Nous remercions T. Hunt, M. Rosenbluth, et le Laboratoire de Lam pour leurs conseils et des discussions utiles. Nous reconnaissons l&#39;appui de G. Spinner et l&#39;Institut d&#39;Electronique et de la nanotechnologie à l&#39;Institut de Technologie de Géorgie. Le soutien financier pour ce travail a été fourni par une subvention du NIH K08-HL093360, UCSF REAC prix, une nanomédecine NIH Centre de développement des Prix PN2EY018244, et le financement du Centre pour la biologie des cellules endothéliales des soins de santé pour enfants d&#39;Atlanta.

1. Fabrication du microdispositif endothéliale <ol><li> Créer un photomasque en soumettant une conception assistée par ordinateur (CAO) dessin du dispositif microfluidique à un fournisseur masque à l&#39;extérieur. Le masque utilisé est composé d&#39;une couche de chrome sur verre sodocalcique. Dans ce cas, la largeur du canal microfluidique était de 30 um. </li><li> Nettoyer une plaquette de silicium nu avec piranha (10:1 rapport de l&#39;acide sulfurique et de peroxyde d&#39;hydrogène) pendant 15 minutes et plonger dans l&#39;acide fluorhydrique pendant 30 secondes. Rincer à l&#39;eau désionisée (DI) de l&#39;eau pendant environ 10 secondes. </li><li> Utilisation d&#39;un tournette, de spin Microchem SU-8 2025 photorésist sur la plaquette à une hauteur de 30 um. Pour SU-8 2025, une vitesse de rotation rpm 3000 est recommandé. Les autres viscosités de SU-8 sont disponibles qui permettront d&#39;atteindre cette hauteur. Les instructions complètes pour SU-8 utilisation sont disponibles à <a href="http://www.microchem.com" target="_blank">www.microchem.com</a> </li><li> Placer la galette avec SU-8 sur une plaque chauffante à 95 ° C pour les5 minutes pour chasser l&#39;excès de solvant. Remarque: Un four va sécher la plaquette différente de celle d&#39;une plaque chauffante et n&#39;est pas recommandée. </li><li> Placer un masque à la forme caractéristique désirée sur la plaquette, et exposer à la lumière UV (160 mJ / cm 2 mesurée à 365 nm) dans un alignement de masque (Karl Suss, MA-6). Cette croix relie la résine photosensible. </li><li> Placez la tranche de retour sur une plaque chauffante à 95 ° C pendant 5 minutes supplémentaires pour encore accélérer la polymérisation de la SU-8. </li><li> Plonger la plaquette dans SU-8 Developer, composée principalement de PGMEA (propylène glycol acétate d&#39;éther méthylique) pendant 4 minutes pour enlever le non-réticulé SU-8. </li><li> Rincer la plaquette récemment mis au point avec de l&#39;alcool isopropylique à 100% (IPA) pendant 10 s. La plaquette peut ensuite être séché à l&#39;aide d&#39;azote sous pression ou en permettant au solvant de s&#39;évaporer de la tranche dans une hotte de fumée propre pendant plusieurs minutes. </li><li> Cassette les bords de la plaquette à motifs sec avec SU-8 dans une boîte de Pétri pour préévacuer le mouvement. </li><li> Appliquer une ml de Sigmacote à la plaquette à l&#39;aide d&#39;une pipette, recouvrir la partie supérieure de la boîte de Pétri, et plaquette de tourbillonnement pour assurer revêtement complet de la plaquette, enlever le couvercle, et permettre plaquette à sécher pendant plusieurs minutes jusqu&#39;à ce que tout le solvant soit évaporé. </li></ol> 2. PDMS (polydiméthylsiloxane) Préparation <ol><li> Mélange polymère PDMS et agent de durcissement dans un rapport 10:01 (p / p) et éliminer les bulles d&#39;air au moyen d&#39;un dessiccateur à vide. La longueur du temps nécessaire pour dégazer le PDMS varie sur la force du système de vide disponible, mais est généralement comprise entre quelques minutes à une heure. Pour une durée de six pouces plat de Pétri, sans préalablement versé PDMS, un volume total de 60 ml de polymère est recommandé. </li><li> Verser le mélange sur la tranche, d&#39;environ 5 mm d&#39;épaisseur. En outre verser sur un plat à fond plat pour créer une feuille mince de PDMS, environ 1 mm d&#39;épaisseur. Durcir à 60 ° C dans un four pendant une nuit. </li><li> Avec un couteau ou un scalpel, découpez autour de la cURED