この作品は、BSに王立協会URF助成UF051616、英国、欧州研究評議会STG助成金243261によってサポートされていました。 MZラボでの作業は、再生医療助成RB1-01417のカリフォルニア工科大学でサポートされています。

1。神経前駆細胞の分離<ol><li> E14-16マウスと冷たいDMEM/F12培地の場所から全体の脳を解剖する。 35ミリメートルのペトリ皿に解剖顕微鏡および転送の脳の下にあるすべての髄膜を除去します。 </li><li>機械的に組織の断片に脳を分離し、15 mlのチューブにそれらを転送するために微細な鉗子を使用し、破片を除去するために3分間800rpmでサンプルを遠心します。 </li><li> DMEM/F12含むbFGFおよびEGFを追加し、1 mlのピ​​ペットでひいて粉にする。 </li><li>単一の細胞懸濁液を得るために、セルストレーナーを介して細胞懸濁液を渡します。 </li><li> 2から5×10 4細胞/ mlのフラスコに細胞をプレートしてから、6日毎3日ごとに、通路の細胞を完全培地の変更を実行します。 </li><li>少なくとも5つの継代後、ダイジェストは単一の神経細胞にトリプシンとEDTAを使用してpoly-D-lysine/laminin-coated electrotactic室（2に後述するように調製した）上に成長。 、N2-サプリメントを含む成長培地を使用します。bFGFおよびEGFはすべての回でNPCの特性を維持します。 </li></ol> 2。 electrotactic室の準備<ol><li>ダイヤモンドペンを使用して半分にオートクレーブ処理22×22ミリメートルNo.1の厚さのカバーを割ってガラスの22×11ミリメートルストリップを準備します。 </li><li>接着でよくインテリア寸法22×10 mmの自立ガラスを作成します。一緒に4垂直に高真空用シリコーングリースを持つ22×11ミリメートルストリップを立っている。井戸は完全に一晩乾燥し、硬化することができます。 </li><li>次の日、シリコーングリースを使用して、100 mmの培養皿の底に、10 mmのギャップを残して互いに平行に、2つの22×11 mmのガラス板を取り付けます。センターに最も近いことが三方にグリースを皿の底に取り付けられたそれぞれの端に22×22 mmのカバースリップを配置することにより、これらのストリップとの間の領域、ではなく、を封印。 </li><li>カバー内部の壁はシードのために限られたスペースを作成するように、スリップの上にも、ステップ2.2で調製したガラスを配置しプレートの底に細胞。シリコーングリースの耐水すべての関節。その後順次、ポリ-D-リジンでコートこの狭い領域をラミニン：チャンバー内に1ミリリットルポリ-D-リジンを追加して、ポリ-D-リジンプレートの底にバインドできるようにするために5分間のままに、チャンバを洗う滅菌したPBSで2回、その後20μg/ mlを得、プレートの底をカバーするために使用する滅菌PBSにラミニンを薄める。一晩室温で残す。 </li><li>次の日、収穫細胞と1×104細胞を含む懸濁液1 mlを準備します。それは完全に乾燥空気、細胞懸濁液1ミリリットルに置き換えることができ、よくガラスからラミニンを削除します。添付ファイルを可能にするために4時間の最低37℃インキュベーターで皿を置きます。 </li><li>細胞が十分にコンフルエントになったらチャンバーからすべてのメディアを削除します。慎重によくガラスを削除します。シリコーングリース、オートクレーブ22×22 mmのカバーガラスで、慎重に取り付けることによって、細胞上の屋根を形成する2つの22×11 mmのストリップ間のブリッジ。乾燥を避けるために媒体の数滴を持つ細胞をカバーしています。 </li><li>室の屋根上の皿の一方の端から他に実行する2つの水密性シリコーングリースの障壁を作成することで、チャンバーの各端部に分離された媒体の貯水池を形成します。フロースルー1培地リザーバから他のを確保し、新鮮培地でチャンバーを記入してください。細胞の回復を可能にするために12時間インキュベーターに皿を返します。 </li></ol> 3。 electrotactic室への電界の印加<ol><li>二つの穴は、移行室の各貯水池の上に配置1をドリルで皿を覆うように蓋を準備します。 </li><li> 25mMのHEPES緩衝液を含む培地でチャンバー内にすべての媒体を交換して温度制御された撮像システムに皿を転送します。タイムラプス、マルチポジション記録のための実験パラメータを設定します。陰極と陽極であるように、チャンバーの位置を合わせますそれぞれ左右と上、EFベクトルが実行されるように水平に顕微鏡下に表示およびイメージングシステムに記録されます。 </li><li> Steinberg社のソリューション（58 mM塩化ナトリウム、0.67 mMの塩化カリウム、0.44 mMのカルシウム（NO 3）2·4H 2 0、1.3 mMのMgSO 4を 0.7 H 2 Oおよび4.6 mMのTrizma塩基、pH7.4）で2つのビーカーを記入してください。 2％（W ​​/で満たされた、あらかじめ用意したガラスの橋を（〜13センチ、直径〜3ミリメートルのガラス管と、ブンゼン炎で加熱することによりU字型に曲げ）を使用して、各媒体タンクに別々のビーカーを接続蓋の穴を通過v）のSteinberg&#39;s寒天ソリューションを提供しています。 