This work was funded by start-up funds provided to Dr. Silviya Zustiak by Saint Louis University as well as by a President&#8217;s Research Fund (PRF) grant awarded to Dr. Silviya Zustiak by Saint Louis University. We thank Naveed Ahmed and Keval Shah for technical assistance.

ヒドロゲル関連ソリューションおよびアリコートの調製<ol><li>ポリアクリルアミドゲル前駆体溶液の調製。 <ol><li>アクリルアミド（A）を混合することにより、ポリアクリルアミドゲル前駆体溶液を製造（w / vの40％、Mは、rは 71.08グラム/モル）、架橋剤ビスアクリルアミド（B）（2％w / vの、Mは、154.17グラム/モルrの ）、およびデ表1に指定されたボリュームの割合でイオン水。 注：これらのソリューションは、大きなバッチで調製し、数ヶ月まで4℃で保存することができる。 <ol><li>注意：アクリルアミドは特に、とき粉末状の、吸入または摂取時に有毒である：従って、好ましくは、毒性のリスクを減らすためにw / v溶液40パーセントを使用します。そのような手袋、ゴーグル、そして白衣として、防護服を着ている間だけハンドル。冷蔵庫の中の光耐性、しっかりと密閉容器（&lt;23℃、換気の良い場所）に保管してください。 </li><li>注意：ビス ​​- アクリルアミドは特に、吸入または摂取時に有毒である、時粉末形態で：従って、好ましくは、毒性のリスクを減らすために2％w / v溶液を使用しています。そのような手袋、ゴーグル、そして白衣として、防護服を着ている間だけハンドル。冷蔵庫の中の光耐性、しっかりと密閉容器（&lt;4℃、換気の良い場所）に保管してください。 </li></ol></li></ol></li><li>調製およびN -Sulfosuccinimidyl -6-（4&#39;-アジド-2&#39;-ニトロフェニルアミノ）ヘキサノエートの使用アリコート（スルホSANPAHは、Mは、492.40グラム/モルrを）。注意：スルホSANPAHは深刻な眼刺激を引き起こす。手袋と適切な保護眼鏡または保護面を扱う。ストア受信時に-20℃で前分注する。 <ol><li>スルホ - SANPAHを保存するには：50 mg / mlのでdimethylsulfoxane（DMSO）にスルホSANPAHを溶解する。チューブあたり20μlに50マイクロ遠心チューブにストック溶液を分注し。ドライアイス上または液体窒素中でフラッシュ凍結-80℃で、店舗（最大架橋剤効率を維持するために任意であるが好ましい工程）。 注：一定分量は、CAnは、数ヶ月間保存すること。 </li><li>スルホ - SANPAH使用するには：簡単にアリコートを解凍し、脱イオン水480μlの希釈する。すぐに使用してください。スルホSANPAHは水に迅速に加水分解する：ので、すぐに上記の手順をすべて実行するために注意を取る。 </li></ol></li><li>過硫酸アンモニウム（M rは 228.18グラム/モル）のアリコートを調製する。 注：過硫酸アンモニウムは、目、皮膚、呼吸器への刺激の原因となる。取り扱いの際に適切な個人保護具を着用する。化学ヒュームフードの過硫酸アンモニウム粉末を扱う。ドライ、風通しの良い場所に保管して粉末。分注した後、希釈溶液は、作業台の上に使用することができる。 <ol><li> w / vの10％の最終の硫酸アンモニウム濃度を達成するために、脱イオン水に過硫酸アンモニウムを溶解する。 -20℃で小分けし、保存。使用直前に解凍します。 </li></ol></li><li> I型コラーゲン溶液の調製。 <ol><li>そうストックを希釈することによって0.2 mg / mlのコラーゲン溶液を調製pH7.4での1×リン酸緩衝生理食塩水（PBS）中リュー。簡単に氷の上に保管してくださいまたは希釈するとすぐに使用しています。 </li></ol></li></ol> 2.ハイドロゲル準備（図1を参照してください） <ol><li>疎水性スライドガラスの調製。 <ol><li>ガラス板上に疎水性溶液を数滴を置き、表面全体に広がってティッシュペーパーを使用しています。空気が乾燥してみましょうと疎水性コーティングを均等に再びティッシュペーパーで拭いてください。 注：RTで可燃性のキャビネットに保管して疎水性のソリューション。取り扱う際は保護具を着用。換気の良い場所で作業します。 </li></ol></li><li>可撓性プラスチック支持体の作製。 <ol><li>疎水性コーティングされたガラス板のサイズに合わせて柔軟なプラスチック支持体を切断した。 </li><li>軽くそのような外科用メスなどの鋭利な工具で表面を引っ掻くことにより、柔軟なプラスチック支持体の疎水性側をマークします。 注： - 完全に透過的である - ゲル一度乾く、スクラッチマークは、柔軟なプラスチック製の支持体側がゲルを含む区別しやすくなります。 注：可撓性プラスチック支持体は、疎水性および親水性の側面を有する。蒸着後、ポリアクリルアミドは、恒久的に簡単に後続のヒドロゲルの取り​​扱いを容易にプラスチック支持体の親水性側に付着する。 </li></ol></li><li>ゲルの調製（例えば容積5mlのゲル前駆体溶液のために与えられている）。 NOTE：ゲルの初期厚さが0.5mmである場合を5ml〜40のゲルを生成するのに十分であろう。ゲルの厚さを小さくすれば、よりゲルを製造することができる。 <ol><li> 50ミリリットルコニカルチューブに所望の最終濃度（ 表1）のポリアクリルアミド前駆体溶液の4972.5μLを置 ​​きます。キャップで30分間脱ガスチャンバ内の場所を開け。 NOTE：酸素ラジカル捕捉剤として作用し、存在する、重合を阻害する。 </li><li>過硫酸アンモニウムw / vの10パーセントの25μlを加え、最終的に達成するために（1.3を指すように参照してください。）0.05％の過硫酸アンモニウムの濃度。 </li><li> N、N、N &#39;、N&#39;-テトラメチルエチレンジアミンの2.5μl加え、0.5％の最終TEMED濃度を達成するために脱気したゲル溶液に（TEMED、Mは116.24グラム/モルをR）。 注：RTで可燃性のキャビネットに保管TEMED。適切な個人保護具を身に着けているときに、化学ドラフト内で取り扱ってください。それは非常に空気や水分に敏感であるように、しっかりと不活性ガス下で閉じておいてください。 </li><li> 5回 - ダウン3をピペッティングにより穏やかに溶液を混合する。ゲル前駆体溶液への酸素の拡散を回避するためにボルテックスしないでください。 </li><li>疎水性コーティングされたスライドガラスを有する可撓性プラスチック支持体とサンドイッチの親水性側上にゲル溶液をピペット。所望の厚さ（ 例えば、0.5ミリメートル）のシリコーンスペーサーを2スライドを区切ります。ゲル化の指標として使用する50mlの円錐管中でポリマー前駆体溶液の少量（約100μl）を残す。 注意：任意のスペーサーを使用することができる。 120μmで - 例えば、パラフィルムの単一のストリップは、100の最後の膨潤ゲルの厚さを与えます。 </li><li>重合時にさえヒドロゲル表面を確認するために柔軟なプラスチック支持体/ゲルサンドイッチの上に別のガラス板を置きます。 </li><li>所望であれば、より少ない時間でより高い重量％のゲルのために使用することができるゲルを、45分間重合してみよう。ゲルが重合したことを確認するため、50ミリリットルコニカルチューブ中の残留溶液を観察する。