NIHの助成金HD052670でサポートされています。

1。 ECMタンパク質とプレートのコーティング<ol><li>無菌条件下で、プレート当たり冷滅菌1X PBS 12 mlでラミニン120μgを混合します。 </li><li> Bioflex未処理の板を取り、各ウェルに溶液の2ミリリットルを追加します（ラミニンの最終濃度は2μg/ cm 2である）。また、他のECMタンパク質は、コラーゲン-1 [10μgの/ cm 2]と、フィブロネクチン[を5μg/ cm 2]と、ビトロネクチン[0.5μgの個/ cm 2]またはエラスチン[10μgの個/ cm 2]のように、使用することができます。コー​​ティング技術は、それがラミニンコーティングのために説明されているものに似ています。井戸は完全に溶液で覆われていることを確認してください。 </li><li>プラスチック製のプレートをラップし、ウェルの底に吸着ラミニンを可能にするために一晩、平らな面に4℃で置く。まだ良いECMの機能を維持しながら、これらの条件において、プレートは少なくとも1週間保存することができます。 </li><li>翌日、無菌条件下で、1X PBSでウェルを3回洗浄します。 </li><l私は&gt;を各ウェルに1×PBSで1％のBSAを1mlを加え、37℃でインキュベート膜上の非特異的結合部位をブロックするために1hの°C。の</li><li>非吸着タンパク質を除去するために1X PBSでプレートを3回すすいでください。 </li><li>細胞は、ステップ2.14から隔離されるまで37℃DMEM中℃でプレートを保持します。 </li></ol> 2。胎児の齧歯類の肺II型上皮細胞の分離分離前の日は、オートクレーブと準備ができて、異なるサイズのナイロンメッシュを使用して、画面のコップを持っています。 <ol><li>妊娠のE17-19の上でマウス/ラット時限妊婦から胎児肺を取得します。 DMEM培地を含む50 mlのコニカルチューブに組織を移し、氷の上に置きます。 </li><li> 50 mlコニカルチューブに消化バッファーを作成します。（10ミリリットル）。このボリュームには、マウス/ラットから約20胎児からの肺組織が使用されます。 8.5ミリリットルDMEM 0.5ミリリットル500MM pH7.4のHEPES 5 mgのコラゲナーゼ1 5 mgのコラゲナーゼ1A 1ミリリットル鶏血清、不活化熱</li><li>すべてのコンポーネントが、無菌フード内0.2ミクロンのシリンジフィルターを通して溶解し、フィルターされるまでよく混ぜる。 37℃の水浴中で消化緩衝液を含むコニカルチューブ℃を置く</li><li>ステップ2.1からコニカルチューブからDMEM培地を除去し、滅菌シャーレに組織を転送します。 </li><li> 1mm以上の組織片は、以下のように、ステップ2.3から予め温めておいた消化緩衝液を含むコニカルチューブに組織を転送するために滅菌済みのハサミかカミソリの刃でも肺をミンチ。 </li><li>上下にピペッティングによって組織を消化する。 10ミリリットルピペットで100倍 5ミリリットルピペットで100倍 パスツールピペットで100倍</li><li>室温で5分間1300 rpmでホモジネートを遠心分離します。 </li><li>慎重に吸引して上清を除去し、ペレットをDMEMを15ml加え、20％FBSで細胞を含む再懸濁する。 </li><li> 100画面のコップを取り出し、 - 、30 - 、15ミクロンのナイロンメッシュと場所番目滅菌した150mlのビーカーにEM。 </li><li>ステップ2.8から細胞ホモジネートを追加し、順次フィルタ100を介して - 、30 - 、15ミクロンのメッシュスクリーンカップ。 </li><li>非上皮細胞のろ過を容易にするために、DMEM +10％FBSで数回洗浄した後、15ミクロンメッシュ - 凝集30から非濾過し、細胞を収集します。 </li><li>主に線維芽細胞が含まれている15ミクロンメッシュからろ液を捨てる。 </li><li> 30分（フラスコ当たりの細胞懸濁液のapproximatelly10 ml）を75-cm 2のフラスコにステップ2.11から細胞をインキュベートすることにより、さらにII型細胞を精製する。 </li><li>ペレット細胞に室温で5分間1300 rpmで遠心し上清を収集します。 </li><li>慎重に吸引して上清を除去し、ペレットを無血清DMEM中で細胞を含む再懸濁する。再懸濁の量は、処理された組織の量に依存します。およそ、私たちプレートは、細胞と同等の阿智のために（2 ml）を各ウェルに1つの胎児から得られた次の日にコンフルエントに百分の60から70の間で前夜。 </li><li>ラミニンまたはフィブロネクチンでプレコートBioflex 6ウェルプレート上にプレートの細胞懸濁液。 </li></ol> 3。胎児のげっ歯類の肺線維芽細胞の分離<ol><li> 2.11までのII型細胞の単離のために上記と同じ手順に従ってください。 </li><li>線維芽細胞が付着できるようにするために30〜60分間37℃で75-cm 2の上に、プレートを、15ミクロンメッシュ（おおよその体積は10〜15 mlで前洗浄せず）からのろ液を取る。 </li><li> supernantantを吸引除去し、無血清DMEMと交換して、一晩インキュベートする。 </li><li>次の日、0.4mMのEDTAおよびプレートそれらのフィブロネクチンでプレコートBioflexプレート上で0.25％（wt / vol）のトリプシンで細胞を収穫します。 </li></ol> 4。肺細胞に機械的刺激を提供するための実験系<ol><li>種子無血清DMEMでBioflex培養プレート上で細胞（約50％コンフルエント）（2ミリリットル/ウェル）し、それらを添付してSPREう実験前少なくとも6時間の広告。一般的に私たちの研究室では、機械的歪みを適用する前に、約24時間の培養中の細胞を維持しています。特定のセルの数は実験に必要な場合は、分離した後、II型細胞は無血清DMEM 75-cm 2のフラスコと次の日に保持され、細胞をトリプシン処理し、計数し、その後に播種されています。 </li><li>翌日、メディアは新鮮な無血清DMEM（2ミリリットル/ウェル）とbioflexプレートに置き換えられますがFlexcell FX-5000ひずみユニットに搭載されている。単分子膜は、実験の開始前に超えない80％コンフルエントでなければなりません。 </li><li>等二軸ひずみが膜に適用されます。株のレジメンは（説明を参照してください）in vivoでの機械的な力のシミュレーションによって異なります。 </li><li>並列で培養した無延伸膜上に培養した細胞を対照として使用されています。 </li><li>実験の終わりに、単分子膜は、遺伝子発現の変更（例えば、界面活性プロテインCのために）分析するために処理することができますリアルタイム-PCR、ウエスタンブロット法によるタンパク質の豊富さなどによっても、組織化単分子膜は、免疫細胞化学実験のために固定することができます。この手法では、固定した後、シラスティック膜は板から切り出しており、抗体を用いた透過性とインキュベーションの前に取り付け剤としての水の10〜20μlを用いてスライドガラス上にマウントされている。上清はまた、成長因子、サイトカインなどの存在を調査するために使用することができます</li></ol> 5。代表的な結果 E18胎児マウスのII型細胞の図1と図2には、show代表的な位相コントラストの写真は、この原稿で説明する手法を用いて単離された。 図3は、その機械的ひずみがマーカーとして界面活性剤、タンパク質-Cを使って胎児のII型上皮細胞の分化を誘導を示しています。 <img src="/files/ftp_upload/3543/3543fig1.jpg" /> 図1。担当者resentative位相コントラストの写真は、右胎児のII型細胞の凝集外観を示す分離後に撮影。 <img src="/files/ftp_upload/3543/3543fig2.jpg" /> 図2：E18胎児のII型上皮細胞は、ここで説明したように単離され、ラミニンでコーティングされたbioflexプレート上に播種した。無延伸細胞はパラホルムアルデヒドで固定した後の写真は、次の日に撮影された。細胞の純度は90であることが決定された±5％をSP-Cの上皮細胞の形態や免疫染色の顕微鏡分析によって。 <img src="/files/ftp_upload/3543/3543fig3.jpg" /> 図3。胎児II型上皮細胞を16時間40サイクル/分で5％、環状の歪みにさらされた。そのひずみを示す界面活性プロテインC（SP-C）mRNAの発現）ノーザンブロットは、異なるECM基板を用いたII型細胞の分化を誘導する。 + / - 記号は、sへの暴露を表すかどうかそれぞれ電車、。データは+ /を意味するとして表示されます- SEM、n = 3で、* P &lt;0.05 B）蛍光免疫細胞化学画像は機械的ひずみにさらされるかどうか（コントロール）胎児のII型細胞でSP-C蛋白質レベル（緑）を実証する。核はDAPI（青）で対比染色した。バーは、そのメカニカルストレッチを示す3つの実験から10μmである。C）ウェスタンブロットの結果は、SP-C蛋白質を向上させます。 N = 3、* P &lt;0.05。

