JP5941454B2 - 対象の造血系を増強させるための医薬組成物 - Google Patents
対象の造血系を増強させるための医薬組成物 Download PDFInfo
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Description
骨髄、胎盤組織および脂肪組織からの接着性間質細胞(ASC)の作製および培養
接着性細胞を、3D担体を含有するバイオリアクターにおいて培養して、特異的な細胞マーカー発現プロフィルにより特徴づけられる3D−ASC細胞を作製した。成長効率を細胞計数によって調べた。これらの細胞の分化能を、分化培地で培養することによって調べた。
骨髄間質細胞−骨髄(BM)間質細胞を、開心術またはBM生検を受ける血液学的に健康なドナーの吸引された胸骨骨髄から得た。骨髄吸引物をハンクス平衡塩溶液(HBSS;GIBCO BRL/Invitrogen、Gaithersburg、MD)で3倍希釈し、Ficoll−Hypaque(Robbins Scientific Corp.、Sunnyvale、CA)密度勾配遠心分離に供した。その後、骨髄単核細胞(1.077gm/cm3未満)を集め、HBSSで3回洗浄し、成長培地[10%のFCS(GIBCO BRL)、10−4Mのメルカプトエタノール(Merck、White House Station、NJ)、Pen−Strep−ナイスタチン混合物(100U/ml:100ug/ml:1.25un/ml;Beit Ha’Emek)、2mMのL−グルタミン(Beit Ha’Emek)が補充されたDMEM(Biological Industries、Beit Ha’emek、イスラエル)]に再懸濁した。個々のドナーからの細胞を、培養培地を毎週交換しながら、37℃(5%CO2)で組織培養フラスコ(Corning、Acton、MA)において別々にインキュベートした。細胞を、0.25%トリプシン−EDTA(Beit Ha’Emek)を使用して3〜4日毎に剥がした。2〜40回の継代培養の後、60〜80%のコンフルエンスに達したとき、細胞を、分析のために、または、バイオリアクターにおける培養のために集めた。
PluriX(商標)バイオリアクターシステムは生理学的な類似する微小環境をもたらす。
接着性細胞のための効率的な培養条件を与えるために、生理学的に類似する微小環境(図1aに示す)が、PluriX(商標)バイオリアクター(Pluristem、Haifa、イスラエル;担体が図1gに例示され、播種前が図1bに示される)を使用して人工的に作製された。図1c〜fに示されるように、播種後の20日(図1c〜d、それぞれ150倍および250倍に拡大)および40日(図1e〜f、それぞれ350倍および500倍に拡大)において、骨髄から作製された3D−ASC細胞が首尾良く培養され、3Dマトリックス上で拡大培養された。
HSCの生着を改善する胎盤由来の3D−ASCの能力の評価
3D−ASCがHSC生着を支持することを、亜致死量の放射線が照射された免疫欠損NOD−SCIDマウス、または、化学療法により前処理された免疫欠損NOD−SCIDマウスにおいて検出されるヒト造血細胞(hCD45+)のレベルによって評価した。
CD34+細胞の単離−臍帯血サンプルを出産時に無菌条件下で採取し(Bnei Zion Medical Center、Haifa、イスラエル)、単核細胞を、Lymphoprep(Axis−Shield PoC As、Oslo、ノルウェー)密度勾配遠心分離を使用して分画し、冷凍保存した。解凍した単核細胞を洗浄し、抗CD34抗体とインキュベートし、midiMACS(Miltenyl Biotech、Bergish Gladbach、ドイツ)を使用して単離した。2つ以上のサンプルからの細胞を、所望の数(50000〜100000個の細胞)を達成するためにプールした。
3D−ASCは放射線照射マウスにおいてHSCの生着を改善した−ヒトCD34+造血細胞と、胎盤組織または脂肪組織に由来する3D−ASCとを、放射線照射されたNOD−SCIDマウスに同時移植した。生着効率を同時移植後4週間で評価し、HSCが単独で移植されたマウスと比較した。表2および図6に示すように、3D−ASCおよびUCB CD34+細胞の同時移植は、UCB CD34+細胞だけにより処置されたマウスと比較したとき、レシピエントマウスのBMにおける相当高い生着率およびより高いレベルのヒト細胞をもたらした。
