著者はXvivo囲まれた細胞培養系を使用することを学んで彼らの助けのためのBiospherixのスタッフ、特にマット・フリーマンを承認したいと思います。実験室でのインフラストラクチャを設定する際に自分の仕事のためのマイルズ＆ケリー建設会社、Inc.のスタッフ、特にラス・ヒューズ。実験室の改造を調整中で自分の仕事のための設備とサポートサービスの小児病院オレンジ郡の部門のスタッフは、特にアダムLukhardとデヴィンHugie。データ管理インフラストラクチャおよびリモートアクセスを設定する際に彼らの助けのための情報システムのオレンジ郡部門の小児病院のスタッフは、特にベトナムトラン。オレンジカウンティエグゼクティブ・マネジメントチームの小児病院プロジェクトの彼らの長年の支援、特に博士マリア・ミノンやブレントDethlefsため。この作品は、子供のオレンジ郡の病院や再生Medicinためのカリフォルニア工科大学によって資金を供給されましたPHSへの補助金TR3-05476を通じて電子。すべての著者は、この作品にも同様に貢献しました。

<ol>
	<li>Brick, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Autism+Spectrum+Disorders+Stem+Cell+Resource+at+Children's+Hospital+of+Orange+County:+Implications+for+Disease+Modeling+and+Drug+Discovery.">The Autism Spectrum Disorders Stem Cell Resource at Children's Hospital of Orange County: Implications for Disease Modeling and Drug Discovery.</a> Stem Cells Transl.Med. 3 (11), 1275-1286 (2014).</li><li>Nethercott, H. E., Brick, D. J., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+induced+pluripotent+stem+cells+by+lentiviral+transduction.">Derivation of induced pluripotent stem cells by lentiviral transduction.</a> Methods Mol.Biol. 767, 67-85 (2011).</li><li>Pistollato, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+and+HIF1alpha+repress+the+differentiative+effects+of+BMPs+in+high-grade+glioma.">Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma.</a> Stem Cells. 27 (1), 7-17 (2009).</li><li>Pistollato, F., Chen, H. L., Schwartz, P. H., Basso, G., Panchision, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+tension+controls+the+expansion+of+human+CNS+precursors+and+the+generation+of+astrocytes+and+oligodendrocytes.">Oxygen tension controls the expansion of human CNS precursors and the generation of astrocytes and oligodendrocytes.</a> Mol.Cell Neurosci. 35, 424-435 (2007).</li><li>Raval, J. S., Koch, E., Donnenberg, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+non-viable+airborne+particles+correlates+with+airborne+colonies+and+represents+an+acceptable+surrogate+for+daily+assessment+of+cell-processing+cleanroom+performance.">Real-time monitoring of non-viable airborne particles correlates with airborne colonies and represents an acceptable surrogate for daily assessment of cell-processing cleanroom performance.</a> Cytotherapy. 14 (9), 1144-1150 (2012).</li><li>Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+stem+cell+therapies+for+neurological+diseases.">The potential of stem cell therapies for neurological diseases.</a> Expert.Rev.Neurother. 6, 153-161 (2006).</li><li>Schwartz, P. H., Brick, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+therapies+for+the+lysosomal+storage+diseases+-+the+quintessential+neurodegenerative+diseases.">Stem cell therapies for the lysosomal storage diseases - the quintessential neurodegenerative diseases.</a> Curr.Stem Cell Res.Ther. 3 (2), 88-98 (2008).</li><li>Schwartz, P. H., Brick, D. J., Nethercott, H. E., Stover, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+human+embryonic+stem+cell+culture.">Traditional human embryonic stem cell culture.</a> Methods Mol.Biol. 767, 107-123 (2011).</li><li>Stover, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Process-based+expansion+and+neural+differentiation+of+human+pluripotent+stem+cells+for+transplantation+and+disease+modeling.">Process-based expansion and neural differentiation of human pluripotent stem cells for transplantation and disease modeling.</a> J Neurosci.Res. 91 (10), 1247-1262 (2013).</li><li>Stover, A. E., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+human+pluripotent+stem+cells+to+feeder-free+conditions+in+chemically+defined+medium+with+enzymatic+single-cell+passaging.">Adaptation of human pluripotent stem cells to feeder-free conditions in chemically defined medium with enzymatic single-cell passaging.</a> Methods Mol.Biol. 767, 137-146 (2011).</li><li>Yoshida, Y., Takahashi, K., Okita, K., Ichisaka, T., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+enhances+the+generation+of+induced+pluripotent+stem+cells.">Hypoxia enhances the generation of induced pluripotent stem cells.</a> Cell Stem Cell. 5 (3), 237-241 (2009).</li></ol>

