JP7117020B2 - 臍帯間葉系幹細胞MSCsの培養法 - Google Patents
臍帯間葉系幹細胞MSCsの培養法 Download PDFInfo
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Description
前記初代培養手順で得られたP1初代細胞を収集し、残留培地を除去し、単細胞懸濁液を調製し、遠心分離により細胞ペレットを得て、この細胞ペレットに無血清培地を加え、低酸素条件下で継代まで培養し、P2~P3継代まで継続的に培養し、後続の各継代培養中にリグストラジン塩酸塩とシェンマイ注射液を加え、細胞が所定の細胞密度に増殖した場合、細胞を消化・収集する継代培養と、
前記継代培養手順で収集された細胞に表現型検査を実施したところ、CD31、HLA-DR、CD34及びCD45が陰性で、CD44、CD73、CD90及びCD105が陽性である細胞が標的MSCs細胞であり、使用に備える表現型検査を備える。
本発明に係る臍帯間葉系幹細胞MSCsの培養法は、低酸素の無血清条件下でMSCsを培養し、インビトロで培養されたMSCsの増殖とコロニー形成能力を向上させ、血管内の低酸素環境での増殖に適応する能力を向上させ、人体への注射後のMSCsの体内への移植率を高め、MSCs製剤を人体に注射した後、残存する動物血清により引き起こす恐れがある血清病を回避することができる。また、革新的に細胞培養液に、気を益し免疫力を高め、陰虚を解消し体液の分泌を促す漢方薬のシェンマイ注射液と、血行を促進し淤血を解消するという特性を持つ漢方薬のセンキュウからのリグストラジン塩酸塩を加え、性質が安定しているリグストラジン塩酸塩は、MSCs細胞の集塊、静脈内注射後の接着・集塊、赤血球連銭形成、細胞塊塞栓症の発生を効果的に防止し、臍帯間葉系幹細胞MSCsが心筋梗塞を予防・治療する能力を大幅に高める。
以下の方法で得られたMSC幹細胞。
一、ドナースクリーニング
1、ドナーは、サンプル採取前の1か月以内に、エイズウィルス(HIV)、B型肝炎ウイルス(HBV)、C型肝炎ウイルス(HCV)、トレポネーマパリダム(TP)、サイトメガロウイルス(CMV)、EBウイルス及び貪食T細胞ウイルス(HLTV)を含むが、これらに限定されない重症感染症検査のために採血し、7つの感染症検査のいずれかで、病原体を保有していることを示したドナー(以前にトレポネーマパリダムに感染したドナーを含む)が除外された。
上記のすべての条件を満たすものは適格ドナーである。
1、サンプル採取
スクリーニングに合格したドナーとサンプル寄付契約を締結し、ドナーが満期妊娠後、帝王切開により生産を行った。胎盤と臍帯組織は、専用滅菌サンプリングバッグに詰められ、適量の冷蔵された組織保護液を注射してから密封され、48時間以内に冷蔵輸送(2~8℃)で実験室に届けた。輸送中にサンプリングバッグが破れないこと、バッグ内の液体が漏れていないこと、サンプルがX線やγ線などの高エネルギー放射線に被ばくしたことがないことを確保する。
2.1 実験室に届けられたサンプルは、無菌の生物学的安全キャビネットで開かれ、事前に冷却された洗浄液で臍帯組織の血痕を洗浄した。
2.2 臍帯を2~3cmのものに切り、数回洗浄した。
2.3 組織鉗子で臍帯組織から動脈と静脈の血管を剥がし、臍帯からホウォートンゼリー組織を剥がし、事前に冷却された組織洗浄液に入れ、ホウォートンゼリー組織に表皮組織が混入できない。
1、初代培養:
1.1 ホウォートンゼリー組織をすべて剥がした後、事前に冷却された組織保護液で数回洗浄し、50ml遠心チューブに入れ、大きさが3×3×3mm3以下になるように組織を切った。
1.2 適量の無血清培地(100U/mlペニシリン/ストレプトマイシンを含む)(友康生物社製MSC幹細胞無血清専用培地)を細かく切り取った組織に加え、平均してT75培養フラスコで接種し、各フラスコにそれぞれ10mlの培養液を加え、酸素濃度3~10%で37℃の恒温二酸化炭素インキュベーターに入れ、酸素濃度5%の低酸素濃度下で培養することが好適である。
1.3 初代培養の組織は5日目に全部の培地を交換し、その後の3日目に半分の培地を交換した。通常、10日目に組織ブロックから遊走する細胞が現れ、15日目に細胞密度90%に増殖し継代基準に達することができる。
2.1 継代する培養フラスコ内の専用培地を収集し、3000rpmで10分間遠心分離し、上清を収集し使用に備えた。
