Nous sommes profondément reconnaissants à Dr. Takeshi Ikeya et le personnel technique dans Toyo Gosei, Tokyo, Japon pour fournir des plaques de culture microélectrodes thermosensibles ainsi que des conseils scientifiques. Nous remercions également Mme Ogura Satomi, Mme Sae Suzuki, Mme Asuka Miyoshi et Mme Katsue Morii d&#39;assistance technique avec des expérimentations animales. Ce travail a été soutenu financièrement en partie par la JSPS KAKENHI Grant-in-Aid pour la recherche scientifique, le Centre du Programme d&#39;innovation (COI) et le programme d&#39;innovation de l&#39;Agence S Japan Science and Technology (JST), et de la JSPS Core- à-Core Programme, A. Réseaux de recherche avancée.

Toutes les études sur les animaux ont été menées avec l&#39;approbation du Comité soin et l&#39;utilisation des animaux de l&#39;Université de Tokyo, Tokyo, Japon. 1. Préparation des cellules <ol><li> Pour hépatocytes primaires, suivre le protocole pour l&#39;isolement des hépatocytes de rat par un processus de digestion de collagénase en deux étapes modifiée 13,14. <ol><li> Anesthésier Sprague Dawley (SD) des rats (mâles, âgés de 5 semaines) sous anesthésie par inhalation avec de l&#39;isoflurane. Placer un rat dans une chambre reliée à un appareil d&#39;anesthésie à l&#39;isoflurane pour fournir de la chambre. Sortez le rat après l&#39;endormissement, et le mettre sur la table d&#39;opération avec une ventilation en utilisant un masque. Contrôler l&#39;isoflurane écouler approximativement à 0,4 à 0,7 L / min en vérifiant les conditions du rat. Perfuser le foie de Sprague Dawley (SD) des rats (mâles, âgées de 5 semaines) à partir de la veine porte hépatique avec une solution spéciale composée de 8 g / L de chlorure de sodium (NaCl), 400 mg / L de chlorure de potassium (KCl), 78 mg / Lle dihydrogénophosphate de sodium dihydrate (NaH 2 PO 4 · 2H 2 O), 151 mg / l phosphate disodique dodécahydraté (Na 2 HPO 4 · 12H 2 O), 2,38 g / L d&#39;acide 2- [4- (2-hydroxyéthyl) -1 pipérazinyl] éthanesulfonique (HEPES), 190 mg / L d&#39;acide éthylèneglycol tétraacétique (EGTA), 350 mg / L de carbonate acide de sodium (NaHCO 3), et 900 mg / L de glucose. </li><li> Faire circuler une solution de collagénase à travers le foie. REMARQUE: La solution est composé de 500 mg / L collagénase, 9,8 g / saline tamponnée de L Hank, 2,38 g / ml d&#39;HEPES, 556 mg / ml de chlorure de calcium hydraté (CaCl 2 · H 2 O), 350 mg / L de NaHCO 3, et l&#39;inhibiteur de trypsine / L de 50 mg, avec le pH ajusté à 7,2. </li><li> Retirer soigneusement le foie, et hacher le doucement sur un plat en utilisant une lame de scalpel, ajouter milieu de Eagle modifié par Dulbecco (DMEM) supplémenté avec 10% de sérum bovin fœtal (FBS), et filtrer la suspension de cellules à travers un 100 ummaille de nylon. Pour retirer les débris supplémentaires, centrifuger la suspension cellulaire à 20 g pendant 1 min. A ce stade, les hépatocytes sont dans le surnageant. </li><li> Répétez l&#39;étape de centrifugation deux fois (un total de trois centrifugations). Enfin, centrifuger les hépatocytes à 50 g pendant 3 min pour les récupérer sous la forme d&#39;une pastille. </li><li> Re-suspendre les hépatocytes à une concentration de 4 x 10 5 cellules / ml dans un milieu de culture spécial composé de DMEM supplémenté avec 10% de FBS, 1% de Pen-Strep-Glut (PSQ), 1% de diméthylsulfoxyde (DMSO), 0,1 umol / L dexaméthasone, 0,5 pg / ml d&#39;insuline, 10 mmol / L nicotinamide, 0,2 mmol / L ascorbate phosphorylé (Asc-2P), et 10 ng / ml de facteur de croissance épidermique humain (hEGF) 15. Ce milieu spéciale est obligatoire pour la préservation de la fonction hépatique dans des conditions in vitro. </li></ol></li><li> Pour obtenir CSM de rats, euthanasier Sprague Dawley (SD) des rats (mâles, âgés de 5 semaines) par l&#39;administration excessive de isoflurane. Réséquer les fémurs et tibias, et de recueillir les moelle osseuse en insérant une aiguille 22 G dans le corps de l&#39;os de la nettoyer avec 10 ml de DMEM complété avec 10% de