The authors declare that they have no competing financial interests.

彼らはバックチャンバを顕微鏡とする処理チャンバにインキュベーターから移動すると、CPF内で増殖した細胞は、酸素や二酸化炭素の濃度に変化は見られません。各チャンバー内の条件は、細胞をインキュベーターから削除される前にセルが保持されている特定のインキュベーターに一致していることが重要です。装置内の雰囲気は、連続的にHEPAろ過され、酸素と二酸化炭素濃度に関してはカスタマイズ可能です。細胞は、それぞれ、標準のPSCまたはNSCのための濃度は、5％と9％で成長させることができます。または代替の濃度が異なる細胞タイプまたは特定の実験のために選択することができます。したがって、この装置は、医療グレードの酸素、二酸化炭素、及び窒素（ 図4）の一定の源が供給されます。これらのガスのすべての3つは、一定の供給を確保ガス固有のマニホールドシステムによって供給されています。装置はまた、からなる校正ガス混合物が供給され酸素10％（±0.01％）、二酸化炭素。マニホールドシステムは、セル製造施設の外に収容され、ガスは、天井を介して施設にパイプされています。校正ガスは、施設内に収容されています。装置は、さらにまた、天井を通して、ハウス真空が供給されています。ユニットを送信する電子監視システム及び無線を使用して、すべてのマニホールドの出力圧力が常に監視されています。任意の圧力が範囲から外れる場合には、セル生産設備のオペレータは、自動的に電話をし、適切な処置をとることができるよう通知されます。 装置の電力要件は、6つの専用の120 V回路天井から下降し、一定の供給を確保するために、病院のバックアップ発電機に接続することによって満たされます。装置の動作は、無停電電源装置から給電PCベースのコンピュータにソフトウェアを介して制御されます。これらの電源とコンピュータの手配でも公共の電源システムに障害が発生した場合に継続的にシステム関数ことを確認してください。装置を制御するソフトウェアは、酸素と二酸化炭素濃度の制御、ならびに温度、湿度、及びチャンバ圧力を可能にするユーザーフレンドリーなグラフィカル・インターフェース（ 図1）を有しています。すべてのこれらのパラメータの値は、連続的に、すべての装置パラメータの実行記録を提供するために記録されています。このデータは、それらの完全性を保護するために毎晩、リモートサーバー上にバックアップされます。コンピュータとソフトウェアが評価および/または任意のパラメータを変更するには、管理ユーザーがリモートでアクセスすることができます。また、コンピュータとソフトウェアは、装置パラメータの対話的な評価を可能にし、ローカルユーザーとトラブルシューティング、リモートからアクセスすることができます。追加の警報送信ユニットを装置に接続されたセル生産設備のオペレータは装置の任意の範囲外の状態が通知されるように。リモートアクセスCapabilitiesは、ログインして範囲外の状態を具体的に評価することができます。 装置は、モジュラーシステムの両方マクロとミクロの意味で設計されています。このようなインキュベータや処理室のような個々の細胞培養モジュールは、互いに対してそれらの寸法および要件に関して、ならびにそれらのレイアウトにカスタマイズすることができます。