APS est soutenu par DebRA la British Skin Foundation International et. Yzn est soutenu par A * STAR - Université de PhD Programme de partenariat Dundee.

Cette étude a été réalisée conformément à la Déclaration de principes d&#39;Helsinki et a été approuvé par les comités d&#39;éthique appropriés. Une. Préparation des milieux et réactifs <ol><li> Préparation de 200x de L-ascorbique 2-phosphate actions <ol><li> Dissoudre 29 mg de L-acide ascorbique-2-phosphate par 5 ml de milieu Eagle modifié de (DMEM) la solution de Dulbecco et filtrer sur filtre de 0,22 um de la membrane. Magasin de 0,25 ml aliquotes stériles -20 ° C. </li><li> Ajouter 0,25 ml aliquote de 200x acide L-ascorbique-2-phosphate magasin pour chaque 50 ml de milieu de fibroblastes (DMEM avec 1% de L-glutamine et du sérum bovin foetal à 10%) le jour nécessaire, pour une concentration finale de 0,1 mM de L acide ascorbique-2-phosphate. </li></ol></li><li> Préparation des milieux de croissance des kératinocytes <ol><li> Isolement et culture des kératinocytes primaires du CCN ont été décrits précédemment 21. Préparation des milieux de croissance des kératinocytes est décritey ainsi. </li><li> En bref, préparer les médias comme suit: 300 ml de DMEM et 100 ml de Ham F-12 supplémenté avec 10% de FBS 0,4 mg / ml d&#39;hydrocortisone 5 mg / ml d&#39;insuline 10 ng / ml d&#39;EGF 5 mg / ml de transferrine 8,4 ng / ml de toxine cholérique 13 ng / ml liothyronine Solution de pénicilline-streptomycine 1x </li></ol></li></ol> 2. Construction in vitro de fibroblastes dérivés matrice maternelle en acide L-ascorbique 2-phosphate Supplement médias <ol><li> Semences 200000 fibroblastes par puits dans des plaques à 6 puits (20 000 cellules / cm 2) dans un milieu supplémenté avec de fibroblastes de l&#39;acide L-ascorbique-2-phosphate. Réinsérez tous les 2-3 jours avec 2-5 ml de milieu. (Remarque: La réalimentation de la fréquence et le volume peuvent être ajustés pour répondre aux besoins individuels des différentes cellules Voir &quot;Discussions&quot;.). </li><li> Une épaisse couche de cellules incluses dans la matrice extracellulaire se formera à la fin de 6 semaines, visibles à l&#39;œil nu ( <strong&gt; Figure 1). Pour libérer cette couche de la plaque de culture de tissu, grattez délicatement la circonférence de la matrice avec une pointe de micropipette de 1 ml puis pousser les bords de la matrice vers le centre du puits. Cette couche de cellules et de la matrice (matrice native) doivent être facilement détachable de la surface de la plaque, et maintenant être flottant dans le support. Étaler la matrice native dans les médias à éviter repliant sur lui-même. </li><li> Soit la matrice native flotteur et remodeler pendant 5 jours, l&#39;évolution des médias tous les 2-3 jours. En raison de la résistance à la traction et le remodelage intrinsèque à l&#39;intérieur de la matrice, la matrice se contracte et devient considérablement réduite à une matrice native plus petit, mais plus épaisse, et est prêt à être utilisé pour le dosage d&#39;invasion. </li></ol> 3. Invasion analyse avec tumeur CSC kératinocytes <ol><li> Ramassez la matrice native délicatement avec une pince émoussée et transfert à Nylon net. Une fois sur le net en nylon, propager la matrice doucement pour mentir aussi plate que possible en utilisant un 1 ml micropipettepointe et le forceps émoussé. </li><li> Préparer les cylindres de clones stériles en étalant une petite quantité de vaseline stérile sur une extrémité. </li><li> Placez les cylindres de clones sur la matrice d&#39;origine, avec le côté de la vaseline bas. Cela permet d&#39;assurer un joint étanche entre la matrice et les anneaux native clonales. </li><li> Ajouter cellules SCLC aux cylindres clonales (250 000 cellules dans 100 ul de milieu de croissance des kératinocytes). </li><li> Retirez les cylindres clonales après 6 heures lorsque les cellules SCLC se sont installés sur la matrice maternelle. </li><li> Soulevez le net en nylon avec la matrice maternelle et cellules SCLC à l&#39;interface air-liquide sur support plié en treillis métallique en acier inoxydable. </li><li> Ajouter un média de croissance de kératinocytes, complétée avec de l&#39;acide ascorbique jusqu&#39;à ce que le niveau de support touche le fond de la matrice native. </li><li> Changer de support tous les 2-3 jours et la récolte à 7 et 14 jours après ensemencement des cellules SCLC. </li></ol> 4. La récolte des cultures 3D et préparation pour histologie <ol><li> Fixer dans 4% paraformaldehyde pendant une nuit à température ambiante. </li><li> Bissecter les échantillons et les incorporer dans la cire de paraffine pour fixés au formol blocs embarqués, avec la surface coupée à l&#39;extérieur. </li><li> Sinon, incorporer les échantillons bissectés dans l&#39;OCT et enclenchez-gel immédiatement dans de l&#39;azote liquide pour les blocs de tissus congelés frais. </li><li> Couper 4 sections um sur microtome et déparaffinage (si nécessaire). Colorer à l&#39;aide de l&#39;hématoxyline et de l&#39;éosine norme. Pour visualiser les cellules SCLC, anticorps monoclonal de souris contre la kératine 14 (LL001, en interne) peut être utilisé en immunohistochimie coloration. </li></ol>