FBS. Recueillir les cellules par filtration à travers un tamis en nylon de 100 um. </li><li> Ensemencer les cellules sur 10 boîtes de culture cm en utilisant du DMEM contenant 10% de FBS et 1% de pénicilline / streptomycine. Pour les expériences de sphéroïde, utiliser MSC dans les 5 passages. </li></ol> 2. Préparation de sphéroïdes cellulaires 3D <ol><li> Obtenir dans le commerce des plaques de culture microélectrodes, sur lequel les zones adhésives cellulaires sont régulièrement disposés à 100 um de diamètre de façon bidimensionnelle, entouré par des zones non adhésives revêtues par la matrice de PEG. NOTE: Pour la transplantation de cellules, une couche supplémentaire avec le PIPAAm de polymère thermosensible est nécessaire pour permettre le détachement des cellules en refroidissant les plaques (voir étape 5.1). Les fluctuations de température peuvent induire des changements dans la chimie de ce polymère10.8. A 37 ° C, PIPAAm est légèrement hydrophobe, ce qui permet aux cellules d&#39;être cultivées dans des conditions normales. Une diminution de la température en dessous de 32 ° C entraîne une hydratation rapide du polymère, conduisant au détachement spontané des cellules. </li><li> Ensemencer les cellules souches mésenchymateuses ou des hépatocytes sur des plaques à 12 puits à une densité de microélectrodes de 4 x 10 5 cellules / puits les andincubate à 37 ° C dans une atmosphère humidifiée contenant 5% de CO 2. Les cellules seront accumuler sur les zones d&#39;adhérence microélectrodes et de former progressivement sphéroïdes rondes en 2 jours. Remarque: Pour la préparation de cellules de contrôle dans une culture monocouche, en utilisant des plaques à 12 puits normales et ensemencer les cellules à une densité identique, suivant une procédure similaire à celle décrite ci-dessus. </li></ol> 3. Préparation de Polyplex nanomicelles <ol><li> Synthétiser un copolymère séquence de PEG-pAsp (DET) (poly [N &#39;- [N - (2-aminoéthyl) -2-aminoéthyl] aspartamide]) (PEG Mw = 12,000, le degré de polymérisation (DP) du PASP (DET) Segment = 59), et un homopolymère qui est composé de seulement le segment cationique [PASP (DET)] (DP = 55), suivant les procédures décrites précédemment par les 11 auteurs, 16, 17. </li><li> Préparer une solution mixte du copolymère séquencé (DET) PEG-pAsp et PASP (DET) homopolymère dans un rapport molaire égal de groupes amino résiduels dans 10 mM de tampon HEPES (pH 7,3) en ajustant la concentration en polymère de 33,3 ug / ml et 19,1 ug / ml, respectivement. REMARQUE: Les concentrations de polymère décrits ci-dessus sont des valeurs représentatives pour la préparation nanomicelles avec un taux résiduel molaire des groupes acides totaux dans les deux polymères aux groupes phosphate dans le ADNp (N / P) de 10. Le rapport N / P peut varier selon le type de cellule et le but (pour plus de détails, voir Discussion). L&#39;utilisation combinée de ces deux polymères peut atteindre à la fois un blindage efficace de PEG et le fonctionnement du PASP (DET) afin d&#39;améliorer endosomiqueéchapper (pour plus de détails, voir discussion) 18. </li><li> Préparer la luciférase ADNp codant Gaussia, GL4 luciférase, ou érythropoïétine en clonant le segment exprimant les gènes respectifs dans le plasmide pCAG-GS (http://www.cdb.riken.jp/pcs/protocol/vector/map/m36.html ) pour obtenir une expression sous la CAG promoteur / amplificateur en utilisant un kit commercial selon le protocole du fabricant. Amplifier l&#39;ADNp dans une souche d&#39;Escherichia coli compétent et le purifier en utilisant un système de purification d&#39;ADN plasmide exempt d&#39;endotoxine. Déterminer la concentration d&#39;ADNp à une absorbance de 260 nm pour obtenir une solution / ml 150 ug dans du tampon HEPES 10 mM (pH 7,3). </li><li> Pour préparer les nanomicelles polyplexe, bien mélanger la solution d&#39;ADNp (150 ug / ml dans du tampon HEPES 10 mM) et la solution de prémélange des deux polymères dans un rapport de 2: 1 (en volume). </li></ol> 4. Gene transfection dans