さらに、個々のモジュールの制御機能の多くは、それ自体は、モジュール、そのような個々の雰囲気ガスコントローラは、例えば、容易にシステムに重大な影響を与えることなく交換することができることです。 そのような細胞培養物の顕微鏡可視化および操作のための1つのような特殊な処理チャンバは、容易にシステムに適合されます。細胞は生きて染色することができ、そしてコロニーをt内部同じ大気条件に切開することができるように、位相差および蛍光顕微鏡の両方がシステム（ 図6）内にあります彼はインキュベーター。処理室の側壁で密封されたグロメットを介して、ケーブルの配線は、通常はカート（ 図6）に、このような電源やコンピュータなどの機器は、装置の外に保つことができます。 セル生産設備内の処理室は、従来のBSCとは異なる空気流パターンを有します。従来のBSCで、空気の流れは、中央排気口から流下した後、キャビネットの床の前方及び後方部分に二つの異なる吸気口によって取り込まれている2つの別々の流れ、に分かれます。これとは対照的に、CPFは、天井の前方部分で単一の通気口があります。空気が下向きにし、それがその後、吸気口の中に上向きに描かれている室の奥に向かって流れます。 CPFは、本質的に非常にきれいであるが、このユニークな気流パターンは、技術者がわずかに汚染のリスクを低減するために、それらの技術を調整しなければならないことを意味します。従来のBSC、ラボワーカーの場合と同様にhouldの回避は、オープン細胞培養プレートとメディアボトルの上流に手を置きます。しかし、上流の方向は、CPFに変更されましたセル生産設備の研究室自体はかなり標準であり、-20℃の冷凍庫を装備しています、-80℃の冷凍庫、4℃の冷蔵庫、遠心分離、および水浴。研究室では、便利なハンズフリー操作用のフット・コントロールとのシンクがあります。この研究室では、機能の臨床セル生産施設となるようにするためには、しかし、いくつかの追加の修正がまだなされなければなりません。まず、装置自体が揮発性有機化合物、微粒子、及び汚染除去のために使用される二酸化塩素の濃度を監視する能力を有するようにアップグレードされなければなりません。第二に、FACS機を含む処理チャンバは、バッファモジュールを介して収容し、装置の残りの部分に接続されてもよいです。これは、細胞選別とTRの精製を可能にします適切な環境条件下でansplantable細胞集団。最後に、装置全体が柔らかい壁クリーンルーム内に収容されなければなりません。これは装置5のための国際標準化機構（ISO）クラス8環境を提供します。 CPFの高い無菌性およびコンピュータ制御の性質は、細胞ベースの治療法との良好な製造プロセスと将来のアプリケーションのための理想的なシステムになります。汚染の危険性が大幅に緩和されるが、より重要なことには、細胞増殖の条件は、自動的に記録され、コンピュータシステムによってアーカイブされます。ガス濃度、温度、湿度、およびシステムへのアクセスのすべてのイベントの偏差は厳密に文書化されています。製品の品質上の問題を調査するときに非常に役立つことができます。しかし、まだ制限があります。任意および全ての試薬 ​​および消耗品（ 例えば、培地成分、ピペット、プレート）の使用量は、別々に文書化されなければなりません。加えますitionally、CPFの監視システムによって文書の変数とは全く無関係である生じる可能性がある（ヒューマンエラーの多くの形態を含む）の潜在的な問題が多数あります。このように、高度に訓練された人材とタスクの詳細なマニュアル文書化の必要性は、所定の位置に残ります。