<ol>
	<li>Bell, E., Ehrlich, H. P., Buttle, D. J., Nakatsuji, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Living+tissue+formed+in+vitro+and+accepted+as+skin-equivalent+tissue+of+full+thickness.">Living tissue formed in vitro and accepted as skin-equivalent tissue of full thickness.</a> Science. 211 (4486), 1052-1054 (1981).</li><li>Asselineau, D., Bernhard, B., Bailly, C., Darmon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+morphogenesis+and+induction+of+the+67+kD+keratin+polypeptide+by+culture+of+human+keratinocytes+at+the+liquid-air+interface.">Epidermal morphogenesis and induction of the 67 kD keratin polypeptide by culture of human keratinocytes at the liquid-air interface.</a> Exp. Cell Res. 159 (2), 536-539 (1985).</li><li>Pruni&#233;ras, M. M., R&#233;gnier, M. M., Woodley, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+Cultivation+of+Keratinocytes+with+an+Air-Liquid+Interface.">Methods for Cultivation of Keratinocytes with an Air-Liquid Interface.</a> J. Invest. Dermatol. 81 (1 Suppl), 28-33 (1983).</li><li>Prunieras, M., R&#233;gnier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+procedure+for+culturing+human+epidermal+cells+on+allogenic+or+xenogenic+skin:+preparation+of+recombined+grafts.">New procedure for culturing human epidermal cells on allogenic or xenogenic skin: preparation of recombined grafts.</a> Ann. Chir. Plast. 24 (4), 357-362 (1979).</li><li>Gaggioli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast-led+collective+invasion+of+carcinoma+cells+with+differing+roles+for+RhoGTPases+in+leading+and+following+cells.">Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells.</a> Nature. 9 (12), 1392-1400 (2007).</li><li>Bhowmick, N. A., Neilson, E. G., Moses, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+fibroblasts+in+cancer+initiation+and+progression.">Stromal fibroblasts in cancer initiation and progression.</a> Nature. 432 (7015), 332-337 (2004).</li><li>Erez, N., Truitt, M., Olson, P., Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-Associated+Fibroblasts+Are+Activated+in+Incipient+Neoplasia+to+Orchestrate+Tumor-Promoting+Inflammation+in+an+NF-&#954;B-Dependent.">Cancer-Associated Fibroblasts Are Activated in Incipient Neoplasia to Orchestrate Tumor-Promoting Inflammation in an NF-&#954;B-Dependent.</a> Manner. Cancer Cell. 17 (2), 135-147 (2010).</li><li>Harkness, R. D., Marko, A. M., Muir, H. M., Neuberger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metabolism+of+collagen+and+other+proteins+of+the+skin+of+rabbits.">The metabolism of collagen and other proteins of the skin of rabbits.</a> Biochem. J. 56 (4), 558-569 (1954).</li><li>Ehrmann, R. L., Gey, G. 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+growth+of+cells+on+a+transparent+gel+of+reconstituted+rat-tail+collagen.">The growth of cells on a transparent gel of reconstituted rat-tail collagen.</a> J. Natl. Cancer Inst. 16 (6), 1375-1403 (1956).</li><li>Kleinman, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basement+membrane+complexes+with+biological+activity.">Basement membrane complexes with biological activity.</a> Biochemistry. 25 (2), 312-318 (1986).</li><li>Vukicevic, S., Kleinman, H. K., Luyten, F. P., Roberts, A. B., Roche, N. S., Reddi, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+multiple+active+growth+factors+in+basement+membrane+Matrigel+suggests+caution+in+interpretation+of+cellular+activity+related+to+extracellular+matrix+components.">Identification of multiple active growth factors in basement membrane Matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components.</a> Exp. Cell Res. 202 (1), 1-8 (1992).