Sphéroïdes <ol><li> Incuber les cellules (hépatocytesou MSC) pendant 72 heures après l&#39;ensemencement sur des plaques microélectrodes pour permettre la formation de sphéroïdes matures. Pour la transfection de gènes, ajouter 100 ul de la solution de polyplex nanomicelle (contenant 10 pg d&#39;ADNp) dans chaque puits après avoir remplacé le milieu de culture avec 1 ml de milieu frais. Poursuivre l&#39;incubation avec la solution de nanomicelle pendant 24 heures. </li><li> Pour un contrôle en utilisant un réactif de transfection à base de lipides, mélanger les solutions ADNp avec le réactif à un rapport en poids de réactif / de l&#39;ADNp 3. Ajuster la dose finale d&#39;ADNp à être égale à la fois pour le réactif à base de lipides et les méthodes de nanomicelle. </li></ol> 5. Récupération et la transplantation de Sphéroïdes cellulaires <ol><li> Remplacez le milieu de culture avec 200 pi de PBS froide et placer les plaques sur de la glace. NOTE: En général, les sphéroïdes peuvent se détacher dans environ 15 min et peut être récupéré sous la forme d&#39;une suspension pour la transplantation. </li><li> Aspirer délicatement les cellules dans le 200 pisuspension en utilisant une seringue avec une aiguille 23 G ou 27 G pour injections in vivo. </li></ol> 6. Évaluation de l&#39;expression transgénique <ol><li> Pour l&#39;évaluation in vitro de l&#39;expression de la luciférase de Gaussia sécrétée dans le milieu de culture, de recueillir de 50 à 100 ul de milieu précisément 24 heures après le remplacement par du milieu frais. Estimer l&#39;expression de la luciférase en utilisant un système de dosage de la luciférase de Renilla commercial et un luminomètre selon le protocole du fabricant. NOTE: La luciférase Gaussia reste stable dans le milieu de culture pour plus d&#39;une semaine. Ainsi, pour évaluer l&#39;efficacité en temps réel de l&#39;expression du transgène, remplacer le milieu avec une douce avant de recueillir le milieu de l&#39;échantillon. Le calendrier de la changement de milieu peut être flexible. </li><li> Pour l&#39;évaluation de l&#39;expression du transgène dans des animaux hôtes in vivo après transplantation de cellules, anesthésier souris BALB / c nu (femelle; 7 semainesancienne) sous anesthésie par inhalation avec de l&#39;isoflurane. <ol><li> Placer une souris dans une chambre reliée à un appareil d&#39;anesthésie à l&#39;isoflurane pour fournir de la chambre. Sortez la souris après l&#39;endormissement, et le mettre sur la table d&#39;opération avec une ventilation en utilisant un masque. Contrôler l&#39;isoflurane écouler approximativement à 0,2 à 0,5 L / min en vérifiant les conditions de la souris. </li><li> Injecter 200 ul de la suspension cellulaire contenant des sphéroïdes transfectées avec les ADNp exprimant la luciférase GL4 (comme décrit en 4.1) dans le tissu sous-cutané de la région abdominale. </li><li> Immédiatement après l&#39;injection de D-luciférine (150 mg / kg; voie intraveineuse), mesurer l&#39;expression de la luciférase en utilisant le système d&#39;imagerie IVIS selon le protocole du fabricant. </li></ol></li><li> Pour évaluer les effets thérapeutiques de la transplantation de cellules, injecter 200 ul de la suspension cellulaire contenant des sphéroïdes transfectées avec les ADNp codant pour l&#39;érythropoïétine dans latissu sous-cutané de la région abdominale. <ol><li> Recueillir des échantillons de sang par des saignements sous-maxillaire pour obtenir environ 200 pi de sang 19. Mesurer le taux d&#39;hémoglobine et l&#39;hématocrite utilisant un échantillon de sang analyseur. REMARQUE: Le nombre de cellules pour la transplantation est réglementée par le nombre ensemencées sur les plaques. Malheureusement, il est difficile de déterminer le nombre de cellules exacte parce que le nombre à l&#39;intérieur sphéroïdes ne peut être mesurée. </li></ol></li><li> Après des expériences, placez la souris sur un coussin chauffant relié à un contrôleur de température jusqu&#39;à ce que l&#39;éveil de l&#39;anesthésie. </li></ol>