dispositif de PDMS et le retirer de la plaquette. En outre, couper une mince feuille de PDMS légèrement plus grand que l&#39;appareil. </li><li> Créer des trous d&#39;entrée et de sortie dans le dispositif PDMS l&#39;aide d&#39;un poinçon de trou de 1,0 mm. Cela peut être accompli en utilisant soit un vice broches, dispositif de Harris Uni-Core, ou similaire. </li><li> Nettoyer les surfaces de l&#39;appareil et la feuille à l&#39;aide du scotch. </li><li> Utilisation d&#39;un détergent à plasma, exposer les surfaces du dispositif PDMS et la feuille PDMS à plasma d&#39;oxygène pendant 30 s. Le plasma d&#39;oxygène crée des espèces réactives à la surface exposée de la liaison lorsque PDMS qui met en contact physique. </li><li> Connecter plus petite tubulure à une petite longueur (quelques centimètres) de tube grande, qui à son tour est reliée à une seringue à aiguille émoussée point, rempli de 50 pg / ml de fibronectine de plasma humain dans du PBS. Le tube et une aiguille sont dimensionnés de telle sorte qu&#39;une friction est créé entre le tube. Insérer plus petite tube dans l&#39;entrée de PDMS et un microdispositifppliquer pression à la seringue pour remplir complètement les canaux avec une solution de la fibronectine et de créer une petite goutte 100 pi à l&#39;orifice de sortie, afin de s&#39;assurer que les canaux restent mouillées. Incuber le dispositif à 37 ° C pendant 40-60 min. </li><li> Connecter une seringue remplie de PBS frais à l&#39;aiguille pointe émoussée. Appliquer une pression à la seringue pour rincer l&#39;appareil avec du PBS. </li></ol> 3. L&#39;ensemencement du dispositif microfluidique avec les cellules endothéliales <ol><li> Préparer 1.000.000 cellules / ml de cellules humaines endothéliales de veine ombilicale (HUVEC) dans les médias de croissance endothéliale avec 8% de dextran. Une gamme de 500.000 à 2.000.000 cellules / ml a été utilisé avec succès. Addition de dextrane dans le milieu de chargement de cellules augmente la viscosité du fluide, ce qui diminue la vitesse des cellules endothéliales qui entrent le système fibronectine revêtu microfluidique. Ce, à son tour, augmente la probabilité que les cellules adhèrent et de la culture avec succès au sein de la microdispositif. </li><li> Celiez la nouvelle seringue et le tube comme à l&#39;étape 2.7. mais avec une plus tube (environ 1 mètre de longueur). </li><li> Pour optimiser les performances de ce système, il est essentiel à ce stade pour éviter toute fuite ou de bulles dans le système de perfusion ensemble; toute fuite de la solution ou la présence de bulles dans les médias va changer le débit et d&#39;empêcher l&#39;ensemencement avec succès des cellules endothéliales. Par conséquent, adhérente de la tubulure est essentiel pour éliminer les fuites entre les connexions. Bulles dans la seringue et / ou des tubes doivent être éliminés avant que les cellules sont introduites dans le microdispositif et le système de perfusion entière doit être amorcée avec la solution de cellules avant de fixer le tuyau à la microfluidique afin d&#39;éviter l&#39;introduction de bulles d&#39;air. </li><li> En utilisant une pompe seringue, injecter la suspension de cellules dans le dispositif PDMS à un débit volumétrique de 1,23 l / min pendant 2 heures à 37 ° C et 5% de CO 2. Des résultats optimaux ont été obtenus lorsque la pompe à seringue était à la<html> même hauteur que le dispositif PDMS. Pour l&#39;entrée, le long tube, qui peut être enroulé dans l&#39;incubateur, en sorte que les cellules et le milieu sont suffisamment chauffé avant de venir en contact avec le dispositif. Dans notre système, le débit volumétrique de 1,23 l / min se rapproche à une vitesse médiane du ~ 1 mm / s dans les plus petites microcanaux, correspondant à une contrainte de cisaillement mur de ~ 1 dyne / cm 2 à ces canaux à l&#39;aide de sang total. </li><li> Utilisation de la pompe même seringue et le tube long, perfuser un milieu de croissance frais pour 2-8 jours à taux de 1,23 l / min. Un dispositif de retenue doit avoir une