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.

Detta protokoll följer LUMC och AMC riktlinjer för mänskliga forskningsetisk kommitté. Tissue Collection 1. kirurgiska ingrepp och Anmärkning: Även om olika tekniker för kirurgisk excision av Dupuytrens kontraktur existerar, är den aktuella guldstandard partiell fasciectomy 15. De flesta patienter behandlas under dagen-kirurgi klinik. <ol><li> Utför operation under allmän eller regional anestesi enligt patientens och anestesiolog preferense...

<ol>
	<li>Shih, B., Bayat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scientific+understanding+and+clinical+management+of+Dupuytren+disease.">Scientific understanding and clinical management of Dupuytren disease.</a> Nat Rev Rheumatol. 6 (12), 715-726 (2010).</li><li>Bisson, M. A., McGrouther, D. A., Mudera, V., Grobbelaar, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+different+characteristics+of+Dupuytren's+disease+fibroblasts+derived+from+either+nodule+or+cord:+expression+of+&#945;-smooth+muscle+actin+and+the+response+to+stimulation+by+TGF-&#946;1.">The different characteristics of Dupuytren's disease fibroblasts derived from either nodule or cord: expression of &#945;-smooth muscle actin and the response to stimulation by TGF-&#946;1.</a> The Journal of Hand Surgery. 28 (4), 351-356 (2003).</li><li>Brickley-Parsons, D., Glimcher, M. J., Smith, R. J., Albin, R., Adams, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+changes+in+the+collagen+of+the+palmar+fascia+in+patients+with+Dupuytren's+disease.">Biochemical changes in the collagen of the palmar fascia in patients with Dupuytren's disease.</a> The Journal of Bone &amp; Joint Surgery. 63 (5), 787-797 (1981).</li><li>Berndt, A., Kosmehl, H., Katenkamp, D., Tauchmann, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appearance+of+the+myofibroblastic+phenotype+in+Dupuytren's+disease+is+associated+with+a+fibronectin,+laminin,+collagen+type+IV+and+tenascin+extracellular+matrix.">Appearance of the myofibroblastic phenotype in Dupuytren's disease is associated with a fibronectin, laminin, collagen type IV and tenascin extracellular matrix.</a> Pathobiology. 62 (2), 55-58 (1994).</li><li>Karkampouna, S., 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=Novel+ex+vivo+culture+method+for+the+study+of+Dupuytren's+disease:+effects+of+TGF&#946;+type+1+receptor+modulation+by+antisense+oligonucleotides.">Novel ex vivo culture method for the study of Dupuytren's disease: effects of TGF&#946; type 1 receptor modulation by antisense oligonucleotides.</a> Mol Ther Nucleic Acids. 3, e142 (2014).</li><li>Kruithof-de Julio, 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=Canonical+Wnt+signaling+regulates+Nkx3.1+expression+and+luminal+epithelial+differentiation+during+prostate+organogenesis.">Canonical Wnt signaling regulates Nkx3.1 expression and luminal epithelial differentiation during prostate organogenesis.</a> Developmental Dynamics. 242 (10), 1160-1171 (2013).</li><li>Krause, C., Kloen, P., ten Dijke, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+transforming+growth+factor+&#946;+and+mitogen-activated+protein+kinase+pathways+mediate+fibrotic+traits+of+Dupuytren's+disease+fibroblasts.">Elevated transforming growth factor &#946; and mitogen-activated protein kinase pathways mediate fibrotic traits of Dupuytren's disease fibroblasts.</a> Fibrogenesis &amp; Tissue Repair. 4 (1), 14 (2011).</li><li>Gorman, D. B., Wu, Y., Seney, S., Zhu, R. D., Gan, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+expression+is+not+correlated+with+beta-catenin+dysregulation+in+Dupuytren's+Disease.">Wnt expression is not correlated with beta-catenin dysregulation in Dupuytren's Disease.</a> J Negat Results Biomed. 5, 13 (2006).</li><li>Streuli, C. H., Schmidhauser, C., Kobrin, M., Bissell, M. 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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 kritiska stegen i odling ex vivo human bindväv är omedelbar användning av vävnaden efter kirurgiskt avlägsnande för att säkerställa lönsamhet; vävnaden bör förbli i medium eller saltlösning vid alla tillfällen; upprätthålla en steril kultur; tvärsektioner av vävnad borde ha maximalt 200 um tjocklek; inrättats av ex vivo odlingssystemet är optimal när vävnaden är i kontakt med mediet men inte helt nedsänkt. Medium bör läggas enbart på utsidan kammaren i transwell i en ...

