私たちは深く感熱マイクロパターン培養プレートと同様に科学的助言を提供するための東洋合成工業、東京、日本の博士武池谷と技術スタッフに感謝します。また、動物実験での技術支援のためにさんさとみ小倉さん沙鈴木さんあすか三好さんとかつえ森井に感謝します。この作品は、財政的に日本学術振興会科研費科学研究費補助金によって部分的にサポートされていた、イノベーション（COI）プログラムのセンターと独立行政法人科学技術振興機構（JST）からのS-イノベーションプログラム、およびJSPSコア - のコアプログラム、A.先端研究ネットワーク。

すべての動物実験は、東京大学の動物実験委員会、東京、日本の承認を得て実施しました。 1.細胞の調製<ol><li>初代肝細胞の場合は、変更された二段階コラゲナーゼ消化プロセス13,14によってラットから肝細胞を単離するためのプロトコルに従ってください。 <ol><li>イソフルラン吸入麻酔下でのスプラーグドーリー（SD）ラット（雄、5週齢）を麻酔。チャンバーのためにイソフルランを提供するために、麻酔装置に接続されたチャンバ内にラットを置きます。眠りに落ちる後のラットを取り出し、マスクを使用して換気を手術台の上に置きます。ラットの条件をチェックすることにより、0.7リットル/分 - イソフルランが0.4で約フロー制御。 8グラム/ L塩化ナトリウムからなる特殊な溶液（NaCl）で、400 mg / Lの塩化カリウム（KCl）、78ミリグラムで肝門脈のスプラーグドーリー（SD）ラット（雄性、5週齢）の肝臓を灌流/ Lリン酸二水素ナトリウム二水和物（のNaH 2 PO 4·2H 2 O）、151 mg / Lのリン酸水素二ナトリウム十二し（Na 2 HPO 4·12H 2 O）、2.38グラム/ Lの2- [4-（2-ヒドロキシエチル）-1-ピペラジニル]エタンスルホン酸（HEPES）、190 mg / Lのエチレングリコール四酢酸（EGTA）、350 mg / Lの炭酸水素ナトリウム（NaHCO 3）、および900 mg / Lのグルコース。 </li><li>肝臓を通してコラゲナーゼ溶液を循環させます。 注：溶液を、500 mg / Lのコラゲナーゼで構成されている、9.8グラム/ Lハンクス緩衝塩2.38グラム/ mlのHEPES、556 mg / mlの塩化カルシウム水和物（のCaCl 2·H 2 O）、350ミリグラム/ LのNaHCO 3、そしてpHが50 mg / Lのトリプシン阻害剤は、7.2に調整しました。 </li><li>慎重に肝臓を取り出し、手術用メスの刃を使用して皿に静かにミンチ、追加ダルベッコ改変イーグル培地、10％ウシ胎児血清（FBS）を補充した（DMEM）、および100ミクロンを介して細胞懸濁液をフィルタリングナイロンメッシュ。追加の破片を除去するために、1分間20×gで細胞懸濁液を遠心してください。このステップでは、肝細胞は、上清中にあります。 </li><li>二回（3遠心分離の合計）遠心分離工程を繰り返します。最後に、遠心3分間50×gで肝細胞をペレットの形でそれらを回収します。 </li><li> 10％FBSを補充したDMEMから成る特殊な培養培地中に4×10 5細胞/ mlの濃度、1％ペニシリン-ストレプトマイシン-供給過剰（PSQ）、1％ジメチルスルホキシドに肝細胞を再懸濁（DMSO）、0.1モル/ Lのデキサメタゾン、0.5 / mlのインスリン、10ミリモル/ Lのニコチンアミド、0.2ミリモル/ Lのリン酸化アスコルビン酸（ASC-2P）、および10 ngの/ mlのヒト上皮増殖因子（hEGF）15。この特別な培地は、 インビトロ条件下で肝機能を維持するために必須です。 </li></ol></li><li>ラットMSCを得るためには、isoflurの過剰投与によりスプラーグドーリー（SD）ラット（雄、5週齢）を安楽死させますANE。