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BD+Biosciences+-+Discover+Labware.">BD Biosciences - Discover Labware.</a> SPC-356230 Rev 5.0 at Rev 5.0. , .</li><li>Hughes, C. S., Postovit, L. M., Lajoie, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+a+complex+protein+mixture+required+for+optimal+growth+of+cell+culture.">Matrigel: a complex protein mixture required for optimal growth of cell culture.</a> Proteomics. 10 (9), 1886-1890 (2010).</li><li>Ng, Y. Z., Dayal, J. H., South, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Predisposition+to+Cutaneous+Squamous+Cell+Carcinoma.">Genetic Predisposition to Cutaneous Squamous Cell Carcinoma.</a> , .</li><li>Christiano, A. M. A., Greenspan, D. S. D., Lee, S. S., Uitto, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+human+type+VII+collagen.+Complete+primary+sequence+of+the+alpha+1(VII)+chain+and+identification+of+intragenic+polymorphisms.">Cloning of human type VII collagen. Complete primary sequence of the alpha 1(VII) chain and identification of intragenic polymorphisms.</a> J. Biol. Chem. 269 (32), 20256-20262 (1994).</li><li>Hovnanian, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+linkage+of+recessive+dystrophic+epidermolysis+bullosa+to+the+type+VII+collagen+gene.">Genetic linkage of recessive dystrophic epidermolysis bullosa to the type VII collagen gene.</a> J. Clin. Invest. 90 (3), 1032-1036 (1992).</li><li>Ryyn&#228;nen, M. M., Ryyn&#228;nen, J. J., Sollberg, S. S., Iozzo, R. V. R., Knowlton, R. G. R., Uitto, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+linkage+of+type+VII+collagen+(COL7A1)+to+dominant+dystrophic+epidermolysis+bullosa+in+families+with+abnormal+anchoring+fibrils.">Genetic linkage of type VII collagen (COL7A1) to dominant dystrophic epidermolysis bullosa in families with abnormal anchoring fibrils.</a> J. Clin. Invest. 89 (3), 974-980 (1992).</li><li>Ng, Y. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast-derived+dermal+matrix+drives+development+of+aggressive+cutaneous+squamous+cell+carcinoma+in+patients+with+recessive+dystrophic+epidermolysis+bullosa.">Fibroblast-derived dermal matrix drives development of aggressive cutaneous squamous cell carcinoma in patients with recessive dystrophic epidermolysis bullosa.</a> Cancer Res. 72 (14), 3522-3534 (2012).</li><li>Larouche, D., Paquet, C., Fradette, J., Carrier, P., Auger, F. A., Germain, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+15.">Chapter 15.</a> Methods Mol. Biol. 482, 233-256 (2009).</li><li>Pouliot, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstructed+human+skin+produced+in+vitro+and+grafted+on+athymic+mice).">Reconstructed human skin produced in vitro and grafted on athymic mice).</a> Transplantation. 73 (11), 1751-1757 (2002).</li><li>Purdie, K. J., Pourreyron, C., South, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+squamous+cell+carcinoma+lines.">Isolation and culture of squamous cell carcinoma lines.</a> Methods Biol. 731, 151-159 (2011).</li><li>Pinnell, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+collagen+biosynthesis+by+ascorbic+acid:+a+review.">Regulation of collagen biosynthesis by ascorbic acid: a review.</a> Yale J. Biol. Med. 58 (6), 553-559 (1985).</li><li>Hata, R., Senoo, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-ascorbic+acid+2-phosphate+stimulates+collagen+accumulation,+cell+proliferation,+and+formation+of+a+three-dimensional+tissuelike+substance+by+skin+fibroblasts.">L-ascorbic acid 2-phosphate stimulates collagen accumulation, cell proliferation, and formation of a three-dimensional tissuelike substance by skin fibroblasts.</a> J. Cell. Physiol. 138 (1), 8-16 (1988).</li></ol>

