著者らは、溶媒蒸気の溶接に関する彼らの実質的な助けのためにダークヘルマン、オリバーウェント、ジークフリートホーン、ムートグートツァイト、とイェルクボーンを感謝したい。さらに、我々は彼らの技術支援のためにマイケルハルトマン、アレックスGerwald、とダニエルライゼンに感謝します。

プロセスのシーケンス＃1：ホットエンボス、加工および溶剤蒸気の溶接そのキュービックデザインのCellChipは、ホ​​ットエンボス加工やマイクロ射出成形によって複製されます。このため、我々は、チップの逆形状と微細加工真鍮の金型を使用してください。コンテナ - 1156コンテナまでの規則的なアレイ状に配置されたが - 300μmの辺の長さを持つ立方体のデザインを持っている。ホットエンボス加工の場合は、レプリケーションプロセスは、従来のWUM02（Jenoptik Mikrotechnik、ドイツ）で行われます。ツールは2つの円形の金属板で構成されています。最初のステップでは、薄いPMMAプレート（Lucryl、G77Q11、BASFが）開いたツールの下のプレートの中央に配置されます。微細金型のインサートはcentrically上部のプレートに取り付けられている。その後、ツールは、金型の避難のために閉鎖し、ポリマーのガラス転移温度より高い温度に加熱される。それらが完全に正確に金型の形状を複製いっぱいになるまで一緒にプレートを押すことで、粘性のポリマーは、真空にキャビティにプッシュされます。ツールを冷却した後、微細ポリマー部分がdemouldedすることができます。プロセスは、実際に金型の空洞を埋めるために必要以上のポリマーの質量が必要です。ポリマーの剰余金は、一部の離型を容易にするために使用することができる残留層を形成する。しかし、容器の底には3μmより小さい直径の孔を作成するには、厚い残留層がダウンして薄くする必要がある、あるいは完全に多孔質膜で取り外し交換。細孔の統合のプロセスを簡素化するために、複製されたCellChipの背面は完全にダイヤモンドの工場で機械加工により除去される。このため、部品が冷却取付板に固定されており、さらに、脆弱な構造が損傷に対してそれらを保護するために、脱イオン水で凍結されています。 最後の工程で、最終的に商業的なイオンのトラックエッチメンブレン（ポリカーボネート、厚さ10μm、孔径3μmの、2 × 10 6ポア/ cm ²の、ピーパーフィルター社）はコンテナの配列の後ろに結合している今の両方を開設上部と下部。接合プロセスは、気密、加熱室[図行わ溶剤蒸気の溶接プロセスです。チャンバーを排気した後、1]、上側のプランジャーと統合された真空チャックと可動側プレートで構成される。4は最大4つの機械加工CellChipsとトラックエッチメンブレンには、気化溶剤を並列にさらされている。その後、成形品と膜は、上部プランジャーで一緒に押されている。露出（&lt;15秒）の短い期間の後、チャンバーは再びそれによって溶媒を除去する排気される。短い接触時間のために、材料に近い表面のみが溶解され、機械的負荷に起因するバルク構造の変形を避けることができます。最後に、チャンバーが開かれ、溶剤溶接CellChipsを除去し、細胞培養[図のために調製することができる。 2]。 <a href="/files/ftp_upload/old/699_2008_5_12_7/Giselbrecht-Fig1.png"><img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_7/Giselbrecht-Fig1.png" alt="図1" width="100" /></a> 図1。 <a href="/files/ftp_upload/old/699_2008_5_12_8/Giselbrecht-Fig2.png"><img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_8/Giselbrecht-Fig2.png" alt="図2" width="100" /></a> 図2。 プロセスのシーケンス＃2：重イオン照射、microthermoformingとトラックのエッチング（SMARTプロセス） SMARTと呼ばれる新しいプロセスは、機能性膜のような微細構造を製造するため、最近開発されたマイクロテクノロジーです。5テクノロジが&quot;microthermoforming&#39;と呼ばれる、microtechnical熱成形プロセスに基づいている。