Name: human dupuytren's ex vivo culture for the study of myofibroblasts and extracellular matrix interactions
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Description: Watch this Scientific Journal Video about Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions at JoVE.com

The authors are thankful to the nurses at the AMC that have facilitated the tissue collection. We would also like to acknowledge A.M.A. van der Laan for excellent technical support with the SHG and the two-photon imaging.

このプロトコルは、人間の研究倫理委員会のLUMCとAMCガイドラインに従っています。 1.手術手順及び組織収集注：デュピュイトラン拘縮の外科的切除のための様々な技術が存在するが、現在のゴールドスタンダードは、部分的な筋膜切除術15です。ほとんどの患者は、一日手術クリニックで治療されている。 <ol><li>患者と麻酔専門医の好みに...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+myotome:+a+muscle+with+no+innervation.">The mammalian myotome: a muscle with no innervation.</a> Evolution &amp; Development. 10 (6), 746-755 (2008).</li><li>Deries, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+remodeling+accompanies+axial+muscle+development+and+morphogenesis+in+the+mouse.">Extracellular matrix remodeling accompanies axial muscle development and morphogenesis in the mouse.</a> Developmental Dynamics. 241 (2), 350-364 (2012).</li><li>Martins, G. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+3D+Cell+Rearrangements+Guided+by+a+Fibronectin+Matrix+Underlie+Somitogenesis.">Dynamic 3D Cell Rearrangements Guided by a Fibronectin Matrix Underlie Somitogenesis.</a> PLoS ONE. 4 (10), e7429 (2009).</li><li>Yokomizo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+three-dimensional+imaging+of+internally+localized+immunostained+cells+within+mouse+embryos.">Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryos.</a> Nat. Protocols. 7 (3), 421-431 (2012).</li><li>Karkampouna, S., de Julio, M. 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ex vivoでの人間の結合組織を培養する最も重要なステップは実行可能性を確保するための外科的除去後の組織の直接の使用である。組織は常に培地または生理食塩溶液中に留まるべきである。無菌培養を維持する。組織の横断切片は、最大200μmの厚さを有するべきである。組織培地と接触しているが、完全に浸水しないときのex vivo培養系でセットアップすることが最適である。?...

デュピュイトラン病（DD）、良性の線維増殖性疾患が原因の手のひらの上で結節及びコードの形成に指の永久的な屈曲を引き起こす。病気の広がりは、北欧の白人の中でも特に高いが、疾患の根底にある遺伝的病因は、1不明のまま。 DDの主な特徴は、恒久的に細かい動きを破壊し、腱や手のひらの皮膚と指の間のスペースを占有しているタフな繊維組織を形成する細胞外マトリックス（ECM）タンパク質（ 例えば、コラーゲン）の過剰生産である手2の、3。疾患の再発が線維1,4の原因として、基礎となる遺伝的変化を示唆している。有効な治療は、細胞および分子レベルで直接制御できない線維性のメカニズムを標的とすることができる。 線維症に関する我々の最近の研究は、の開発に私たちをリードしてきました新規な薬物検査の潜在的なヒト線維性組織の短期培養を可能にする三次元培養系。このシステムは、2D線維芽細胞培養の制限アプローチを克服し、線維症5の仲介にTGFβ経路活性化のエクソンスキッピングによって達成部分的なダウンレギュレーションの役割を、定義するために貢献してきました。 私たちは、筋線維芽細胞と周囲のECM 5、6の間の相互作用を研究するために培養したDD患者からex vivoでの人間の切除標本の方法を開発した。DD結合組織線維化の研究だけでなく、他の線維性の疾患は、切除した外科の病理組織学的分析に依存している標本、組織及び初代培養または細胞選別手順の確立からの線維芽細胞を単離する。彼らは実験者による病気の性質または治療的介入の外因性の操作を許可しないので、これらのアプローチは非常に静的です。加えて、一次のceLL培養は、培養条件に適応する傾向があり、それらの遺伝子発現特性にも早期の継代（継代3間および6）7、8の間に、すべての通過時の生体内の状況から本質的に異なっている。私たちは、廃棄物の外科的材料を維持するために管理している患者特有の特性の研究及び抗線維症または抗炎症性薬剤化合物のスクリーニングを可能にする期間のex vivoでの培養条件。 システムは、DD線維芽細胞ならびに他の細胞型9を培養する際に、以前に観察されたように、取り付けの際に組織の変質を防止するため、プラスチックを含む培地と組織の接触を可能にするではなく、ニトロセルロース膜に基づいている。 DD組織自体は、これらのタンパク質を大量に生成するのでないコラーゲンゲルまたは他のECMタンパク質基質は、必要とされない。これは、ネイティブのECM微小環境と売上高の罪の維持のために有利であるCEのマトリックス基質は組織構造および機能10、11の重要な調節因子である。例えば、フィブロネクチン、ラミニンおよびコラーゲンなどのECMタンパク質は、のように、同様の上皮細胞12に頂端-基底極性のために示された線維芽細胞の前後極性、13に影響を与える可能性。極性細胞は、細胞の移動および遺伝子発現、 例えば、膜上のα1β1インテグリンのアクセシビリティは、私が14型コラーゲンへの細胞接着に影響を与えるを決める外分子の非対称な分布を有する。この3Dモデルの主な目標は、天然の微小環境を維持することであったため、人工ECMマトリクス基板は使用しなかった。 簡単に言えば：切除標本を均等に無菌環境で切断され、nitrocellularメンブレン上に置いた。注射による投与の治療が必要とされる場合、それらは、膜上に配置された後に組織が注入される。治療は、INJを経由して投与するために必要としない場合ection次に、化合物（1％ウシ胎児血清（FCS）、1％ペニシリン - ストレプトマイシン（P / S）を、ダルベッコ改変イーグル培地（DMEM））を培養培地に添加される。以前5に記載のように培養物を、30％ショ糖溶液を介して処理組織を4％パラホルムアルデヒド（PFA）で固定した後10日目の最大、OCTコンパウンドに包埋し、-80℃で保存のために維持される。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium</td>
			<td>Invitrogen</td>
			<td>11965-084</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal calf serum (FCS)</td>
			<td>Gibco</td>
			<td></td>
			<td>high glucose, heat inactivated</td>
		</tr>
		<tr>
			<td>penicillin-streptomycin</td>
			<td>Invitrogen</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture inserts</td>
			<td>Millicell</td>
			<td>PIHA01250</td>
			<td>0.45um pore size, 12mm diameter</td>
		</tr>
		<tr>
			<td>anti-collagen type I</td>
			<td>Southern Biotech</td>
			<td>1310-08</td>
			<td></td>
		</tr>
		<tr>
			<td>anti- &#945; smooth muscle actin</td>
			<td>Sigma</td>
			<td>A2547</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-collagen type III</td>
			<td>Southern Biotech</td>
			<td>1330-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor555 Donkey Anti-Mouse IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A-31570</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Donkey Anti-Goat IgG (H+L) Antibody</td>
			<td>Invitrogen</td>
			<td>A-11055</td>
			<td></td>
		</tr>
		<tr>
			<td>TOPRO-3</td>
			<td>Invitrogen</td>
			<td>T3605</td>
			<td></td>
		</tr>
		<tr>
			<td>methylsalicylate</td>
			<td>Sigma</td>
			<td>M6752</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Tek OCT</td>
			<td>Sakura</td>
			<td>25608-930</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsccope glass coverslips</td>
			<td>Menzel-Glaser</td>
			<td>BB024060A1</td>
			<td>24x60mm</td>
		</tr>
		<tr>
			<td>Microscope SuperFrost slides</td>
			<td>Menzel-Glaser</td>
			<td>AA00000102E</td>
			<td>76x26mm</td>
		</tr>
		<tr>
			<td>VECTASHIELD HardSet Mounting Medium</td>
			<td>Vector laboratories</td>
			<td>H-1400</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica TCS SP5 II confocal microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>Argon-488, 514 nm and HeNe-633 nm laser lines</td>
		</tr>
		<tr>
			<td>Zeiss 710 NLO upright confocal microscope</td>
			<td>Jena, Germany</td>
			<td></td>
			<td>equipped with femtosecond Spectra - Physics Deep See MP laser (Santa Clara, United States) using a Plan -Apochromat 20x/1.0 NA 
			water-immersion objective.</td>
		</tr>
	</tbody>
</table>