残りの溶液がゲル化していれば、それはガラス板上にゲル化が同様に開始された可能性がある。しかし、金型の早期開口部が完全な重合を防止することに注意。 </li><li>ゲルが形成されると、上に共有結合したポリアクリルアミドゲルを有する可撓性プラスチック支持体から剥離し、空気乾燥ゲル側を上向きにして設定。 NOTE：対流または熱乾燥は、このステップを加速するためにも使用することができる。可撓性プラスチック支持体上に乾燥した後、ゲルをストアすることができ無期限dは。 <ol><li>ポリアクリルアミドゲルは、気泡のために形成されなかった柔軟なプラスチック支持体、のいずれかの裸のスポットをマークします。ゲルが乾燥したら、これは柔軟なプラスチック支持体に溶け込むようマークゲルはまだ水和されている間。 </li></ol></li></ol></li></ol> 3.マルチウェルプレートアセンブリ、コラーゲンコーティング、および滅菌<ol><li>マルチウェルプレートアセンブリ。 <ol><li>乾燥させた後、所望の形状にPAゲルをカット。 <ol><li> 96ウェルプレートの場合は、〜6ミリメートルの直径を有する大型穴パンチを使用しています。正方形または長方形の形状にゲルをカットする大型ペーパーカッターを使用してください。代わりに、はさみを使用しています。 </li></ol></li><li>製造元の指示に従って、96ウェルプレート当たりPDMSの約500μlのを準備します。マルチウェルプレートの底にゲルを接着するために、各ウェルの中心にポリジメチルシロキサン（PDMS）の小滴（〜5μl）を配置します。鉗子を使用して、1つのポリアクリルアミドを配置ダウン各ウェル、可撓性プラスチック支持体側の電子ゲル。 PDMSを硬化できるようにするには、4時間の最小37℃で組み立てられたプレートを残す。 </li></ol></li><li>ポリアクリルアミドゲルのコラーゲンコーティング。 <ol><li>転送ピペットを用いて、少量プレイス - スルホ - SANPAHの（7 8μl）をゲル表面に均一にコーティングするために左右によくスワールそれぞれに（1.2.2を指すように参照）。スルホ - SANPAHは水中で安定していないので、速やかに作業します。 </li><li> 5分間 - 高強度UVランプ（365nmの強度= 37ミリワット、λ= 302）の下でウェルプレートを置きます。過剰スルホ - SANPAHを除去してPBSでゲルを洗浄します。 </li><li> 0.2ミリグラムのピペットを50μl/ mlのI型コラーゲン溶液（1.4ポイントを参照）を各ウェルに。 4℃で少なくとも2時間またはO / N RTで​​覆われたプレートのままにしておきます。 NOTE：コラーゲンコーティングのプロセスをスピードアップするために、ピペットは、コラーゲン溶液を追加するために使用することができる。一般的に、1 - 溶液2滴が完了するのに十分であるべきであるLYゲル表面をカバーしています。 </li><li>コラーゲン結合を可能にするために、少なくとも2時間室温でウェルプレートにしておきます。 </li><li> 2時間、組織培養フード中の過剰コラーゲン溶液を除去し、UV（λ= 200 nm）の下で殺菌するためにPBSで洗浄すること。 </li><li>水和平衡化するO / N（培地組成はセクション4.1を参照のこと）完全培地中でゲルを浸す。最大2日間すぐに播種細胞ま​​たは冷蔵庫に店舗に使用します。 </li></ol></li></ol> PA剛性アッセイ4.細胞播種注：一般的な哺乳動物細胞株のための代表的なものの、ここで説明するプロトコルは、特に乳癌MDA-MB-231細胞株で使用される（ 図4及び図5を参照）。 <ol><li> 37℃で5分間、5％トリプシン/ EDTAに暴露することにより組織培養フラスコから細胞を収集する。 〜培養フラスコ領域の各1cm 2当たりのトリプシン/ EDTA80μlのを使用します。