肉眼から適応されたこの中央のプロセスステップにおいて、6,7シート熱成形プロセスを閉じ込め、加熱された高分子薄膜を金型キャビティ[図へのガスの圧力によって、その軟化、ゴム弾性状態で形成される。 3]。ホットエンボス加工または射出成形とは異なり、このプロセスは、プライマリ形成ではなく、ポリマーを溶融されていません。フィルムは永久的な材料の凝集固体状態ではまだ形成されているという事実のために、高い横方向の解像度を持つ材料の変更は、第一の平面高分子膜上に生成することができ、成形過程で保存されます。 microthermoformingステップの後、これらの変更は、さらに選択的に湿式化学処理により、例えば、処理することができます。 <img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_9/Giselbrecht-Fig3.png" alt="図3" width="400" height="217" /> 図3 原則としてSMARTプロセスは、以下の3つのプロセスの手順で構成されます。 <ol><li>プリプロセスの平面薄いポリマーフィルム上で高度に解決した変更のパターンの作成</li><li> 3D SHAP（パターン）を失うことなくmicrothermoformingによるフィルムのINGの修正</li><li>薄肉微細部品の最終的な官能化のためのポストプロセス（オプション） </li></ol>我々は現在、多孔CellChipsの製造に適用されているSMARTプロセスは、以下の工程[図を含みます。 4]。薄いポリマーフィルム、ポリカーボネート（Pokalon OG461Gl、50μm以下、LoFoハイテク映画社、ドイツ）からの例は、、（ダルムシュタット、GSIの加速器施設での加速、重イオン（例えばキセノン、AuまたはUのイオンなど）を照射し、約のエネルギーを持つドイツ）。 1 GeVのと工具を冷却した後、106 8の順にフルーエンスは、薄肉部がdemouldedすることができます。イオン/ cm ²。フィルムを貫通する場合は、各イオンは、潜在的なトラックと呼ば​​れる、変更された材料のほぼまっすぐな道を生成します。前処理されたフィルムはその後、25 × 25薄肉microcontainers、300μmの直径と深さとそれぞれの配列に熱成形されています。プロセスは、現在、修正されたホットエンボスプレス[図で実行されます。高分子フィルムを2枚の金属板との間にクランプされている5]、。上部のプレートは、微細加工金型を装備し、小さい方が圧力と真空コネクタが含まれています。フィルムは、最大5 MPaまでの圧力で窒素による金型の以前に排気したマイクロキャビティに引き伸ばされている。フィルムは、アニールトラックを防止するため、そのガラス転移温度近傍に形成されています。 <img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_10/Giselbrecht-Fig4.png" alt="図4" width="400" height="119" /> 図4 <a href="/files/ftp_upload/old/699_2008_5_12_11/Giselbrecht-Fig5.png"><img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_11/Giselbrecht-Fig5.png" alt="図5" width="100" /></a> 図5 ポストプロセスでは、イオントラックを選択的に適切なエッチング媒体（例えば、5モル/ L水酸化ナトリウム、10％（w / v）のメタノール）に全体の微細構造を浸漬することによって毛穴にエッチングされています。エッチング時間を制御し、そのような濃度、温度および特殊添加剤（例えば、エッチプロモーター）などの条件を、エッチングすることにより、結果として得られる細孔の大きさと形状は、[図を調整することができます。 6]。 <a href="/files/ftp_upload/old/699_2008_5_12_12/Giselbrecht-Fig6.png"><img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_12/Giselbrecht-Fig6.png" alt="図6" width="100" /></a> 図6