大腿骨と脛骨を切除し、DMEM 10mlでそれをフラッシュするために、骨の軸に22 G針を挿入することによって、骨髄を収集し、10％FBSを補充しました。 100μmのナイロンメッシュを通して濾過することにより細胞を収集します。 </li><li> 10％FBSおよび1％ペニシリン/ストレプトマイシンを含むDMEMを用いて10cmの培養皿に細胞をシード。回転楕円体の実験のために、5通路内でMSCを使用します。 </li></ol> 3Dスフェロイドの作製<ol><li>市販の細胞接着性領域は規則的にPEGマトリックスによりコーティング非接着性領域によって囲まれ、二次元的に直径100μmで配列されたマイクロパターン培養プレートを得ます。 注：細胞移植のために、感熱性ポリマーPIPAAmと追加のコーティングは、プレートを冷却することにより、細胞剥離を可能にするために必要である（ステップ5.1参照）。温度変動は、このポリマーの化学的性質の変化を誘導することができます8-10。 37℃では、PIPAAmは、細胞が正常な条件下で培養することができるように、わずかに疎水性です。 32℃以下の温度の低下は、細胞の自発的な離脱につながる、ポリマーの急速な水和になります。 </li><li>ウェル5％CO 2を含む加湿雰囲気中で37℃でそれらをandincubate / 4×10 5細胞の密度で12ウェルマイクロパターンプレート上の肝細胞やMSCをシード。細胞は、マイクロパターンの接着領域に蓄積され、徐々に2日間で丸スフェロイドを形成することになります。 注：単層培養で対照細胞を調製するために、通常の12ウェルプレートを使用し、上記と同様の手順に従って、同一の密度で細胞を播種。 </li></ol>ポリプレックスナノミセルの調製<ol><li>ブロック共重合体を合成し、PEG-PASP（DET）（ポリ[N &#39; - [N - （2-アミノエチル）-2-アミノエチル]アスパルトアミド]）（PEG Mwは12、000、重合度PASP（DET）のセグメント（DP）= 59）、および以前に著者ら11によって記載された手順に従って、唯一のカチオン性セグメント[PASP（DET）（DP = 55）から構成されているホモポリマー、 16、17。 </li><li> 33.3μgのにポリマーの濃度を調整することにより、10mMのHEPES緩衝液（pH 7.3）中の残留アミノ基の等モル比でPEG-PASP（DET）ブロック共重合体の混合溶液とPASP（DET）ホモポリマーを準備/ mlおよびそれぞれ19.1μgの/ mlで、。 注：上述のポリマー濃度変化させることができる10 N / P比のpDNA（N / P比）中のリン酸基への2つのポリマーの全アミノ基の残存モル比でナノミセルを調製するための代表的な値であります細胞のタイプと目的に応じて（詳細については、議論を参照）。 2つのポリマーの併用は、エンドソームを高めるために効果的なPEGシールドとPASP（DET）の機能の両方を達成することができますエスケープ18（詳細については、説明を参照）。 </li><li> pCAG-GSプラスミドにそれぞれの遺伝子を発現するセグメントをクローニングすることによりpDNAをコードするガウシアルシフェラーゼ、GL4ルシフェラーゼ、またはエリスロポエチンの準備（http://www.cdb.riken.jp/pcs/protocol/vector/map/m36.html ）を製造者のプロトコールに従って市販のキットを用いてCAGプロモーター /エンハンサー下式を得ます。コンピテント大腸菌株でのpDNAを増幅し、エンドトキシンフリーのプラスミドDNA精製システムを使用して精製します。 10mMのHEPES緩衝液（pH 7.3）で150 / mlの溶液を得た260 nmでの吸光度でのpDNAの濃度を決定します。 </li><li> （体積比）1：ポリプレックスナノミセルを調製するために、十分のpDNA溶液（10mMのHEPES緩衝液中の150μgの/ mlで）および2の比率で2つのポリマーのプレミックス溶液を混合します。 </li></ol>スフェロイドに4遺伝子のトランスフェクション<ol><li>細胞（肝細胞をインキュベート成熟したスフェロイドの形成を可能にするためにマイクロパターンプレート上に播種後72時間、またはMSCS）。遺伝子トランスフェクションのために、新鮮な培地1 mlの培養液を交換した後、各ウェルに（pDNAを10μgのを含む）ポリプレックスナノミセル溶液100μlを添加します。 24時間ナノミセル溶液でインキュベーションを続けます。 </li><li>脂質ベースのトランスフェクション試薬を使用して制御するために、3の重量比試薬/のpDNAで試薬とのpDNA溶液を混合脂質ベースの試薬とナノミセル法の両方のために等しくなるようにpDNAの最終用量を調整します。 </li></ol>スフェロイドの5回復と移植<ol><li>チルド200μlのPBSと培地を交換し、氷上にプレートを配置します。 注意：一般的に、スフェロイドは、約15分で取り外すことができ、移植のための懸濁液の形態で回収することができます。 </li><li>ゆっくり200μlの細胞を吸引in vivoでの注射用23 Gまたは27 G針で注射器を使用してサスペンション。 </li></ol>導入遺伝子発現の6評価<ol><li>正確に24時間新鮮な培地と交換した後、培地100μL -培養液中に分泌型ガウシアルシフェラーゼの発現の in vitroでの評価のために、50を収集します。商業ウミシイタケルシフェラーゼアッセイシステムと製造業者のプロトコルに従って、ルミノメーターを用いてルシフェラーゼ発現を推定します。 注：ガウシアルシフェラーゼは、1週間以上培養培地中で安定なままです。このように、導入遺伝子発現のリアルタイムの効率を評価するためのサンプル媒体を収集する前に、新鮮なものを含む培地を交換してください。培地交換のタイミングは柔軟であることができます。 </li><li>細胞移植後の宿主動物における導入遺伝子発現の in vivo評価のために、BALB / cヌードマウス（雌を麻酔; 7週間イソフルラン吸入麻酔下に古いです）。 <ol><li>チャンバーのためにイソフルランを提供するために、麻酔装置に接続されたチャンバ内にマウスを置きます。眠りに落ちる後にマウスを取り出し、マスクを使用して換気を手術台の上に置きます。マウスの状態をチェックすることによって、0.5リットル/分 - イソフルランは0.2で約フロー制御。 </li><li>腹部の皮下組織に（4.1で説明したように）GL4ルシフェラーゼ発現のpDNAをトランスフェクトしたスフェロイドを含む200μlの細胞懸濁液を注入します。 </li><li> （150ミリグラム/ kgで、静脈内経路）直後D-ルシフェリンを注入した後、製造業者のプロトコルに従ってIVISイメージングシステムを使用して、ルシフェラーゼ発現を測定します。 </li></ol></li><li>細胞移植の治療効果を評価するために、中にエリスロポエチンをコードするpDNAを用いてトランスフェクトされたスフェロイドを含む200μlの細胞懸濁液を注入腹部の皮下組織。 <ol><li>血液19の約200μLを得るために、顎下出血によって血液サンプルを収集します。血液サンプル分析装置を用いてヘモグロビンおよびヘマトクリット値を測定します。 注：移植のための細胞数をプレート上に播種した数によって調節されます。スフェロイド内の数字は測定できないので、残念ながら、それは、正確な細胞数を決定することは困難です。 </li></ol></li><li>実験の後、麻酔から覚醒するまで、温度コントローラーに接続された加熱パッドの上にマウスを置きます。 </li></ol>