Standard stem cell culture techniques suffer from several environmental constraints that place undue stresses on the cells and expose the cells to unacceptable risks of contamination. Among the stresses that cells may endure under standard cell culture conditions are precipitous changes in the levels of carbon dioxide and oxygen concentrations3,4. This occurs when the cells are moved from the incubator to the biosafety cabinet and/or microscope which may not be optimal for the cells. Previous studies have confirmed the advantages of culturing both pluripotent and neural stem cells in hypoxic conditions4,11, and for best results, these conditions need to be continuous. Moreover, risks of cellular contamination are higher as the laboratory environment and personnel impinge upon the cells at almost every step of their culture and manipulation. Traditional clean rooms comprise one effective method to greatly decrease contamination risks but they are expensive, have a large footprint and fail to address stressors related to carbon dioxide and oxygen concentrations. 
A cell production facility that can address both contamination risks and gas concentrations and that can be qualified to meet cGMP criteria9 provides high quality cells for basic science research as well as clinical applications1,6,7. Such a cell production facility consists, at a minimum, of the following components: a process chamber, which acts as a heated workspace for the feeding and manipulation of cell cultures; a laminar flow hood, for the initial sterilization of reagents, tubes, and tools; two buffering airlock chambers in between the hood and the process chamber; two cell culture incubators accessible from the process chamber; a microscope chamber adjacent to the process chamber; and finally, computer software to set and monitor the conditions within these modules. Using this basic infrastructure, a wide variety of tasks can be performed, such as standard feeding and passaging of pluripotent stem cells and multipotent neural stem cells, as well as more specialized methods like Sendai virus-based reprogramming, in vitro migration studies, and differentiation of neural stem cells for electrophysiological characterization.

<table>
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xvivo System</td>
			<td>Biospherix</td>
			<td>custom made</td>
			<td></td>
		</tr>
		<tr>
			<td>Xvivo Software</td>
			<td>Biospherix</td>
			<td>version i.o.2.1.2.1</td>
			<td></td>
		</tr>
		<tr>
			<td>O2 Manifold</td>
			<td>Amico</td>
			<td>P-M2H-C3-S-U-OXY</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Manifold</td>
			<td>Amico</td>
			<td>M2H-C3-D-U-CO2</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 Manifold</td>
			<td>Western Innovator</td>
			<td>CTM75-7-2-2-BM</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope with DP21 camera and fluorescence</td>
			<td>Olympus Corporation</td>
			<td>CKX41</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 Glutamax</td>
			<td>Life Technologies</td>
			<td>10565-018</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro hESC Supplement</td>
			<td>Life Technologies</td>
			<td>A100006-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Millipore</td>
			<td>SCR005</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Sodium</td>
			<td>Hyclone</td>
			<td>9236</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast Growth Factor 2</td>
			<td>R&#38;D Systems</td>
			<td>AFL233</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Protide</td>
			<td>PP1130</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s-based Cell dissociation Buffer</td>
			<td>Life Technologies</td>
			<td>13150-016</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor</td>
			<td>R&#38;D Systems</td>
			<td>AFL236</td>
			<td></td>
		</tr>
		<tr>
			<td>Oct-3/4 Antibody</td>
			<td>Millipore</td>
			<td>AB3209</td>
			<td></td>
		</tr>
		<tr>
			<td>TRA-1-60 Antibody</td>
			<td>Millipore</td>
			<td>MAB4260</td>
			<td></td>
		</tr>
		<tr>
			<td>SSEA4 Antibody</td>
			<td>Millipore</td>
			<td>MAB4304</td>
			<td></td>
		</tr>
		<tr>
			<td>BIT-9500 Serum Supplement</td>
			<td>Stemcell Technologies</td>
			<td>9500</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Consumable Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2mL Serological pipet</td>
			<td>VWR</td>
			<td>89130-894</td>
			<td></td>
		</tr>
		<tr>
			<td>5mL Serological pipet</td>
			<td>Olympus Plastics</td>
			<td>12-102</td>
			<td></td>
		</tr>
		<tr>
			<td>10mL Serological pipet</td>
			<td>Olympus Plastics</td>
			<td>12-104</td>
			<td></td>
		</tr>
		<tr>
			<td>25mL Serological pipet</td>
			<td>Olympus Plastics</td>
			<td>12-106</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL Serological pipet</td>
			<td>Olympus Plastics</td>
			<td>12-107</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Corning</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 flask</td>
			<td>TPP</td>
			<td>90026</td>
			<td></td>
		</tr>
		<tr>
			<td>T-75 flask</td>
			<td>TPP</td>
			<td>90076</td>
			<td></td>
		</tr>
		<tr>
			<td>20uL pipet tips</td>
			<td>Eppendorf</td>
			<td>22491130</td>
			<td></td>
		</tr>
		<tr>
			<td>200uL pipet tips</td>
			<td>Eppendorf</td>
			<td>22491148</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 pipet tips</td>
			<td>Eppendorf</td>
			<td>22491156</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovials</td>
			<td>Thermo Scientific</td>
			<td>5000.102</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>BDH</td>
			<td>BDH1164-4LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Sanimaster 4</td>
			<td>Ecolab</td>
			<td>65332960</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Clorox</td>
			<td>A714239</td>
			<td></td>
		</tr>
	</tbody>
</table>

