Authors thank Mr. Kotaro Okubo for the kind assistance with SEM imaging. This work was supported by the Japan Society for the Promotion of Science Grant-in-Aid for Basic Research (B) (20310069), Grant-in-Aid for Research Activity Start-up (25880021), and by research grants from the Kurata Memorial Hitachi Science and Technology Foundation and the Nippon Sheet Glass Foundation for Materials Science and Engineering.

1. Préparation de TiO 2 revêtues de verre Coverslip <ol><li> Nombre les lamelles à l&#39;aide d&#39;une pointe à tracer de diamant. Cela permet non seulement de garder une trace de chaque lamelles, mais aussi pour veiller à ce que le bon côté de l&#39;échantillon est orientée vers le haut. Nettoyer les lamelles couvre-objet, d&#39;abord sous exécutant ddH 2 O, puis en les immergeant dans une solution de piranha (H 2 SO 4: H 2 O 2 = 4: 1). Après 10 minutes, rincez soigneusement les lamelles, 8 fois en ddH 2 O. Sécher les lamelles sous N2 flux. </li><li> Ensemble de TiO 2 dans la cible de radiofréquence (RF) de système de pulvérisation. Fixez les lamelles sur un support de l&#39;équipement de pulvérisation à l&#39;aide d&#39;un ruban de polyimide de l&#39;échantillon. Placez le porte-échantillon dans la chambre de pulvérisation. Évacuer la chambre jusqu&#39;à ce que la pression atteint 2,0 × 10 -4 Pa. </li><li> Introduire du gaz Ar dans la chambre et régler la pression de dépôt à 4,0 mTorr. Tout en gardant l&#39;obturateur fermé, augmenter progressivementla puissance RF à 70 W. </li><li> Ouvrir le volet et par pulvérisation cathodique pendant 15 min pour obtenir un film d&#39;une épaisseur de 120 à 150 nm (Figure 1: Etape A). Taux de croissance du film doit être dérivé pour chaque machine. </li></ol> 2. Revêtement de surface avec Cell-répulsif Film <ol><li> Rendre hydrophile la surface de TiO 2 par traitement de l&#39;échantillon avec du plasma O 2, en suivant les instructions fournies par le fabricant du réacteur à plasma. Nous traitons l&#39;échantillon pendant 5 min à 200 W avec O 2 débit de 100 sccm. Immerger l&#39;échantillon dans le trou DDH 2 O et de confirmer que la surface est superhydrophile. Séchez soigneusement la surface sous le N 2 flux. </li><li> Préparez 1 mM octadécyltrichlorosilane (OTS) solution par addition de 39,6 OTS pi à 100 ml de toluène. Immerger l&#39;échantillon dans la solution pendant 1 heure à température ambiante. Effectuer cette étape l&#39;intérieur d&#39;un sac à gants 2 -filled N (Figure 1: Étape B). </li><li> Pour éliminer les molécules physisorbées, sonicate l&#39;échantillon dans le toluène, l&#39;acétone, l&#39;éthanol, et ddH 2 O pendant 5 minutes chacune, dans cet ordre. Rincer l&#39;échantillon quatre fois en frais ddH 2 O et sécher la surface sous le N 2 flux. La surface doit être hydrophobe avec un angle de 100-110 ° de contact. </li></ol> 3. Ex-Situ structuration de surface <ol><li> Travailler dans une hotte à flux laminaire, tirer plusieurs traces de rayures avec un abonné de diamant sur la surface. Les marques aident à garder la trace des régions traitées et également mettre au point des microscopes. Stériliser le TiO 2 revêtu OTS en plongeant l&#39;échantillon dans de l&#39;éthanol 70% pendant 5 min. Puis rincer l&#39;échantillon deux fois dans stérilisée ddH 2 O. </li><li> Placer l&#39;échantillon dans un plat de 35 mm, et ajouter 2 ml de milieu de croissance de PC12 (voir l&#39;étape 4.2). Incuber pendant plus de 3 heures dans un incubateur à CO 2 (37 ° C). Cette procédure est destinée à laisser sérumalbumines absorbent sur la surface. Albumines adsorbées inhibent l&#39;adsorption ultérieure d&#39;autres proteins et des cellules (Figure 1: l&#39;étape c). </li><li> En attendant, mettre en place le microscope inversé à fluorescence. <ol><li> Allumez la lampe à arc, insérez le cube de filtre UV, et définir l&#39;objectif de 20X. </li><li> Mesurer l&#39;intensité lumineuse I (W cm -2) au moyen d&#39;un compteur d&#39;intensité UV, et de calculer la durée d&#39;irradiation t (en secondes) pour une dose de d (en cm -2 J) en tant que: t = d / I. Par exemple, pour irradier à une dose de 200 J cm -2 utilisant une source lumineuse de 600 mW cm -2, 333 secondes d&#39;irradiation est nécessaire. </li><li> Utilisez un micromètre et fermer le diaphragme de champ pour définir la taille de la région à irradier, par exemple, 200 um. </li></ol></li><li> Après l&#39;incubation de trois heures, de compléter le milieu avec 200 ul de 3,0 mg ml-1 de type IV collagène (Col-IV; concentration finale de 300 ug ml-1). </li><li> Transférer le plat de 35 mm de la platine du microscope. Trouver le scratch marche, focaliser