この作品は、NIHの助成金EB007278によってサポートされていました。

BFCの作成 BFCsは4&quot;シリコンマスターウエハ上に成形ポリジメチルシロキサン（PDMS）で作られています。 マスターのウェハの作成<ol><li> 130℃で30分間Siウェハを脱水することにより開始℃に</li><li>スピンコーター上にウエハを配置、それぞれ、40〜30ミリメートルの機能の高さを得るために30秒のためにウェーハ、300回転/秒の3000から4000回転までのランプ、そしてスピンにSU8 ​​- 2050（マイクロケム）を注ぐ。 </li><li>スピンした後、65℃ホットプレート上にウエハを配置、直ちに℃で5〜6分間、その後65℃にランプダウン95に温度をランプアップ</li><li>暗視野マスクを使用して、コンタクトアライナで200ミリジュール/ cm 2のUV用量にウェハを公開。 </li><li> 65ウェーハを置き° Cホットプレート、直ちに° C 4-5分間95に温度をランプアップし、65℃の下のそれをランプ</li><li> PMアセテートとイソプロパノールのウェーハを開発する。 </li><li>からPDMSを防ぐために、ヘキサメチルジシロキサン（信越MicroSi）、または（トリデカフルオロ-1,1,2,2 - テトラヒドロ）-1 - トリクロロシランを（t2492 - KG、UCT特産、ブリストル、ペンシルバニア州）、使用して30分間ウェーハをSilanize Siのマスターのウェハに付着。 </li></ol>成形チップ<ol><li> 10:1の比でミックスPDMS（ダウコーニング、Sylgard 184）、マスターのSiウェハ（ウェハ当たり約15 g）を介してそれを注ぐ、そしてそれは、室温で一晩硬化させることができます。 </li><li> Siウェーハオフ硬化PDMSピールとカミソリの刃を使用して各チップを切り出した。 </li><li>扱いやすいの1x1センチカットの顕微鏡のスライドへの結合の各チップを。 </li></ol>使用するためのBFCの準備<ol><li>実験中にPDMSにメディアの吸収を防ぐために、PBSでBFC一晩浸漬する。 </li><li>キムワイプ（Kimtech科学）のチップを乾燥させます。 </li><li> 7.5％の低下を置くウシ血清アルブミン（BSA）（〜200mL）をBSAを分散させ、井戸からの気泡を除去するためにピペットチップでそれをこすりしBFCのパターン化表面上に、と。 </li><li>少なくとも0.5時間BFC表面にBSAを残す。理想的にはBSAの痂皮を防ぐために15分毎に撹拌。 </li><li> 35x10 mmのTCPSディッシュ（ファルコン、35〜3001）の内側BFCに合わせて、¾&quot;バインダークリップが（オフィスデポ）チップをクランプするために皿の縁に合うれるようにダウンTCPS皿の半分ほどの縁切ると一緒に料理。 </li><li> PDMSシート（シリコン特選商品）や他のシリコンからスペーサーガスケットを（20&#39;20ミリメートル外側の縁、15&#39;15ミリメートル内縁、及び250〜500ミリメートルの厚さで枠状）カットし、皿にそれを適用するピンセットを使って。 </li></ol> BFCとパターニング細胞<ol><li> 1分間UV光下皿、ガスケット、BSAでコーティングされたチップ、2バインダークリップを滅菌する。 </li><li> BSAを吸引し、200 mLのPBSで2回BFCをすすぐ。 </li><li>あなたのセルラインに必要な場合にはカットTCPSディッシュにゼラチンを追加します。そうでない場合は、（下記参照）チップに適用する前に、いくつかのメディアでTCPS表面を濡らし。これにより、チャンバー内に形成される気泡を防ぎます。 </li><li> PBSを吸引後、細胞溶液のピペットを200mL（〜1 × 10 6細胞/ mL）BFC表面に。細胞は5〜10分間のために解決しましょう​​。 </li><li> 〜15 °の角度で角の1つにBFCを傾けて、ゆっくりと200mLのピ​​ペットで細胞溶液をオフピペット。その後、BFC平らに置き、反対側のBFCのコーナーにPBSまたはブランクメディアを100 mLを加え。間だけでなく地域からのすべてのセルをクリアするには、この方法で、必要に応じて追加の2-4回、BFCをすすいでください。 </li><li>チップの表面上にメディアのピペットを200mL。 TCPSからゼラチン/培地を吸引除去する。その後、事前に接液皿を逆さにしてゆっくりと皿に、BFCの上を押してください。 </li><li>それを密封するためにチャンバーの両側に、それぞれのクリップバインダーを適用します。裏返し時にその料理が水平を保つようにバインダークリップから金属製プロングを取り外します。 </li><li>最後に、すぐに不要な動きを避けながら上にセットアップを反転し、インキュベーターに入れてください。 </li></ol>

<ol>
	<li>Rosenthal, A., Macdonald, A., Voldman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Patterning+Chip+for+Controlling+the+Stem+Cell+Microenvironment.">Cell Patterning Chip for Controlling the Stem Cell Microenvironment.</a> Biomaterials. 28, 3208-3216 (2007).</li></ol>