culturing human pluripotent and neural stem cells in an enclosed cell culture system for basic and preclinical research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

本稿では、いくつかの詳細にセル生産施設、クローズドシステムの内部で細胞を培養するユニークな側面について説明します。セル生産設備の大部分は、小さなフットプリントを有し、容易に6×7.5 m 2の部屋（ 図2）内に収容することができるカスタムメイドの、囲まれた、細胞培養装置を含みます。時間がない時に実験者や環境にさらさ装置内の細胞です。この装置は、複数のモジュールで構成さ：処理チャンバ、層流フード、フード処理室、二つの細胞培養インキュベーター、処理室に隣接顕微鏡室との間で2緩衝エアロックチャンバ（ 図3）。システムは、環境変数を制御および監視することを可能にするソフトウェア（ 図1）によって制御されます。このソフトウェアを実行しているコンピュータは、無停電電源装置です。ガスは番目に供給されます酸素、窒素及び二酸化炭素（ 図4）のためのマニホールドの集合によって電子システム。アクティブセットと予備セット - デバイス用のガスがタンクごとの2セットを持つマニホールドシステムによって供給されています。アクティブセットがドローダウンされた場合、マニホールドは、自動的に予備セットとスタッフの順序交換タンクに切り替わります。電源は、バックアップ発電システムです。 多能性神経幹細胞は、新規の機器に固有の追加の合併症と以前に開発されたプロトコル9を使用して、この施設で、低酸素条件下で増殖させることができます。多能性幹細胞は、次いで、単離し、拡張され、完全にトランスフェクトされたコロニーを同定するために多能性特異的抗体を用いて、センダイウイルスを使用して導出することができます。 （ 図5AおよびB）は 、これらのiPS細胞から、神経幹細胞及び神経細胞は、デュアルSMAD阻害を用いて導出することができ、それらの表現型は、USIを確認しました<html> NG NSC特異的抗体（ 図5C及びD）。 <img alt="図1" src="/files/ftp_upload/53685/53685fig1.jpg" /> セル生産設備については、図1のグラフィカルインタフェース。（A）CPFのグラフィカルな表現は、デフォルト画面で、（ 図1に2F） 図3。バッファモジュール1の上にマウスのクリックで示したCAD図面と一致しましたO 2の値が（ 図3の3）処理室に一致するように設定され、その制御画面（B）を開きます。同様に、プロセスチャンバまたはインキュベータ1をクリックすると、値は適宜変更または監視することができるそれぞれのコントロール画面（それぞれCおよびDを 、）、起動します。<html>目標確認= &quot;_空白&quot;&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。 <img alt="図2" src="/files/ftp_upload/53685/53685fig2.jpg" /> セル生産設備2.図。（A）右フロントビューから見たシステム。天井に電力とガスの接続と右に顕微鏡のアクセサリーやコンピュータとカートに注意してください。 （B）右で見られるバッファモジュールへのアクセスドアを持つ層流フード。後方から見た（C）は、2つのインキュベータ（黒ドア）を有するプロセスチャンバ。 （D）システムの右側を通るビュー顕微鏡を示す距離で見られるバッファ室へのドアで監視。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53685/53685fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> トン &quot;FO：キープtogether.withinページ=&quot; 1 &quot;&gt; <img alt="図3" src="/files/ftp_upload/53685/53685fig3.jpg" /> セル生産設備の図3. A CAD図面は装置に入るすべてのアイテムは最初の層流フード内で乾燥させ、アルコールと空気で拭いている（1）。アイテムはその後UCPCモジュールに渡される前にフロントバッファモジュール（2F）内の適切な雰囲気に移行、またはされているプロセスチャンバー（3）。細胞は、2つのインキュベータ（4,5）で培養します。ライブ細胞染色、コロニーピッキング、一般的な細胞形態および移行解析の評価は、UPCモジュールで行われ、または顕微鏡商工会議所（6）。最後に、廃棄物はリアバッファモジュール（2R）を介してシステムを出る。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53685/53685fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> &lt;<html> / P&gt; <img alt="図4" src="/files/ftp_upload/53685/53685fig4.jpg" /> ガスと電力の図4.ソース。（A）は、実験室のすべてのインキュベーターを供給するようにCO 2マニホールドは、4×4のセットアップです。 （B）O 2マニホールドは、2×2の設定のみを供給し、セル生産設備があります。システムのための窒素はまた、自動的に（左下）cryofreezersを埋める（右へ）液体窒素マニホールドに取り付けられた気化器（ちょうど終了記号以下のC、）で生成されます。マニホールドは、160 L液体窒素タンクの二対によって供給されている2×2の設定です。 CO 2、O ​​2、およびN 2は、閉止弁（D）を介して天井から供給されます。ハウス真空はまたこの場所で供給されます。システムに電源を供給する電源ケーブルの一対だけ供給ガスの閉止弁の背後に見られます。F = &quot;https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53685/53685fig4large.jpg&quot;ターゲット= &quot;_空白&quot;&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。 <img alt="図5" src="/files/ftp_upload/53685/53685fig5.jpg" /> センダイウイルスと低酸素症に派生図5.多能性幹細胞。（A）SC187-SF4-2I0-E3 iPS細胞iPS細胞（命名法のStover ら 9で見つかった）、位相コントラスト10X。 （B）SC88.1-UH1-2I0性IPSC AF-594で標識したTRA-1-60、1でライブ染色：メディアで100希釈。すべてのiPS細胞は、センダイウイルスで5％のO 2のセル生産設備に導入しました。 （C）チャンバースライドで5％O 2で成長したSC68.1-UH0-2I0-M0S13-N2G6 IPSC由来のNSCの位相コントラスト。次いで、細胞を4％パラホルムアルデヒドで固定し、抗ヒトネスチン一次抗体で染色し、アレックス-488二次抗体ました。すべてのスケールバーは100であります1;メートル<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53685/53685fig5large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research at JoVE.com

