灌流液との間の組成の比較が可能になった。全ドナーのデータは秘密保持されている。
臍帯及び胎盤の瀉血後、胎盤を室温にて滅菌断熱容器に入れ、分娩後4時間以内に実験室に到達させた。検査の際、器官の断片化もしくは臍血管の剥離などの物理的損傷の形跡があった場合、胎盤を廃棄した。胎盤を、滅菌容器中で2〜20時間、室温(23+/-2℃)で維持するか、又は冷蔵(4℃)した。定期的に、胎盤を25+/-3℃で滅菌食塩水に浸して洗浄し、目に見える表面の血液又は破片を全て除去した。臍帯を、胎盤への挿入部分から約5 cmのところで横切開し、臍血管に、胎盤の二方向灌流及び流出液の回収を可能にする滅菌液体路に接続したテフロンもしくはポリプロピレンカテーテルをカニューレ挿入した。本発明で用いた系により、制御された周囲雰囲気条件下で実施される調整、灌流及び流出液の回収、並びに血管内の圧力及び流速、コア及び灌流液の温度、並びに回収された流出液の容量のリアルタイムなモニタリングの全態様が可能になった。各種の調整プロトコルを、産後24時間に渡って評価し、流出液の細胞組成を、フローサイトメトリー、光学顕微鏡及びコロニー形成ユニットアッセイにより分析した。
胎盤の調整
残存細胞の増殖及び動員にとって生理的に適合する環境をシミュレートし、維持する試みとして、種々の条件下で胎盤を維持した。カニューレを、2U/mlヘパリン(EJkins-Sinn, NJ)を含むIMDM無血清培地(Gibco BRL, NY)でフラッシュした。約150 mLの灌流液が回収されるまで、50 mL／分の流速で、胎盤の灌流を継続した。灌流液のこの容量を「初期画分」と称した。同じ流速で胎盤の灌流を継続したところ、約150 mLの第2の画分が回収され、これを「後期画分」と称した。この手順の過程の間、灌流プロセスを援助し、細胞材料の回収を援助するために胎盤を穏やかにマッサージした。動脈カニューレを通じて重力ドレナージ及び吸引の双方により灌流循環物から流出液を回収した。
両親の同意書を得た後、分娩室から臍帯血と共に胎盤を得て、出産後12〜24時間以内に室温にて処理した。処理前に、膜を除去し、母体由来の部分を洗浄して残存する血液を除去した。血液サンプル回収のために、20ゲージの蝶型針から作製したカテーテルを臍血管にカニューレ挿入した。次いで、ヘパリン処理(2U/mL)したダルベッコの改変イーグル培地(H. DMEM)を用いて、15 mL／分の速度で10分間、胎盤を灌流し、灌流液を1時間以内に母体由来の部分から回収し、有核細胞を計測した。回収される有核細胞数が100個/μLに低減するまで、灌流及び回収手順を1又は2回繰り返した。灌流液をプールし、軽く遠心分離して、血小板、破片及び脱核細胞膜を除去した。次いで、有核細胞を、Ficoll-Hypaque密度勾配遠心分離により単離し、洗浄後、H.DMEMに再懸濁した。接着細胞の単離のために、5〜10 x 106細胞のアリコートを、いくつかのT-75フラスコの各々に入れ、Bio Whittakerから入手した市販のMesenchymal Stem Cell Growth Medium (MSCGM)と共に培養し、組織培養インキュベーター(37℃、5％ CO2)に置いた。10〜15日後、PBSを用いた洗浄により、非接着細胞を除去した後、PBSをMSCGMと交換した。種々の接着細胞型の存在について、特に、線維芽細胞のクラスターの同定及び増殖について、フラスコを毎日試験した。
細胞の回収及び単離
X 00 x gで15分間、室温にて遠心分離することにより、細胞を灌流液から回収する。この手順は、夾雑している破片及び血小板から細胞を分離するのに役立った。細胞ペレットを、2U/mlヘパリン及び2 mM EDTA (Gibco BRL, NY)を含むIMDM無血清培地に再懸濁した。総単核細胞画分を、Lymphoprep (Nycomed Pharma, Oslo, Norway)を用いて製造業者の推奨手順に従って単離し、単核細胞画分を再懸濁した。血球計数器を用いて細胞を計数した。トリパンブルー排除により、生存能力を評価した。間葉細胞の単離を、0.2％ EDTA (Sigma)を含む0.05％トリプシンの溶液を用いて、「示差的トリプシン処理」により達成した。線維芽細胞は約5分以内にプラスチック表面から剥離するが、他の接着集団は20〜30分以上のインキュベーションを必要とするため、示差的トリプシン処理が可能であった。