例えば、トリプシン/ EDTA foの2 mlを使用RA T25細胞培養フラスコ。再懸濁所望の最終細胞濃度で、10％ウシ胎児血清（FBS）および1％ペニシリン/ストレプトマイシンを補充した完全培地中で回収された細胞である。細胞は、アッセイに播種される時間だけでなく、時間の長さを倍にアカウントセルに入れて、適切なサブコンフルエント細胞濃度を選択する。添加した培地を完全にヒドロゲルを沈めるのに十分であることを確認してください。 NOTE：ヒドロゲル培地で予め平衡化されているために十分であるべきで100μlの体積（3.2.6ステップを参照）。ゲルは前のステップでメディアで事前に平衡化されているので、マルチウェルプレートに使用される典型的なメディアのボリュームは十分なはずです。 注：このアッセイは、任意の付着依存性細胞型のために適切であろう。 <ol><li>血球計を使用して、倒立顕微鏡下で細胞数をカウントする。負荷10の各血球計ポートに細胞懸濁液μlの少なくとも8象限から細胞数を平均化する。取得するため最終細胞濃度は、10 4細胞数を掛ける。 注：各血球計象限における細胞数が20である場合、最良の細胞計数結果が得られる - 50。 </li></ol></li><li>培養物を37℃で加湿インキュベーター中でPA剛性アッセイ上に細胞を、5％CO 2。 3日間 - すべての2メディアを変更します。 5分間37℃で5％トリプシン/ EDTAに暴露することによって、必要なときに、細胞を収集する。組織培養フラスコの1cm 2当たりのトリプシン/ EDTAの〜80μlのを使用してください。ヒドロゲルに触れるのではなく、わずかに横マルチウェルプレートを傾けると、各ウェルの側壁にピペットチップに触れることにより、吸引またはピペット媒体：全細胞操作工程の間に、ヒドロゲルの表面を破壊しないように特別な注意を払う。 注：標準的な組織培養処理時のヒドロゲルの表面を損傷しないように十分注意し除き、細胞は、それらが上に播種されたかのように剛性のアッセイは同じように操作することができる上に播種定期的なマルチウェルプレートに。 </li></ol> PA剛性アッセイ上に播種された細胞の5イメージング<ol><li>直接PA剛性アッセイにおける画像セル。 注：すべての顕微鏡 - 倒立蛍光または共焦点顕微鏡は、細胞イメージングのために使用することができる。 NOTE：剛性アッセイを構築するために使用される可撓性プラスチック支持体は、完全に透明であり、autofluoresceまたは細胞画像化を妨害しない。しかし、可撓性プラスチック支持体自体が透明であっても、イメージング機能は対物レンズの作動距離によって制限される。柔軟なプラスチック支持体は、0.23ミリメートルの厚さを有し、10倍の対物レンズの典型的な作動距離が急激に高倍率のために減少〜4mmである。 <ol><li>生細胞イメージングのための顕微鏡のプレートホルダー及び画像中の位置PA剛性アッセイ。 2時間の下でイメージングセッションを保持し、またはより長い撮影時刻のための環境室を備えた顕微鏡を使用しています。 </li><li>ときに生細胞イムエージングが目的ではない、0.1％の界面活性剤を補充した4％ホルムアルデヒド溶液で細胞を固定する。 2時間の最小室温で固定液中で細胞を浸す。二回PBSですすぐ。指定された廃棄物容器内の固定液の廃棄物を捨てる。 注：細胞は、直ちに画像化することができる、または2週間まで4℃でPBS中に沈めて格納。注意：ホルムアルデヒドは吸入と接触した際に有毒である。化学ヒュームフード中で手袋を取り扱ってください。 注記：特定の固定剤は、いくつかの細胞タンパク質に損傷を与えるので、細胞の固定プロトコルは、その後の細胞染色および処理プロトコルに基づいて選択されな​​ければならない。 </li><li>蛍光イメージングのために、染色は、マルチウェルプレート中で直接所望の細胞染色で細胞を固定。画像をすぐに。 </li></ol></li></ol>