Steinberg社のソリューションの各ビーカーに直流電源に接続されたAg / AgCl電極の電極を配置することによって電気回路を完成させます。 </li><li> 0とに切り替えるには、電源の電圧、ダイヤルを設定します。電圧計を使用して、電圧、ダイヤルを回しながらelectrotactic室の両端の電圧を測定し、実験的な要件に合わせて調整します。 </li><li>タイムラプス録画を開始します。培地交換を行い、時間ごと、必要に応じて、電圧を再調整します。必要に応じて新鮮な培地、薬物または化学物質は、貯水池に追加することができます。培地交換を実行するときに、2つのオプションは以下のように考えることができます。 <ol><li>オプション1番 - 一時的に休止タイムラプス録画をするには、慎重に、カバーの蓋を乱す避けるために、チャンバーからガラスブリッジを削除するには、新鮮な培地にすべての媒体を交換して静かに滅菌パスツールピペットを使用して、ガラスの橋を元に戻し、次に録音を再開します。 </li><li>オプションNo.2は - あるいは、カスタムも培地交換の目的に使用することができる4つの穴（2は、移行室の各貯水池の上に配置）とカバーの蓋を作りました。ガラスのブリッジ接続用の2つ、他の2つのメディアを変更する。オプション2は、培地交換時の干渉を受けずに連続記録することができます。 </li></ol></li></ol> 4。器官脊髄スライスの準備<ol><li>ラム解剖2週齢のC57 BL / 6マウスのAR脊髄。 </li><li>マッキルウェーン組織チョッパで500μmの厚さのセクションに脊髄のスライス。 </li><li>独立した解剖顕微鏡下でスライスはそのまま軸/矢脊髄構造を持つスライスを選択します。 </li><li>マトリゲルのタンパク質が生成するために自己集合するまで、30μlのマトリゲル、それらを配置するを含む35ミリメートルのペトリ皿のプレートスライスはできるだけ中心に近い、少なくとも30分間37℃5％CO 2インキュベーター内で、それらを維持する脊髄スライスの表面を覆う薄膜。それは完全に、別の30分、必要に応じて37℃でペトリ皿をインキュベートマトリゲルが組み立てされていることを確認することが非常に重要です。 </li><li>スライス上に直接メディアフローを回避するために、25mMのHEPES緩衝液と非常に穏やかになるように15から20パーセントのウシ胎児血清を含む4〜6ミリリットルDMEM/F-12培地を追加します。スライスが完全にウェルに露出した外植片の表面を残して、培地中に浸漬されていないことを確認空気。週2回培地を変更します。 </li></ol> 5。器官、脊髄スライスにヘキスト33342というラベルの付いたNPCの注入<ol><li> 1でNPC懸濁液を調製×10 6細胞/μlの。 </li><li> 30分間、5μMHoechst 33342で培地中の細胞懸濁液をプレインキュベートする。 </li><li>顕微鏡下で徐々に脊髄のスライスに懸濁液2μlを顕微注入するキャピラリーガラス管を使用しています。キャピラリーガラス管は、マトリゲル（顕微鏡下でピンク色の部分）を通過し、脊髄スライス（顕微鏡下で灰色の組織）の内部に到達していることを確認し、上で実行する細胞懸濁液を避けるために、少なくとも30秒間、脊髄スライスの内部に残る。インキュベーターに脊髄スライスを含むペトリ皿（37℃、5％CO 2）を入れ、一晩そのままにしておきます。 </li><li>次の日、に記載されているメソッドを使用してelectrotacticチャンバー内のヘキスト33342で標識されたNPCを（含まれている脊髄のスライスには500mV / mmのEFを適用する3）。 </li></ol> 6。代表的な結果 NPCは、彼らが陰極に向かって非常に指示細胞遊走（図1）を示した生理的な排出係数の範囲にさらされたとき。同じ観測はまた器官脊髄スライスを ex vivoモデルの単一細胞レベル、 生体内条件下で模倣し、3D環境（図2）で行われた。 <img alt="図1" src="/files/ftp_upload/3453/3453fig1.jpg" /> 図1。 NPCには、EFSの指示への移行を示しています 。排出係数にさらされたときのPCは陰極に向かって非常に向け移行を示し、赤線と青の矢印は細胞運動の軌跡と方向（）を表しています。 Bは、NPCの移行パスを示しています。バー：50μmである。 <img alt="図2" src="/files/ftp_upload/3453/3453fig2.jpg" /> 図2。移植されたNPCは器官脊髄スライスのカソードに向け移行を示す </stロン&gt;。 （A）ヘキスト33342で標識されたNPCは、EF治療の開始点での器官、脊髄スライスに移植した。 NPCはEFの極性が反転された時点で、2.5時間のためのカソード、（B）に向かって一方向に移行しました。 EFの極性を変更すると、新しい陰極（C）に向かって走電性の鋭い逆転を引き起こした。 （D）タイムラプス録画の終了時に脊髄スライス内に​​移植されたNPCのイメージ。 （E）脊髄スライス内に​​移植されたNPCの三次元再構成。 3Dスキャニングのセクションでは、スライスの下部中央からの開始と終了、厚さは300μmであった。点線は反転、初めに移植された細胞の同じ人口の相対的な位置を示しており、EF治療のエンドポイント（A - それぞれC、A）。アローヘッドは、ヘキスト33342標識されたNPCの同じ人口を示しています。バー：50μmである。