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利害の衝突が宣言されません。

本稿では、我々は胎児のII型上皮細胞と線維芽細胞を分離し、Flexcellひずみ装置を使用して機械的な刺激にそれらを公開する方法について説明します。我々は、上皮細胞の分化1,2を評価し、受容体とストレッチ3月9日によって活性化シグナル伝達経路を研究するためにこのテクニックを使用しています。さらに、このメソッドはまた、機械的損傷10,11によって誘導され?...

<table><tbody><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>D5648</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Collagenase 1</td><td>Sigma-Aldrich</td><td>C0130</td><td></td></tr><tr><td>Collagenase 1A</td><td>Sigma-Aldrich</td><td>C9891</td><td></td></tr><tr><td>Chicken serum</td><td>Sigma-Aldrich</td><td>C5405</td><td></td></tr><tr><td>Screen cups</td><td>Sigma-Aldrich</td><td>CD1-1KT</td><td></td></tr><tr><td>Syringe filters</td><td>Fisher Scientific</td><td>09-754-25</td><td></td></tr><tr><td>100 micron nylon mesh</td><td>Small Parts, Inc.</td><td>CMN-100-D</td><td></td></tr><tr><td>30 micron mesh</td><td>Small Parts, Inc.</td><td>CMN-30-D</td><td></td></tr><tr><td>15 micron mesh</td><td>Dynamic Aqua-Supply Ltd.</td><td>NTX15</td><td></td></tr><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>L2020</td><td></td></tr><tr><td>Collagen-1</td><td>Collagen Biomed</td><td>PC0701</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma-Aldrich</td><td>F1141</td><td></td></tr><tr><td>Vitronectin</td><td>Sigma-Aldrich</td><td>V-0132</td><td></td></tr><tr><td>Elastin</td><td>Sigma-Aldrich</td><td>E-6402</td><td></td></tr><tr><td>Bioflex plate</td><td>Flexcell International</td><td>BF-3001U</td><td>Uncoated</td></tr><tr><td>Flexcell Strain Unit</td><td>Flexcell International</td><td>FX-5000</td><td></td></tr></tbody></table>

an experimental system to study mechanotransduction in fetal lung cells

反復的な呼吸のような動きによって、流体の膨張によって子宮内で生成された機械的な力は、通常の肺の開発のために重要である。肺開発の主要なコンポーネントは、肺サーファクタントの主要な源、肺胞II型上皮細胞の分化である。これらの細胞はまた、肺胞腔、宿主防御、および損傷修復に体液の恒常性に参加しています。さらに、遠位の肺実質細胞が直接、出生後の機械的人工換気中の誇張されたストレッチを公開することができます。しかし、肺の細胞が肺の開発に影響を与えると肺傷害を促進するために機械的刺激を感知する正確な分子·細胞メカニズムは完全に理解されていません。ここでは、げっ歯類の胎児の肺からのII型細胞と線維芽細胞を単離するためのシンプルかつ高純度のメソッドを提供します。その後、我々は、機械的な力をシミュレートし、胎児の細胞に機械的な刺激を提供するためには、in vitroシステム、Flexcellひずみユニットで説明胎児肺の開発や肺損傷。この実験システムでは、ストレッチにさらされる胎児肺細胞では分子·細胞メカニズムを調べるための優れたツールを提供しています。このアプローチを使用して、私たちの研究室では、胎児の肺の開発と肺傷害にメカノに参加し、いくつかの受容体とシグナル伝達タンパク質を同定しました。

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An Experimental System to Study Mechanotransduction in Fetal Lung Cells

機械的な力は、肺の開発と肺傷害において重要な役割を果たしています。ここでは、げっ歯類の胎児の肺II型上皮細胞と線維芽細胞を分離するとを使用して機械的刺激にそれらを公開する方法について説明します。<em&gt; in vitroで</em&gt;システム。

胎児肺細胞におけるメカノトランスを研究するための実験システム

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key ...