<ol>
	<li>Knedlitschek, G., Schneider, F., Gottwald, E., Schaller, T., Eschbach, E., Weibezahn, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tissue-like+culture+system+using+microstructures:+influence+of+extracellular+matrix+material+on+cell+adhesion+and+aggregation.">A tissue-like culture system using microstructures: influence of extracellular matrix material on cell adhesion and aggregation.</a> J Biomech Eng. 121, 35-39 (1999).</li><li>Gottwald, E., Giselbrecht, S., Augspurger, C., Lahni, B., Dambrowsky, N., Truckenm&#252;ller, R., Piotter, V., Gietzelt, T., Wendt, O., Pfleging, W., Welle, A., Rolletschek, A., Wobus, A. M., Weibezahn, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chip-based+platform+for+the+in+vitro+generation+of+tissues+in+three-dimensional+organization.">A chip-based platform for the in vitro generation of tissues in three-dimensional organization.</a> Lab Chip. 7, 777-785 (2007).</li><li>Heckele, M., Schomburg, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+on+micro+molding+of+thermoplastic+polymers.">Review on micro molding of thermoplastic polymers.</a> Journal of Micromechanics And Microengineering. 14, (2004).</li><li>Giselbrecht, S., Gietzelt, T., Guber, A. E., Gottwald, E., Trautmann, C., Truckenm&#252;ller, R., Weibezahn, K. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microthermoforming+as+a+novel+technique+for+manufacturing+scaffolds+in+tissue+engineering+(CellChips.">Microthermoforming as a novel technique for manufacturing scaffolds in tissue engineering (CellChips.</a> IEE Proc.-Nanobiotechnol. 151, 151-157 (2004).</li><li>Giselbrecht, S., Gietzelt, T., Gottwald, E., Trautmann, C., Truckenm&#252;ller, R., Weibezahn, K. -. F., Welle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+tissue+culture+substrates+produced+by+microthermoforming+of+pre-processed+polymer+films.">3D tissue culture substrates produced by microthermoforming of pre-processed polymer films.</a> Biomed Microdevices. 8, 191-199 .</li><li>Truckenm&#252;ller, R., Rummler, Z., Schaller, T., Schomburg, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-cost+thermoforming+of+micro+fluidic+analysis+chips.">Low-cost thermoforming of micro fluidic analysis chips.</a> Journal of Micromechanics and Microengineering. 12, 375-379 (2002).</li><li>Truckenm&#252;ller, R., Giselbrecht, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microthermoforming+of+flexible,+not+buried+hollow+microstructures+for+chip-based+life+sciences+applications.">Microthermoforming of flexible, not buried hollow microstructures for chip-based life sciences applications.</a> IEE Proc.-Nanobiotechnol. 151, 163-166 (2004).</li><li>Fleischer, R. L., Price, P. B., Walker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+tracks+in+solids.">Nuclear tracks in solids.</a> , .</li></ol>