le microscope sur la surface de l&#39;échantillon, et irradier la lumière UV à une dose de 200 J cm -2 (Figure 1: Etape D). La zone d&#39;irradiation UV peut être modifiée, soit en ajustant l&#39;ouverture du diaphragme de champ ou par le remplacement du diaphragme de champ avec un masque métallique de géométrie d&#39;arbitrage. </li><li> Remplacez le milieu avec un milieu de croissance frais (sans Col-IV), et placer l&#39;échantillon dans l&#39;incubateur. </li></ol> 4. Culture Cellulaire <ol><li> Culture de routine de cellules PC12 est effectuée sur une boîte en matière plastique revêtue de collagène. <ol><li> Pour revêtir un plat de culture de tissu de 60 mm avec Col-IV, mouiller d&#39;abord la surface avec le trou DDH 2 O et aspirer tous ddH 2 O. </li><li> Préparer 300 ng ml -1 Col-IV en diluant la solution initiale (3 mg ml -1) 10x avec ddH 2 O. </li><li> Ajouter 200 pi de 300 pg ml -1 Col-IV par 60 mm plat. Laissez la solution se propager à travers la surface. </li><li> À sec til plat dans une hotte à flux laminaire pendant environ 1 heure. </li><li> Rincer la surface deux fois avec 4 ml de tampon phosphate salin de Dulbecco la (D-PBS). Lorsqu&#39;ils ne sont pas immédiatement d&#39;utiliser le plat enduit, rincez le plat une fois avec 4 ml de ddH 2 O. Après aspiration ddH 2 O, stocker le plat dans l&#39;incubateur. Essayez de ne pas le garder stocké pendant plus de deux semaines. </li></ol></li><li> Les cellules PC12 sont cultivées dans un milieu de croissance qui se compose de: moyen (faible taux de glucose) de Eagle modifié par Dulbecco + sérum solution à 1% de pénicilline-streptomycine + 10% de sérum de veau foetal 5% + cheval. Incuber les cellules dans un incubateur à CO 2 (37 ° C, 5% CO 2), et remplacer la moitié du milieu tous les deux jours. Passage cellules avant qu&#39;elles atteignent la confluence. <ol><li> Aspirer le milieu de croissance, et ajouter 2 ml de PBS + (D-PBS + 10 mg ml -1 d&#39;albumine de sérum bovin (BSA) + 10 mM d&#39;EDTA) préchauffé à 37 ° C. Incuber pendant 5 min à 37 ° C. Tapoter doucement le plat de détacher tous les CELLS. </li><li> Recueillir les cellules dans un tube conique de 15 ml. Rincer le plat avec 3 ml de frais D-PBS, préchauffée à 37 ° C. </li><li> Centrifuger le tube à 150 g pendant 4 min. </li><li> Aspirer le surnageant et ajouter 1 ml de milieu de croissance. </li><li> Pour la culture de routine, diviser les cellules 1: 3 à 1: 5. </li></ol></li><li> Pour étaler les cellules sur l&#39;échantillon modifié-UV OTS / TiO 2, comptez densité cellulaire et ajouter 3,0 × 10 5 cellules dans une boîte de 35 mm (Figure 1: Étape E). Incuber le plat au incubateur humidifié (37 ° C, 5% CO 2) pendant 1-2 jours. Les deux facteurs de croissance et naïf nerf (NGF) cellules PC12 -differentiated peuvent être utilisées pour les expériences de mise en forme. Pour la différenciation NGF, 100 ng ml -1 de 7S-NGF est ajouté à la croissance moyenne plusieurs jours avant l&#39;étalement des cellules sur l&#39;échantillon. NGF-différenciation semble augmenter le adhesibility des cellules PC12 et facilite la manipulation, en particulier dans le modelage in situ eXperiments. </li></ol> 5. In-Situ structuration de surface <ol><li> Après 1-2 jours de culture sur la surface modifiée ex-situ, confirment que les cellules attachent croissant et seulement sur ​​la région irradiée aux UV. Mettre en place le microscope, comme décrit dans l&#39;étape 3.4. </li><li> Transférer l&#39;échantillon dans une nouvelle boîte de culture tissulaire de 35 mm contenant du milieu de croissance, 100 ng ml-1 NGF (dans le cas de l&#39;utilisation de cellules différenciées NGF), et 100 ug ml-1 Col-IV. </li><li> Placez le plat sur la platine du microscope. Trouver la position appropriée, et irradier la lumière UV à une dose de 200 J cm -2 (Figure 1: Etape F, G). La région permissive nouvellement créé est indiqué par un astérisque dans la figure 1: Etape G. </li><li> Essayer de terminer le traitement d&#39;un seul échantillon dans les 30 min. Après irradiation, transférer l&#39;échantillon de nouveau dans le milieu sans Col-IV. </li><li> Soyez extrêmement prudent lors de la réalisation du plat avec cultured cellules et le transfert de l&#39;échantillon de plat à plat pour éviter le détachement des cellules motifs. </li></ol><img alt="Figure 1" src="/files/ftp_upload/53045/53045fig1highres.jpg" width="700" /> Figure 1. Représentation schématique de l&#39;ensemble du processus. Voir le texte pour plus de détails. <a href="https://www.jove.com/files/ftp_upload/53045/53045fig1large.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette figure.</a>

