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.

Denne protokol følger LUMC og AMC retningslinjer for menneskelig videnskabsetisk komité. 1. kirurgiske indgreb og Tissue Collection Bemærk: Selv om forskellige teknikker til kirurgisk excision af Kuskefingre findes, den nuværende guldstandarden er den delvise fasciectomy 15. De fleste patienter behandles i dag-klinik. <ol><li> Udføre kirurgi under generel eller regional anæstesi efter patientens og anæstesilægen præferencer og patientens co-...

<ol>
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T. H., Wildin, C. J., Dias, J. J., Page, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+common+practice+in+Dupuytren+surgery.">A review of common practice in Dupuytren surgery.</a> Techniques in Hand &amp; Upper Extremity Surgery. 9 (4), 178-187 (2005).</li><li>Deries, M., Collins, J. J. P., Duxson, M. 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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De mest kritiske trin i dyrkning ex vivo humane bindevæv er den umiddelbare anvendelse af vævet efter kirurgisk fjernelse for at sikre levedygtighed vævet bør forblive i medium eller saltvandsopløsning på alle tidspunkter; opretholde en steril kultur; tværsnit af væv skal have maksimalt 200 um tykkelse; opsætning af ex vivo kultur system er optimal, når vævet er i kontakt med mediet, men ikke fuldt neddykket. Medium bør tilføjes kun på ydersiden kammer transwell i et lille volumen (f....

Dupuytrens sygdom (DD), en godartet fiberproliferativ sygdom forårsager permanent bøjning af fingrene som følge af dannelsen af ​​knuder og ledninger i håndfladen. Selvom sygdomsspredning er særlig høj blandt kaukasiere i Nordeuropa, det underliggende genetiske ætiologi af sygdommen er stadig ukendt 1. Den vigtigste egenskab ved DD er overskydende produktion af ekstracellulær matrix (ECM) proteiner (fx collagen), som danner en hård fibrøst væv optager rummet mellem sener og hud håndfladen og fingrene, permanent forstyrre de fine bevægelser af hånden 2, 3. Den gentagelse af sygdommen tyder underliggende genetiske ændringer som årsag til fibrose 1, 4. En effektiv behandling kunne være at målrette direkte de ukontrollable fibrotiske mekanismer på det cellulære og molekylære niveau. Vores seneste arbejde på fibrose har ført os til at udvikle etroman 3D kultur system, der tillader kortsigtet kultur af human fibrøst væv med potentiale narkotika testning. Dette system har været med til at overvinde den begrænsende tilgang af 2D fibroblastkulturer og definere en rolle for den delvise nedreguleringen, opnået ved exon skipping, af TGF-vejen aktivering i mediering fibrose 5. Vi har udviklet en metode til dyrkning ex vivo human resektion prøver fra DD patienter at studere interaktionen mellem myofibroblaster og den omgivende ECM 5, 6. Studiet af DD bindevæv fibrose samt andre fibrotiske sygdomme beror på histopatologisk analyse af det udskårne kirurgiske prøver, isolering af fibroblaster fra vævet og etablering af primære kulturer eller cellesortering procedurer. Disse metoder er ret statisk, da de ikke tillader eksogen manipulation af ejendomme sygdommen eller terapeutisk intervention ved forsøgslederen. Derudover kan primære cell kulturer en tendens til at tilpasse sig de dyrkningsbetingelser og deres genekspression egenskaber hovedsageligt fra in vivo situationen adskiller sig på hver passage, selv under tidlige passager (blandt passage 3 og 6) 7, 8. Det er lykkedes at fastholde affaldet kirurgisk materiale i ex vivo dyrkningsbetingelser i et tidsrum, der tillader undersøgelse af patient-specifikke egenskaber og screening af anti-fibrotiske eller antiinflammatoriske lægemiddelstoffer. Systemet er baseret på en nitrocellulosemembran som tillader kontakt af vævet med mediet, men ikke med plast, således at forhindre ændring af vævet ved fastgørelse, som tidligere observeret, når dyrkning DD fibroblaster samt andre celletyper 9. Nr kollagen gel eller andre ECM proteinsubstrat er påkrævet, da DD væv selv producerer store mængder af disse proteiner. Dette er fordelagtigt for vedligeholdelse af native ECM mikromiljø og omsætningen since matrixsubstrater er vigtig regulator af væv arkitektur og funktion 10, 11. For eksempel ECM-proteiner, såsom fibronectin, laminin og kollagen, kan påvirke forreste-bageste polaritet af fibroblaster som tilsvarende vist for apikal-basal polaritet i epitelceller 12, 13 . polariserede celler har asymmetrisk fordeling af ekstracellulære molekyler, som bestemmer cellevandring og genekspression, f.eks α1β1 integrin tilgængelighed på membranen påvirker celleadhæsion til type I collagen 14. Da et primært mål for denne 3D-model var at bevare den indfødte mikromiljø, blev der ikke kunstig ECM matrix anvendte substrat. Kort: resektion prøver lige skåret i et sterilt miljø og anbringes på nitrocellular membraner. Hvis behandling administreres via injektion er påkrævet væv injiceres, efter at de er blevet placeret på membranen. Hvis behandlingen ikke kræver at administreres via injfdeling derefter forbindelsen tilsættes til dyrkningsmediet (Dulbeccos Modified Eagles Medium (DMEM), med 1% føtalt kalveserum (FCS), 1% penicillin-streptomycin (P / S)). Kulturerne opretholdes højst ti dage, hvorefter vævet fikseret i 4% paraformaldehyd (PFA), behandles gennem 30% saccharoseopløsning, indlejret i OCT-forbindelse og opbevaret ved -80 ° C, som tidligere beskrevet 5.

