Les auteurs tiennent à remercier les sources de financement suivantes: Instituts de recherche en santé du Canada (IRSC), les Sciences naturelles et du Conseil de recherches en génie (CRSNG).

Une. Programme de développement des LabView <ol><li> Un régulateur de débit massique (MKS M100B Mass-Flo Controller) est fixée à la conduite de gaz d&#39;hélium à assurer un débit de gaz précis. Cette unité est commandée par une tension d&#39;entrée, compris entre 0 et 5 V. Une boucle de commande dans le programme de LabView détermine la tension requise à un moment donné. L&#39;interfaçage entre l&#39;ordinateur et le contrôleur de débit massique est exécutée par un dispositif d&#39;acquisition de données (NI USB-6009), et une alimentation électrique de 12 V alimente le régulateur de débit massique. </li><li> Deux coulisseaux de positionnement linéaires sont utilisés pour contrôler le mouvement de la plaque à 6 puits, une pour chaque direction. Chaque ensemble se compose d&#39;un MA15-série Velmex Assemblée Unislide couplé avec un moteur pas à pas, et les deux assemblées sont contrôlées par un seul Velmex VXM Stepping Motor Controller. Le dispositif de commande permet de pulser manuel de chaque moteur ou accepte une chaîne de commandes série externes, dont la combinaison permet de mouvement p spécifiqueConstantes. Plusieurs modèles peuvent être programmées dans le programme de LabView, obligeant l&#39;utilisateur à ne fournir que la vitesse de la flamme désirée, ainsi que les dimensions de trajectoire. </li></ol> 2. Préparation de l&#39;équipement <ol><li> Ouvrez la fois la bouteille d&#39;hélium et le régulateur. </li><li> Mettre en marche le dispositif de commande du moteur. </li><li> Ouvrez le programme LabView et vérifier toutes les conditions. </li><li> Assurez-vous que la plate-forme est centrée. Pour vérifier, regardez à chaque étape de moteur et vérifier que chaque chariot n&#39;est pas près de chaque extrémité de la scène. Soit que le centre du programme, les voitures de régler manuellement ou en utilisant les contrôles de jogging sur le devant de la commande de moteur. </li><li> Si c&#39;est la première course de la journée, il est recommandé d&#39;exécuter le programme une fois sans cellules, pour assurer l&#39;installation fonctionne correctement. </li></ol> 3. Préparation de la hauteur capillaire Jet Nozzle Gas <ol><li> Retirez le capillaire du support et zéro la molette de hauteur. </li><li> Placez le 6 puits plaque on la plate-forme. </li><li> Remplacer et abaisser le capillaire du jet de gaz au fond du puits, jusqu&#39;à ce qu&#39;il frappe la lamelle. Fixer le capillaire dans le support. </li><li> Utilisation de la molette de hauteur, augmenter le capillaire 3 mm. Marquer cette hauteur sur la base capillaire. </li><li> Soulevez le capillaire à une hauteur sécuritaire, puis retirez la plaque de 6 puits. </li></ol> 4. Cellule Protocole perméabilisation <ol><li> Culture lignée de cellules adhérentes jusqu&#39;à confluence de la couverture de verre glisse dans une plaque à 6 puits en utilisant des techniques de culture standard. Pour les cellules HeLa, semences plaques de 6 puits avec 2 x 10 5 cellules par puits et incuber pendant 48 heures avant le traitement. </li><li> Préparer une quantité appropriée de la solution contenant le vecteur désiré. Pour hrGFPII-1, une concentration de 50 ng / ul dans du PBS 1x fournit un fort signal de fluorescence dans les cellules HeLa 24 heures après la transfection. Pour les études de dextrane, de 80 pg / ml de 10 kDa dextran fluorescent vert est recommandée. Désormais, cette solution sera soumise à uns &quot;de la solution de vecteur&quot;. </li><li> Retirer milieux de culture cellulaire de bien rincer et trois fois avec PBS 1x. </li><li> Pipeter 1370 pi de solution de vecteur préparés à l&#39;étape 4.2 dans le puits. Il devrait en résulter une profondeur de liquide d&#39;environ 1,3 mm. </li><li> Placer le puits contenant la solution de vecteur au centre de la plate-forme. Abaisser la buse d&#39;éjection de gaz au niveau préalablement marquée indiquant une hauteur de buse de 3 mm au-dessus du coulisseau. </li><li> Réglez le débit d&#39;hélium dans le programme LabView et sélectionnez le modèle de mouvement désiré à être utilisé. Sélectionnez &quot;Exécuter&quot; lorsque vous êtes prêt à commencer le protocole de perméabilisation. Il y aura une période initiale de latence au cours de laquelle le contrôleur de débit massique se prépare à fournir le débit nécessaire. Une fois que le débit d&#39;écoulement approprié a été atteint, le programme de LabView va activer les moteurs pas à pas pour déplacer le puits dans le modèle de perméabilisation souhaitée. Une fois la plate-forme s&#39;est arrêté, attendre environ 30 secondes et retirer la solution de vecteur.Remarque: perméabilisation optimale se produit à proximité d&#39;une pression dynamique de 100 à 200 Pa à la sortie de la buse, en fonction du diamètre du capillaire. </li><li> Lavez bien trois fois avec PBS 1x. </li><li> Remplissez bien avec milieu de culture frais. </li><li> Répétez les étapes 4.3 à 4.6 pour le nombre désiré d&#39;expériences. Remarque: Assurez-vous d&#39;inclure des échantillons de contrôle qui consistent en des puits étant remplis avec la solution de vecteur sans le jet de gaz est de fonctionner sur eux. Laissez la solution dans les puits de contrôle pendant environ 1 min, puis suivez les étapes 4.5 et 4.6. </li><li> Si l&#39;exécution d&#39;une expérience de transfection hrGFP, retourner la plaque de 6 puits à l&#39;incubateur pendant 24 heures pour permettre au matériau transfectées pour être transcrit par les cellules. Si l&#39;exécution d&#39;une expérience de perméabilisation dextran, retourner la plaque de 6 puits à l&#39;incubateur pendant 15 min. </li></ol> 5. Imagerie cellulaire <ol><li> Si vous le souhaitez, immédiatement avant l&#39;imagerie, de contraste avec tache direct / morts approprié pour évaluer la mort cellulaire. </li><li> cellules d&#39;image avec appropmicroscope riés d&#39;enquêter transfection / perméabilisation efficacité. </li></ol>

