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.

<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. J., Derynck, R. <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+regulates+expression+of+the+TGF-beta+1+gene.">Extracellular matrix regulates expression of the TGF-beta 1 gene.</a> The Journal of Cell Biology. 120 (1), 253-260 (1993).</li><li>Vogel, V., Sheetz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+force+and+geometry+sensing+regulate+cell+functions.">Local force and geometry sensing regulate cell functions.</a> Nat Rev Mol Cell Biol. 7 (4), 265-275 (2006).</li><li>Gottrup, F., &#197;gren, M. S., Karlsmark, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+for+use+in+wound+healing+research:+A+survey+focusing+on+in+vitro+and+in+vivo+adult+soft+tissue.">Models for use in wound healing research: A survey focusing on in vitro and in vivo adult soft tissue.</a> Wound Repair and Regeneration. 8 (2), 83-96 (2000).</li><li>Gudjonsson, 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=Normal+and+tumor-derived+myoepithelial+cells+differ+in+their+ability+to+interact+with+luminal+breast+epithelial+cells+for+polarity+and+basement+membrane+deposition.">Normal and tumor-derived myoepithelial cells differ in their ability to interact with luminal breast epithelial cells for polarity and basement membrane deposition.</a> Journal of Cell Science. 115 (1), 39-50 (2002).</li><li>Xu, R., Boudreau, A., Bissell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+architecture+and+function:+dynamic+reciprocity+via+extra-+and+intra-cellular+matrices.">Tissue architecture and function: dynamic reciprocity via extra- and intra-cellular matrices.</a> Cancer and Metastasis Reviews. 28 (1-2), 167-176 (2009).</li><li>Jokinen, J., 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=Integrin-mediated+cell+adhesion+to+type+I+collagen+fibrils.">Integrin-mediated cell adhesion to type I collagen fibrils.</a> Journal of Biological Chemistry. 279 (30), 31956-31963 (2004).</li><li>Au-Yong, I. 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. K. r. u. i. t. h. o. f. -. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrosis:+a+novel+approach+for+an+old+problem.">Fibrosis: a novel approach for an old problem.</a> Receptors &amp; Clinical Investigation. 1 (5), (2014).</li><li>Wolf, K., 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=Collagen-based+cell+migration+models+in+vitro+and+in+vivo.">Collagen-based cell migration models in vitro and in vivo.</a> Seminars in Cell &amp; Developmental Biology. 20 (8), 931-941 (2009).</li><li>Freund, I., Deutsch, M., Sprecher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connective+tissue+polarity.+Optical+second-harmonic+microscopy,+crossed-beam+summation,+and+small-angle+scattering+in+rat-tail+tendon.">Connective tissue polarity. Optical second-harmonic microscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon.</a> Biophysical Journal. 50 (4), 693-712 (1986).</li><li>Mohler, W., Millard, A. C., Campagnola, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second+harmonic+generation+imaging+of+endogenous+structural+proteins.">Second harmonic generation imaging of endogenous structural proteins.</a> Methods. 29 (1), 97-109 (2003).</li><li>Boulesteix, 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=Micrometer+scale+ex+vivo+multiphoton+imaging+of+unstained+arterial+wall+structure.">Micrometer scale ex vivo multiphoton imaging of unstained arterial wall structure.</a> Cytometry Part A. 69A (1), 20-26 (2006).</li><li>Tomasek, J. J., Gabbiani, G., Hinz, B., Chaponnier, C., Brown, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myofibroblasts+and+mechano-regulation+of+connective+tissue+remodelling.">Myofibroblasts and mechano-regulation of connective tissue remodelling.</a> Nat Rev Mol Cell Biol. 3 (5), 349-363 (2002).</li><li>Hinz, B., Mastrangelo, D., Iselin, C. E., Chaponnier, C., Gabbiani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+tension+controls+granulation+tissue+contractile+activity+and+myofibroblast+differentiation.">Mechanical tension controls granulation tissue contractile activity and myofibroblast differentiation.</a> The American Journal of Pathology. 159 (3), 1009-1020 (2001).</li><li>Eastwood, M., McGrouther, D. A., Brown, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+culture+force+monitor+for+measurement+of+contraction+forces+generated+in+human+dermal+fibroblast+cultures:+evidence+for+cell-matrix+mechanical+signalling.">A culture force monitor for measurement of contraction forces generated in human dermal fibroblast cultures: evidence for cell-matrix mechanical signalling.</a> Biochimica et Biophysica Acta (BBA) - General Subjects. 1201 (2), 186-192 (1994).</li><li>Verjee, L. S., Midwood, K., Davidson, D., Eastwood, M., Nanchahal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-transcriptional+regulation+of+&#945;-smooth+muscle+actin+determines+the+contractile+phenotype+of+Dupuytren's+nodular+cells.">Post-transcriptional regulation of &#945;-smooth muscle actin determines the contractile phenotype of Dupuytren's nodular cells.</a> Journal of Cellular Physiology. 224 (3), 681-690 (2010).</li></ol>