2D培養のASCおよび3D培養のASCによるリンパ球応答の抑制
接着性細胞、具体的には、3D−ASCは、ヒト臍帯血単核細胞の免疫反応をMLRアッセイにおいて抑制することが見出された。
混合リンパ球反応(MLR)アッセイ−胎盤から作製された、2D由来培養手法のASCおよび3D由来培養手法のASCの免疫抑制特性および免疫特権特性を、応答(増殖性)細胞および刺激(非増殖性)細胞の混合培養における不適合リンパ球の増殖率によって行われるような、HLA座における組織適合性を測定するMLRアッセイによって求めた。ヒト臍帯血(CB)単核細胞(2×105個)を応答細胞として使用し、等量(105個)の放射線照射(3000Rad)されたヒト末梢血由来単球(PBMC)と、あるいは、胎盤から作製された2D培養もしくは3D培養の接着性細胞、または、接着性細胞およびPBMCの組合せと同時培養することによって刺激した。それぞれのアッセイを3回繰り返した。細胞を96ウエルプレートにおいてRPMI1640培地(20%のFBSを含有する)で(加湿5%CO2雰囲気のもと、37℃で)4日間にわたって同時培養した。プレートを、培養の最後の18時間、1μCの3H−チミジンによりパルス処理した。その後、細胞をガラス繊維フィルター上に集め、チミジンの取り込みをシンチレーションカウンターにより定量した。
図8は、PBMCにより刺激されたとき、これらの細胞の増殖の高まりによって表されるようなCB細胞の免疫応答を示す。これは、理論によって拘束されることはないが、おそらくは、HLA不適合性に対する応答におけるT細胞の増殖に関連する。しかしながら、相当低いレベルの免疫応答が、本発明の接着性細胞とインキュベートされたとき、これらの細胞によって示された。そのうえ、PBMCに対するCBの免疫応答が、これらの接着性細胞と同時インキュベートされたとき、実質的に減少した。従って、MSCに類似した様式で、ASCは、GvHDに典型的なドナー細胞のT細胞増殖を減少させる潜在的能力を有することが見出された。両方の培養物(2Dおよび3D)はリンパ球の免疫応答を減少させたが、また、本明細書中上記で記載される3D−ASCの他の利点と一致して、3D ASCはより免疫抑制的である。
Claims (8)
- インスリン依存性糖尿病、多発性硬化症、自己免疫性脳脊髄炎(EAE)、全身性エリテマトーデス(SLE)、リウマチ様関節炎、全身性硬化症、シェーグレン症候群、重症筋無力症(MG)、ギラン・バレー症候群(GBS)、橋本甲状腺炎(HT)、グレーヴズ病および炎症性腸疾患からなる群より選択される自己免疫疾患、または移植片対宿主病(GvHD)の治療において使用するための、治療有効量の接着性の未分化の間質細胞を含む医薬組成物であって、前記接着性の未分化の間質細胞の起源は胎盤組織であり、前記接着性の未分化の間質細胞は、細胞の拡大培養を支持する不織布マトリックスから作られた担体を含む三次元培養条件下で培養されたものである、医薬組成物。
- 前記不織布マトリックスが合成材料から作られる、請求項1に記載の医薬組成物。
- 前記三次元培養条件が3Dバイオリアクターを含む、請求項1に記載の医薬組成物。
- 前記培養を少なくとも3日間行う、請求項1に記載の医薬組成物。
- 前記培養を前記接着性細胞が少なくとも60%のコンフルエンスに達するまで行う、請求項1に記載の医薬組成物。
- 前記接着性細胞がCD73、CD90、CD29およびCD105からなる群から選択される陽性のマーカー発現アレイを含む、請求項1に記載の医薬組成物。
- 前記接着性細胞がCD45、CD80、HLA-DR、CD11b、CD14、CD19、CD34およびCD79からなる群から選択される陰性のマーカー発現アレイを含む、請求項1に記載の医薬組成物。
- 前記接着性の未分化の間質細胞がFlt-3リガンド、IL-6、およびSCFを培養培地中に分泌する、請求項1に記載の医薬組成物。
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