monocouche de cellules endothéliales en croissance à l&#39;intérieur de l&#39;appareil dans les 24-48 heures. Des expériences antérieures ont montré que des monocouches confluentes de manière appropriée d&#39;exprimer la VE-cadhérine à jonctions cellule-cellule dans le dispositif 14. </li><li> Injecter du sang ou de la suspension cellulaire dans le système pour l&#39;expérimentation. </li></ol> 4. Les résultats représentatifs _content &quot;&gt; En utilisant ce protocole, les techniques classiques de microfabrication lithographiques sont utilisés pour créer le moule nécessaire pour produire les canaux microfluidiques que, physiologiquement, imitent l&#39;sizescale de la microvascularisation (figure 1A). En utilisant une technique de perfusion optimisé, les cellules endothéliales alors la culture de semences et de la confluently toute la surface interne du système microfluidique dans les 24-48 heures de ensemencement des cellules (figure 1B). Comme le système microfluidique est transparent, le microdispositif entier peut être placé sur un fond clair / étape microscope à fluorescence pour l&#39;imagerie et la collecte de données. Notre système peut alors être appliquée pour étudier les maladies hématologiques qui impliquent modifiés propriétés biophysiques, comme la drépanocytose, dans lequel la rigidité accrue de globules rouges falciformes et aberrante d&#39;adhérence des leucocytes et des cellules endothéliales contribuent à l&#39;obstruction microvasculaire. Un médicament approuvé cliniquement, l&#39;hydroxyurée, améliore les symptômes, mais sa dDIRECT effet sur les flux microvasculaire est inconnue. Notre test prend en compte la rigidité cellulaire et l&#39;adhérence, et démontre que l&#39;hydroxyurée améliore de manière significative l&#39;écoulement dans la drépanocytose (figure 2). La drépanocytose est un seul exemple d&#39;une demande de la microvascularisation-sur-une-puce, comme ce système est idéalement adapté à l&#39;étude tout processus hématologique dans lequel les cellules sanguines interagir les uns avec les autres cellules et des cellules endothéliales dans la microcirculation. D&#39;autres applications cliniquement pertinents comprennent les troubles inflammatoires, sepsis / poumon blessures, les microangiopathies thrombotiques, le paludisme, et les métastases du cancer tout en plus d&#39;applications de base comprennent la biologie des leucocytes et de cellules souches hématopoïétiques biologie, parmi beaucoup d&#39;autres. <img src="/files/ftp_upload/3958/3958fig1.jpg" alt="Figure 1"/> Figure 1. A) la première PDMS microfluidique appareil avant endothélialisation. Ici, le microdispositif est injecté l&#39;esprith colorant alimentaire pour illustrer la conception sizescale et dans l&#39;ensemble du système. B) microscopie en fond clair montre le système microfluidique est complètement endothélialisée dans les 48 heures de l&#39;ensemencement de cellules en utilisant le protocole décrit ici. <img src="/files/ftp_upload/3958/3958fig2.jpg" alt="Figure 2"/> Figure 2. A) La microvascularisation-sur-une-puce avec microcanaux se rapproche de la taille de veinules post-capillaires (30 um), le site de la plupart des événements drépanocytaires obstructives microvasculaires. Le sang total chez les patients drépanocytaires recevant l&#39;hydroxyurée et de patients ne recevant pas l&#39;hydroxyurée est coulé par deux différents microvascularisation-sur-une-puce. B) Le sang total chez les patients drépanocytaires qui accueillent des flux hydroxyurée avec une relative facilité dans les microcanaux endothélialisée. C) Dans les mêmes conditions hémodynamiques, le sang total chez les patients drépanocytaires qui ne reçoivent pas l&#39;hydroxyurée, cependant, le débit beaucoup plus lent avec microchobstruction annel. Le canal inférieur est complètement obstruée sans écoulement et les vitesses d&#39;écoulement dans les microcanaux autres endothélialisée sont significativement plus faible que dans l&#39;état hydroxyurée. La barre d&#39;échelle dans les trois images est de 30 um.