Les auteurs n&#39;ont rien à révéler.

En raison de la nature de cette expérience, le temps total nécessaire pour l&#39;achèvement peut être jusqu&#39;à deux mois. Pendant tout ce temps, les pratiques de culture de soins et de tissus stérile maximum doivent être utilisés pour prévenir la contamination microbienne. En plus de son rôle en tant que cofacteur dans la synthèse de l&#39;hydroxyproline et de l&#39;hydroxylysine des collagènes, de l&#39;acide ascorbique stimule l&#39;expression d&#...

L&#39;utilisation d&#39;un biomatériau dans une culture de tissu en 3D a permis aux chercheurs d&#39;étudier le comportement des cellules dans le laboratoire dans des conditions physiologiques plus semblables à un environnement in vivo que celle de l&#39;adhérence avec une récapitulé 2D et un substrat en matière plastique. En particulier, de grands progrès ont été réalisés dans la modélisation épithéliums stratifiés avec l&#39;adoption de méthodes de culture 3D à l&#39;interface air-liquide 1-4. De telles techniques imitent fidèlement la différenciation des kératinocytes et l&#39;invasion des cellules tumorales permettant une plus grande souplesse et la fidélité pour les chercheurs étudiant ces processus. Le choix du substrat biomatériau pour imiter l&#39;environnement stromal a principalement impliqué l&#39;utilisation de collagène de type I, Engelbreth-Holm-Swarm matrice de sarcome de la souris et du derme dé-epidermized. Par exemple, les fibroblastes associés au cancer ont été montrés pour contribuer à l&#39;invasion de cancer 5, l&#39;initiation et la progression par l&#39;intermédiaire d&#39;interactions de stroma-épithéliales 6,7 WHEn grandi dans ces substrats. L&#39;étalon-or pour mimer l&#39;environnement stromal dans la peau, les plus grandes et les plus étudiés épithéliums stratifiés à l&#39;aide de ces techniques, est considéré être dé-epidermized derme humain (DED). Préparation du DED implique la suppression de l&#39;épiderme par traitement à la trypsine ou la dissociation physique de cadavre 3,4 de la peau humaine. Toutefois, l&#39;accès à cette peau peut être très difficile pour les laboratoires ne sont pas associés avec des institutions cliniques, et derme malades est presque impossible à obtenir. Comme alternative, les laboratoires utilisent souvent une combinaison de collagène de type I (isolé à partir de queues de rat) et / ou matrice de sarcome Engelbreth-Holm-Swarm souris. Après la découverte en 1927 par Nageotte 8 que le collagène peut facilement être isolé en utilisant de l&#39;acide acétique et la précipitation de sel, son application à la culture de tissu a ensuite été lancée par Huzella et ses collègues 9. revêtement de collagène s&#39;est révélée être supérieure à celle du verre pour la culture de cellules souches et de 29 explants de tissus comme interrogés par Ehrmann Gey et 9. Actuellement, le principal type de collagène utilisé dans la culture de tissu est isolé à partir de tendons de queue de rat, et est généralement acheté auprès de sources commerciales. Cependant, l&#39;inconvénient de fidèles substrat récapitulation est que le collagène de queue de rat n&#39;est pas identique au collagène humain, ou du derme