<ol>
	<li>Hughes, M. A., Brennan, P. M., Bunting, A. S., Shipston, M. J., Murray, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Patterning+on+Photolithographically+Defined+Parylene-C:+SiO2+Substrates.">Cell Patterning on Photolithographically Defined Parylene-C: SiO2 Substrates.</a> J. Vis. Exp. (85), e50929 (2014).</li><li>Kleinfeld, D., Kahler, K. H., Hockberger, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Outgrowth+of+Dissociated+Neurons+on+Patterned+Substrates.">Controlled Outgrowth of Dissociated Neurons on Patterned Substrates.</a> J. Neurosci. 8, 4098-4120 (1988).</li><li>Pensen, D., Heinz, W. F., Werbin, J. L., Hoh, J. H., Haviland, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+Beam+Patterning+of+Fibronectin+Nanodots+that+Support+Focal+Adhesion+Formation.">Electron Beam Patterning of Fibronectin Nanodots that Support Focal Adhesion Formation.</a> Soft Matter. 3, 1280-1284 (2007).</li><li>Tanii, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Organosilane+Monolayer+Template+to+Quantitative+Evaluation+of+Cancer+Cell+Adhesive+Ability.">Application of Organosilane Monolayer Template to Quantitative Evaluation of Cancer Cell Adhesive Ability.</a> Jpn. J. Appl. Phys. 50, 06GL01 (2011).</li><li>Yamamoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-Situ+Guidance+of+Individual+Neuronal+Processes+by+Wet+Femtosecond+Laser+Processing+of+Self-Assembled+Monolayers.">In-Situ Guidance of Individual Neuronal Processes by Wet Femtosecond Laser Processing of Self-Assembled Monolayers.</a> Appl. Phys. Lett. 99, 163701-1610 (2011).</li><li>Yamamoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Neurite+Outgrowth+is+Required+for+Axon+Specification+by+Cultured+Hippocampal+Neurons.">Differential Neurite Outgrowth is Required for Axon Specification by Cultured Hippocampal Neurons.</a> J. Neurochem. 123, 904-910 (2012).</li><li>Shen, K., Qi, J., Kam, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcontact+Printing+of+Proteins+for+Cell+Biology.">Microcontact Printing of Proteins for Cell Biology.</a> J. Vis. Exp. (22), e1065 (2008).</li><li>Johnson, D. M., LaFranzo, N. A., Maurer, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+Two-Dimensional+Patterned+Substrates+for+Protein+and+Cell+Confinement.">Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement.</a> J. Vis. Exp. (55), e3164 (2011).</li><li>Singhvi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+Cell+Shape+and+Function.">Engineering Cell Shape and Function.</a> Science. 264, 696-698 (1126).</li><li>Chen, C. S., Mrksich, M., Huang, S., Whitesides, G. M., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometric+Control+of+Cell+Life+and+Death.">Geometric Control of Cell Life and Death.</a> Science. 276, 1425-1428 (1997).</li><li>Degot, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Visualization+and+Quantitative+Analysis+of+Drug+Effects+using+Micropatterned+Cells.">Improved Visualization and Quantitative Analysis of Drug Effects using Micropatterned Cells.</a> J. Vis. Exp. (46), e2514 (2010).</li><li>Nakanishi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivation+of+a+Substrate+for+Cell+Adhesion+under+Standard+Fluorescence+Microscopes.">Photoactivation of a Substrate for Cell Adhesion under Standard Fluorescence Microscopes.</a> J. Am. Chem. Soc. 126, 16314-16315 (2004).