Dupuytrens sjukdom (DD), orsakar en godartad fibroproliferativa sjukdom permanent böjning av fingrarna på grund av bildandet av knölar och sladdar i handflatan. Även spridningen sjukdomen är särskilt hög bland vita i norra Europa, den underliggande genetiska etiologin av sjukdomen är okänd 1. Det som främst kännetecknar DD är den överproduktion av extracellulärt matrix (ECM) proteiner (t.ex. kollagen), som utgör en tuff fibrös vävnad ockuperar utrymmet mellan senor och hud i handflatan och fingrarna, permanent störa de fina rörelser av handen 2, 3. Den återfall av sjukdomen tyder underliggande genetiska förändringar som orsak till fibros 1, 4. En effektiv behandling kan vara att rikta direkt de okontrollerbara fibrotiska mekanismer på cell- och molekylnivå. Vår senaste arbete med fibros har lett oss till utvecklingen av ennytt 3D kultursystem som möjliggör kortsiktiga kultur av mänskligt fibrotisk vävnad med potential drogtester. Detta system har bidragit till att övervinna den begränsande synsätt av 2D fibroblastkulturer och att definiera en roll för den partiella nedreglering, uppnås genom exon hoppa, av TGF väg aktivering i medier fibros 5. Vi har utvecklat en metod att odla ex vivo human resektion prover från DD patienter för att studera interaktionen mellan myofibroblaster och den omgivande ECM 5, 6. Studiet av DD bindvävsfibros samt andra fibrotiska sjukdomar bygger på histopatologisk analys av utskurna kirurgiska prover, isolering av fibroblaster från vävnaden och etablering av primära odlingar eller cellsorteringsprocedurer. Dessa tillvägagångssätt är ganska statiska eftersom de inte tillåter exogen manipulation av fastigheter sjukdomen eller terapeutiska ingrepp av försöks. Dessutom primära cell kulturer tenderar att anpassa sig till de odlingsbetingelser och deras genuttryck egenskaper skiljer sig väsentligt från situationen in vivo vid varje passage, även under tidiga passager (bland passagen 3 och 6) 7, 8. Vi har lyckats behålla avfallet kirurgiska materialet i ex vivo odlingsbetingelser under en tidsperiod som möjliggör studie av patientspecifika egenskaper och screening av anti-fibrotiska eller anti-inflammatoriska läkemedelsföreningar. Systemet är baserat på ett nitrocellulosamembran som medger kontakt av vävnaden med mediet men inte med plasten således förhindra ändring av vävnaden vid infästning, såsom tidigare observerats vid odling DD fibroblaster samt andra celltyper 9. Ingen kollagengel eller andra ECM proteinsubstrat är nödvändigt, eftersom den DD vävnaden själv producerar stora mängder av dessa proteiner. Detta är fördelaktigt för upprätthållande av nativa ECM mikromiljö och omsättning syndce matris substrat är viktig reglerare av vävnad arkitektur och funktion 10, 11. Exempelvis ECM-proteiner såsom fibronektin, laminin och kollagen, kan påverka fram-bak polaritet fibroblaster som på liknande sätt visade för apikal-basal polaritet i epitelceller 12, 13 . Polarise celler har asymmetrisk fördelning av extracellulära molekyler som bestämmer cellmigration och genuttryck, t.ex. α1β1 grin tillgänglighet på membranet påverkar celladhesion till typ I kollagen 14. Eftersom ett primärt mål för denna 3D-modell var att bevara den naturliga mikroades ingen artificiell ECM matrissubstratet används. I korthet: resektion exemplar är jämnt skuren i en steril miljö och placeras på nitrocellular membran. Om behandling administreras via injektion krävs vävnaderna injiceras efter att de har släppts ut på membranet. Om behandlingen inte kräver att administreras via injektion sedan föreningen sättes till odlingsmediet (Dulbeccos modifierade Eagles medium (DMEM), med en% fetalt kalvserum (FCS), 1% penicillin-streptomycin (P / S)). De kulturerna bibehålls under högst tio dagar, varefter vävnaden fixeras i 4% paraformaldehyd (PFA), bearbetas genom 30% sackaros lösning, inbäddade i OCT förening och förvaras vid -80 ° C, som tidigare beskrivits 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.

Metoden för ex vivo, är 3D kultur av bindväv ett enkelt och robust uppsättning upp system för att förstå sambandet mellan ECM och andra cellkomponenter som utgör DD vävnad och eventuellt andra typer av fibros också. Dessutom detta system möjliggör en tillförlitlig metod för att testa effekterna av föreningar på olika celltyper och deras effekter på fibrotiska lasten 20. Stegen från insamling av kirurgiska avfallsmaterial härrörande från Dupuytrens fib...

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

Dupuytrens sjukdom (DD) är en fibroproliferativa sjukdom i handflatan. Här presenterar vi ett protokoll till kultur resektion exemplar från DD i en tredimensionell (3D) odlingssystem. En sådan kortsiktig kultur systemet tillåter bevarandet av 3D-strukturen och molekylära egenskaper hos fibrotiska vävnaden.

Humant Dupuytrens<em&gt; Ex vivo</em&gt; Kultur för studien av Myofibroblaster och extracellulära matrixinteraktioner

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

Ex vivo kultur Patient Tissue &amp; Undersökning av 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

Mänskliga neuroendokrin tumör cellinjer som en tredimensionell modell för studiet av mänskliga neuroendokrin tumör 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 Människa<em&gt; Ex vivo</em&gt; Aterosklerotiska plack modell för att studera Lesion 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

Förfarande för mänskliga vena Vener<em&gt; Ex Vivo</em&gt; Perfusion och extern Förstärkning

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

Isolering, Frysförvaring och kultur för mänskliga amnion epitelceller för kliniska tillämpningar

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; Modell för att studera Hormone Action 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 och ex vivo Approaches att studera äggstockscancer Metastatisk Colonization av Milky Spot Structures i Peritoneal Fett

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 Microtumors Använda 3D Human Biogel odlingssystemet och patientgenererade glioblastomceller för Kinomic Profilering och läkemedelssvar Testing

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