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The authors declare that they have no competing financial interests.

Dans ce protocole, il est essentiel de maintenir la structure 3D de sphéroïdes lors des étapes d&#39;introduction de gène et la récupération de sphéroïde. Il est essentiel de maintenir un micro-environnements favorables pour les cellules afin d&#39;éviter la mort cellulaire ou la perte de l&#39;activité cellulaire. Par exemple, la sécrétion d&#39;albumine, une fonction innée représentant des hépatocytes, est bien conservés dans les sphéroïdes d&#39;hépatocytes, tandis que les hépatocytes dans la cult...

La thérapie cellulaire de la transplantation a attiré l&#39;attention généralisée pour le traitement de diverses maladies incurables. L&#39;activité et la demi-vie de facteurs bioactifs qui sont sécrétées par les cellules transplantées sont essentiels pour améliorer l&#39;efficacité thérapeutique d&#39;un système de greffe de cellules. La modification génétique des cellules avant transplantation est une technique utile pour réguler et manipuler les fonctions cellulaires, y compris la sécrétion de facteurs bioactifs. Il est également important de maintenir un micro-environnement favorable pour les cellules pour éviter la mort cellulaire ou la perte de l&#39;activité cellulaire. Three-dimensional culture (3D) de cellules de sphéroïde, dans laquelle les interactions cellule-cellule sont bien conservés, est prometteuse à cet effet, par exemple, pour améliorer la sécrétion d&#39;albumine à partir des hépatocytes primaires et favoriser la différenciation de lignées multiples à partir de cellules souches mésenchymateuses (MSC ) 1-7. Dans cette étude, un nouveau système de combinaison de spheroid culture et transfection génique est utilisée pour servir de plate-forme pour une transplantation de cellules génétiquement modifiées. Pour la création de cellules sphéroïdes, un système de culture sphéroïde sur des plaques de culture microélectrodes est utilisé. Sur ces plaques, les zones d&#39;adhésion cellulaire de 100 um de diamètre sont régulièrement disposées de manière bidimensionnelle et sont entourées par des zones non adhésives revêtues par une matrice 3 de PEG. Par ensemencement d&#39;un nombre suffisant de cellules, des matrices de sphéroïdes 3D de 100 um de diamètre correspondant sont formés dans le lit de culture microélectrodes. Les sphéroïdes sont récupérés sans perturber leur structure 3D en utilisant thermosensibles plaques de culture cellulaire, qui a été revêtu d&#39;un polymère thermosensible, poly (iso-propylacrylamide) (PIPAAm) 10/08. L&#39;architecture microélectrodes est construit sur les plaques thermosensibles (construit sur mesure). En abaissant simplement la température des plaques, les sphéroïdes sont détachés du lit de culture et se dispersentd en solution saline tamponnée au phosphate (PBS). Ainsi, un grand nombre de sphéroïdes d&#39;une taille uniforme de 100 um peut être obtenue sous la forme d&#39;une suspension injectable. <img alt="Figure 1" p="" src="/files/ftp_upload/52384/52384fig1.jpg" /> Figure 1. Représentation schématique du système de culture sur une plaque sphéroïde microélectrodes. La modification génétique est obtenue par transfection des gènes en utilisant le support de gène non viral initial, nanomicelle polyplex. Il est composé d&#39;ADN plasmidique (ADNp) et de polyéthylène glycol (PEG) des copolymères séquencés de 11 -polycation. Celles-ci ont une structure noyau-enveloppe caractéristique qui consiste en une coquille PEG et un noyau interne de pDNA condensée, permettant l&#39;introduction du gène sûr et efficace dans les cellules à des fins thérapeutiques 11. <a href="https://www.jove.com/files/ftp_upload/52384/52384fig1large.jpg" target="_blank">S&#39;il vous plaît, cliquez ici pour voir une version plus grande de thi</a>s figure. <img alt="Figure 2" src="/files/ftp_upload/52384/52384fig2.jpg" /> Figure 