</li><li>Kim, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addressable+Micropatterning+of+Multiple+Proteins+and+Cells+by+Microscope+Projection+Photolithography+Based+on+a+Protein+Friendly+Photoresist.">Addressable Micropatterning of Multiple Proteins and Cells by Microscope Projection Photolithography Based on a Protein Friendly Photoresist.</a> Langmuir. 26, 12112-12118 (2010).</li><li>Deka, G., Okano, K., Kao, F. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Photopatterning+of+Cells+In+Situ+by+Q-Switched+Neodymium-Doped+Yttrium+Ortho-Vanadate.">Dynamic Photopatterning of Cells In Situ by Q-Switched Neodymium-Doped Yttrium Ortho-Vanadate.</a> Laser. J. Biomed. Opt. 19, 011012 (2014).</li><li>Robertus, J., Browne, W. R., Feringa, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Control+over+Cell+Adhesive+Properties+using+Molecular-Based+Surface+Engineering+Strategies.">Dynamic Control over Cell Adhesive Properties using Molecular-Based Surface Engineering Strategies.</a> Chem. Soc. Rev. 39, 354-378 (2010).</li><li>Choi, I., Yeo, 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=Self-Assembled+Monolayers+with+Dynamicity+Stemming+from+(Bio)chemical+Conversions:+From+Construction+to+Application.">Self-Assembled Monolayers with Dynamicity Stemming from (Bio)chemical Conversions: From Construction to Application.</a> ChemPhysChem. 14, 55-69 (2013).</li><li>Nakanishi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switchable+Substrates+for+Analyzing+and+Engineering+Cellular+Functions.">Switchable Substrates for Analyzing and Engineering Cellular Functions.</a> Chem. Asian J. 9, 406-417 (2014).</li><li>Yamamoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Situ+Modification+of+Cell-Culture+Scaffolds+by+Photocatalytic+Decomposition+of+Organosilane+Monolayers.">In Situ Modification of Cell-Culture Scaffolds by Photocatalytic Decomposition of Organosilane Monolayers.</a> Biofabrication. 6, 035021 (2014).</li><li>Sekine, K., Yamamoto, H., Kono, S., Ikeda, T., Kuroda, A., Tanii, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+Modification+of+Cell+Scaffold+in+Aqueous+Solution+using+TiO2+Photocatalysis+and+Linker+Protein+L2+for+Patterning+Primary+Neurons.">Surface Modification of Cell Scaffold in Aqueous Solution using TiO2 Photocatalysis and Linker Protein L2 for Patterning Primary Neurons.</a> e-J. Surf. Sci. Nanotech. 13, 213-218 (2015).</li><li>Arima, Y., Iwata, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Surface+Functional+Groups+on+Protein+Adsorption+and+Subsequent+Cell+Adhesion+using+Self-Assembled+Monolayers.">Effects of Surface Functional Groups on Protein Adsorption and Subsequent Cell Adhesion using Self-Assembled Monolayers.</a> J. Mater. Chem. 17, 4079-4087 (2007).</li><li>Fujishima, A., Zhang, X., Tryk, 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=TiO2+Photocatalysis+and+Related+Surface+Phenomena.">TiO2 Photocatalysis and Related Surface Phenomena.</a> Surf. Sci. Rep. 63, 515-582 (2008).</li><li>Sigal, G. B., Mrksich, M., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Surface+Wettability+on+the+Adsorption+of+Proteins+and+Detergents.">Effect of Surface Wettability on the Adsorption of Proteins and Detergents.</a> J. Am. Chem. Soc. 120, 3464-3473 (1998).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Transparent+and+Photo-Patternable+Superhydrophobic+Film.">A Transparent and Photo-Patternable Superhydrophobic Film.</a> Chem. Commun. 2007, 4949-4951 (1039).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+Hippocampal+Neurons.">Culturing Hippocampal Neurons.</a> Nat. Protoc. 1, 2406-2415 (2006).</li></ol>