<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.

Fremgangsmåden til ex vivo 3D kultur af bindevæv er en let og robust sæt op for at forstå relationen mellem ECM og andre cellebestanddele udgør DD væv og potentielt andre typer af fibrose samt. Desuden dette system gør det muligt for en pålidelig metode til at teste virkningen af forbindelser på forskellige celletyper og deres indvirkning på fibrotiske belastning 20. Trinene fra indsamling af kirurgisk affaldsmateriale stammer fra Dupuytrens fibrose, er samlinge...

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

Dupuytrens sygdom (DD) er en fibroproliferativ sygdom i håndfladen. Her præsenterer vi en protokol til kultur resektion prøver fra DD i en tredimensional (3D) dyrkningssystem. Sådanne kortsigtede kultur system tillader bevarelse af 3D-struktur og molekylære egenskaber af fibrøst væv.

Humant Dupuytrens<em&gt; Ex vivo</em&gt; Kultur for Studiet af myofibroblaster og ekstracellulære matrix-interaktioner

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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JoVE Journal

Medicine

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

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

Humane Neuroendokrine tumorcellelinier som en tredimensional model for studiet af den menneskelige Neuroendokrine Tumor Therapy

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

En human<em&gt; Ex Vivo</em&gt; Aterosklerotisk plaque Model til Studieinformationen Læsionssynligheden Biologi

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

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

Procedure for Menneskerettigheder saphenous Veins<em&gt; Ex vivo</em&gt; Perfusion og udvendig forstærkning

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

Isolation, Kryopræservering og dyrkning af humane amnion epitelceller ved kliniske anvendelser

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

En<em&gt; Ex vivo</em&gt; Model til Uddannelse hormonvirkning i Human Breast

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

In vivo og ex vivo Approaches at studere kræft i æggestokkene Metastatisk Kolonisering af Milky Spot Strukturer i peritoneal Adipose

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

Generation af Microtumors Brug 3D Menneskelig Biogel Kultur System og Patient-afledte glioblastomaceller for Kinomic Profilering and Drug respons Testing

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