2. Structure de la nanomicelle polyplexe formé par le complexe d&#39;acides nucléiques et des copolymères à blocs PEG-bloc-polycation. Dans cette étude, le principal avantage de cette technique est que la structure sphéroïde ne soit pas perturbé par transfection génique pendant les nanomicelles. Après les transfections à médiation par des nanomicelle de sphéroïdes d&#39;hépatocytes de rat primaires, l&#39;expression du transgène est obtenu prolongée pendant plus d&#39;un mois avec continu sécrétion d&#39;albumine à partir des hépatocytes à un niveau comparable à celui de sphéroïdes non transfectées 12. L&#39;expression du transgène et de sécrétion d&#39;albumine dans les sphéroïdes sont également maintenus après la récupération à partir des plaques thermosensibles. Il est évident que nanomicelles peuvent faciliter en toute sécurité l&#39;introduction du gène sans altérer les fonctions innées de l&#39;hépatiteatocytes. Ainsi, la combinaison de cellules cultivées sur des plaques sphéroïde micropatterned thermosensibles avec l&#39;introduction du gène en utilisant nanomicelles est une plate-forme prometteuse pour la transplantation de cellules génétiquement modifiées. <a href="https://www.jove.com/files/ftp_upload/52384/52384fig2large.jpg" target="_blank">S&#39;il vous plaît, cliquez ici pour voir une version plus grande de cette figure.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pen-Strep-Glut</td>
			<td>GIBCO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Wako Pure Chemical Industries</td>
			<td>041-18861</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Wako Pure Chemical Industries</td>
			<td>141-01202</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s buffered salt and L-ascorbic acid 2-phosphate (Asc-2P)</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>Human epidermal growth factor (hEGF)</td>
			<td>Toyobo</td>
			<td>PT10015</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-able multi-well plates</td>
			<td>Toyo Gosei</td>
			<td>PP-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermosensitive cell culture plates (Upcell)</td>
			<td>CellSeed Inc</td>
			<td></td>
			<td>The micropatterned architecture is constructed on the thermosensitive plates (custom-built by Toyo Gosei)</td>
		</tr>
		<tr>
			<td>Lipid-based transfection reagent (FuGENE HD)</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Renilla Luciferase Assay System</td>
			<td>Promega</td>
			<td>E2810</td>
			<td></td>
		</tr>
		<tr>
			<td>pGL4 Luciferase Reporter Vector</td>
			<td>Promega</td>
			<td>E6651</td>
			<td></td>
		</tr>
		<tr>
			<td>pDNA expressing Gaussia luciferase</td>
			<td>New England BioLabs</td>
			<td>N8082S</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse erhthropoietin-expressing vector</td>
			<td>Origene</td>
			<td>MC208445</td>
			<td></td>
		</tr>
		<tr>
			<td>pCAG-GS</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Laboratory for Pluripotent Cell Studies, Center for Developmental Biology, RIKEN</td>
		</tr>
		<tr>
			<td>Escherichia coli DH5&#945; competent cells</td>
			<td>Takara</td>
			<td>9057</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotoxin-free plasmid DNA purification system</td>
			<td>Nippon Genetics</td>
			<td>NucleoBond Xtra EF</td>
			<td></td>
		</tr>
		<tr>
			<td>collagenase</td>
			<td>Wako Pure Chemical Industries</td>
			<td>639-00951</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin inhibitor</td>
			<td>GIBCO</td>
			<td>R-007-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Promega</td>
			<td>GloMax&#8482; 96 Microplate Luminometer</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Imaging System</td>
			<td>Xenogen Corp.</td>
			<td>Xenogen IVIS Spectrum in vivo imaging system</td>
			<td></td>
		</tr>
		<tr>
			<td>blood sample analyzer</td>
			<td>Sysmex</td>
			<td>pocH-100i Automated Hematology Analyzer</td>
			<td></td>
		</tr>
	</tbody>
</table>