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12.9K ビュー.  Alpert Medical School of Brown University. 機械的な力は、肺の開発と肺傷害において重要な役割を果たしています。ここでは、げっ歯類の胎児の肺II型上皮細胞と線維芽細胞を分離するとを使用して機械的刺激にそれらを公開する方法について説明します。 in vitroでシステム。

ビデオ: An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

使い方<em&gt;エクスビボ</em&gt;全体の胎児器官の直立飛沫培養物をマウス器官中に発生過程を研究します

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

発生生物学

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

胎児のマウスモデルにおける生物活性剤の気管内投与のための新しい手術アプローチ

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

医学

Live Cell Imaging during Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

メカニカルストレッチ中のライブセルイメージング

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

生物工学

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

マウス胎児肺の文化、分岐の分子機構を評価するシステム

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

生物学

Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent stem cells (hiPSCs) can be differentiated stepwise into 3D multicellular lung organoids, made of both epithelial and mesenchymal cell populations. We recapitulate embryonic developmental cues by temporally introducing a variety of growth factors and small molecules to efficiently generate definitive endoderm, anterior foregut endoderm, and subsequently lung progenitor cells. These cells are then embedded in growth factor reduced (GFR)-basement membrane matrix medium, allowing them to spontaneously develop into 3D lung organoids in response to external growth factors. These whole lung organoids (WLO) undergo early lung developmental stages including branching morphogenesis and maturation after exposure to dexamethasone, cyclic AMP and isobutylxanthine. WLOs possess airway epithelial cells expressing the markers KRT5 (basal), SCGB3A2 (club) and MUC5AC (goblet) as well as alveolar epithelial cells expressing HOPX (alveolar type I) and SP-C (alveolar type II). Mesenchymal cells are also present, including smooth muscle actin (SMA), and platelet-derived growth factor receptor A (PDGFR&#945;). iPSC derived WLOs can be maintained in 3D culture conditions for many months and can be sorted for surface markers to purify a specific cell population. iPSC derived WLOs can also be utilized to study human lung development, including signaling between the lung epithelium and mesenchyme, to model genetic mutations on human lung cell function and development, and to determine the cytotoxicity of infective agents.

The article describes step wise directed differentiation of induced pluripotent stem cells to three-dimensional whole lung organoids containing both proximal and distal epithelial lung cells along with mesenchyme.

肺発生生物学と疾患をモデル化するための人工多能性幹細胞からの3D全肺オルガノイドの生成

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent ...

hiPSC由来内皮細胞の分化と機械的刺激

In Vitro Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides transporting oxygen and nutrients to ensure fetal growth, fetal circulation controls many crucial developmental events taking place within the endothelial layer through mechanical cues. Biomechanical signals induce blood vessel structural changes, establish arteriovenous specification, and control the development of hematopoietic stem cells. The inaccessibility of the developing tissues limits the understanding of the role of circulation in early human development; therefore, in vitro models are pivotal tools for the study of vessel mechanobiology. This paper describes a protocol to differentiate endothelial cells from human induced pluripotent stem cells and their subsequent seeding into a fluidic device to study their response to mechanical cues. This approach allows for long-term culture of endothelial cells under mechanical stimulation followed by&#160;retrieval of the endothelial cells for phenotypical and functional characterization. The in vitro model established here will be instrumental to elucidate&#160;the intracellular molecular mechanisms that transduce the signaling mediated by mechanical cues, which ultimately orchestrate vessel development during human fetal life.