<ol>
	<li>Huttenlocher, A., Horwitz, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+with+electric+potential.">Wound healing with electric potential.</a> N. Engl. J. Med. 356, 303-303 (2007).</li><li>McCaig, C. D., Rajnicek, A. M., Song, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+cell+behavior+electrically:+current+views+and+future+potential.">Controlling cell behavior electrically: current views and future potential.</a> Physiol. Rev. 85, 943-943 (2005).</li><li>Borgens, R. B., Jaffe, L. F., Cohen, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+and+persistent+electrical+currents+enter+the+transected+lamprey+spinal+cord.">Large and persistent electrical currents enter the transected lamprey spinal cord.</a> PNAS. 77, 1209-1209 (1980).</li><li>Shapiro, S., Borgens, R., Pascuzzi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillating+field+stimulation+for+complete+spinal+cord+injury+in+humans:+a+phase+1+trial.">Oscillating field stimulation for complete spinal cord injury in humans: a phase 1 trial.</a> J. Neurosurg. Spine. 2, 3-3 (2005).</li><li>Zhao, M., Song, B., Pu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+signals+control+wound+healing+through+phosphatidylinositol-3-OH+kinase-gamma+and+PTEN.">Electrical signals control wound healing through phosphatidylinositol-3-OH kinase-gamma and PTEN.</a> Nature. 442, 457-457 (2006).</li><li>Yao, L., Shanley, L., McCaig, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+applied+electric+fields+guide+migration+of+hippocampal+neurons.">Small applied electric fields guide migration of hippocampal neurons.</a> J. Cell. Physiol. 216, 527-527 (2008).</li><li>Li, L., El-Hayek, Y. H., Liu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct-current+electrical+field+guides+neuronal+stem&#47;progenitor+cell+migration.">Direct-current electrical field guides neuronal stem&#47;progenitor cell migration.</a> Stem Cells. 26, 2193-2193 (2008).</li><li>Meng, X., Arocena, M., Penninger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+mediated+electrotaxis+of+embryonic+and+adult+neural+progenitor+cells+in+the+presence+of+growth+factors.">PI3K mediated electrotaxis of embryonic and adult neural progenitor cells in the presence of growth factors.</a> Exp. Neurol. 227, 210-210 (2011).</li><li>Bonnici, B., Kapfhammer, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cells+promote+neurite+extension+in+organotypic+spinal+cord+slice:+significance+for+cell+transplantation+therapy.">Bone marrow stromal cells promote neurite extension in organotypic spinal cord slice: significance for cell transplantation therapy.</a> Neurorehabil. Neural Repair. 22, 447-447 (2008).</li><li>Shichinohe, H., Kuroda, S., Tsuji, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cells+promote+neurite+extension+in+organotypic+spinal+cord+slice:+significance+for+cell+transplantation+therapy.">Bone marrow stromal cells promote neurite extension in organotypic spinal cord slice: significance for cell transplantation therapy.</a> Neurorehabil. Neural Repair. 22, 447-447 (2008).</li><li>Song, B., Gu, Y., Pu, j. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+direct+current+electric+fields+to+cells+and+tissues+in+vitro+and+modulation+of+wound+electric+field+in+vivo.">Application of direct current electric fields to cells and tissues in vitro and modulation of wound electric field in vivo.</a> Nature Protocol. 2, 1479-1479 (2007).</li></ol>