2.2 培養フラスコ内の初代細胞を生理食塩水で2回洗浄し、残留培地を除去した。
2.3 3mlの0.05%トリプシンを加え細胞を消化し、培養フラスコを軽くたたいて壁に付着している細胞をすべて落とした。5mlの遠心分離済みの専用培地を加えトリプシンの働きを止め、細胞を軽く吹き付けて単細胞懸濁液を得た。
2.4 細胞懸濁液を収集し、培養フラスコを生理食塩水で2回洗浄し、液体全体を収集し、100μMフィルターで消化されていない組織ブロックを除去した。
2.5 ろ過された単細胞懸濁液を1300rpm×6minで遠心分離し、上清を除去した。
2.6 15mlの生理食塩水を細胞ペレットに加え細胞を再懸濁し、均一に混合した後、少量の液体を取り計数し、1300rpm×6 minで再度遠心分離し、上清を除去した。
2.7 無血清培地(抗生物質なし)を細胞ペレットに加え、500mlごとに細胞懸濁液を調製した。この細胞培養液は、友康生物社の間葉系幹細胞無血清専用培地でもよいし、アメリカンGIBCO社のDMEM、F12、DMEM/F12、RPMI1640基礎培地をベースにサイトカインを加えた培地でもよい。細胞計数の結果により、10000/cm2の密度でT175培養フラスコで接種し、酸素濃度3~10%で37℃の恒温二酸化炭素インキュベーターに入れ、酸素濃度5%の低酸素濃度下で培養することが好適である。
2.8 細胞が細胞密度90%(約3日)に増殖した後、再度継代培養し、P2継代まで継続的に培養した。
2.9 P3継代からP5継代まで、それぞれリグストラジン塩酸塩(ハルビン三聯薬業股分有限公司が準拠している国家規格の第2版増補、執行標準の国薬準字H20041175注射液、人体に直接注射可能)とシェンマイ注射液(雲南植物薬業有限公司が準拠している国家薬品規格(改訂)の公布文書WS3-B-3428-98-2010、執行標準の国薬準字Z53021721、人体に直接注射可能)を加えた。上記のリグストラジン塩酸塩の濃度が40~80Mg/Lであり、シェンマイ注射液の体積パーセンテージが0.5%である。24時間培養し、培地を交換してからさらに1週間培養し、細胞が細胞密度80~90%に増殖したとき細胞を収集し、消化後に収集された細胞を検査した。この手順で得られた生成物に病原体の生物学的検出を実施し、病原学的汚染を排除した。
3.1 上記の収集された細胞に表現型検査を実施し、CD31、HLA-DR、CD34及びCD45が陰性で、CD44、CD73、CD90及びCD105が陽性である細胞は、適格な標的MSC幹細胞である。
3.2 適格なMSC幹細胞を、計数の結果に従って、1×107 /mlの密度で専用凍結保存溶液に再懸濁した。凍結保存溶液に懸濁している単細胞を凍結保存用チューブ(1ml/本)に加えラベルを貼り付けた。ラベル情報は、細胞タイプ、細胞数、凍結保存世代、チューブあたりの凍結保存細胞数、凍結保存日などの情報を含むが、これらに限定されない。密封フィルムで凍結保存用チューブの口を密封し、プログラムフリーザーで-90℃までゆっくりと冷却し、その後、直接-205℃~-185℃(-196℃が好適である)の液体窒素で長期保存した。
この実施形態の臍帯MSC幹細胞のMSC品質管理は、2015年に中国食品医薬品監督管理局によって発行された「幹細胞製剤の品質管理及び臨床前研究に関するガイドライン(試行)」の幹細胞製剤の品質検査基準を参照した。
以下の方法で得られたMSCs幹細胞注射液。
1、準備:
1.1 恒温水槽で再加熱し、水温を37℃で安定させた。
1.2 30分早めに生物学的安全キャビネットのUV消毒と換気を行った。
1.3 蘇生した細胞数に応じて生理食塩水を調製し、遠沈管に入れ、生理食塩水の量を凍結保存溶液全体の10倍以上にした。
あらかじめ定められた要件に従って、液体窒素タンクから凍結保存細胞を取り出し、念のため細胞の種類、細胞コード、細胞数、細胞の世代などの重要な情報を注意深く確認した。
各複合製剤にそれぞれ低酸素MSCsの細胞が(0.5~1.5)×109個含まれ、各複合製剤の前記生理食塩水にそれぞれ10~20UMの低分子量ヘパリンナトリウムが含まれ、各複合製剤にそれぞれ18種のL-複合アミノ酸が含まれている。