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

Basicおよび前臨床研究のための密閉型細胞培養系でヒト多能性および神経幹細胞を培養します

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

culturing-human-pluripotent-neural-stem-cells-an-enclosed-cell

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

10.2K ビュー.  Childrens Hospital of Orange County Research Institute. Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

ビデオ: Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

同軸ノズルを用いて多能性幹細胞のアルギン酸カプセル化

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

発生生物学

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

ヒト多能性幹細胞からメラノサイトのフィーダーフリーの導出

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

再現性の高い小分子ベースの分化プロトコルを使用したヒト多能性幹細胞から心筋細胞の大規模生産

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

トランスポゾンのシステムを用いたマウスの羊水細胞から多能性幹細胞の作製

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人間の胚または誘導多能性幹細胞からの網膜色素上皮細胞の急速な監督の分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

ヒト多能性幹細胞の肝細胞への効率的な分化

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

体節誘導体誘導される人間からの体外世代多能性幹細胞します。

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

ヒト誘導多能性幹細胞由来神経細胞の治療移植によるマウス脳損傷の制御された皮質衝撃モデル

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

フィーダーとゼノフリーの定義条件におけるヒト多能性幹細胞からの血原性内皮分化

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

ヒト胚性幹細胞の単細胞培養を用いた効率的な神経分化

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...