<ol>
	<li>Mezzano, D., Quiroga, T., Pereira, 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+Level+of+Laboratory+Testing+Required+for+Diagnosis+or+Exclusion+of+a+Platelet+Function+Disorder+Using+Platelet+Aggregation+and+Secretion+Assays.">The Level of Laboratory Testing Required for Diagnosis or Exclusion of a Platelet Function Disorder Using Platelet Aggregation and Secretion Assays.</a> Semin. Thromb. Hemost. 35, 242-254 (2009).</li><li>Young, E. W. K., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+microfluidic+cell+culture+in+controlled+microenvironments.">Fundamentals of microfluidic cell culture in controlled microenvironments.</a> Chemical Society Reviews. 39, 1036-1048 (2010).</li><li>Young, E. W. K., Simmons, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macro-+microscale+fluid+flow+systems+for+endothelial+cell+biology.">Macro- microscale fluid flow systems for endothelial cell biology.</a> Lab on a Chip. 10, 143-160 (2010).</li><li>Higgins, J. M., Eddington, D. T., Bhatia, S. N., Mahadevan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sickle+cell+vasoocclusion+and+rescue+in+a+microfluidic+device.">Sickle cell vasoocclusion and rescue in a microfluidic device.</a> Proceedings of the National Academy of Sciences. 104, 20496-20500 (2007).</li><li>Rosano, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+physiologically+realistic+in+vitro+model+of+microvascular+networks.">A physiologically realistic in vitro model of microvascular networks.</a> Biomedical Microdevices. 11, 1051-1057 (2009).</li><li>Meer, A. D. v. a. n. d. e. r., Poot, A. A., Duits, M. H. G., Feijen, J., Vermes, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+Technology+in+Vascular+Research.">Microfluidic Technology in Vascular Research.</a> Journal of Biomedicine and Biotechnology. 2009, (2009).</li><li>Karunarathne, W., Ku, C. -. J., Spence, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dual+nature+of+extracellular+ATP+as+a+concentration-dependent+platelet+P2X1+agonist+and+antagonist.">The dual nature of extracellular ATP as a concentration-dependent platelet P2X1 agonist and antagonist.</a> Integrative Biology. 1, 655-663 (2009).</li><li>Kotz, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+microfluidics+for+neutrophil+genomics+and+proteomics.">Clinical microfluidics for neutrophil genomics and proteomics.</a> Nat. Med. 16, 1042-1047 (2010).</li><li>Rosenbluth, M. J., Lam, W. A., Fletcher, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+cell+mechanics+in+hematologic+diseases+with+microfluidic+biophysical+flow+cytometry.">Analyzing cell mechanics in hematologic diseases with microfluidic biophysical flow cytometry.</a> Lab on a Chip. 8, 1062-1070 (2008).</li><li>Shelby, J. P., White, J., Ganesan, K., Rathod, P. K., Chiu, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+model+for+single-cell+capillary+obstruction+by+Plasmodium+falciparum-infected+erythrocytes.">A microfluidic model for single-cell capillary obstruction by Plasmodium falciparum-infected erythrocytes.</a> Proceedings of the National Academy of Sciences. 100, 14618-14622 (1073).</li><li>Borenstein, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+endothelialized+microvascular+networks+with+circular+cross-sections+in+a+tissue+culture+substrate.">Functional endothelialized microvascular networks with circular cross-sections in a tissue culture substrate.</a> Biomedical Microdevices. 12, 71-79 (2010).</li><li>Nesbitt, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+shear+gradient-dependent+platelet+aggregation+mechanism+drives+thrombus+formation.">A shear gradient-dependent platelet aggregation mechanism drives thrombus formation.</a> Nat. Med. 15, 665-673 (2009).</li><li>Prabhakarpandian, B., Shen, M. -. C., Pant, K., Kiani, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+for+modeling+cell-cell+and+particle-cell+interactions+in+the+microvasculature.">Microfluidic devices for modeling cell-cell and particle-cell interactions in the microvasculature.</a> Microvascular Research. 82, 210-220 (2011).</li><li>Tsai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+modeling+of+the+microvascular+occlusion+and+thrombosis+that+occur+in+hematologic+diseases+using+microfluidic+technology.">In vitro modeling of the microvascular occlusion and thrombosis that occur in hematologic diseases using microfluidic technology.</a> The Journal of Clinical Investigation. , (2011).</li><li>Green, D. A., Murphy, W. G., Uttley, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haemolytic+uraemic+syndrome:+prognostic+factors.">Haemolytic uraemic syndrome: prognostic factors.</a> Clinical &#38; Laboratory Haematology. 22, 11-14 (2000).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