وقد رفعت المؤلفين طلب البراءة لمرض دوبوترين: الثقافة الجهاز 3D. # GB1307200. تتوفر عقود الترخيص والخدمات التجارية.

الخطوات الأكثر أهمية من زراعة الأنسجة الضامة الإنسان خارج الحي هي استخدام الفوري من الأنسجة بعد الاستئصال الجراحي لضمان جدوى. ينبغي أن يظل النسيج في محلول ملحي أو متوسطة في جميع الأوقات؛ الحفاظ على الثقافة العقيمة. يجب المقاطع العرضية من الأنسجة يكون الحد الأق?...

مرض دوبوترين (DD)، وهو مرض يسبب انثناء fibroproliferative حميدة دائمة العضوية في الأصابع بسبب تشكيل عقيدات والحبال في كف اليد. على الرغم من أن انتشار المرض مرتفع بشكل خاص بين القوقازيين من شمال أوروبا، والمسببات الوراثية الكامنة وراء هذا المرض ما زال مجهولا 1. السمة الرئيسية للDD هو إنتاج فائض من المصفوفة خارج الخلية (ECM) البروتينات (مثل الكولاجين)، والتي تشكل النسيج الليفي الصعبة التي تحتل مساحة بين الأوتار والجلد من كف اليد والأصابع، وتعطيل حركات غرامة بشكل دائم من جهة 2، 3، وتكرار المرض تشير التعديلات الوراثية الكامنة كسبب للتليف 1، 4. علاج فعال يمكن أن يكون لاستهداف مباشرة الآليات تليفي لا يمكن السيطرة عليها على المستوى الخلوي والجزيئي. وقد أدى عملنا مؤخرا على التليف بنا إلى تطويررواية نظام الثقافة 3D التي تسمح الثقافة على المدى القصير من أنسجة متليفة الإنسان مع إمكانية اختبار المخدرات. وقد ساعد هذا النظام على التغلب على نهج الحد من الثقافات الليفية 2D و تحديد دور لتنظيم أسفل جزئي، من خلال اكسون تخطي يتحقق، تفعيل مسار TGFβ في التوسط التليف 5. لقد قمنا بتطوير طريقة لثقافة فيفو السابقين العينات استئصال الإنسان من المرضى DD لدراسة التفاعل بين myofibroblasts وECM المحيطة 5 و 6. وتعتمد دراسة DD تليف الأنسجة الضامة وكذلك الأمراض تليفي أخرى على تحليل الأنسجة من رفعه الجراحية العينات وعزل الخلايا الليفية من الأنسجة وإنشاء الثقافات الأولية أو إجراءات خلية الفرز. هذه المناهج هي ثابتة تماما لأنها لا تسمح التلاعب خارجي من خصائص مرض أو التدخل العلاجي من قبل المجرب. وبالإضافة إلى ذلك، CE الأساسيالثقافات ليرة لبنانية تميل إلى التكيف مع الظروف الثقافة والجينات خصائص التعبير عنها تختلف أساسا عن الوضع في الجسم الحي على كل مرور، حتى خلال الممرات الأولى (من بين مرور 3 و 6) 7، 8. لقد نجحنا في الحفاظ على المواد الجراحية النفايات في السابق الظروف الثقافة الجسم الحي لفترة زمنية تسمح دراسة خصائص المريض محددة وفحص المركبات عقار مضاد للمتليفة أو المضادة للالتهابات. ويستند هذا النظام على غشاء