トリプシン処理、及びTrypsin Neutralyzing Solution (TNS, Bio Whitaker)を用いるトリプシン中和の後、剥離した線維芽細胞を回収した。細胞をH.DMEM中で洗浄し、MSCGMに再懸濁した。Becton-Dickinson FACSCalibur装置を用いてフローサイトメトリーを実施し、骨髄由来MSC(間葉幹細胞)の公知のマーカーに基づいて選択されたFITC及びPE標識モノクローナル抗体はB.D. and Caltag laboratories (S. San Francisco, CA)から購入し、SH2、SH3及びSH4抗体産生ハイブリドーマはAM. Cul.から購入し、それらの培養上清中のMoAbの反応性をFITC又はPE標識F(ab)'2ヤギ抗マウス抗体により検出した。市販の誘導及び維持培養培地(Bio Whittaker)を、製造業者の説明書に従って用いて、系譜への分化が実施された。
胎盤幹細胞の単離
培養フラスコ中の接着細胞の顕微鏡観察により、形態学的に異なる細胞型が示された。紡錘形細胞、大きな核及び多数の核周囲小胞を有する円形細胞及びいくつかの突起を有する星状細胞のうちの一つを介して、細胞はフラスコに接着していた。これらの接着細胞をさらに特徴づける試みは為されていないが、同様の細胞は骨髄、臍帯血及び末梢血の培養物中に観察され、従って、天然では非幹細胞であると考えられた。クラスターとして最後に出現する線維芽細胞は、MSCの候補であり、示差的トリプシン処理により単離され、これを第2のフラスコ中で継代培養した。トリプシン処理後、高度に顆粒化される円形細胞を位相差顕微鏡観察したところ、実験室で作製されるか、又はBio Whittakerから購入した骨髄由来MSCと区別できなかった。継代培養した場合、胎盤由来細胞は、その初期段階とは対照的に、数時間以内に接着し、特徴的な線維芽細胞の形状を受け継ぎ、参照する骨髄由来MSCと同一の増殖パターンを示した。さらに、継代培養及び栄養補充中に、緩やかに結合した単核細胞を洗浄除去し、培養物を均質に維持し、目に見える非線維芽細胞夾雑物を含まないようにした。
フローサイトメトリー
初期及び後期画分の精製された単核細胞上のCD-34、CD-38、及び他の幹細胞関連表面マーカーの発現を、フローサイトメトリーにより評価した。簡単に述べると、細胞をPBS中で洗浄した後、抗CD34フィコエリトリン及び抗CD38フルオレセインイソチオシアネート(Becton Dickinson, Mountain View, CA)で二重染色した。
例示の目的で本発明の特定の実施形態を本明細書に記載してきたが、特許請求の範囲に定義されるような本発明から逸脱することなく、本発明の詳細の多くの変更が為され得るということが当業者には明らかであろう。
図1は、胎盤を灌流した後、灌流液を回収するための、胎盤の静脈及び動脈のカニューレ挿入の断面図である。 図2a〜eは、排液され灌流された胎盤の回収、クランピング、灌流、回収及び保存を示す図である。 図2a〜eは、排液され灌流された胎盤の回収、クランピング、灌流、回収及び保存を示す図である。 図2a〜eは、排液され灌流された胎盤の回収、クランピング、灌流、回収及び保存を示す図である。 図2a〜eは、排液され灌流された胎盤の回収、クランピング、灌流、回収及び保存を示す図である。 図2a〜eは、排液され灌流された胎盤の回収、クランピング、灌流、回収及び保存を示す図である。 図3は、バイオリアクターとして使用するための、デバイス中の灌流された胎盤の断面図である。 図4は、灌流された胎盤から回収された細胞を分別するための選択スキームである。

Claims (36)

  1. 単離し血液を除去した哺乳動物の胎盤から胎盤幹細胞を回収する方法であって、
    該胎盤を、灌流液を用いて灌流するが、ただし該胎盤は、灌流前に臍帯血を排液し、さらに洗い流して残存血液を除去したものであり、かつ、該胎盤の臍動脈および臍静脈の一方または両方に灌流液を通過させることによりその灌流を行うこと；および
    該胎盤から胎盤幹細胞および灌流液を回収すること；
    を含む、前記方法。
  2. 単離し血液を除去した哺乳動物の胎盤からCD34+ 胎盤幹細胞を回収する方法であって、