このようなマイクロ射出成形または熱エンボス加工のようなポリマーのmicroreplicationの確立された方法は、微細構造を製造するのに適しているが、彼らはCellChipのために必要とされる様に、統合と高度に制御された多孔度を有する微細構造を製造するのに本当に効果的ではありません。バルキーな構造は、例えば、その後のレーザー穿孔のために壁の厚さを減らすために高価な機械加工を必要とするか、壁に?...

microfabrication of chip-sized scaffolds for three-dimensional cell cultivation

微細加工技術を使用すると、三次元細胞培養のための再現可能な幾何学と一定の品質の足場を作るための前提条件です。これらの技術は、製造業のためだけでなく、さまざまなアプリケーションのためだけでなく、利点の広いスペクトルを提供しています。形成された細胞のクラスターのサイズと形状は、微細加工足場の正確で再現性のあるアーキテクチャの影響を受け、したがって、栄養素とガスの拡散パスの長さがcontrolled.1することができますこれは紛れもなくのアポトーシスと壊死を防ぐために有用なツールですできます不十分な栄養とガスの供給や携帯代謝物の除去に起因する細胞。 CellChipと呼ばれる私たちのポリマーチップは、中央の微細構造の面積は2 × 2cmの外寸を持っています。このエリアは、キュービックデザインの300メートルの一辺​​の長さ（CP -またはCF -チップ）のまたはラウンドデザイン（R -チップ）は300 mの直径と深さの典型的なディメンションを持つ1156 microcontainersまでの配列に分割されます。 2 これまでのところ、ホットエンボス加工やマイクロインジェクション成形は、（部品のその後の面倒な機械加工との併用）微細チップの製造に使用した。基本的には、マイクロ射出成形は、今まで、しかしポリマーmicrostructures.3の大量生産のための可能な、唯一のポリマーベースのレプリケーション技術の一つであり、両方のテクニックは、世代と溶融粘度のポリマーの処理に起因する特定の不必要な制限があります。非常に薄い壁や穴に統合。 CellChipの場合には、薄底の層は、ポリマーを穿孔し、容器のマイクロ流体灌流により培養液の例で細胞を供給するために定義されたサイズの小さな孔を提供するために必要です。 これらの制約を克服するために、我々はダウンスケーリングされた熱成形プロセスに基づいて新しいmicrotechnicalアプローチを開発して製造コストを削減するために。高度に多孔性と薄肉ポリマーチップの製造のために、我々は、重イオン照射、microthermoformingとトラックのエッチングの組み合わせを使用してください。このいわゆる&quot;SMART&quot;プロセス（熱成形による基板変形と複製）に薄い高分子膜は、いわゆる&quot;潜在的なトラックは&quot;その後、ゴム弾性状態のフィルムを3つに形成されて導入し、数百MeVの高エネルギー重投射物に照射されていますトラックを変更したり、アニーリングなしの次元の部品。成形工程の後に、選択的化学エッチングは、最終的に調整可能な直径の円筒状の孔にトラックを変換します。

Watch this Scientific Journal Video about Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation at JoVE.com

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

我々は、三次元細胞培養用多孔性ポリマーチップの微細加工のための2つのプロセスを提示する。最初のものは、溶剤蒸気の溶接プロセスと組み合わせて熱エンボス加工です。もう一つは製造の大幅な簡素化につながるイオン飛跡技術と組み合わせた最近開発microthermoformingプロセスを使用しています。

三次元細胞培養用チップサイズの足場の微細加工

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

microfabrication-chip-sized-scaffolds-for-three-dimensional-cell

Research

JoVE Journal

Biology

11.7K ビュー.  Karlsruhe Research Centre. 我々は、三次元細胞培養用多孔性ポリマーチップの微細加工のための2つのプロセスを提示する。最初のものは、溶剤蒸気の溶接プロセスと組み合わせて熱エンボス加工です。もう一つは製造の大幅な簡素化につながるイオン飛跡技術と組み合わせた最近開発microthermoformingプロセスを使用しています。

ビデオ: Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

灌流マイクロバイオリアクターのチップベースの三次元細胞培養

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

生物学

Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research

A Nature Editorial in 2003 asked the question "Good-bye, flat biology?" What does this question imply? In the past, many in vitro culture systems, mainly monolayer cultures, often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.
The last generation of CellChips -- micro-thermoformed containers -- a specific technology was developed, which offers the additional possibility to modify the whole surface of the 3D formed containers. This allows a surface-patterning on a submicron scale with distinct signalling molecules. Sensors and signal electrodes may be incorporated. Applications range from basic research in cell biology to toxicology and pharmacology. Using biodegradable polymers, clinical applications become a possibility. Furthermore, the last generation of micro-thermoformed chips has been optimized to allow for cheap mass production.

In the past many in vitro culture systems -- mainly monolayer cultures -- often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.

インタビュー：幹細胞研究のためのバイオリアクターと浮上し、修正3D -足場

A Nature Editorial in 2003 asked the question  "Good-bye, flat biology?"  What does this question imply? In the past, many in vitro culture systems,  ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

組織工学と細胞培養のための繊維状重合体の足場をエレクトロ

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

三次元混合細胞スフェロイド共培養から乳腺上皮細胞の分離

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

DNA損傷応答タンパク質の2次元および3次元生細胞イメージング

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

マウス胎児腸全体培養システム<em&gt;生体外</em&gt;シグナル伝達経路の操作と絨毛開発の3次元ライブイメージング

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

三次元ハイドロゲルでイメージングをFRET

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

受動的な明快さクリア マウス卵巣の血管の 3次元再構築

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

裏打ちクリア: 組織の脂肪組織の三次元イメージング法をクリア

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

インタクトオルガノイドの単一細胞分解能3次元イメージング

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...