Name: human dupuytren's ex vivo culture for the study of myofibroblasts and extracellular matrix interactions
Uploaded: 2015-04-18T14:00:00
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.

Bu protokol, insan araştırma etik kurul LUMC ve AMC yönergeleri takip eder. 1. Cerrahi Prosedür ve Doku Koleksiyonu Not: Dupuytren kontraktürü cerrahi eksizyon için çeşitli teknikler mevcut olmasına rağmen, mevcut altın standart kısmi fasiyektomi 15 olduğunu. Hastaların çoğu günübirlik cerrahi kliniğinde tedavi edilir. <ol><li> Hasta ve anesteziyoloji tercihlerine ve hastanın komorbidite göre genel veya bölgesel anestezi altın...

<ol>
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ex vivo insan bağ dokusu kültürleme en kritik adımlar canlılığı sağlamak için cerrahi müdahaleden sonra dokusunun hemen kullanımı vardır; Doku her zaman orta ya da tuzlu su çözeltisi içinde kalmalıdır; steril kültür korumak; doku enine kesitleri en fazla 200 mikron kalınlığına sahip olmalıdır; Doku ortamı ile temas içinde olan, ancak tamamen su olmadığında, ex vivo kültür sistemi kurmak en uygunudur. Orta sadece küçük bir hacim içinde transwell dış bölme (ö...

Dupuytren hastalığı (DD), benign fibroproliferatif hastalık nedeniyle avucunun içinde nodüller ve kord oluşumuna parmakların fleksiyonu kalıcı olur. Hastalık yayılmış Kuzey Avrupa Kafkasyalılar arasında özellikle yüksek olmasına rağmen, hastalığın altta yatan genetik nedenleri 1 bilinmemektedir. DD temel özelliği, tendon ve el ve parmakların avuç derisi arasındaki boşluğu işgal zor bir fibröz doku oluşturan hücre dışı matriks (ECM) proteinleri (örneğin, kolajen), aşırı üretim sürekli ince hareketleri kesintiye el 2, 3. Hastalığın nüks fibrozis 1, 4 nedeni olarak altta yatan genetik değişiklikler göstermektedir. Etkili bir tedavi doğrudan hücresel ve moleküler düzeyde kontrol edilemeyen fibrotik mekanizmaları hedef olabilir. Fibrozis Bizim son çalışma gelişmesine bizi açmıştıruyuşturucu testi potansiyeli ile insan fibrotik doku kısa vadeli kültürünü tanır yeni 3D kültür sistemi. Bu sistem 2B fibroblast kültürleri sınırlayıcı yaklaşımı aşmak ve fibrozis 5 aracılık TGFβ yolu aktivasyonu ekson atlama elde kısmi aşağı düzenlenmesi için bir rol, tanımlamak için yardımcı olmuştur. Biz DD hastaların ex vivo insan rezeksiyon örnekleri miyofibroblastlar arasındaki etkileşimi ve çevresindeki ECM 5, 6 çalışma kültürüne bir yöntem geliştirdik. DD bağ dokusu fibrozis çalışma yanı sıra diğer fibrotik hastalıklar, cerrahi eksize histopatolojik analizi dayanır Numuneler, doku ve primer kültürlerinde ya da hücre sıralama prosedürlerin kuruluşundan fibroblastların izolasyonu. Onlar deneyci tarafından hastalık özelliklerine veya terapötik müdahale eksojen manipülasyon izin vermez çünkü bu yaklaşımlar oldukça statik bulunmaktadır. Ek olarak, birincil cell kültürleri kültür koşullarına uyum ve gen ifadesi özellikleri hatta erken geçişleri sırasında, her pasajda üzerine esasen in vivo durumdan farklı eğilimi (geçit 3 arasında ve 6) 7, 8. Biz atık cerrahi malzeme korumak başarmış hastaya özgü özellikleri ve bunların anti-fibrotik veya iltihap-önleyici ilaç bileşimlerinin tarama izin veren bir zaman süresi için ex vivo kültür koşulları. Sistem DD fibroblastlar gibi diğer hücre tipleri 9 kültürlenmesi, önceden görüldüğü gibi, bağlantı üzerine doku değişimini önlemek, böylece, orta değil plastik doku teması sağlayan bir nitroselüloz zar üzerine dayanır. DD dokulardaki bu proteinlerin, büyük miktarlarda üretim için herhangi kolajen jel veya başka bir ECM protein alt-tabakası, gereklidir. Bu yerli ECM mikroçevresinin ve ciro günah bakımı için avantajlıdırce matriks substrat doku yapısının ve fonksiyonunun 10, 11 arasında önemli bir regülatör bulunmaktadır. Örneğin, bu gibi fibronektin, laminin ve kolajen gibi ECM proteinlerinin yanı Benzer epitel hücreleri 12 apikal-bazal polaritesi için gösterilen fibroblast ön-arka polarite, 13 etkileyebilir . Polarize hücreler, hücre göçü ve gen ekspresyonu, örneğin, zar üzerinde α1β1 integrini erişilebilirlik bir 14 kolajen tip hücre yapışmasını etkilediği tespit dışı moleküllerin asimetrik dağılımına sahiptir. Bu 3D modeli temel amacı yerli mikroçevreyi korumak için olduğundan, hiçbir yapay ECM matris substrat kullanılmıştır. Özet olarak: reseksiyon örneklerinden eşit steril bir ortamda kesilir ve nitrocellular zarlar üzerine yerleştirilir. Enjeksiyon yoluyla yönetilmektedir tedavi gerekiyorsa onlar membranın yerleştirilmiş sonra dokular enjekte edilir. Tedavi inj ile idare edilecek gerektirmiyorsaection daha sonra, bileşik (% 1 fetal buzağı serumu (FCS),% 1 penisilin-streptomisin (P / S), Dulbecco Modifiye Eagle Ortamı (DMEM)) kültür ortamına ilave edilir. Daha önce tarif edildiği gibi 5 kültürleri,% 30 sukroz çözeltisi ile işlenmiş doku% 4 paraformaldehit (PFA) içerisinde sabitlenir ve bundan sonra on gün kadar, OCT bileşiği içine gömülmüş ve -80 ° C&#39;de saklandı tutulur.