The authors have nothing to disclose.

Dans notre protocole actuel, TiO 2 film a été formé par RF-pulvérisation magnétron. Nous privilégions cette méthode de dépôt, car il nous permet de préparer de façon reproductible un film photocatalytique de TiO 2 avec une rugosité sous-nm. Bien que les processus de dépôt par pulvérisation sont familiers aux spécialistes des matériaux et des ingénieurs en électronique, il peut ne pas être tout à fait accessible aux biologistes. Dans ce cas, TiO 2 film spin-enduit ser...

Semiconductor processus de lithographie et de ses dérivés tels que la photolithographie - 1,2, la lithographie par faisceau d&#39;électrons 3-6 et microcontact impression 7-10 - sont devenus un outil créé en biologie cellulaire pour cultiver des cellules vivantes dans une position définie et la géométrie. Procédé de formation de motifs repose sur l&#39;utilisation de substrats micro-usinés, comprenant des micro-île de revêtement permissive de cellules dans un arrière-plan non permissive. Un tel substrat sert de matrice à motif cellules. Ces technologies nous ont fourni les nouvelles méthodes pour modifier des cellules et leur fonction à un niveau mono et multi-cellulaire, pour extraire les propriétés intrinsèques des cellules, et d&#39;augmenter le débit de base de cellules criblage de médicaments 11. Le degré de liberté dans un patron de cellules augmenterait considérablement si la géométrie du motif de modèle pourrait être modifié in situ, à savoir, tandis que les cellules sont cultivées sur des sta tête. Les procédés classiques de formation de motif ne peut pas être directement appliquées ici, étant donné qu&#39;ils traitent les échantillons dans une atmosphère ou sous vide. Par conséquent diverses nouvelles techniques de modification de surface ont été proposées, qui sont basées, par exemple, sur des composés photo-réactifs 12,13 ou ablation laser 5,14, juste pour en nommer quelques-uns. Les méthodes proposées ont été bien examiné par Robertus et al. 15, et plus récemment par Choi et al. 16 et 17 par Nakanishi. Ici, dans cet article, nous décrivons un nouveau protocole de modification in situ surface, qui tire parti de la dégradation photocatalytique de molécules organiques sur un dioxyde de titane (TiO 2) de surface 18,19. Dans ce procédé, un film de TiO 2 est insérée entre le substrat de verre et le film organique qui assure l&#39;interface des cellules, et le film organique est décomposé in situ en irradiant localement ultraviolet (UV)la lumière pour une région d&#39;intérêt (λ &lt;388 nm). Nous montrons que le nouveau protocole peut être utilisé pour créer micropatterns de protéines de la matrice extracellulaire et des cellules vivantes ex situ et in situ. TiO 2 est biocompatible, chimiquement stable, et optiquement transparents, caractéristiques de ce qui le rend sympathique à introduire dans des expériences de culture cellulaire. Ce protocole fournit une alternative de matériaux à base scientifique pour modifier les échafaudages de culture cellulaire en milieu de culture cellulaire.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass coverslip</td>
			<td>Warner Instruments</td>
			<td>CS-15R15</td>
			<td>15 mm, #1.5 thickness</td>
		</tr>
		<tr>
			<td>Diamond scriber</td>
