0日目 - 実験の日前に滅菌<ol><li>プラスチック浴槽に材料を入れ、2時間、70％アルコールで消毒。 <ul><li>チェンバース</li><li>蓋</li><li>フレーム（全3枚） </li><li>ネジ</li><li>ゴム製のガスケット</li><li>鉗子</li><li>はさみ</li><li>六角レンチ</li></ul></li><li> Aquet石鹸と蒸留水でメンブレンをクリーニング。 </li><li> 10分間70％アルコールで膜を超音波処理してください。 </li><li>プラスチック製の四角い皿にきれいな膜を置きます。パターニングされた膜を使用している場合、パターン化された側は表向き（スプレー溝を下に実行するために液体の膜と時計の片面にアルコール）であることを確認してください。 </li><li> 2時間70％アルコールで覆われた膜を残す。 </li><li>明日の朝オートクレーブ用アルミ箔へのパッケージの手袋。 </li><li>明日のオートクレーブ用蒸留水で2％（重量/体積）ゼラチン溶液（2g/100mL）を作る。 </li><li> 70％アルコールで2時間滅菌した後、一晩UV用フードにすべての高分子材料と膜（非オートクレーブ材）に置きます： <ul><li>チェンバース</li><li>蓋</li><li>フレーム（全3枚） </li><li>メンブレン</li></ul></li><li>オートクレーブ可能な袋に残りの材料をパック。 <ul><li>ネジ</li><li>ゴム製のガスケット</li><li>鉗子</li><li>はさみ</li><li>六角レンチ</li></ul></li></ol> 1日目 - ストレッチチャンバーの組立<ol><li>オートクレーブ鉗子、はさみ、20分、合計で240 ° Fで小さなオートクレーブを使用して六角レンチ、ネジ、ガスケット、手袋、およびゼラチン</li><li> UVからメンブレンを取り出して、約1分間O 2プラズマ（上にパターン側）で扱います。 </li><li> TCのフードでは、ゼラチンとプラズマ処理膜のパターン化された領域をカバー。 UV下分間コート30。 </li><li> PBS 2Xで各メンブレンを洗浄する。第二洗浄した後、チャンバーに簡単に組み立てのために、それらが滑りやすい保つために膜にいくつかのPBSを残す。 （も簡単に組立用ガスケットの潤滑にこのPBSを使用する必要があります）。 </li><li>チャンバー内に膜をアセンブル（組み立て時のオートクレーブ手袋を着用） <ol><li> 1本のネジを使用してフレームの2つの主要部分を接続します。 </li><li>フレームを裏返し、フレームに向かってゼラチンでコーティングされた側面が（パターン化された領域が中心になるはず）ように上に膜を置きます。 </li><li>各側のガスケットを使用してフレームに膜を固定します。 </li><li>そのような膜をリッピングすることはなく、穏やかな、均等に力を使って、でガスケットを押してください。 </li></ol></li><li> T -バーに組み立てフレームを取り付けます。 </li><li>逆さまにチャンバー内にフレームと場所を反転します。 30分間フレームと膜のUV背面側</li><li>再びフレームを反転し、チャンバーにネジ（制御室用の変更なし、しかしストレッチチャンバー用、チャンバーの下部にフレームのエンドピースを接続する2本のネジを使用する必要が、となる1本のネジを削除する必要があります。 ）一緒にフレームの2つの部分を保持する。 30分間UV正面側。膜は、次の手順に進む前に、完全に乾いてから、または他の細胞溶液は種まき時にオフに滑りができることを確認してください。 </li><li>種子の細胞。 3膜の1枚=エリアの面積は、その膜ごとコンフルエントプレートの1 / 3を使用してください。膜セルあたりの溶液1 mLを使用してください。 1.5mlのか以上は解決策を続けることは困難になります。のみパターン化された領域に細胞溶液を入れ、そして周りのソリューションを広めるためにピペットチップを使用してください。チャンバーをカバーし、細胞がフードで30分をRTに接続することができます。 </li><li>インキュベーターにチャンバーを移動し、細胞が1以上の時間のために添付してみましょう。細胞溶液をオフスリップさせることを避けるためにチャンバーを移動するには十分注意してください！解決策は、この時点で落ちる場合、そのチャンバー内の細胞が台無しにされます。 </li><li>フードにチャンバーを戻すと20 mLのメディアを追加。 </li><li>アルミ箔（また、アルコールをスプレー）で覆われてアルコール洗浄浴槽の場所室。インキュベーター（10％CO 2）を延長するためにチャンバーを移動します。 </li><li>ストレッチマシンにチャンバーを固定します。細胞はさらに一晩接続し、次の日にストレッチを始めましょう。 （ 注 ：ストレッチマシンに室を置くとき、チャンバーを配置する前に&quot;ゼロ&quot;の位置にマシンをロックしてから、ギアまわりのラバーバンドを締めるために覚えてマシンを起動する前にギアのロックを解除することを忘れないでください。）。 </li></ol>