<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.

ex vivo yöntemi, bağ dokusu 3D kültürü de fibrozis ECM ve DD doku oluşturan diğer hücre bileşenleri ve potansiyel diğer türleri arasındaki ilişkiyi anlamak için kolay ve sağlam bir set up sistemi olduğunu. Ayrıca bu sistem, güvenilir bir yöntem, farklı hücre tipleri ve fibrotik yük 20 üzerindeki etkileri üzerinde bileşiklerin etkisini test etmek için izin verir. dupuytren fibroz türetilen cerrahi atık maddelerin toplanması ile ilgili adım kü...

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

Dupuytren hastalığı (GG) avucunun bir fibroproliferatif hastalıktır. Burada, biz üç-boyutlu (3D) kültür sistemi DD kültür rezeksiyon örneklerinde bir protokol mevcut. Bu tür kısa vadeli kültür sistemi 3D yapı korunması ve fibrotik doku moleküler özelliklerini tanır.

İnsan Dupuytren en<em&gt; Ex vivo</em&gt; Miyofibroblastların Çalışması ve Ekstrasellüler Matriks Etkileşimleri Kültür

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

Hasta Doku ve Gen Teslimat Sınav Ex Vivo Kültür

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

İnsan Nöroendokrin Tümör Terapi Çalışmaları Üç Boyutlu Model İnsan nöroendokrin tümör hücre hatlarında

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 ...

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Bir İnsan<em&gt; Ex Vivo</em&gt; Aterosklerotik Plak Model Lezyon Biyoloji Eğitim için

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

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

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 ...

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

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İnsan Safen Ven Prosedürü<em&gt; Ex vivo</em&gt; Perfüzyon ve Dış Takviye

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

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

İzolasyon, Kriyoprezervasyon ve Kültür Klinik Uygulamaları İnsan Amniyon Epitel Hücreleri

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

Bir<em&gt; Ex vivo</em&gt; Model İnsan Meme Hormon Eylem Eğitim için

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

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

Vivo ve Ex Vivo Periton yağ içinde Samanyolu Nokta Yapıların Yumurtalık Kanseri Metastatik Kolonizasyon Eğitim Yaklaşımlar içinde

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 Profil Yöntemi ve İlaç Tepki Testi için 3D İnsan BIOGEL Kültür Sisteminin Kullanılması Microtumors ve Hasta kaynaklı Glioblastom Hücreleri Üretimi

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