humain, où le type III collagènes I et sont présents comme constituants principaux, et isolé de queue de rat collagène est toujours fragmenté. Engelbreth-Holm-Swarm matrice de sarcome de souris est un mélange de protéines gélatineuse sécrétée par les cellules de sarcome de souris en culture Engelbreth-Holm-Swarm 10. Les principaux constituants sont la laminine, collagène de type IV, le sulfate d&#39;héparine protéoglycanes, entactine et nidogène et les rapports exacts de ces protéines peuvent varier d&#39;un lot à l&#39;. Mis à part pro structurelleprotéines, cette matrice contient également des niveaux significatifs de facteurs de croissance tels que la transformation de β du facteur de croissance, facteur de croissance épidermique, de l&#39;insuline-like growth factor 1, bovin facteur de croissance des fibroblastes, et le facteur de croissance dérivé des plaquettes qui pourrait altérer le comportement cellulaire 11,12. Pointant vers la complexité de Engelbreth-Holm-Swarm matrice de sarcome de la souris, un total de 1 851 protéines ont été identifiées dans une étude protéomique récente 13. Compte tenu de la nature riche et complexe de cette matrice, la prudence a été informé lors de l&#39;interprétation et la comparaison des différentes expériences avec l&#39;utilisation de celui-ci 11. Nos laboratoires ont un vif intérêt dans les maladies génétiques de la peau, en particulier ceux qui ont une prédisposition à développer un cancer cutané spinocellulaire (SCLC) 14. Dans le cas d&#39;récessif bulleuse dystrophique épidermolyse (de EBDR), une maladie grave avec cloques mutations germinales dans le gène COL7A1 <sjusqu&#39;à&gt; 15-17, nous avons déterminé que le microenvironnement de la peau chez ces patients est la tumeur la promotion 18. Au cours de cette étude, nous n&#39;avons pas pu évaluer la tumeur promouvoir propriétés de fibroblastes dermiques intégrés dans le collagène I / Engelbreth-Holm-Swarm matrice de sarcome de la souris et étudiés les moyens d&#39;évaluer propre, natif matrice de cellules. Pour ce faire, nous avons modifié la technique précédente du laboratoire de Lucie Germain travailler sur la peau humaine équivalents 19,20. La technique de Germain a pu reconstituer la peau humaine avec la membrane basale bien organisée, utilisant des kératinocytes humains et des fibroblastes primaires cultures en l&#39;absence d&#39;un échafaudage ou synthétique cadavérique. Dans ce document, les étapes à suivre pour récapituler cutané micro-environnement stromal de la tumeur (matrice native) proviennent directement des fibroblastes du stroma primaires in vitro sont décrits 18. Matrices autochtones produit par culture à long terme des fibroblastes ont été utilisés comme un équivalent dermique de dosage pour l&#39;invasion des cellules SCLC. Nous présentons des données utilisant la matrice native issus soit de la matrice extracellulaire sécrétée par les fibroblastes EBDR (déficientes en collagène de type VII (C7)) ou à partir de fibroblastes de EBDR rétrovirale transduites avec un collagène de type VII construction exprimant et de démontrer l&#39;effet profond d&#39;une seule collagène sur la tumeur l&#39;invasion des cellules.

