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>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;i&gt;Xvivo System&lt;/i&gt;</td><td>Biospherix</td><td>custom made</td><td></td></tr><tr><td>&lt;i&gt;Xvivo&lt;/i&gt; Software</td><td>Biospherix</td><td>version i.o.2.1.2.1</td><td></td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt; Manifold</td><td>Amico</td><td>P-M2H-C3-S-U-OXY</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Manifold</td><td>Amico</td><td>M2H-C3-D-U-CO2</td><td></td></tr><tr><td>N&lt;sub&gt;2&lt;/sub&gt; 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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</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&amp;amp;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&#039;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&amp;amp;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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Consumable Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2 ml Serological pipet</td><td>VWR</td><td>89130-894</td><td></td></tr><tr><td>5 ml Serological pipet</td><td>Olympus Plastics</td><td>12-102</td><td></td></tr><tr><td>10 ml Serological pipet</td><td>Olympus Plastics</td><td>12-104</td><td></td></tr><tr><td>25 ml Serological pipet</td><td>Olympus Plastics</td><td>12-106</td><td></td></tr><tr><td>50 ml 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>20 &amp;micro;l pipet tips</td><td>Eppendorf</td><td>22491130</td><td></td></tr><tr><td>200 &amp;micro;l pipet tips</td><td>Eppendorf</td><td>22491148</td><td></td></tr><tr><td>1,000 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

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

ヒト多能性のための新しい文化モデルは、プラセンタ馴化培地中のゼラチンの細胞増殖幹

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

発生生物学

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

ヒト神経幹細胞を継代

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

生物学

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

廃棄されたヒト胎児脳組織から神経幹細胞の生成

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

神経科学

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

小分子の誘導と多能性ヒト胚性幹細胞からヒト神経前駆細胞と神経細胞の効率的な導出

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

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 ...

自動培養システムによる多能性幹細胞分化の誘導

An Automated Culture System for Maintaining and Differentiating Human-Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, including the elucidation of rare disease pathologies, the development of new medicines, and regenerative medicine aiming to restore damaged organs. Despite this, the social implementation of hiPSCs is still limited. This is partly because of the difficulty of reproducing differentiation in culture, even with advanced knowledge and sophisticated technical skills, due to the high sensitivity of iPSCs to minute environmental changes. The application of an automated culture system can solve this issue. Experiments with high reproducibility independent of a researcher's skill can be expected according to a shared procedure across various institutes. Although several automated culture systems that can maintain iPSC cultures and induce differentiation have been developed previously, these systems are heavy, large, and costly because they make use of humanized, multi-articulated robotic arms. To improve on the above issues, we developed a new system using a simple x-y-z axis slide rail system, allowing it to be more compact, lighter, and cheaper. Furthermore, the user can easily modify parameters in the new system to develop new handling tasks. Once a task is established, all the user needs to do is prepare the iPSC, supply the reagents and consumables needed for the desired task in advance, select the task number, and specify the time. We confirmed that the system could maintain iPSCs in an undifferentiated state through several passages without feeder cells and differentiate into various cell types, including cardiomyocytes, hepatocytes, neural progenitors, and keratinocytes. The system will enable highly reproducible experiments across institutions without the need for skilled researchers and will facilitate the social implementation of hiPSCs in a wider range of research fields by diminishing the obstacles for new entries.

著者スポットライト:iPS細胞培養の自動化による再現性の向上 

Here, we present a protocol for an automated cell culture system. This automated culture system reduces labor and benefits the users, including researchers unfamiliar with handling induced pluripotent stem (iPS) cells, from the maintenance of iPS cells to differentiation into various types of cells.

ヒト人工多能性幹細胞の維持・分化のための自動培養システム

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

ヒト多能性のための代替培養はセル生産、メンテナンス、および遺伝子解析幹

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

感染と自然免疫を研究するためのマウス脳胚混合細胞培養

Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of neurons, astrocytes, oligodendrocytes, and microglia. Due to increasing efforts to replace and reduce animal use, a variety of in vitro cell culture systems have been developed to explore innate cell properties, which allow the development of therapeutics for CNS infections and pathologies. Whilst certain research questions can be addressed by human-based cell culture systems, such as (induced) pluripotent stem cells, working with human cells has its own limitations with regard to availability, costs, and ethics. Here, we describe a unique protocol for isolating and culturing cells from embryonic mouse brains. The resulting mixed neural cell cultures mimic several cell populations and interactions found in the brain in vivo. Compared to current equivalent methods, this protocol more closely mimics the characteristics of the brain and also garners more cells, thus allowing for more experimental conditions to be investigated from one pregnant mouse. Further, the protocol is relatively easy and highly reproducible. These cultures have been optimized for use at various scales, including 96-well based high throughput screens, 24-well microscopy analysis, and 6-well cultures for flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. This culture method is a powerful tool to investigate infection and immunity within the context of some of the complexity of the CNS with the convenience of in vitro methods.

著者スポットライト:感染と自然免疫を研究するためのマウス胚脳からの混合ニューロン細胞培養とグリア細胞培養の確立  

This protocol presents a unique way of generating central nervous system cell cultures from embryonic day 17 mouse brains for neuro(immuno)logy research. This model can be analyzed using various experimental techniques, including RT-qPCR, microscopy, ELISA, and flow cytometry.

感染と自然免疫を研究するためのマウス胚性脳からの混合神経細胞およびグリア細胞培養の確立

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of ...

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

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ヒト多能性のための新しい文化モデルは、プラセンタ馴化培地中のゼラチンの細胞増殖幹

ヒト神経幹細胞を継代

廃棄されたヒト胎児脳組織から神経幹細胞の生成

小分子の誘導と多能性ヒト胚性幹細胞からヒト神経前駆細胞と神経細胞の効率的な導出

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

ヒト人工多能性幹細胞の維持・分化のための自動培養システム

ヒト人工多能性幹細胞の維持・分化のための自動培養システム

ヒト人工多能性幹細胞の維持・分化のための自動培養システム

ヒト多能性のための代替培養はセル生産、メンテナンス、および遺伝子解析幹

感染と自然免疫を研究するためのマウス胚性脳からの混合神経細胞およびグリア細胞培養の確立