<ol>
	<li>Banes, A. J., Gilbert, J., Taylor, D., Monbureau, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+vacuum-operated+stress-providing+instrument+that+applies+static+or+variable+duration+cyclic+tension+or+compression+to+cells+in+vitro.">A new vacuum-operated stress-providing instrument that applies static or variable duration cyclic tension or compression to cells in vitro.</a> J Cell Sci. 75, 35-42 (1985).</li><li>Park, J. S., Chu, J. S., Cheng, C., Chen, F., Chen, D., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+equiaxial+and+uniaxial+strain+on+mesenchymal+stem+cells.">Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells.</a> Biotechnol. Bioeng. 88, 359-368 (2004).</li><li>Kurpinski, K., Chu, J., Hashi, C., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anisotropic+mechanosensing+by+mesenchymal+stem+cells.">Anisotropic mechanosensing by mesenchymal stem cells.</a> Proc. Natl. Acad. Sci. U S A. 103, 16095-16100 (2006).</li></ol>

ベインズら。最初はセル1に機械的な力を提供する柔軟なエラストマー材を使用してin vitroでの細胞の機械的な刺激のためのシステムを使用することを報告した。この時以来、この設計上の多くのバリエーションが考案され、利用されています。いくつかのラボは、特注の装置を使用しながら、いくつかのメカニカルストレッチシステムでは、名前&quot;Flexercell&quot;（Flexcellインターナショナル株式会?...

メディア上の注意：これらの一軸伸張の実験では、バイオリアクターのセットアップの複雑さに起因する汚染が発生しがちです。これに対抗するために、我々は抗生物質と抗真菌剤のさまざまな組み合わせを試してみました。抗生物質の組み合わせと一緒に通常必要な1％のfungizone、。どちらペン/連鎖球菌の組み合わせを使用し、またはカナマイシンおよびゲンタマイシンの組み合わせを（それが耐性変異株の細菌につながる可能性があるので一緒に3つ以上の抗生物質の組み合わせ/カクテルを使用してしようとしないで、そしてまた、細胞に有害かもしれない）を使用して。

mechanical stimulation of stem cells using cyclic uniaxial strain

生体組織の開発と保守における機械的な力の役割は、骨リモデリング、筋肥大、および平滑筋細胞の可塑性など、いくつかの機械的に安定化の現象を含め、文書化されています。しかし、関与する力がしばしば監視し、生体内で制御することが非常に複雑で困難です。より良い細胞に対する機械的な力の影響を調べるために、私たちは弾性膜上で培養した接着細胞に一軸周期引っ張り歪みを印加するためのin vitro法で開発している。このメソッドは、所望の力を適用するには電動カムローターシステムによるカスタム設計のバイオリアクターを利用しています。ここでは、歪みの方向に向い細胞へのマイクロパターン形状を持つシリコン膜を、この場合は、を含む、各&quot;ストレッチチャンバー&quot;のさまざまなコンポーネントを、組み立てる方法を示すステップバイステップのビデオのプロトコルを提示する。我々はまた、チャンバを滅菌メンブレン上に細胞を播種、バイオリアクターにチャンバーをラッチし、機械的なパラメータ（つまり、大きさとひずみの速度）を調整するための手順について説明します。この特定のプロトコルで説明した手順は、歪みの方向に対して平行に配向さ10μm幅のチャネルをシリコン膜上にヒト間葉系幹細胞を播種するための固有のものです。しかし、このシステムで提示方法および材料は、このテーマのバリエーションの数を収容するのに十分な柔軟性があります：ひずみ速度、大きさ、持続時間、細胞の種類、膜の地形、膜のコーティングなどはすべて、目的のアプリケーションに合わせることができますか成果。これは、in vitroで細胞に適用される一軸引張ひずみの影響を調査するための堅牢な方法です。