gene transfection toward spheroid cells on micropatterned culture plates for genetically-modified cell transplantation

Pour améliorer l&#39;efficacité thérapeutique de la transplantation de cellules, un système de transplantation, de sphéroïdes injectables génétiquement modifiés a été développé. Les sphéroïdes de cellules sont préparés dans un système de culture sur des plaques de microélectrodes recouvertes d&#39;un polymère thermosensible. Un certain nombre de sphéroïdes sont formées sur les plaques, correspondant aux zones d&#39;adhésion cellulaire de 100 um de diamètre, régulièrement disposés de manière bidimensionnelle, entourées par des zones non adhésives qui sont recouvertes par un polyéthylène glycol de matrice (PEG). Les sphéroïdes peuvent facilement être récupérés sous forme d&#39;une suspension liquide en abaissant la température des plaques, et leur structure est bien maintenu en les faisant passer à travers des aiguilles d&#39;injection avec un calibre suffisamment grand (plus de 27 G). La modification génétique est obtenue par transfection des gènes en utilisant le support d&#39;origine non virale gène, nanomicelle polyplexe, qui est capable d&#39;introduire des gènes dans des cellules sans perturber la structure du sphéroïde. Pour primsphéroïdes d&#39;hépatocytes aires transfectées avec un gène de la luciférase exprimant la luciférase est durablement obtenus chez les animaux transplantés, ainsi que la fonction des hépatocytes conservés, comme indiqué par l&#39;expression de l&#39;albumine. Ce système peut être appliqué à une variété de types de cellules comprenant des cellules souches mésenchymateuses.

Gene transfection de la luciférase de Gaussia exprimant ADNp a été effectuée dans les sphéroïdes formés par les hépatocytes ou les cellules souches mésenchymateuses à l&#39;aide nanomicelles Polyplex ou le réactif de transfection à base de lipide de commande 12. Les nanomicelles induites pratiquement aucun changement dans la structure de sphéroïde par rapport à sphéroïdes non transfectées microélectrodes sur les plaques, tandis que le réactif de contrôle perturbé de manière significativ...

Watch this Scientific Journal Video about Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation at JoVE.com

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

Transfection génique vers les cellules Spheroid sur microélectrodes plaques de culture destinés à la transplantation de cellules génétiquement modifiées

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

gene-transfection-toward-spheroid-cells-on-micropatterned-culture

Research

JoVE Journal

Bioengineering

8.7K vues.  The University of Tokyo. This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

Vidéo: Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Surfaces Micropatterned pour étudier les interactions acide hyaluronique avec les cellules cancéreuses

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Recherche

Bio-ingénierie

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

Amélioration de la visualisation et l&#39;analyse quantitative des effets des drogues utilisant des cellules Micropatterned

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Fabrication d&#39;hydrogels pour les systèmes de culture Micropatterned neuronaux utilisant la photolithographie dynamique projection de masque

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

Rotation systèmes de culture cellulaire de la culture de cellules humaines: les cellules du trophoblaste humain comme modèle

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

Cytométrie de masse: Protocole pour Tuning quotidien et échantillons cellulaires course sur un cytomètre de masse CyTOF

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

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 ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

Stretching cellules Micropatterned sur une membrane PDMS

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

Microfluidique génipine dépôt Technique de la culture avancée de microélectrodes vasculaires musculaires Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

Segmentation de la croissance des cellules endothéliales dans des plaques à 6 puits sur un agitateur orbital pour les études mécanobiologiques

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Une méthode de culture pour maintenir les cellules souches hématopoïétiques humaines quiescentes

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...