我々は、開示することは何もありません。

我々が使用するプロトコルは、以前の研究5,11に基づいています。標準化された寸法精度のカスタム設計electrotacticチャンバー内で培養した細胞またはスライスに、寒天橋、Steinberg社のソリューション、及びAg / AgCl電極電極を介してEFを適用しながら、これらのメソッドを使用して、安定した文化や電流条件を維持することができる。チャンバーの深さが異なるサンプルに対応するため?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> FGF-基本的な組換えヒト</td><td>インビトロジェン</td><td> PHG0026 </td><td> 20 ng / mLの</td></tr><tr><td> EGF遺伝子組換えヒト</td><td>インビトロジェン</td><td> PHG0311 </td><td> 20 ng / mLの</td></tr><tr><td> N2-サプリメント（100X）液体</td><td>インビトロジェン</td><td> 02048 </td><td></td></tr><tr><td> DMEM/F12培地（高グルコース） </td><td>インビトロジェン</td><td> 31330-095 </td><td></td></tr><tr><td>ポリ-D-リジン</td><td>ミリポア</td><td> -003-E </td><td></td></tr><tr><td>ナチュラルマウスラミニン</td><td>インビトロジェン</td><td> 23017-015 </td><td></td></tr><tr><td>成長因子の減少基底膜マトリックス（マトリゲル） </td><td> BDバイオサイエンス</TD&gt; <td> 354230 </td><td></td></tr><tr><td> HEPES緩衝液</td><td>ギブコ</td><td> 15630 </td><td></td></tr><tr><td>マッキルウェーン組織チョッパー</td><td>ミクル研究所エンジニアリングのCo株式会社</td><td> TC752-PD </td><td></td></tr><tr><td>ダウコーニング高真空用シリコーングリース</td><td> Sigma-Aldrich社</td><td> Z273554 </td><td></td></tr></tbody></table>

electric field-controlled directed migration of neural progenitor cells in 2d and 3d environments

内因性電界（EFS）は、生体内で自然に発生し、中枢神経系の1,2のことを含め、組織/臓器の開発と再生時に重要な役割を果たしている。これらの内因性排出係数は、細胞や組織の電気抵抗と組み合わせてイオン輸送の細胞調節によって生成されます。それが適用され、EF治療は、動物およびヒトにおける3,4 -脊髄損傷の機能的な修復を促進することが報告されている。特に、EF主導細胞遊走は、神経前駆細胞（NPCに）7,8を含む細胞の種類5,6、多種多様な、で実証されている。直流（DC）排出係数の適用は、ほとんどの研究室で一般的に使用可能なテクニックではありません。我々は以前5,11細胞や組織文化へのDCの排出係数の適用のための詳細なプロトコルを記述している。ここでは2Dを設定するには、計算された電界強度に基づいて、標準的な方法のデモビデオを提示dは3DはNPCのための環境、および2Dの単一細胞の成長条件、および3Dの器官脊髄スライスの両方でEFの刺激に対する細胞応答を調査する。 cytoarchitectonic組織の組織が ​​十分にこれらの培養9,10内に保存されているため、脊髄cordsliceは、移植後、NPC ex vivoでの動作を研究するための理想的な受信者の組織である。さらに、このex vivoでのモデルはまた、単一細胞レベルでの経時的記録を用いたin vivo で細胞を追跡することは技術的に可能ではありません手順をすることができます。それは、2D環境ではないだけでなく、 生体内環境では真似の3D器官の状態で細胞の挙動を評価することが決定的に重要である。このシステムは 、in vitro 及び ex vivo における単一細胞遊走の3Dトラッキングと組織又は器官培養にカバーガラスベースディッシュを用いた高分解能イメージングを可能にし、viで上に移動する前の中間ステップことができますVOのパラダイム。

Watch this Scientific Journal Video about Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments at JoVE.com