パターンのマウス胚性幹細胞（mESCs）にBFC 1を使用して記述するここに示されているプロトコルやビデオが、BFCsはパターンに興味のある実質的に任意のセル型を使用することができます。一つ加える必要が唯一の変更は、マイクロウェルの大きさを変更することです。我々の経験で、最高の動作する​​別の細胞型、マイクロウェルの直径と結合されていない細胞の直径よりも大きい10ミクロンに等?...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>PDMS</td>
<td>&nbsp;</td>
<td>Dw Corning</td>
<td>Sylgard 184</td>
<td>&nbsp;</td>
<tr>
<td>Silicon master mould</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture dishes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>35-3001</td>
<td>35 mm</td>
</tr>
<tr>
<td>3/4" binder clips</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>hexamethyldisiloxane</td>
<td>&nbsp;</td>
<td>Shin-Etsu MicroSi</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>Isopropanol</td>
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<td>BSA</td>
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patterning of embryonic stem cells using the bio flip chip

拡散性シグナル伝達および細胞間接触（juxtacrineシグナリング）で構成された細胞間相互作用は、腫瘍の増殖、幹細胞の分化、および幹細胞の自己複製のような多数の生物学的プロセスにおいて重要である。メソッドの数は現在、in vitroでの細胞内シグナル伝達を調節するために存在します。拡散性シグナル伝達の変調総量の一つの方法は、培養中の細胞の播種密​​度を変化させることです。細胞播種、実際の細胞間の間隔と細胞間接触の量にかなりの変化でこの結果、及び地域の環境を処方することができないのランダムな性質に起因する。変調細胞のシグナル伝達のためのより具体的なアプローチは、特定のシグナル伝達タンパク質をノックダウンする分子阻害剤や遺伝的アプローチを使用することですが、これらのメソッドの両方が最良の分子の数が少ないが操作するのに適しています。ここで、我々は、基板上に所望の位置でセルの異なる数字を配置することで細胞のクラスタのローカル環境を調節する調節する細胞間シグナル伝達への新しいアプローチを示しています。このメソッドは、セル間の局所拡散性とjuxtacrineシグナリングを制御するための補完的な方法を提供します。我々の手法はバイオフリップチップ（BFC）、細胞の単一細胞または小数値のいずれかを保持するためにマイクロウェルの数百から数千、各サイズを含む微細加工シリコンチップを使用しています。我々は、単に井戸の配列にそれらをピペッティングし、配列からアンロードされた細胞を洗浄することにより、細胞をチップにロードする。細胞は彼らのパターニングを維持し、井戸のそして基板上に落ちるそれによってチップは、その後、基板上に反転されます。細胞が接続した後、チップは（または上左）削除することができます。細胞のパターニングへのこのアプローチは、その点でユニークです：1）このように細胞が増殖して移行できるように、基板の化学的性質を変更しないこと、2）任意の基板上にパターン形成を可能にする、組織培養ポリスチレン、ガラス、マトリゲルを含む、とさらにフィーダー細胞層、および3）それらの島々の中に置かれた細胞と細胞外マトリックスの島々の創造を可能にする、従来のマイクロコンタクトプリンティングと互換性があります。このビデオでは、我々はBFCを用いて組織培養ポリスチレン上にマウス胚性幹細胞のパターニングを示す。

Watch this Scientific Journal Video about Patterning of Embryonic Stem Cells Using the Bio Flip Chip at JoVE.com

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

我々は、基板上に所望の位置にセルを配置するための簡単​​な方法を示しています。このメソッドは、パターン基板上への細胞で満たされたマイクロウェルを含有するシリコーンチップを反転して細胞を。このメソッドは、セル間の拡散性とjuxtacrineシグナリングを変調する新しい方法を提供します。

バイオフリップチップを用いた胚性幹細胞のパターニング

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

patterning-of-embryonic-stem-cells-using-the-bio-flip-chip

Research

JoVE Journal

Biology

9.4K ビュー.  MIT - Massachusetts Institute of Technology. 我々は、基板上に所望の位置にセルを配置するための簡単​​な方法を示しています。このメソッドは、パターン基板上への細胞で満たされたマイクロウェルを含有するシリコーンチップを反転して細胞を。このメソッドは、セル間の拡散性とjuxtacrineシグナリングを変調する新しい方法を提供します。

ビデオ: Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

ヒト胚性幹（ES）細胞の増殖

生物学

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

マウス胚性幹細胞から造血幹細胞の導出

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

CD4 + Tリンパ球キャプチャHIVのための使い捨てマイクロ流体チップを用いた

RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit

Bio - Rad社のTotal RNA Kitを使​​用してNeuroprecursor細胞からのRNA抽出

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

免疫手術でヒト胚性幹細胞の導出

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

ヒト胚性幹細胞からクロマチン免疫沈降

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

ハンギングドロップ法を用いたマウス胚性幹（MES）細胞のin vitroにおける分化

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

ヒト胚性幹細胞の文化とメンテナンス

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

ヒト胚性幹細胞を凍結融解

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

マウスES胚細胞の単離と導出

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...