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The authors have nothing to disclose.

もともと電気泳動のために開発され、ポリアクリルアミドゲルは、28は、現在日常的に細胞の形態、運動性、および他の細胞特性のうち通信3,24,29上の基板の剛性の影響を研究するために細胞培養基質として使用される。ポリマー前駆体濃度の単純な変化- （300キロパスカル0.3）1（ 図2、表1も参照17,25,26参照 ）、ポリアクリルアミド、体内のすべて...

体内のほとんどの組織は、ソフト軟骨100キロパスカルに脳0.1キロパスカルの範囲のヤング率を有する軟質の粘弾性材料であり、まだ、 インビトロの細胞研究のほとんどは、約1 GPaの弾性率を有する組織培養ポリスチレン（TCP）上で実施される。1この剛性のミスマッチが大幅に細胞がその環境に対応方法に影響します。研究の成長体は、幹細胞を含む種々の細胞型、2,3の運命の基板の剛性の影響を解明するこうして専用である。4その結果、複数のヒドロゲルは剛性依存性細胞の理解を助けるために開発されている基板の剛性が細胞の運命に大きな影響を持っていることの証拠が増えている一方で、ポリアクリルアミド（PA）、5-7ポリエチレングリコール（PEG）、8,9-ジメチルシロキサン（PDMS）、10およびアルギン酸塩を含む、生物。11、ほとんどの研究は、上実施されるsの数が少ない小規模amples。細胞型または環境条件のアレイ用基板の剛性の影響に関する系統的、多次元研究は稀である。12 いくつかの有望なハイスループットヒドロゲル技術は、PEGに基づくマイクロアレイ、アガロースヒドロゲルマイクロビーズを製造するための13のマイクロ流体デバイス、14またはミクロ及び剛性がマイクロロッドの直径および高さによって変調されたナノロッドを含む、開発されてきた。15ただし、そのような基板を製造するための技術が洗練された研究室の限られた数に利用可能である。剛性変調細胞応答に関与する多くの研究が実施が安価で簡単であるだけでなく、ポリアクリルアミド（PA）ゲルを利用するだけでなく、ヤング率、すなわち、0.3の生理学的に関連する範囲を示す-を300kPaにPAを製造する16-22しかしながら、既存の方法細胞培養用ゲルは、労働集約的で、その結果、分取され小さなバッチでARED。細胞基質としてPAゲルの調製に関連する困難のいくつかは、ゲルを用意しなければならない要件から生じる：1）は、酸素の非存在下で完全な重合を可能にする、2）平滑な表面を有する均一なセルを可能にする付着及び拡散、および3）永久フローティング防止するための細胞培養皿の底に固定された。 いくつかのグループが、大きなバッチでの細胞培養のためのPAゲルを製造することを試みてきた。ゼムラーらパンチ、次いで「切断」であり、96ウェルプレートに入れ、PAゲルを調製した厚さのシート23は、しかしながら、この方法は、より堅いゲル、 すなわち、&gt; 1キロパスカルのヤング率で、より柔らかいために制限されゲルは、カットすることは困難であり、容易に損傷を受けた「スティッキー」である。 MIH らは、ゲルが直接ガラスボトムウェルプレート中で重合することができ、より洗練された技術を開発した。 <sアップ&gt; 6これは、官能化ガラス底プレート中にゲル溶液を注ぐとカスタムカバーガラス配列でそれらを「挟み込む」ことによりゲルを形成することによって達成された。6にもかかわらず、非常に有望な、わずかなエッジ効果はまだ、この技術を用いて観察された。さらに、この技術は、多くの研究室だけでなく、高価なガラスボトムマルチウェルプレートにすぐにアクセスすることはできませんカスタムデザインの配列が必要です。 本論文では、簡単に任意の研究室によって採用することができ、マルチウェルプレートにPAゲルを組み立てるための簡単​​で安価な方法について説明します。撥PAゲルに疎水一つ、共有結合、堆積時PAゲルに結合する親水オン、 - ここで、柔軟なプラスチック支持体は、2つの側面を有し、利用される。 PAゲルシートは、可撓性プラスチック支持体に固定され永久に堆積された後は、任意の厚さまたは剛性のゲルを処理し、任意の所望の形状にして切断を可能にする。このAPPRoachは、PA結合溶液で、ガラスカバースリップまたは高価なガラスボトムマルチウェルプレートのウェルのいずれかを他の方法で市販されていないサイズのカスタムプラスチック「カバーガラス」を生成するだけでなく、ガラス表面-御馳走事前に必要がなくなるだけでなく、退屈で時間のかかる工程。最後に、均一なPAゲルシートは、大きなバッチで調製し、数ヶ月デ水和格納することができる。 要約すると、ここで提示アッセイは、いくつかの態様において、既存の方法の改良である。まず、マルチウェルプレートアセンブリのプロセスが効率的であり、必要な材料の全体的なコストが低い。第二に、ヒドロゲルは、単一の均質ゲルフィルムに大きなバッチで製造される。最終的に、市販されている唯一の材料が必要とされる。アッセイの有用性は、細胞形態の基板の剛性の影響を探索し、面積を広げることによって示されている。