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

このプロトコルは、細胞を追跡することができますカスタム設計electrotactic室で2Dと3D環境を確立するために使用されるメソッドを示しています<em&gt;生体/ ex vivoでの</em&gt;直流（DC）電界（EFS）を指示する走電性および他の細胞応答を調べるために、単一細胞レベルでの経時的記録を使用します。

2Dと3D環境での神経前駆細胞の電界制御監督移行

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

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JoVE Journal

Bioengineering

11.6K ビュー.  Cardiff University. このプロトコルは、細胞を追跡することができますカスタム設計electrotactic室で2Dと3D環境を確立するために使用されるメソッドを示しています生体/ ex vivoでの直流（DC）電界（EFS）を指示する走電性および他の細胞応答を調べるために、単一細胞レベルでの経時的記録を使用します。

ビデオ: Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

3次元自己組織化ペプチドハイドロゲル中のヒト神経前駆細胞の栽培

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

生物工学

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

シングルセルおよび3D閉じ込めにおける単一胚を注文：ハイコンテントスクリーニングのための新しいデバイスを

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

マルチチャンネルデュアル電界マイクロ流体チップを用いて、肺癌細胞の走電性の研究

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

2Dと3Dの肌の改善<em&gt;インビトロ</em分子クラウディングの使用&gt;モデル

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

2D and 3D Matrices to Study Linear Invadosome Formation and Activity

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor cells have to cross anatomical barriers to invade and migrate through the surrounding tissue in order to reach blood or lymphatic vessels. This requires the interaction between cells and the extracellular matrix (ECM). At the cellular level, many cells, including the majority of cancer cells, are able to form invadosomes, which are F-actin-based structures capable of degrading ECM. Invadosomes are protrusive actin structures that recruit and activate matrix metalloproteinases (MMPs). The molecular composition, density, organization, and stiffness of the ECM are crucial in regulating invadosome formation and activation. In vitro, a gelatin assay is the standard assay used to observe and quantify invadosome degradation activity. However, gelatin, which is denatured collagen I, is not a physiological matrix element. A novel assay using type I collagen fibrils was developed and used to demonstrate that this physiological matrix is a potent inducer of invadosomes. Invadosomes that form along the collagen fibrils are known as linear invadosomes due to their linear organization on the fibers. Moreover, molecular analysis of linear invadosomes showed that the discoidin domain receptor 1 (DDR1) is the receptor involved in their formation. These data clearly demonstrate the importance of using a physiologically relevant matrix in order to understand the complex interactions between cells and the ECM.

This protocol describes how to prepare a 2D mixed matrix, consisting of gelatin and collagen I, and a 3D collagen I plug to study linear invadosomes. These protocols allow for the study of linear invadosome formation, matrix degradation activity, and the invasion capabilities of primary cells and cancer cell lines.

線状インベーゾームの形成および活性を研究するための2Dおよび3Dマトリックス

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

三次元培養細胞遊走を特徴付ける傷の環境

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

プライマリのグリア細胞の in Vitro Neuroinflammation のモデリングのための三次元ハイドロゲル文化を改善

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.

This protocol demonstrates the use of a robotic platform for microinjection into single neural stem cells and neurons in brain slices. This technique is versatile and offers a method of tracking cells in tissue with high spatial resolution.

ロボットマイクロインジェクションを用いた脳スライスにおける単一神経幹細胞とニューロンの操作

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, ...

Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell culture system that was used for a long-term cell differentiation study using microscopy. The microfluidic system consists of the following major components: an optically transparent electrotactic chip, a transparent indium-tin-oxide (ITO) heater, a culture media-filling pump, an electrical power supply, a high-frequency power amplifier, an EF multiplexer, a programmable X-Y-Z motorized stage, and an inverted phase-contrast microscope equipped with a digital camera. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This work involves the differentiation of neural stem and progenitor cells (NPCs) induced by direct current (DC) pulse stimulation. In the stem cell maintenance medium, the mouse NPCs (mNPCs) differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. The results suggest that simple DC pulse treatment could control the fate of mNPCs and could be used to develop therapeutic strategies for nervous system disorders. The system can be used for cell culture in multiple channels, for long-term EF stimulation, for cell morphological observation, and for automatic time-lapse image acquisition. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.

In this study, we present a protocol for the differentiation of neural stem and progenitor cells (NPCs) solely induced by direct current (DC) pulse stimulation in a microfluidic system.

マイクロ流体デバイスにおける電界誘発神経前駆細胞分化

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

生体組織の刺激のための電界・磁場装置

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...