Notre système de microdispositif endothélialisée est le mieux adapté lorsqu&#39;il est utilisé en conjonction avec les expériences in vivo, et son approche réductionniste peut aider à élucider les mécanismes biophysiques des processus hématologiques qui sont observés chez les humains et les modèles animaux. En outre, notre système n&#39;est pas sans limites. Par exemple, nos canaux microfluidiques sont carrées en coupe. Bien que techniquement microcanaux circulaires peuvent être fabriqu...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> aiguille à pointe émoussée </td><td> OK International </td><td> 920 050-TE </td><td> Précision TE aiguille de calibre 20 1/2 x &quot;, rose </td></tr><tr><td> dextrane </td><td> Sigma-Aldrich </td><td> 31392 </td><td></td></tr><tr><td> La fibronectine </td><td> Sigma-Aldrich </td><td> F0895 </td><td></td></tr><tr><td> Perforatrice (étau broches) </td><td> Innovations techniques </td><td></td><td></td></tr><tr><td> Les cellules endothéliales de cordon ombilical (HUVEC) </td><td> Lonza </td><td> CC-2519 </td><td></td></tr><tr><td> Plasma cleaner </td><td> Plasma </td><td> PDC-326 </td><td></td></tr><tr><td>Polydiméthylsiloxane (PDMS) </td><td> Fisher Scientific </td><td> NC9285739 </td><td> Sylgard 184 KIT élastomère de silicone </td></tr><tr><td> Sigmacote </td><td> Sigma-Aldrich </td><td> SL2 </td><td></td></tr><tr><td> SU-8 2025 </td><td> Microchem </td><td> Y111069 </td><td></td></tr><tr><td> SU-8 Developer </td><td> Microchem </td><td> Y020100 </td><td></td></tr><tr><td> Pompe à seringue </td><td> Harvard Apparatus </td><td> 70-3008 </td><td> PHD-ULTRA </td></tr><tr><td> tube (plus) </td><td> Instrument Company Cole-Parmer </td><td> 06418-02 </td><td> Tygonreg microbore tube, 0,020 &quot;x 0,060 ID&quot; OD </td></tr><tr><td> tubulure (plus petit) </td><td> Instrument Company Cole-Parmer </td><td> 06417-11 </td><td> PTFE microbore tube, 0,012 &quot;x 0,030 ID&quot; OD </td></tr></tbody></table>