human dupuytren's ex vivo culture for the study of myofibroblasts and extracellular matrix interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

エキソビボの方法は、結合組織の三次元培養は、同様にECMとDDの組織を構成する他の細胞成分および線維症の潜在的に他の種類との関係を理解するための簡単かつ堅牢なアップ方式である。さらにこのシステムは、異なる細胞型および線維性負荷20への影響に対する化合物の効果を試験するための信頼性の高い方法を可能にする。 デュピュイトラン線維症?...

Watch this Scientific Journal Video about Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions at JoVE.com

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

デュピュイトラン病（DD）は、手のひらの線維増殖性疾患である。ここでは、三次元（3D）培養系においてDDから培養切除標本のプロトコルを提示する。このような短期培養系は、3次元構造の保存および線維性組織の分子特性を可能にする。

人間デュピュイトラン<em&gt;エクスビボ</em筋線維芽細胞と細胞外マトリックス相互作用の研究のために&gt;文化

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

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Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Ex Vivo Culture of Patient Tissue &amp; Examination of Gene Delivery

Ex Vivo Culture of Patient Tissue & Examination of Gene Delivery

遺伝子デリバリーの患者の組織及び審査のex vivoでの文化

This video describes the use of patient tissue as an ex vivo model for the study of gene delivery.  Fresh patient tissue obtained at the time of surgery ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

人間の神経内分泌腫瘍療法の研究のための三次元モデルとしての人間の神経内分泌腫瘍細胞株

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

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A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

ヒューマン<em&gt;生体外</em&gt;動脈硬化性プラークのモデルは、病変生物学を研究する

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic ...

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

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The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

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人間の伏在静脈のための手順<em&gt;生体外</em&gt;灌流および外部補強

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal ...

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臨床応用のためのヒト羊膜上皮細胞の単離、凍結保存と文化

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

An Ex vivo Model to Study Hormone Action in the Human Breast

An Ex vivo Model to Study Hormone Action in the Human Breast

アン<em&gt;エクスビボ</emヒト乳癌でホルモンの作用を研究するために&gt;モデル

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

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生体内および生体外では腹膜脂肪にミルキースポット構造の卵巣がん転移性コロニー形成を研究するためのアプローチで

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

Kinomicプロファイリングと薬物反応検査のための3D人間バイオゲル培養システムと患者由来の神経膠芽腫細胞を用いた微小腫瘍の発生

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...