著者スポットライト:流体媒介性機械的刺激下で培養されたhiPSC由来内皮細胞の研究 

Described here is a simple workflow to differentiate endothelial cells from human pluripotent stem cells followed by a detailed protocol for their mechanical stimulation. This allows for the study of the developmental mechanobiology of endothelial cells. This approach is compatible with downstream assays of live cells collected from the culture chip after mechanical stimulation.

イン・ビトロ 発生メカノバイオロジー研究のための胎児血管オンチップモデル

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides ...

Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such as aspiration or coverslip compression have been used to mechanically stimulate whole embryos. These approaches limit experimental design as they are imprecise, require manual handling, and can process only a couple of embryos simultaneously. Microfluidic systems have great potential for automating such experimental tasks while increasing throughput and precision. This article describes a microfluidic system developed to precisely compress whole Drosophila melanogaster (fruit fly) embryos. This system features microchannels with pneumatically actuated deformable sidewalls and enables embryo alignment, immobilization, compression, and post-stimulation collection. By parallelizing these microchannels into seven lanes, steady or dynamic compression patterns can be applied to hundreds of Drosophila embryos simultaneously. Fabricating this system on a glass coverslip facilitates the simultaneous mechanical stimulation and imaging of samples with high-resolution microscopes. Moreover, the utilization of biocompatible materials, like PDMS, and the ability to flow fluid through the system make this device capable of long-term experiments with media-dependent samples. This approach also eliminates the requirement for manual mounting which mechanically stresses samples. Furthermore, the ability to quickly collect samples from the microchannels enables post-stimulation analyses, including -omics assays which require large sample numbers unattainable using traditional mechanical stimulation approaches. The geometry of this system is readily scalable to different biological systems, enabling numerous fields to benefit from the functional features described herein including high sample throughput, mechanical stimulation or immobilization, and automated alignment.

The present protocol describes the design, fabrication, and characterization of a microfluidic system capable of aligning, immobilizing, and precisely compressing hundreds of Drosophila melanogaster embryos with minimal user intervention. This system enables high-resolution imaging and recovery of samples for post-stimulation analysis and can be scaled to accommodate other multicellular biological systems.

ハイスループットマイクロ流体圧縮システムによる多細胞生物のメカノ刺激

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such ...

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.

胎児肺細胞におけるメカノトランスを研究するための実験システム

要約

さらに動画を探す

この動画の章

使い方

胎児のマウスモデルにおける生物活性剤の気管内投与のための新しい手術アプローチ

メカニカルストレッチ中のライブセルイメージング

マウス胎児肺の文化、分岐の分子機構を評価するシステム

肺発生生物学と疾患をモデル化するための人工多能性幹細胞からの3D全肺オルガノイドの生成

イン・ビトロ発生メカノバイオロジー研究のための胎児血管オンチップモデル

イン・ビトロ発生メカノバイオロジー研究のための胎児血管オンチップモデル

イン・ビトロ発生メカノバイオロジー研究のための胎児血管オンチップモデル

ハイスループットマイクロ流体圧縮システムによる多細胞生物のメカノ刺激

マウス胎児腸全体培養システム

胎児肺細胞におけるメカノトランスを研究するための実験システム

要約

さらに動画を探す

この動画の章

使い方

胎児のマウスモデルにおける生物活性剤の気管内投与のための新しい手術アプローチ

メカニカルストレッチ中のライブセルイメージング

マウス胎児肺の文化、分岐の分子機構を評価するシステム

肺発生生物学と疾患をモデル化するための人工多能性幹細胞からの3D全肺オルガノイドの生成

イン・ビトロ 発生メカノバイオロジー研究のための胎児血管オンチップモデル

イン・ビトロ 発生メカノバイオロジー研究のための胎児血管オンチップモデル

イン・ビトロ 発生メカノバイオロジー研究のための胎児血管オンチップモデル

ハイスループットマイクロ流体圧縮システムによる多細胞生物のメカノ刺激

マウス胎児腸全体培養システム

イン・ビトロ発生メカノバイオロジー研究のための胎児血管オンチップモデル

イン・ビトロ発生メカノバイオロジー研究のための胎児血管オンチップモデル

イン・ビトロ発生メカノバイオロジー研究のための胎児血管オンチップモデル