Watch this Scientific Journal Video about Mechanical Stimulation of Stem Cells Using Cyclic Uniaxial Strain at JoVE.com

Mechanical Stimulation of Stem Cells Using Cyclic Uniaxial Strain

それは広く体内に機械的な力が細胞の分化と増殖に影響を与えることができることが理解される。ここでは、柔軟なマイクロパターン基板上に培養した細胞を食い止めるために一軸周期引っ張り歪みを配信するための特注のバイオリアクターの使用方法を示すビデオプロトコルを提示する。

繰返し一軸性歪みを用いた幹細胞の機械的刺激

The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated ...

mechanical-stimulation-of-stem-cells-using-cyclic-uniaxial-strain

Research

JoVE Journal

Biology

12.7K ビュー.  University of California, Berkeley. それは広く体内に機械的な力が細胞の分化と増殖に影響を与えることができることが理解される。ここでは、柔軟なマイクロパターン基板上に培養した細胞を食い止めるために一軸周期引っ張り歪みを配信するための特注のバイオリアクターの使用方法を示すビデオプロトコルを提示する。

ビデオ: Mechanical Stimulation of Stem Cells Using Cyclic Uniaxial Strain

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 ...

生物学

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

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 ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

集積光と原子間力顕微鏡による研究機械的刺激に対する細胞応答を生きる

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the differentiation of ES cells is the formation of embryoid bodies (EBs) aggregates 2, 3. EB formation is based on spontaneous aggregation when ES cells are cultured in non adherent plates. Three-dimensional EB recapitulates many aspects of early mammalian embryogenesis and differentiate into the three germ layers: ectoderm, mesoderm and endoderm 4.
Immunofluorescence and in situ hybridization are widely used techniques for the detection of target proteins and mRNA present in cells of a tissue section 5, 6, 7. Here we present a simple technique to generate high quality cryosections of embryoid bodies. This approach relies on the spatial orientation of EB embedding in OCT followed by the cryosection technique. The resulting sections can be subjected to a wide variety of analytical procedures in order to characterize populations of cells containing certain proteins, RNA or DNA. In this sense, the preparation of EB cryosections (10&mu;m) are essential tools for histology staining analysis (e.g. Hematoxilin and Eosin, DAPI), immunofluorescence (e.g. Oct4, nestin) or in situ hybridization. This technique can also help to understand aspects of embryogenesis with regards to the maintenance of the tri-dimensional spherical structure of EBs.

Pluripotent stem cells growing in suspension differentiate into embryoid bodies (EBs). Here we demonstrate how to obtain high quality EB cryosections useful for studying cellular and molecular aspects of embryogenesis, while preserving their organization as aggregates.

胚様体の凍結切片を使用して、多能性幹細胞の解析

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the ...

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. 
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

同系ゼブラフィッシュにおける限界希釈細胞移植を用いた腫瘍増殖細胞の頻度を定量化

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

原子間力顕微鏡を用いた肺組織のマイクロメカニカル特性

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

相間細胞核の力学特性を探査する生物物理学的なアッセイ：基板の歪みのアプリケーションおよびマイクロニードル操作

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

GFPをレトロウイルスベクターを用いた誘導多能性幹細胞（性IPSC）にヒト体細胞を再プログラミングする

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

軟骨アガロースゲルの機械的刺激

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

細胞単層に機械的刺激によって誘発されるカルシウム波の伝播：ウシ角膜内皮細胞の例

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...