<ol>
	<li>Landry, J., Bernier, D., Ouellet, C., Goyette, R., Marceau, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroidal+aggregate+culture+of+rat+liver+cells:+histotypic+reorganization,+biomatrix+deposition,+and+maintenance+of+functional+activities.">Spheroidal aggregate culture of rat liver cells: histotypic reorganization, biomatrix deposition, and maintenance of functional activities.</a> J Cell Biol. 101 (3), 914-923 (1985).</li><li>Yuasa, C., Tomita, Y., Shono, M., Ishimura, K., Ichihara, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+cell+aggregation+for+expression+of+liver+functions+and+regeneration+demonstrated+with+primary+cultured+hepatocytes.">Importance of cell aggregation for expression of liver functions and regeneration demonstrated with primary cultured hepatocytes.</a> J Cell Physiol. 156 (3), 522-530 (1993).</li><li>Otsuka, H., 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=Two-dimensional+multiarray+formation+of+hepatocyte+spheroids+on+a+microfabricated+PEG-brush+surface.">Two-dimensional multiarray formation of hepatocyte spheroids on a microfabricated PEG-brush surface.</a> Chembiochem. 5 (6), 850-855 (2004).</li><li>Wang, W., 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=3D+spheroid+culture+system+on+micropatterned+substrates+for+improved+differentiation+efficiency+of+multipotent+mesenchymal+stem+cells.">3D spheroid culture system on micropatterned substrates for improved differentiation efficiency of multipotent mesenchymal stem cells.</a> Biomaterials. 30 (14), 2705-2715 (2009).</li><li>Bartosh, T. 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=Aggregation+of+human+mesenchymal+stromal+cells+(MSCs)+into+3D+spheroids+enhances+their+antiinflammatory+properties.">Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties.</a> Proc Natl Acad Sci U S A. 107 (31), 13724-13729 (2010).</li><li>Frith, J. E., Thomson, B., Genever, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+three-dimensional+culture+methods+enhance+mesenchymal+stem+cell+properties+and+increase+therapeutic+potential.">Dynamic three-dimensional culture methods enhance mesenchymal stem cell properties and increase therapeutic potential.</a> Tissue Eng Part C Methods. 16 (4), 735-749 (2010).</li><li>Nakasone, Y., Yamamoto, M., Tateishi, T., Otsuka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte+spheroids+underlayered+with+nonparenchymal+cells+for+biomedical+applications.">Hepatocyte spheroids underlayered with nonparenchymal cells for biomedical applications.</a> IEICE Transactions on Electronics. E94, 176-180 (2011).</li><li>Nishida, K., 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=Corneal+reconstruction+with+tissue-engineered+cell+sheets+composed+of+autologous+oral+mucosal+epithelium.">Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium.</a> N Engl J Med. 351 (12), 1187-1196 (2004).</li><li>Ohashi, K., 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=Engineering+functional+two-+and+three-dimensional+liver+systems+in+vivo+using+hepatic+tissue+sheets.">Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets.</a> Nat Med. 13 (7), 880-885 (2007).</li><li>Sekine, H., 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=Cardiac+cell+sheet+transplantation+improves+damaged+heart+function+via+superior+cell+survival+in+comparison+with+dissociated+cell+injection.">Cardiac cell sheet transplantation improves damaged heart function via superior cell survival in comparison with dissociated cell injection.</a> Tissue Eng Part A. 17 (23-24), 2973-2980 (2011).</li><li>Itaka, K., Kataoka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+prospects+of+polyplex+nanomicelles+for+plasmid+DNA+delivery.">Progress and prospects of polyplex nanomicelles for plasmid DNA delivery.</a> Curr Gene Ther. 11 (6), 457-465 (2011).</li><li>Endo, T., Itaka, K., Shioyama, M., Uchida, S., Kataoka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfection+to+spheroid+culture+system+on+micropatterned+culture+plate+by+polyplex+nanomicelle:+a+novel+platform+of+genetically-modified+cell+transplantation.">Gene transfection to spheroid culture system on micropatterned culture plate by polyplex nanomicelle: a novel platform of genetically-modified cell transplantation.</a> Drug Deliv and Transl Res. 2 (5), 398-405 (2012).</li><li>Howard, R. B., Christensen, A. K., Gibbs, F. A., Pesch, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enzymatic+preparation+of+isolated+intact+parenchymal+cells+from+rat+liver.">The enzymatic preparation of isolated intact parenchymal cells from rat liver.</a> J Cell Biol. 35 (3), 675-684 (1967).</li><li>Berry, M. N., Friend, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-yield+preparation+of+isolated+rat+liver+parenchymal+cells:+a+biochemical+and+fine+structural+study.">High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study.</a> J Cell Biol. 43 (3), 506-520 (1969).</li><li>Tateno, C., Yoshizato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+cultivation+of+adult+rat+hepatocytes+that+undergo+multiple+cell+divisions+and+express+normal+parenchymal+phenotypes.">Long-term cultivation of adult rat hepatocytes that undergo multiple cell divisions and express normal parenchymal phenotypes.</a> Am J Pathol. 148 (2), 383-392 (1996).</li><li>Kanayama, N., 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=A+PEG-based+biocompatible+block+catiomer+with+high+buffering+capacity+for+the+construction+of+polyplex+micelles+showing+efficient+gene+transfer+toward+primary+cells.">A PEG-based biocompatible block catiomer with high buffering capacity for the construction of polyplex micelles showing efficient gene transfer toward primary cells.</a> ChemMedChem. 1 (4), 439-444 (2006).</li><li>Itaka, K., Ishii, T., Hasegawa, Y., Kataoka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+polyamino+acid-based+polycations+as+safe+and+effective+gene+carrier+minimizing+cumulative+toxicity.">Biodegradable polyamino acid-based polycations as safe and effective gene carrier minimizing cumulative toxicity.</a> Biomaterials. 31 (13), 3707-3714 (2010).</li><li>Uchida, S., 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=PEGylated+Polyplex+With+Optimized+PEG+Shielding+Enhances+Gene+Introduction+in+Lungs+by+Minimizing+Inflammatory+Responses.">PEGylated Polyplex With Optimized PEG Shielding Enhances Gene Introduction in Lungs by Minimizing Inflammatory Responses.</a> Mol Ther. 20 (6), 1196-1203 (2012).</li><li>Golde, W. T., Gollobin, P., Rodriguez, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+simple,+and+humane+method+for+submandibular+bleeding+of+mice+using+a+lancet.">A rapid, simple, and humane method for submandibular bleeding of mice using a lancet.</a> Lab Anim (NY). 34, 39-43 (2005).</li><li>Uchida, S., 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=An+injectable+spheroid+system+with+genetic+modification+for+cell+transplantation+therapy.">An injectable spheroid system with genetic modification for cell transplantation therapy.</a> Biomaterials. 35 (8), 2499-2506 (2014).</li></ol>