<table border="0" cellpadding="0" cellspacing="0" width="352">
	<colgroup>
		<col span="4" style="width:66pt" width="88" />
	</colgroup>
	<tbody>
		<tr height="64" style="height:48.0pt">
			<td class="xl63" height="64" style="height:48.0pt;width:66pt" width="88">L-Ascorbic acid 2-phosphate</td>
			<td class="xl63" style="width:66pt" width="88">Sigma</td>
			<td class="xl63" style="width:66pt" width="88">A8960</td>
			<td class="xl64" style="width:66pt" width="88"></td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl65" height="42" style="height:31.5pt;width:66pt" width="88">DMEM with l-glutamine,</td>
			<td class="xl67" rowspan="2" style="border-bottom:1.0pt solid black;
 border-top:none;width:66pt" width="88">Life Technologies</td>
			<td class="xl67" rowspan="2" style="border-bottom:1.0pt solid black;
 border-top:none;width:66pt" width="88">11995-073</td>
			<td class="xl67" rowspan="2" style="border-bottom:1.0pt solid black;
 border-top:none;width:66pt" width="88"></td>
		</tr>
		<tr height="106" style="height:79.5pt">
			<td class="xl63" height="106" style="height:79.5pt;width:66pt" width="88">4,500 mg/L d-glucose, 110 mg/L sodium pyruvate</td>
		</tr>
		<tr height="85" style="height:63.75pt">
			<td class="xl63" height="85" style="height:63.75pt;width:66pt" width="88">100x Penicillin-Streptomycin</td>
			<td class="xl63" style="width:66pt" width="88">Life Technologies</td>
			<td class="xl63" style="width:66pt" width="88">15070</td>
			<td class="xl64" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl63" height="43" style="height:32.25pt;width:66pt" width="88">Vaseline</td>
			<td class="xl63" style="width:66pt" width="88">VWR</td>
			<td class="xl63" style="width:66pt" width="88">PROL28908.290</td>
			<td class="xl64" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl63" height="43" style="height:32.25pt;width:66pt" width="88">Clonal cylinders</td>
			<td class="xl63" style="width:66pt" width="88">Sigma</td>
			<td class="xl63" style="width:66pt" width="88">Z370789</td>
			<td class="xl64" style="width:66pt" width="88"></td>
		</tr>
		<tr height="148" style="height:111.0pt">
			<td class="xl63" height="148" style="height:111.0pt;width:66pt" width="88">Nylon Net Filter Disc Hydrophilic 100um 25um diameter 100/pk</td>
			<td class="xl63" style="width:66pt" width="88">Millipore</td>
			<td class="xl63" style="width:66pt" width="88">NY1H02500</td>
			<td class="xl64" style="width:66pt" width="88"></td>
		</tr>
		<tr height="127" style="height:95.25pt">
			<td class="xl63" height="127" style="height:95.25pt;width:66pt" width="88">Bent stainless steel wire mesh support</td>
			<td class="xl63" style="width:66pt" width="88">Made in house</td>
			<td class="xl63" style="width:66pt" width="88"></td>
			<td class="xl66" style="width:66pt" width="88">Dimensions were made so that the mesh would fit into 6-well plates</td>
		</tr>
	</tbody>
</table>

tissue engineering of tumor stromal microenvironment with application to cancer cell invasion