    該胎盤を、灌流液を用いて灌流するが、ただし該胎盤は、灌流前に臍帯血を排液し、さらに洗い流して残存血液を除去したものであり、該CD34+ 胎盤幹細胞は臍帯血から得られるものではなく、かつ、該胎盤の臍動脈および臍静脈の一方または両方に灌流液を通過させることによりその灌流を行うこと；および
    該胎盤からCD34+ 胎盤幹細胞および灌流液を回収すること；
    を含む、前記方法。
  3. 前記胎盤が、残存血液の除去後であって灌流前の少なくとも４時間にわたって4℃〜25℃で培養されている、請求項１記載の方法。
  4. 前記胎盤が、残存血液の除去後であって灌流前の少なくとも12時間にわたって4℃〜25℃で培養されている、請求項１記載の方法。
  5. 前記胎盤が、残存血液の除去後であって灌流前の少なくとも24時間にわたって4℃〜25℃で培養されている、請求項１記載の方法。
  6. 約30 ml〜約150 mlの第１の容量の前記灌流液を用いて灌流を行う、請求項１記載の方法。
  7. 約30 ml〜約150 mlの第２の容量の前記灌流液を用いる灌流を続けて行うことをさらに含み、その第２の容量は前記の第１の容量とは別に回収される、請求項６記載の方法。
  8. 灌流を複数回行う、請求項１記載の方法。
  9. 灌流を、それぞれの回について、約30 ml〜約150 mlの容量の前記灌流液を用いて行う、請求項８記載の方法。
  10. 前記灌流液から胎盤幹細胞を分離することをさらに含む、請求項６記載の方法。
  11. 灌流液が凝固防止剤を含む、請求項１記載の方法。
  12. 灌流液が凝固防止剤を含む、請求項６記載の方法。
  13. 灌流液が凝固防止剤を含む、請求項８記載の方法。
  14. 灌流液がヘパリン、エチレンジアミン四酢酸（EDTA）またはクレアチンリン酸デキストロース（CPDA）を含む、請求項１記載の方法。
  15. 単離した哺乳動物胎盤が、成功裡の分娩後に残存する分娩後胎盤である、請求項１に記載の方法。
  16. 灌流を、残存血液の除去後の少なくとも４時間行う、請求項２記載の方法。
  17. 灌流を、残存血液の除去後の少なくとも12時間行う、請求項２記載の方法。
  18. 灌流を、残存血液の除去後の少なくとも24時間行う、請求項２記載の方法。
  19. 約30 ml〜約150 mlの第１の容量の前記灌流液を用いて灌流を行う、請求項２記載の方法。
  20. 約30 ml〜約150 mlの第２の容量の前記灌流液を用いる灌流を続けて行うことをさらに含み、その第２の容量は前記の第１の容量とは別に回収される、請求項２記載の方法。
  21. 灌流を複数回行う、請求項２記載の方法。
  22. 灌流を、それぞれの回について、約30 ml〜約150 mlの容量の前記灌流液を用いて行う、請求項２１記載の方法。
  23. 灌流液が凝固防止剤を含む、請求項２記載の方法。
  24. 灌流液が凝固防止剤を含む、請求項１９記載の方法。
  25. 灌流液が凝固防止剤を含む、請求項２１記載の方法。
  26. 灌流液がヘパリン、エチレンジアミン四酢酸（EDTA）またはクレアチンリン酸デキストロース（CPDA）を含む、請求項２記載の方法。
  27. 単離した哺乳動物胎盤が、成功裡の分娩後に残存する分娩後胎盤である、請求項２に記載の方法。
  28. 前記胎盤幹細胞が多能性である、請求項１または２記載の方法。
  29. 前記胎盤幹細胞を前記灌流液から分離することをさらに含む、請求項７記載の方法。
  30. 前記胎盤幹細胞を最大48時間の期間にわたり回収する、請求項６記載の方法。
  31. 前記胎盤幹細胞を最大48時間の期間にわたり回収する、請求項７記載の方法。
  32. CD34+ 胎盤幹細胞を、CD34+ 胎盤幹細胞以外の細胞および前記灌流液から分離することをさらに含む、請求項２記載の方法。
  33. CD34+ 胎盤幹細胞を、前記灌流液から分離することをさらに含む、請求項１９記載の方法。
  34. CD34+ 胎盤幹細胞を、前記灌流液から分離することをさらに含む、請求項２０記載の方法。
  35. 前記CD34+ 胎盤幹細胞を最大48時間の期間にわたり回収する、請求項１９記載の方法。
  36. 前記CD34+ 胎盤幹細胞を最大48時間の期間にわたり回収する、請求項２０記載の方法。
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