			<td>Ogura Jewel Industry</td>
			<td>D-Point Pen</td>
			<td></td>
		</tr>
		<tr>
			<td>RF sputtering system</td>
			<td>ANELVA</td>
			<td>SPC350</td>
			<td></td>
		</tr>
		<tr>
			<td>TiO2 sputtering target</td>
			<td>Kojundo Chemical Lab</td>
			<td>Titanium (IV) oxide, target</td>
			<td>Purity, 99.9%</td>
		</tr>
		<tr>
			<td>Plasma reactor</td>
			<td>Yamato</td>
			<td>PR301</td>
			<td></td>
		</tr>
		<tr>
			<td>n-octadecyltrichlorosilane 
			(OTS)</td>
			<td>Aldrich</td>
			<td>104817</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Wako</td>
			<td>204-01866</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-culture dish (35 mm)</td>
			<td>Greiner</td>
			<td>627160</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-culture dish (60 mm)</td>
			<td>BD Falcon</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>Type-IV collagen</td>
			<td>Nitta Gelatin</td>
			<td>Cellmatrix Type IV</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium (DMEM)</td>
			<td>Gibco</td>
			<td>11885-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>12483-020</td>
			<td>Heat-inactivate and pass through a 0.22 mm filter before use</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Gibco</td>
			<td>26050-088</td>
			<td>Pass through a 0.22 mm filter before use</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (100x)</td>
			<td>Nacalai tesque</td>
			<td>26253-84</td>
			<td></td>
		</tr>
		<tr>
			<td>7S nerve growth factor (NGF)</td>
			<td>Alomone Labs</td>
			<td>N-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Dojindo</td>
			<td>N001</td>
			<td>Stock solution in 0.5 M</td>
		</tr>
		<tr>
			<td>TiO2 nanoparticle</td>
			<td>Tayca</td>
			<td>TKD-701</td>
			<td></td>
		</tr>
	</tbody>
</table>

photopatterning proteins and cells in aqueous environment using tio2 photocatalysis

Les contaminants organiques adsorbés à la surface du dioxyde de titane (TiO 2) peuvent être décomposés par photocatalyse sous rayonnement ultraviolet (UV). Nous décrivons ici un nouveau protocole employant la photocatalyse du TiO 2 à modifier localement affinité des cellules de la surface du substrat. Pour cette expérience, un film mince de TiO 2 est revêtu par pulvérisation sur une lamelle de verre, et la surface de TiO 2 a ensuite été modifié par une monocouche organosilane dérivé de l&#39;octadécyltrichlorosilane (OTS), qui inhibe l&#39;adhérence cellulaire. L&#39;échantillon a été immergé dans un milieu de culture cellulaire, et concentre la lumière UV a été irradié à une région de forme octogonale. Quand un neuronales cellules PC12 lignée de cellules ont été étalées sur l&#39;échantillon, les cellules adhèrent seulement sur la zone irradiée aux UV. On montre en outre que cette modification de surface peut également être réalisée in situ, autrement dit, même lorsque les cellules sont en croissance sur le substrat. Modification appropriée de la surface nécessaire une matrice extracellulaire protein collagène d&#39;être présent dans le milieu au moment de l&#39;irradiation UV. La technique présentée ici peut éventuellement être utilisé dans plusieurs types de cellules pour la construction de structuration des systèmes de co-culture ou de manipuler arbitrairement cellules dans la culture.