The authors declare that they have no competing financial interests.

このプロトコルでは、遺伝子導入及びスフェロイド回収工程中のスフェロイドの3次元構造を維持することが重要です。細胞が細胞死または細胞活性の損失を回避することが有利な微小環境を維持するために不可欠です。従来の単層培養中の肝細胞が急速に数日12を播種した後、それらの分泌能力を失いつつ例えば、アルブミン分泌、肝細胞の代表的な先天性機能は、よく、肝細胞ス?...

細胞移植療法は、様々な難治性疾患を治療するために広く注目を集めています。移植された細胞によって分泌される生物活性因子の活性及び半減期は、細胞移植システムの改良された治療効果のために不可欠です。移植前の細胞の遺伝子改変は、生物活性因子の分泌などの細胞機能を調節して操作するための有益な技術です。これは、細胞死または細胞活性の喪失を回避するための細胞のための良好な微環境を維持することも重要です。細胞間相互作用が十分に保持された3次元（3D）スフェロイド細胞培養物は、（初代肝細胞のアルブミン分泌を改善し、間葉系幹細胞の多系列分化を促進するために、例えば、この目的のためにMSCを約束しています）1-7。 spheroiのこの研究では、新規な組み合わせシステムD培養および遺伝子トランスフェクションは、遺伝的に改変された細胞移植のためのプラットフォームとして機能するように使用されます。スフェロイド細胞を作成するための、マイクロパターン培養プレート上でスフェロイド培養系を用いています。これらのプレート上で、100ミクロン直径の細胞接着領域が規則的に二次元的に配列され、PEGマトリックス3によって被覆された非接着性領域によって囲まれています。細胞の十分な数を播種することにより、直径100μmの3Dスフェロイドのアレイは、マイクロパターン培養ベッドに対応して形成されています。 スフェロイドは、感熱性細胞培養プレートを使用して、3D構造を破壊せずに回収される感熱性ポリマー、ポリ（イソ-プロピル）（PIPAAm）8-10でコーティングしたもの。マイクロパターンアーキテクチャは、感熱板（カスタムビルド）上に構築されています。単にプレートの温度を下げることにより、スフェロイド培養ベッドから離脱して分散されていますリン酸中のdは、緩衝生理食塩水（PBS）。従って、100μmの均一なサイズを有する球状の多数の注射懸濁液の形態で得ることができます。 <img alt="図1" p="" src="/files/ftp_upload/52384/52384fig1.jpg" /> 図1マイクロパターンプレート上でスフェロイド培養システムの概略図。遺伝的改変は、元の非ウイルス遺伝子キャリア、ポリプレックスナノミセルを用いた遺伝子導入によって達成されます。これは、プラスミドDNA（pDNAを）およびポリエチレングリコール（PEG）-polycationブロック共重合体11で構成されています。これらは、治療目的のために11細胞への安全かつ効果的な遺伝子導入を可能にする、PEGシェルと凝縮pDNAの内部コアからなる特徴的なコア-シェル構造を有している。 <a href="https://www.jove.com/files/ftp_upload/52384/52384fig1large.jpg" target="_blank">THIの拡大版を表示するには、こちらをクリックしてください</a>のフィギュア。 <img alt="図2" src="/files/ftp_upload/52384/52384fig2.jpg" /> 核酸およびPEG-ブロック-ポリカチオンブロック共重合体の複合体によって形成された。本研究ではポリプレックスナノミセルの図2の構造は 、この技術の主な利点は、回転楕円体の構造はナノミセルによる遺伝子トランスフェクションの際に破壊されないことです。ラット初代肝細胞スフェロイドのナノミセル媒介性トランスフェクションの後、長期の導入遺伝子の発現は、トランスフェクトしていないスフェロイド12のそれに匹敵するレベルで肝細胞からの連続アルブミン分泌と1ヶ月以上得られます。スフェロイドからの導入遺伝子発現およびアルブミン分泌も感熱プレートからの回復後も維持されています。これは、ナノミセル安全HEPの生来の機能を損なうことなく、遺伝子導入を容易にすることができることは明らかですatocytes。このように、ナノミセルを用いた遺伝子導入と感熱マイクロパターンプレート上で培養したスフェロイド細胞の組み合わせは、遺伝的に改変された細胞の移植のための有望なプラットフォームです。 <a href="https://www.jove.com/files/ftp_upload/52384/52384fig2large.jpg" target="_blank">この図の拡大版を表示するには、こちらをクリックしてください。</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pen-Strep-Glut</td>
			<td>GIBCO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Wako Pure Chemical Industries</td>
			<td>041-18861</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Wako Pure Chemical Industries</td>