<table border="0" cellpadding="0" cellspacing="0" width="898">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Reagents</td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">40% Acrylamide</td>
			<td style="width:200px;">Bio-Rad</td>
			<td style="width:187px;">161-0140</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">2% Bis-acrylamide</td>
			<td style="width:200px;">Bio-Rad</td>
			<td style="width:187px;">161-0142</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">Ammonium Persulfate</td>
			<td style="width:200px;">Bio-Rad</td>
			<td style="width:187px;">161-07000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">TEMED</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">T9281</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">Sulfo-SANPAH</td>
			<td style="width:200px;">Thermo Scientific</td>
			<td style="width:187px;">22589</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">Collagen Type 1, from Rat tail, 3.68 mg/mL</td>
			<td style="width:200px;">BD Biosciences</td>
			<td style="width:187px;">354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:200px;">Fisher Scientific&#160;</td>
			<td style="width:187px;">BP231-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">Hydrophobic solution - Repel Silane&#160;</td>
			<td style="width:200px;">GE Healthcare Bio-Sciences</td>
			<td style="width:187px;">17-1332-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">PBS (1x), pH 7.4</td>
			<td style="width:200px;">HyClone</td>
			<td style="width:187px;">SH30256.01</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:303px;">Polydimehylsiloxane (PDMS) [Slygard 182 Elastomer Kit]</td>
			<td style="width:200px;">Elsworth Adhesives</td>
			<td style="width:187px;">3097358-1004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Tyrpsin/EDTA (10x)</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">44174</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">RPMI-1640 Medium (1x)</td>
			<td style="width:200px;">HyClone</td>
			<td style="width:187px;">SH30027-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Fetal Bovine Serum</td>
			<td style="width:200px;">HyClone</td>
			<td style="width:187px;">SH30073-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Penicillin Streptomycin</td>
			<td style="width:200px;">MP Biomedicals</td>
			<td style="width:187px;">1670046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Detergent - Triton-X</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Formaldehyde 37% Solution</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">F1635</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">A2153</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">BSA-based cell adhesion blocking kit - ECM Cell Adhesion Array Kit</td>
			<td style="width:200px;">Chemicon International</td>
			<td style="width:187px;">ECM540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;"></td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Disposable lab equipment</td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">flexible plastic support - GelBond PAG Film for Polyacrylamide Gels</td>
			<td style="width:200px;">GE Healthcare Bio-Sciences</td>
			<td style="width:187px;">309819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Glass Plates</td>
			<td style="width:200px;">Slumpys</td>
			<td style="width:187px;">GBS4100SFSL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">50 mL Conicals</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">3181345107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">15 mL Conicals</td>
			<td style="width:200px;">FALCON</td>
			<td style="width:187px;">352097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Micro centrifuge tubes</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">2 mL: 02681258</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">96-well plate (flat bottom)</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">12565501</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:303px;">Disposable Pipettes (1 mL, 2mL, 5mL, 10mL, 25 mL, 50mL)</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">1 mL: 13-678-11B, 2mL: 05214038, 5mL(FALCON): 357529, 10mL: 13-678-11E, 25mL: 13-678-11, 50mL: 13-678-11F</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Glass Transfer Pipettes</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">5 3/4&#34;: 1367820A, 9&#34;:136786B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Pipette Tips (1-200uL, 101-1000uL)</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">2707509</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Plastic Standard Disposable Transfer Pipettes</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:187px;">13-711-9D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Parafilm</td>
			<td style="width:200px;">PARAFILM&#160;</td>
			<td style="width:187px;">PM992</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Powder Free Examination Gloves</td>
			<td style="width:200px;">Quest</td>
			<td style="width:187px;">92897</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Silicone spacers - Silicone sheet, 0.5 mm thick/13 cm x 18 cm</td>
			<td style="width:200px;">Grace Bio-Labs</td>
			<td style="width:187px;">JTR-S-0.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;"></td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Large/non-disposable lab equipment</td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Light and Flourescent Microscope (Axiovert 200M)</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:187px;">3820005619</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microscope Software</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:187px;">AxioVision Rel. 4.8.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">UV oven</td>
			<td style="width:200px;">UVITRON</td>
			<td style="width:187px;">UV1080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Vacuum chamber/degasser</td>
			<td style="width:200px;">BelArt</td>
			<td style="width:187px;">999320237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Vacuum pump for degasser</td>
			<td style="width:200px;">KNF Lab</td>
			<td style="width:187px;">5097482</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Tissue Culture Hood</td>
			<td style="width:200px;">NUAIRE</td>
			<td style="width:187px;">NU-425-600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Chemical Fume Hood</td>
			<td style="width:200px;">KEWAUNEE</td>
			<td style="width:187px;">99151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Inverted Microscope (Axiovert 25)</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:187px;">663526</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Incubator</td>
			<td style="width:200px;">NUAIRE</td>
			<td style="width:187px;">NU-8500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Pipette Aid</td>
			<td style="width:200px;">Drummond Scientific Co.</td>
			<td style="width:187px;">P-76864</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Hemacytometer</td>
			<td style="width:200px;">Bright-Line</td>
			<td style="width:187px;">383684</td>
			<td></td>
		</tr>
	</tbody>
</table>