La figure 2A montre une image au microscope électronique à balayage en coupe transversale (SEM) de la pellicule de TiO 2 déposée par pulvérisation cathodique. De l&#39;observation, l&#39;épaisseur du film a été estimée à environ 150 nm. Notable ici est la planéité de la couche de TiO 2 déposée. Une analyse plus poussée par microscopie à force atomique (AFM) a révélé que la rugosité racine carrée moyenne (RMS) de la surface était de 0,2 nm (figure</stro...

Watch this Scientific Journal Video about Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis at JoVE.com

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

Photopatterning protéines et des cellules dans une solution aqueuse Environnement Utilisation de TiO<sub&gt; 2</sub&gt; Photocatalyse

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe ...

photopatterning-proteins-cells-aqueous-environment-using-tio2

Research

JoVE Journal

Bioengineering

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

7.8K vues.  Tohoku University. We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

Vidéo: Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

La fabrication des électrochimique de l&#39;ADN des Biocapteurs pour la détection sans réactif d&#39;acides nucléiques, protéines et petites molécules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Recherche

Bio-ingénierie

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology 1. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes 2, 3. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice 4, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast 5. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes 6, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.
In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.

Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these protocols easily and to utilize negative stain EM as a routine assay, in addition to other biochemical assays, for evaluating their protein samples.

Visualisation protéines et de complexes macromoléculaires par EM négatif Tache: de la préparation de grille pour l&#39;acquisition d&#39;images

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural ...

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

Imagerie des cellules vivantes des cellules exprimant des protéines par fluorescence Migration-étiquetés dans une matrice tridimensionnelle

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native &beta;-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFD&alpha; and &beta;, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFD&alpha; and &beta; improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via &beta;-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

Une boîte à outils pour permettre la conversion d&#39;hydrocarbures en milieu aqueux

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

Time-lapse d&#39;imagerie par fluorescence de la croissance racinaire d&#39;Arabidopsis avec la manipulation rapide de l&#39;environnement root en utilisant le RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

La co-culture de cellules utilisant une solution aqueuse Modélisation des systèmes biphasiques

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Transplantation de progéniteurs myoangioblastes dérivés de cellules souches pluripotentes induites dans des modèles murins de régénération musculaire

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

Luminescence Resonance Energy Transfer pour étudier les changements conformationnels dans des protéines membranaires exprimés dans les cellules de mammifères

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In conventional lab-on-a-chip-platforms, solvents and samples are passed through defined microfluidic channels with complex flow control installations. The droplet actuation platform presented here is a promising alternative. With it, it is possible to move a liquid drop (microreactor) on a planar surface of a reaction platform (lab-in-a-drop). The actuation of microreactors on the hydrophobic surface of the platform is based on the use of magnetic forces acting on the outer shell of the liquid drops which is made of a thin layer of superhydrophobic magnetite particles. The hydrophobic surface of the platform is needed to avoid any contact between the liquid core and the surface to allow a smooth movement of the microreactor. On the platform, one or more microreactors with volumes of 10 &#181;L can be positioned and moved simultaneously. The platform itself consists of a 3 x 3 matrix of electrical double coils which accommodate either neodymium or iron cores. The magnetic field gradients are automatically controlled. By variation of the magnetic field gradients, the microreactors' magnetic hydrophobic shell can be manipulated automatically to move the microreactor or open the shell reversibly. Reactions of substrates and corresponding enzymes can be initiated by merging the microreactors or bringing them into contact with surface immobilized catalysts.

Lab-in-a-drop reaction systems allow the versatile implementation of complex reactions in a microfluidic scale. An automated actuation platform consisting of a 3 x 3 matrix of electromagnetic coils was developed and successfully used to merge two 10 &#181;L microreactors and thereby initiate an enzymatic reaction in the resulting liquid marbles.

Aqueux gouttelettes servis de microréacteurs enzymatiques et leur déclenchement électromagnétique

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Une méthode polyvalente de patterning Les protéines et les cellules

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...