النيتروسليلوز الذي يسمح اتصال من النسيج مع المتوسط ​​ولكن ليس مع البلاستيك، وبالتالي، ومنع تغيير النسيج على المرفق، كما لوحظ سابقا عند زراعة الخلايا الليفية DD وكذلك أنواع الخلايا الأخرى 9. لا يلزم جل الكولاجين أو غيرها من ECM البروتين الركيزة، منذ الأنسجة DD نفسها تنتج كميات كبيرة من هذه البروتينات. هذا هو مفيد لصيانة المكروية ECM الأم ودوران الخطيئةركائز م المصفوفة هي المنظم المهم العمارة الأنسجة وظيفة 10 و 11 على سبيل المثال، والبروتينات ECM مثل فبرونيكتين، laminin والكولاجين، قد تؤثر قطبية الأمامي الخلفي من الخلايا الليفية كما هو موضح بالمثل لقطبية قمية-القاعدية في الخلايا الظهارية 12، 13 . خلايا الاستقطاب لها توزيع غير متوازن من جزيئات الخلية والذي يحدد الهجرة الخلية والتعبير الجيني، على سبيل المثال، α1β1 الوصول إنتغرين على الغشاء يؤثر التصاق الخلية إلى النوع الأول من الكولاجين 14. منذ كان الهدف الأساسي من هذا النموذج 3D للحفاظ على المكروية الأم، تم استخدام أي مصطنعة ECM مصفوفة الركيزة. في سطور: يتم قطع العينات بتر قدم المساواة في بيئة معقمة ووضعها على الأغشية nitrocellular. إذا كان مطلوبا العلاج تدار عن طريق الحقن يتم حقن الأنسجة بعد أن يكونوا قد وضعت على الغشاء. إذا لا تتطلب العلاج إلى أن تدار عن طريق حقنةection ثم يضاف المركب إلى وسائل الإعلام الثقافة (Dulbecco لتعديل متوسطة النسر (DMEM)، مع 1٪ مصل العجل الجنين (FCS)، 1٪ البنسلين، الستربتومايسين (P / S)). يتم الحفاظ على الثقافات لمدة أقصاها عشرة أيام وبعد ذلك يتم إصلاح الأنسجة في 4٪ بارافورمالدهيد (PFA)، معالجتها من خلال 30٪ من محلول السكروز، جزءا لا يتجزأ في أكتوبر مركب وتخزينها في -80 ° C، كما هو موضح سابقا 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.

طريقة فيفو السابقين، والثقافة 3D من النسيج الضام هو انشاء نظام سهل وقوي لفهم العلاقة بين ECM ومكونات الخلايا الأخرى المكونة للنسيج DD وأنواع أخرى من المحتمل أن التليف كذلك. وعلاوة على ذلك يسمح هذا النظام طريقة موثوقة لاختبار آثار المركبات على أنواع مختلفة من الخلا?...

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&#039;s Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

مرض دوبوترين (DD) هو مرض fibroproliferative من كف اليد. هنا، نقدم بروتوكول لعينات ثقافة الاستئصال الجزئي من DD في ثلاثي الأبعاد (3D) نظام الثقافة. مثل هذا النظام الثقافة على المدى القصير يسمح الحفاظ على هيكل 3D والخصائص الجزيئية للأنسجة متليفة.