simple polyacrylamide-based multiwell stiffness assay for the study of stiffness-dependent cell responses

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 &#8211; 100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn&#8217;t require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses.

ポリアクリルアミド（PA）ヒドロゲルは広く剛性依存性細胞応答を試験するために使用される。17,24アクリルアミド（A）とビスアクリルアミド（B）の種々の濃度を混合することにより、一つのほとんどの軟組織の剛性の範囲に及ぶPAゲルを作ることができるボディ- 0.3 -ここで300kPaのヤング率1が、ポリアクリルアミドゲルの調製が面倒であり、時間は、多くの場合、例えば薬?...

Watch this Scientific Journal Video about Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses at JoVE.com

Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

剛性依存性細胞応答の研究のための簡単​​なポリアクリルアミド系マルチウェル剛性アッセイ

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a ...

simple-polyacrylamide-based-multiwell-stiffness-assay-for-study

Research

JoVE Journal

Bioengineering

19.4K ビュー.  Saint Louis University. Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

ビデオ: Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

エンジニア心臓組織の移植後免疫反応の評価（EHT）のための生物発光イメージング

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

生物工学

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

3D楕円体の生成のための簡単​​なハンギングドロップ細胞培養プロトコル

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

器官型コラーゲンIアッセイ：3次元コンテキストでのセルの挙動を評価するための可鍛性プラットフォーム

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca2+-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca2+ levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca2+-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.

This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

FlexStation 3を用いてTRPチャネルの機能のハイスループットスクリーニングへの媒体のためのセルベースカルシウムアッセイ

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

用接着周波数アッセイその場で</em細胞間のインタフェースにおけるクロス接合分子間相互作用の>速度論解析

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

セルの剛性を推定するために基板に接続された細胞のマイクロピペット吸引

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

における損傷応答のライブイメージングや研究のためのマイクロ流体チップを使用<em&gt;ショウジョウバエ</em&gt;幼虫

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D相互作用の研究のための3Dコラーゲンゲルおよびマイクロチャネルの作製<em&gt;インビボ</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

A Simple and Scalable Fabrication Method for Organic Electronic Devices on Textiles

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices cannot be worn under everyday conditions. Therefore, textiles are commonly considered the best substrate to accommodate electronic devices in wearable use. In this paper, we describe how to selectively pattern organic electroactive materials on textiles from a solution in an easy and scalable manner. This versatile deposition technique enables the fabrication of wearable organic electronic devices on clothes.

In this paper, we present a protocol to selectively deposit organic materials on textiles, which allows for the direct integration of organic electronic devices with wearables. The fabricated devices can be fully integrated in textiles, respecting their mechanical appearance and enabling sensing capabilities.

テキスタイル上の有機電子デバイスのためのシンプルでスケーラブルな製造方法

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

化学誘引物質勾配に対するマルチセル応答の3D 解析

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

剛性依存性細胞応答の研究のための簡単​​なポリアクリルアミド系マルチウェル剛性アッセイ

剛性依存性細胞応答の研究のための簡単なポリアクリルアミド系マルチウェル剛性アッセイ