			<td>141-01202</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s buffered salt and L-ascorbic acid 2-phosphate (Asc-2P)</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>Human epidermal growth factor (hEGF)</td>
			<td>Toyobo</td>
			<td>PT10015</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-able multi-well plates</td>
			<td>Toyo Gosei</td>
			<td>PP-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermosensitive cell culture plates (Upcell)</td>
			<td>CellSeed Inc</td>
			<td></td>
			<td>The micropatterned architecture is constructed on the thermosensitive plates (custom-built by Toyo Gosei)</td>
		</tr>
		<tr>
			<td>Lipid-based transfection reagent (FuGENE HD)</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Renilla Luciferase Assay System</td>
			<td>Promega</td>
			<td>E2810</td>
			<td></td>
		</tr>
		<tr>
			<td>pGL4 Luciferase Reporter Vector</td>
			<td>Promega</td>
			<td>E6651</td>
			<td></td>
		</tr>
		<tr>
			<td>pDNA expressing Gaussia luciferase</td>
			<td>New England BioLabs</td>
			<td>N8082S</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse erhthropoietin-expressing vector</td>
			<td>Origene</td>
			<td>MC208445</td>
			<td></td>
		</tr>
		<tr>
			<td>pCAG-GS</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Laboratory for Pluripotent Cell Studies, Center for Developmental Biology, RIKEN</td>
		</tr>
		<tr>
			<td>Escherichia coli DH5&#945; competent cells</td>
			<td>Takara</td>
			<td>9057</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotoxin-free plasmid DNA purification system</td>
			<td>Nippon Genetics</td>
			<td>NucleoBond Xtra EF</td>
			<td></td>
		</tr>
		<tr>
			<td>collagenase</td>
			<td>Wako Pure Chemical Industries</td>
			<td>639-00951</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin inhibitor</td>
			<td>GIBCO</td>
			<td>R-007-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Promega</td>
			<td>GloMax&#8482; 96 Microplate Luminometer</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Imaging System</td>
			<td>Xenogen Corp.</td>
			<td>Xenogen IVIS Spectrum in vivo imaging system</td>
			<td></td>
		</tr>
		<tr>
			<td>blood sample analyzer</td>
			<td>Sysmex</td>
			<td>pocH-100i Automated Hematology Analyzer</td>
			<td></td>
		</tr>
	</tbody>
</table>