前記複合アミノ酸注射液は18種のアミノ酸で構成され、L-イソロイシン、L-アルギニン、L-ロイシン、L-アスパラギン酸、L-リジン、L-システイン、L-グルタミン酸、L-メチオニン、L-ヒスチジン、L-フェニルアラニン、L-プロリン、L-スレオニン、L-セリン、L-トリプトファン、L-チロシン、L-バリン、L-グリシン、L-アラニンを含み、100mlの複合アミノ酸注射液あたり10±2gのアミノ酸を含む。(前記複合アミノ酸は、複合アミノ酸注射液18AA-IIIであり、市販で入手可能)
細胞を再懸濁し、集塊した細胞を40μMフィルターで除去し、単細胞懸濁液を得た。
心筋梗塞の治療におけるMSCs幹細胞注射液の応用。
本発明の臍帯間葉系幹細胞MSCs製剤は用途が広く、現在、実施形態1のMSCsを実施形態2に示す複合製剤に調製し、心筋梗塞患者の治療に適用することでその効果を観察している。
1、方法
対照群(MSC幹細胞培養時に、リグストラジン塩酸塩とシェンマイ注射液を加えなかった)と投与群(実施形態2の複合製剤)の被験者の末梢血を顕微鏡で観察した。
結果を図10~11に示し、図10は対照群の被験者の末梢血を示し、図11は投与群の被験者の末梢血を示した。図10から、対照群では、被験者の末梢血に細胞の接着・集塊、赤血球連銭形成、細胞塊塞栓が発生したことがわかった。図11から、血行を促進し淤血を解消し、気を益し陰虚を解消するという効果のあるリグストラジン塩酸塩とシェンマイ注射液で培養されたMSC幹細胞製剤を使用すると、被験者の赤血球形態が正常であり、集塊が発生しなかったことがはっきりとわかった。
上記の製剤は、仏山地域で心筋梗塞や重度の冠動脈疾患の数十例に使用されており、代表的な症例は2001年に発病した以来今まで生き残った。その症例は以下の通りである。
Claims (4)
- MSCsの培養手順を備え、前記MSCsの培養手順が、インビトロ培養により得られた臍帯のホウォートンゼリー組織を採取し、無血清培地で低酸素条件下で継代基準に達するまで培養する初代培養と、
前記初代培養手順で得られたP1初代細胞を収集し、残留培地を除去し、単細胞懸濁液を調製し、遠心分離により細胞ペレットを得て、この細胞ペレットに無血清培地を加え、低酸素条件下で継代まで培養し、P2~P3継代まで継続的に培養し、後続の各継代培養中にリグストラジン塩酸塩と国薬準字Z53021721のシェンマイ注射液を加え、細胞が所定の細胞密度に増殖した場合、細胞を剥離・収集する継代培養と、
前記継代培養手順で収集された細胞に表現型検査を実施したところ、CD31、HLA-DR、CD34及びCD45が陰性で、CD44、CD73、CD90及びCD105が陽性である細胞が標的MSCs細胞であり、使用に備える表現型を備え、
前記初代培養手順では、前記低酸素条件が、酸素濃度3~10%の二酸化炭素インキュベーターで培養することであり、前記継代基準が細胞密度80~90%に増殖することであり、
前記継代培養手順では、前記低酸素条件が、酸素濃度3~10%の二酸化炭素インキュベーターで培養することであり、添加された前記リグストラジン塩酸塩の濃度が40~80Mg/Lであり、添加された前記シェンマイ注射液の体積パーセンテージが0.5%であることを特徴とする臍帯間葉系幹細胞MSCsの培養法。 - 前記MSCsの培養手順では、前記無血清培地が、間葉系幹細胞無血清培地、またはDMEM、F12、DMEM/F12、RPMI1640培地をベースにサイトカインを加えた培地から選択されることを特徴とする請求項1に記載の臍帯間葉系幹細胞MSCsの培養法。
- 前記継代培養手順では、リグストラジン塩酸塩とシェンマイ注射液を加えてから、P4~P6継代まで継続的に培養することを特徴とする請求項1に記載の臍帯間葉系幹細胞MSCsの培養法。
- 前記MSCs培養手順の前に、インビトロ培養により得られた臍帯組織を採取し、冷蔵輸送を行い、サンプルが高エネルギー放射線に被ばくしたことがないことを確保し、組織内の血管を洗浄・剥離し、組織内のホウォートンゼリー組織を採取するMSCs調製手順を備え、前記MSCs培養手順の後に、得られた標的MSCsに細胞同定及び純度検出、細胞増殖活性検出、細菌とマイコプラズマ検出、エンドトキシン検出、外因性病原因子検出、異常免疫反応試験、腫瘍原性試験及び/または添加成分の残留含有量検出を実施する品質管理を備えることを特徴とする請求項1~3のいずれかに記載の臍帯間葉系幹細胞MSCsの培養法。
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