الإنسان دوبوترين<em&gt; فيفو السابقين</em&gt; الثقافة لدراسة Myofibroblasts وخارج الخلية مصفوفة التفاعلات

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 ...

human-dupuytren-s-ex-vivo-culture-for-study-myofibroblasts

Research

JoVE Journal

Medicine

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

10.3K Views.  Leiden University Medical Center.  مرض دوبوترين (DD) هو مرض fibroproliferative من كف اليد. هنا، نقدم بروتوكول لعينات ثقافة الاستئصال الجزئي من DD في ثلاثي الأبعاد (3D) نظام الثقافة. مثل هذا النظام الثقافة على المدى القصير يسمح الحفاظ على هيكل 3D والخصائص الجزيئية للأنسجة متليفة. 

Video: 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

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 is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial.

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

This article describes the culture of patient tissue slices for gene delivery studies and subsequent analysis of gene expression using IVIS bioluminescence imaging.

فيفو السابقين الثقافة من الأنسجة للمريض وفحص الجينات التسليم

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

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 malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

الإنسان الخلية السرطانية الهرموني العصبي خطوط بمثابة نموذج ثلاثي الأبعاد لدراسة الإنسان علاج ورم الغدد الصم العصبية

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

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic lesions. Mouse models are an important tool to investigate inflammatory processes in atherogenesis, but these models suffer from the phenotypic and functional differences between the murine and human immune system. In vitro cell experiments are used to specifically evaluate cell type-dependent changes caused by a substance of interest, but culture-dependent variations and the inability to analyze the influence of specific molecules in the context of the inflammatory compound in atherosclerotic lesions limit the impact of the results. In addition, measuring levels of a molecule of interest in human blood helps to further investigate its clinical relevance, but this represents systemic and not local inflammation. Therefore, we here describe a plaque culture model to study human atherosclerotic lesion biology ex vivo. In short, fresh plaques are obtained from patients undergoing endarterectomy or coronary artery bypass grafting and stored in RPMI medium on ice until usage. The specimens are cut into small pieces followed by random distribution into a 48-well plate, containing RPMI medium in addition to a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. After incubation, the plaque pieces can be shock frozen for mRNA isolation, embedded in Paraffin or OCT for immunohistochemistry staining or smashed and lysed for western blotting. Furthermore, cells may be isolated from the plaque for flow cytometry analysis. In addition, supernatants can be collected for protein measurement by ELISA. In conclusion, the presented ex vivo model opens the possibility to further study inflammatory lesional biology, which may result in identification of novel disease mechanisms and therapeutic targets.

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory process. This manuscript illustrates an easy to use ex vivo model to investigate fresh carotid or coronary artery plaques. The ex vivo model allows for the investigation of potential substances on the inflammatory milieu in human atherosclerotic lesions and results can be analyzed by various methods.

والإنسان<em&gt; فيفو السابقين</em&gt; تصلب الشرايين البلاك نموذج لدراسة علم الأحياء الآفات

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

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex&#160;vivo perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex&#160;vivo conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mechanisms leading to the development of intimal hyperplasia (IH) and vein graft failure are still poorly understood. This study describes an ex&#160;vivo system to perfuse human veins under controlled flow and pressure. Furthermore the efficiency of external mesh reinforcement to limit the development of IH was evaluated.

إجراءات الصافن عروق الإنسان<em&gt; فيفو السابقين</em&gt; الإرواء والتعزيز الخارجي

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

An Ex vivo Model to Study Hormone Action in the 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 cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

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

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

ل<em&gt; السابقين فيفو</em&gt; نموذج لدراسة هرمون العمل في الثدي البشري

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

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

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

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

في فيفو وفيفو السابقين المداخل لدراسة سرطان المبيض النقيلي الاستعمار من الهياكل التبانة بقعة في البريتوني الدهنية

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

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

جيل من Microtumors عن طريق نظام الثقافة 3D Biogel الإنسان وخلايا ورم أرومي دبقي المستمدة من المريض لKinomic التنميط والدواء استجابة اختبار

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

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

الاستراتيجية غشائي أنبوب خلية تشكيل الفحص: مقارنة خارج الخلية مصفوفة وعامل نمو مخفض خارج الخلية مصفوفة

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

خطوات الإعداد لقياس مفاعليه في الماوس الشرايين الشبكية السابقين فيفو

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.