JP6851311B2 - 回転式撹拌培養法による血小板の製造方法 - Google Patents
回転式撹拌培養法による血小板の製造方法 Download PDFInfo
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Description
システムのような薬剤応答性の遺伝子発現誘導システムを用いる場合、強制発現する工程においては、対応する薬剤、例えば、テトラサイクリンまたはドキシサイクリンを培地に含有させ、これらを培地から除くことによって強制発現を抑制してもよい。
1−1.iPS細胞からの造血前駆細胞の調製
ヒトiPS細胞(TKDN SeV2:センダイウイルスを用いて樹立されたヒト胎児皮膚繊維芽細胞由来iPS細胞)から、Takayama N., et al. J Exp Med. 2817-2830 (2010)に記載の方法に従って、血球細胞への分化培養を実施した。即ち、ヒトES/iPS細胞コロニーを20ng/mL VEGF (R&D SYSTEMS)存在下でC3H10T1/2フィーダー細胞と14日間共培養して造血前駆細胞(Hematopoietic Progenitor Cells;HPC)を作製した。培養条件は20% O2、5% CO2で実施した(特に記載がない限り、以下同条件)。
遺伝子導入システムは、レンチウイルスベクターシステムを利用した。レンチウイルスベクターは、Tetracycline制御性のTet-on(登録商標)遺伝子発現誘導システムベクターである。LV-TRE-mOKS-Ubc-tTA-I2G(Kobayashi, T., et al. Cell 142, 787-799 (2010))のmOKSカセットをc-MYC、BMI1、BCL-xLに組み替えることで作製した。それぞれ、LV-TRE-c-Myc-Ubc-tTA-I2G、LV-TRE-BMI1-Ubc-tTA-I2G、およびLV-TRE-BCL-xL-Ubc-tTA-I2Gとした。
予めC3H10T1/2フィーダー細胞を播種した6 well plate上に、上記の方法で得られたHPCを5x104cells/wellずつ播種し、レンチウイルス法にてc-MYCおよびBMI1を強制発現させた。このとき、細胞株1種類につき6 wellずつ使用した。即ち、それぞれMOI 20になるように培地中にウイルス粒子を添加し、スピンインフェクション(32℃ 900rpm、60分間遠心)で感染させた。本操作は、12時間おきに2回実施した。
上記の方法でcMYC及びBMI1ウイルス感染を実施した日を感染0日目として、以下の通り、cMYC及びBMI1遺伝子導入型巨核球細胞を培養することで、巨核球自己増殖株をそれぞれ作製した。BMI1遺伝子、c-MYC遺伝子の強制発現は、培地にドキシサイクリン(clontech #631311) 1μg/mLを加えることにより行った。
ピペッティングにて上記の方法で得られたウイルス感染済み血球細胞を回収し、1200rpm、5分間遠心操作を行って上清を除去した後、新しい分化培地で懸濁して新しいC3H10T1/2フィーダー細胞上に播種した(6well plate)。感染9日目に同様の操作をすることによって継代を実施した。細胞数を計測後1×105 cells/2mL/wellでC3H10T1/2フィーダー細胞上に播種した(6well plate)。
感染2日目と同様の操作を実施した。細胞数を計測後3×105cells/10mL/100mm dishでC3H10T1/2フィーダー細胞上に播種した(100mm dish)。
ウイルス感染済み血球細胞を回収し、細胞1.0×105個あたり、抗ヒトCD41a-APC抗体(BioLegend)、抗ヒトCD42b-PE抗体(eBioscience)、抗ヒトCD235ab-pacific blue(BioLegend)抗体をそれぞれ2μL、1μL、1μLずつを用いて抗体反応した。反応後に、FACS Verse(BD)を用いて解析した。感染14日目において、CD41a陽性率が50%以上であった細胞を、巨核球自己増殖株とした。
前記感染14日目の巨核球自己増殖株に、レンチウイルス法にてBCL-xLを遺伝子導入した。MOI 10になるように培地中にウイルス粒子を添加し、スピンインフェクション(32℃ 900rpm、60分間遠心)で感染させた。BCL-xL遺伝子の強制発現は、培地にドキシサイクリン (clontech #631311) 1μg/mLを加えることにより行った。
・感染14目〜感染18日目
前述の方法で得られたBCL-xLを遺伝子導入した巨核球自己増殖株を回収し、1200rpm、5分間遠心操作を行った。遠心後、沈殿した細胞を新しい分化培地で懸濁した後、新しいC3H10T1/2フィーダー細胞上に2×105cells/2mL/wellで播種した(6well plate)。
細胞数を計測後、3×105 cells/10mL/100mm dishで播種した。
細胞数を計測後、1×105 cells/10mL/100mm dishで播種した。以後、4-7日毎に継代を行い、維持培養を行った。
FBS(シグマ#172012 lot.12E261)15%
L-Glutamin (Gibco #25030-081) 2mM
ITS (Gibco #41400-045) 100倍希釈
MTG (monothioglycerol, sigma #M6145-25ML) 450μM
アスコルビン酸 (sigma #A4544) 50μg/mL
Puromycin (sigma #P8833-100MG) 2μg/mL
SCF (和光純薬 #193-15513) 50ng/mL
TPO様作用物質 200ng/mL
培養条件は、37℃、5%CO2とした。
FBS 15%
L-Glutamin (Gibco #25030-081) 2mM
ITS (Gibco #41400-045) 100倍希釈
アスコルビン酸 (sigma #A4544) 50μg/mL
SCF (和光純薬 #193-15513) 50ng/mL
TPO様作用物質 200ng/mL
ADAM阻害剤 15μM
SR1 750nM
ROCK阻害剤 5μM
3−1.血小板の測定