endothelialized microfluidics for studying microvascular interactions in hematologic diseases

Les progrès des techniques de microfabrication ont permis la production de bon marché et reproductible des systèmes microfluidiques pour mener des expériences biologiques et biochimiques à l&#39;1,2 micro-et nanométrique. En outre, la microfluidique ont également été spécifiquement utilisés pour l&#39;analyse quantitative des processus hématologiques et microvasculaire, en raison de leur capacité de contrôler facilement l&#39;environnement dynamique fluidique et des conditions biologiques 3-6. En tant que tel, les chercheurs ont plus récemment utilisé des systèmes microfluidiques pour étudier la déformabilité des globules, l&#39;agrégation des cellules du sang, le flux sanguin microvasculaire, et le sang interactions cellulaires de cellules endothéliales-6-13. Toutefois, ces systèmes microfluidiques soit ne comprennent pas les cellules endothéliales en culture ou étaient plus que la pertinente sizescale à microvasculaires processus pathologiques. Une plate-forme microfluidique avec des cultures de cellules endothéliales qui récapitule avec précision le cellulaire, physique, et hemodynenvironnement amique de la microcirculation est nécessaire d&#39;approfondir notre compréhension de la pathophysiologie sous-jacente biophysique des maladies hématologiques qui impliquent le système microvasculaire. Nous rapportons ici une méthode pour créer un «endothélialisée&quot; modèle in vitro de la microvascularisation, en utilisant un processus simple et unique masque de microfabrication en conjonction avec standards endothéliales techniques de culture cellulaire, pour étudier les interactions pathologiques microvasculaires biophysiques qui se produisent dans la maladie hématologique. Ce &quot;système microvasculaire-sur-une-puce» fournit au chercheur un test robuste, qui contrôle étroitement biologique ainsi que les conditions biophysiques et est exploité à l&#39;aide d&#39;une seringue électrique standard et clair / microscopie à fluorescence. Des paramètres tels que les conditions hémodynamiques microcirculatoires, le type de cellules endothéliales, le type de cellules sanguines (s) et concentration (s), la drogue ou de concentration inhibitrice etc, tout cela peut être facilement contrôlé. En tant que tel, notre microsystème fournitune méthode pour étudier les processus quantitativement la maladie dans laquelle l&#39;écoulement microvasculaire est altérée en raison de modifications dans l&#39;adhésion cellulaire, l&#39;agrégation et la déformabilité, d&#39;une capacité disponible avec les tests actuels.

Watch this Scientific Journal Video about Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases at JoVE.com

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Procédé pour la culture d&#39;une monocouche de cellules endothéliales sur toute la surface intérieure 3D d&#39;un dispositif microfluidique avec microvasculaire taille canaux (&lt;30 um) est décrite. Cette<em&gt; In vitro</em&gt; Microvascularisation modèle permet l&#39;étude des interactions biophysiques entre les cellules sanguines, cellules endothéliales, et les facteurs solubles dans les maladies hématologiques.

Microfluidique endothélialisée pour étudier les interactions microvasculaires dans Hémopathies

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

endothelialized-microfluidics-for-studying-microvascular-interactions

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Bioengineering

15.8K vues.  Emory University School of Medicine. Procédé pour la culture d'une monocouche de cellules endothéliales sur toute la surface intérieure 3D d'un dispositif microfluidique avec microvasculaire taille canaux (

Vidéo: Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

Mélangeurs microfluidiques pour étudier repliement des protéines

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

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Bio-ingénierie

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Détermination de la vitesse de transport des xénobiotiques et des nanomatériaux à travers le placenta en utilisant l&#39;<em&gt; Ex vivo</em&gt; Human perfusion placentaire modèle

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

Procédure à suivre pour le développement de la multi-profondeur circulaire transversale endothélialisées microcanaux-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

L&#39;utilisation de puces microfluidiques pour l&#39;imagerie en direct et l&#39;étude des réponses blessures en<em&gt; Drosophila</em&gt; Larves

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

L&#39;impression submergé de cellules sur une surface modifiée à l&#39;aide d&#39;un flux continu Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

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. ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Multicolor détection Fluorescence pour Droplet microfluidique utilisant des fibres optiques

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

Isolement des murins dérivés tissu adipeux microvasculaire fragments comme Vascularization unités pour l&#39;ingénierie tissulaire

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

Interfaçage Microfluidics microélectrode tableaux pour étudier la Communication neuronale et la Propagation des signaux axonale

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

Organoïdes neuronaux humains pour étudier le cancer du cerveau et les maladies neurodégénératives

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...