Cultures organotypiques 3D des cellules épithéliales sur une matrice incorporée avec les cellules mésenchymateuses sont largement utilisés pour étudier la différenciation des cellules epitheliales et l&#39;invasion. Type de queue de rat collagène et / ou de la matrice dérivée de cellules de sarcome de souris Engelbreth-Holm-Swarm ont été traditionnellement utilisé comme substrats pour modéliser la matrice ou stromale microenvironnement dans lequel les cellules mésenchymateuses (fibroblastes en général) sont remplies. Bien que des expériences à l&#39;aide de ces matrices sont très instructifs, on peut affirmer qu&#39;en raison d&#39;une présence dominante d&#39;une seule protéine (comme en collagène de type I) ou une teneur élevée en sous-sol composants de la membrane et des facteurs de croissance (tels que dans la matrice dérivée de la souris des cellules de sarcome), ces substrats ne reflètent pas la meilleure contribution de composition de matrice faite par les cellules stromales eux-mêmes. Pour étudier matrices indigènes produites par les fibroblastes dermiques primaires isolées de patients avec une tumeur sujettes, trouble génétique cloques (dystrophique récessiveépidermolyse bulleuse), nous avons adapté un protocole de matrice native existante pour étudier l&#39;invasion des cellules tumorales. Les fibroblastes sont induites à produire leur propre matrice sur une période prolongée dans la culture. Cette matrice native est ensuite détaché de la boîte de culture et les cellules épithéliales sont ensemencés sur elle avant que l&#39;ensemble de co-culture est portée à l&#39;interface air-liquide. Différenciation et / ou l&#39;invasion cellulaire peuvent ensuite être évaluées au cours du temps. Cette technique permet d&#39;évaluer les interactions des cellules épithéliales-mésenchymateuses dans un environnement 3D sans la nécessité d&#39;une matrice synthétique ou étrangère avec le seul inconvénient étant le laps de temps prolongé requis pour produire la matrice native. Nous décrivons ici l&#39;application de cette technique pour évaluer la capacité d&#39;une seule molécule exprimée par les fibroblastes, le collagène de type VII, d&#39;inhiber l&#39;invasion des cellules tumorales.

<html> Cette technique ouvre la possibilité d&#39;examiner et de comparer le comportement invasif des cellules tumorales (dans ce cas SCLC) sous différents environnements stromales 3D. En utilisant cette technique, non seulement des matrices indigènes C7-déficientes que récapitulées l&#39;environnement EBDR cutanée peuvent être générés, mais aussi des matrices supplémentaires qui ont été génétiquement modifiées pour surexprimer C7 18. Comme le montre la figure 2, l&#39;invasio...

Watch this Scientific Journal Video about Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion at JoVE.com

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

L&#39;ingénierie tissulaire natif matrice dérivé des fibroblastes est un nouvel outil pour générer un substrat du stroma qui supporte la prolifération cellulaire et la différenciation epitheliale. Voici un protocole application de cette méthodologie pour évaluer l&#39;impact de différents types de cellules stromales de la biologie des cellules tumorales est présenté.

Ingénierie tissulaire de la tumeur stromale Microenvironnement avec application de Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

tissue-engineering-tumor-stromal-microenvironment-with-application-to

Research

JoVE Journal

Bioengineering

9.7K vues.  University of Dundee.  L'ingénierie tissulaire natif matrice dérivé des fibroblastes est un nouvel outil pour générer un substrat du stroma qui supporte la prolifération cellulaire et la différenciation epitheliale. Voici un protocole application de cette méthodologie pour évaluer l'impact de différents types de cellules stromales de la biologie des cellules tumorales est présenté. 

Vidéo: Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

Dispositif microfluidique pour Recréer un microenvironnement des tumeurs<em&gt; In vitro</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Recherche

Bio-ingénierie

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

Réalisé cellulaire auto-assemblage pour fabriquer des anneaux de tissus dérivés de cellules pour l&#39;analyse biomécanique et génie tissulaire

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

Tridimensionnelle modèle de culture cellulaire pour mesurer les effets de l&#39;écoulement des fluides interstitiels sur l&#39;invasion tumorale

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Les tissus d&#39;ingénierie muscle squelettique de myoblastes murins cellules progénitrices et application d&#39;une stimulation électrique

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

génie tissulaire d&#39;un être humain 3D<em&gt; In vitro</em&gt; Système de tumeur de test

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

Ingénierie à base de fibrine constructions de tissu de myofibroblastes et application des contraintes et de déformation à Provoquer Organisation cellulaire et collagène (Re)

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

À long terme intravitale immunofluorescence imagerie de tissus composants matriciels avec épifluorescence et deux microscopie biphotonique

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microenvironnements sandwich à exploiter cellulaires / Interactions matérielles

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

Modèle in vitro de physiologiques et pathologiques Blood Flow avec application aux enquêtes de cellules vasculaires Rénovation

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Génie tissulaire par Intrinsic vascularisation dans un<em&gt; In Vivo</em&gt; Tissue Chambre Ingénierie

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...