正常血小板、劣化血小板、血小板の生理活性の測定のために、1.5 mLマイクロチューブに希釈液 900 μLを添加し、そこに培養上清100 μLを添加し、混合した。希釈混合した培養上清200 μLをFACSチューブに分注し、以下の標識抗体或はタンパク質を添加して染色を行った。異常血小板の測定のために、培養上清 100 μLをFACSチューブに分注し、以下の標識抗体或はタンパク質を添加して染色を行い、フローサイトメーター分析直前にAnnexin V binding buffer(BD)で5倍希釈し、分析した。
正常血小板および劣化血小板の測定
0.5μL 抗CD41抗体 APC標識(Bio Legend 303710)
0.5μL 抗CD42a抗体PB標識(eBioscience 48-0428-42)
0.5μL 抗CD42b抗体PE標識(eBioscience 12-0428-42)
血小板の生理活性の測定
0.5μL 抗CD42a抗体PB標識(eBioscience 48-0428-42)
0.5μL 抗CD42b抗体PE標識(eBioscience 12-0428-42)
0.5μL 抗CD62p抗体APC標識(Bio Legend 304910)
10μL 抗PAC-1抗体FITC標識(BD 303704)
異常血小板数の測定
0.5μL 抗CD41抗体 APC標識(Bio Legend 303710)
0.5μL 抗CD42a抗体PB標識(eBioscience 48-0428-42)
5μL Annexin V FITC標識(BD, 556419)
血小板の刺激は、PMA 0.2 μM (Phorbol 12-myristate 13-acetate, sigma #P1585-1MG)、又は、ADP 100 μM(sigma #A2754)およびThrombin 0.5 U/mL(sigma)で室温にて行った。刺激30分後にBD社FACSverceにて測定を実施した。
CD42a陽性の血小板画分における、刺激前後のPAC-1陽性率及びCD62p陽性率を測定し、比較評価した。
結果を図1に示す。PMAもしくはADP/Thrombin刺激時のPAC-1陽性率のMFIは、BCPによる撹拌培養の場合、125mLシェーカーフラスコでの振とう培養条件の場合よりも、約2〜3倍向上した。
また、図2に示すとおり、BCPによる撹拌培養では、シェーカーフラスコでの振とう培養に比較して、CD62p陽性率も2倍以上高く、生理活性の高い血小板が得られたことが示された。
前記3−1.の方法によって、各処理サンプルをフローサイトメーターを用いて分析し、CD41a陽性CD42b陽性の粒子数を正常血小板数、CD41a陽性CD42b陰性の粒子数を劣化血小板数として、算出した。
結果を図3に示す。正常血小板の産生効率は、振とう培養の場合に比べて、BCPによる撹拌培養の場合、約1.7倍に増加した。一方、劣化した血小板は、振とう培養の場合に比べて、BCPによる撹拌培養の場合、約0.75倍に減少した。
前記3−1.の方法によって、各処理サンプルをフローサイトメーターを用いて分析し、アネキシンV陽性の粒子数を異常血小板数とした。結果を図4に示す。振とう培養の場合に比べて、BCPによる撹拌培養の場合、異常血小板数は、約0.7倍に減少した。
BCP撹拌培養装置の撹拌回転数を変えたときの血小板生産効率の違いを測定した。撹拌回転数を100rpm、120rpm、140rpmとして、CD41a陽性CD42b陽性粒子を測定した結果を図5に示す。
最も回転数が高い140rpmのときに、血小板(CD41a陽性CD42b陽性)の生産量が最も高くなった。
Claims (7)
- 血小板の製造方法であって、
培養容器内の培養液中で巨核球細胞を培養する工程を含み、
前記培養工程において、前記培養液を撹拌羽根で撹拌する、方法。 - 前記撹拌羽根が、平羽根タービン型翼、プロペラ型翼、矢じり羽根型、曲がり羽根型翼又は円盤付き片平羽根タービン型翼である、請求項1に記載の方法。
- 前記培養容器が、密閉型バイオリアクターである、請求項1又は2に記載の方法。
- 前記撹拌子を、回転数50rpm以上で回転させる、請求項1から3のいずれか1項に記載の方法。
- 前記巨核球細胞が、
巨核球細胞より未分化な細胞において、癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子からなる群より選択される遺伝子の少なくとも1つを強制発現した後、当該強制発現を解除した細胞である、請求項1から4のいずれか1項に記載の方法。 - 血小板製剤の製造方法であって、
請求項1から5のいずれか1項に記載の方法で巨核球細胞に血小板を産生させ、培養物から血小板を回収する工程と、
前記血小板から血小板以外の血球系細胞成分を除去する工程と、を含む方法。 - 血液製剤の製造方法であって、
請求項6に記載の方法で血小板製剤を製造する工程と、
前記血小板製剤を他の成分と混合して血液製剤を得る工程と、を含む方法。
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CA2998463A1 (en) | 2017-03-23 |
JPWO2017047492A1 (ja) | 2018-07-05 |
RU2018113461A (ru) | 2019-10-18 |
SG11201801939SA (en) | 2018-04-27 |
US10570372B2 (en) | 2020-02-25 |
KR102641631B1 (ko) | 2024-02-29 |
RU2756000C2 (ru) | 2021-09-24 |
EP3351627A4 (en) | 2019-03-27 |
WO2017047492A1 (ja) | 2017-03-23 |
KR20180044427A (ko) | 2018-05-02 |
US20180258395A1 (en) | 2018-09-13 |
EP3351627A1 (en) | 2018-07-25 |
CN108026511B (zh) | 2021-09-24 |
RU2018113461A3 (ja) | 2020-02-20 |
CN108026511A (zh) | 2018-05-11 |
AU2016324365A1 (en) | 2018-04-12 |
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