gene transfection toward spheroid cells on micropatterned culture plates for genetically-modified cell transplantation

細胞移植の治療効果を改善するために、遺伝的に改変された、注射可能なスフェロイドの移植システムを開発しました。細胞スフェロイドは、感熱性ポリマーでコーティングされたマイクロパターン上でプレート培養系で調製されます。スフェロイドの数は、ポリエチレングリコール（PEG）マトリックスによって被覆されている非接着性領域によって囲まれ、定期的に二次元的に配列された直径100μmの細胞接着領域に対応する、プレート上に形成されています。スフェロイドは、容易にプレートの温度を低下させることにより液体懸濁液として回収することができ、その構造は、ウェル（27 G上の）十分に大きな口径の注射針に通して維持されます。遺伝子改変は、球状構造を破壊することなく細胞に遺伝子を導入することが可能である元の非ウイルス遺伝子キャリア、ポリプレックスナノミセルを用いた遺伝子導入によって達成されます。プリムのためのアルブミンの発現によって示されるように、ルシフェラーゼを発現する遺伝子でトランスフェクト進肝細胞スフェロイドは、ルシフェラーゼは持続、保存肝細胞機能と共に、移植動物において得られます。このシステムは、間葉系幹細胞を含む種々の細胞型に適用することができます。

ガウシアルシフェラーゼを発現するpDNAの遺伝子トランスフェクションは、ポリプレックスナノミセルまたは対照脂質ベースのトランスフェクション試薬12を使用して、肝細胞またはMSCのことで形成された球状体で実施しました。対照試薬が大幅にトランスフェクション（ 図3）の翌日の構造を破壊し、一方ナノミセルは、マイクロパターンプレート上の非トランスフェク?...

Watch this Scientific Journal Video about Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation at JoVE.com

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

遺伝的に改変された細胞移植のためのマイクロパターン培養プレート上スフェロイド細胞に対する遺伝子導入

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

gene-transfection-toward-spheroid-cells-on-micropatterned-culture

Research

JoVE Journal

Bioengineering

8.7K ビュー.  The University of Tokyo. This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

ビデオ: Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

癌細胞とヒアルロン酸の相互作用を研究するマイクロパターン表面

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

生物工学

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

マイクロパターン細胞を用いた薬物の効果の改善可視化と定量分析

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

動的なマスク投影リソグラフィーを用いたニューラル培養システムのためのマイクロパターンハイドロゲルの作製

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

ヒト細胞培養のための回転細胞培養システム：モデルとしてのヒト栄養膜細胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

ミササイトメトリー：CyTOFマスメーターで毎日のチューニングと実行した細胞サンプルのためのプロトコル

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

筋肉再生のマウスモデルにおける誘導多能性幹細胞由来メソアンジオ芽細胞様筋原性前駆細胞の移植

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

PDMS膜上のマイクロパターン細胞を伸ばす

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

マイクロパターン血管筋薄膜の拡張文化ためのマイクロ流体ゲニピン堆積技術

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

メカノ生物学的研究のための軌道シェーカー上の6ウェルプレートにおける内皮細胞の成長のセグメント化

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

静止ヒト造血幹細胞を維持する培養法

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...