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	<li>Rosenberg, S. A., 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=Gene+transfer+into+humans--immunotherapy+of+patients+with+advanced+melanoma,+using+tumor-infiltrating+lymphocytes+modified+by+retroviral+gene+transduction.">Gene transfer into humans--immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction.</a> The New England Journal of Medicine. 323, 570-578 (1990).</li><li>Yang, Y., 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=Cellular+immunity+to+viral+antigens+limits+E1-deleted+adenoviruses+for+gene+therapy.">Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy.</a> Proceedings of the National Academy of Sciences of the United States of America. 91, 4407-4411 (1994).</li><li>Chen, C., Okayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transformation+of+mammalian+cells+by+plasmid+DNA.">High-efficiency transformation of mammalian cells by plasmid DNA.</a> Molecular and cellular biology. 7, 2745-2752 (1987).</li><li>Walev, I., 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=Delivery+of+proteins+into+living+cells+by+reversible+membrane+permeabilization+with+streptolysin-O.">Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O.</a> Proceedings of the National Academy of Sciences of the United States of America. 98, 3185-3190 (2001).</li><li>Felgner, P. L., 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=Lipofection:+a+highly+efficient,+lipid-mediated+DNA-transfection+procedure.">Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.</a> Proceedings of the National Academy of Sciences of the United States of America. 84, 7413-7417 (1987).</li><li>Hamm, A., Krott, N., Breibach, I., Blindt, R., Bosserhoff, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transfection+method+for+primary+cells.">Efficient transfection method for primary cells.</a> Tissue engineering. 8, 235-245 (2002).</li><li>Rauth, S., Kucherlapati, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+DNA+Transferred+into+Mammalian-Cells.">Expression of DNA Transferred into Mammalian-Cells.</a> J. Bioscience. 6, 543-567 (1984).</li><li>Spandidos, 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=Electric+field-mediated+gene+transfer+(electroporation)+into+mouse+Friend+and+human+K562+erythroleukemic+cells.">Electric field-mediated gene transfer (electroporation) into mouse Friend and human K562 erythroleukemic cells.</a> Gene analysis techniques. 4, 50-56 (1987).</li><li>Chu, G., Hayakawa, H., Berg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+for+the+efficient+transfection+of+mammalian+cells+with+DNA.">Electroporation for the efficient transfection of mammalian cells with DNA.</a> Nucleic Acids Research. 15, 1311-1326 (1987).</li><li>Fechheimer, 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=Transfection+of+mammalian+cells+with+plasmid+DNA+by+scrape+loading+and+sonication+loading.">Transfection of mammalian cells with plasmid DNA by scrape loading and sonication loading.</a> Proceedings of the National Academy of Sciences of the United States of America. 84, 8463-8467 (1987).</li><li>Koch, S., Pohl, P., Cobet, U., Rainov, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+enhancement+of+liposome-mediated+cell+transfection+is+caused+by+cavitation+effects.">Ultrasound enhancement of liposome-mediated cell transfection is caused by cavitation effects.</a> Ultrasound in Medicine &amp; Biology. 26, 897-903 (2000).</li><li>Greenleaf, W. J., Bolander, M. E., Sarkar, G., Goldring, M. B., Greenleaf, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+cavitation+nuclei+significantly+enhance+acoustically+induced+cell+transfection.">Artificial cavitation nuclei significantly enhance acoustically induced cell transfection.</a> Ultrasound in Medicine &amp; Biology. 24, 587-595 (1998).</li><li>Yang, N. S., Burkholder, J., Roberts, B., Martinell, B., McCabe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+and+in+vitro+gene+transfer+to+mammalian+somatic+cells+by+particle+bombardment.">In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment.</a> Proceedings of the National Academy of Sciences of the United States of America. 87, 9568-9572 (1990).</li><li>Hallow, D. 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=Shear-induced+intracellular+loading+of+cells+with+molecules+by+controlled+microfluidics.">Shear-induced intracellular loading of cells with molecules by controlled microfluidics.</a> Biotechnology and Bioengineering. 99, 846-854 (2008).</li><li>Chouinard-Pelletier, G. L., Guay, M., Coulombe, D., Leask, S., L, R., Jones, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+inert+gas+jets+to+measure+the+forces+required+for+mechanical+gene+transfection.">Use of inert gas jets to measure the forces required for mechanical gene transfection.</a> Biomedical Engineering Online. 11, (2012).</li><li>Leduc, M., Coulombe, S., Leask, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atmospheric+Pressure+Plasma+Jet+Deposition+of+Patterned+Polymer+Films+for+Cell+Culture+Applications.">Atmospheric Pressure Plasma Jet Deposition of Patterned Polymer Films for Cell Culture Applications.</a> Ieee T. Plasma Sci. 37, 927-933 (2009).</li><li>Lu, 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=Microfluidic+shear+devices+for+quantitative+analysis+of+cell+adhesion.">Microfluidic shear devices for quantitative analysis of cell adhesion.</a> Analytical Chemistry. 76, 5257-5264 (2004).</li><li>Sankin, G. N., Yuan, F., Zhong, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsating+tandem+microbubble+for+localized+and+directional+single-cell+membrane+poration.">Pulsating tandem microbubble for localized and directional single-cell membrane poration.</a> Physical Review Letters. 105, 078101 (2010).</li></ol>

Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

Jet de gaz inerte perméabilisation est une technique utile pour la transfection de cellules adhérentes. Il offre la possibilité de transférer des biomolécules dans des cellules à l&#39;aide de forces mécaniques, ce qui élimine le besoin pour des produits chimiques potentiellement nocives ou des vecteurs viraux. La technique pourrait permettre aux chercheurs et aux cliniciens un moyen efficace et relativement simple pour la transfection des cellules précisément. La sélectivité de la perméabilisation est éga...

Avec l&#39;évolution de la biomédecine et la compréhension de la mécanique des cellules, la livraison de biomolécules dans les cellules est devenu vital pour de nombreux domaines de recherche et les traitements médicaux. Différentes techniques ont été mises au point pour introduire les molécules étrangères à travers la membrane plasmique et dans le cytosol de la cellule. Ceux-ci peuvent généralement être classés comme étant soit: techniques virales, chimiques ou mécaniques. Techniques virales peuvent transfecter matériel génétique tel que l&#39;ARN et de l&#39;ADN en utilisant des vecteurs viraux 1. Techniques virales ont tendance à avoir des rendements élevés dans certaines lignées cellulaires, mais ils ont le potentiel pour les réactions immunitaires et inflammatoires 2. Procédés de transfection chimique communs comprennent la précipitation au phosphate de calcium 3, les exotoxines bactériennes 4 et 5 lipofection. Ces techniques se sont révélées être efficaces, cependant, certains problèmes se posent tels que la toxicité cellulaire et la non-spécificité. En outre, des techniques telles que phosphat de calciume précipitations ont été trouvés pour avoir des efficacités de transfection allant jusqu&#39;à 70%, mais seulement dans certaines lignées cellulaires, rarement dans des cellules primaires 6. C&#39;est la note que l&#39;augmentation des efforts de recherche sont mis dans des cellules primaires, en particulier dans le cas de la conception de divers traitements pour une utilisation clinique et l&#39;étude du fonctionnement de l&#39;ADN. Perturbation temporelle de la membrane cellulaire par l&#39;intermédiaire des stimuli mécaniques est une alternative pour introduire des molécules dans des cellules étrangères. Les techniques comprennent: une micro-injection des molécules 7, électroporation en utilisant un champ électrique pour rompre la membrane de 8,9, sonoporation utilisant des ondes ultrasonores pour perturber la membrane cellulaire de 10 à 12, le bombardement de particules tel que démontré par le «canon à gènes», où l&#39;on tire des particules liées avec gènes dans la cellule 13, et, plus récemment, l&#39;application d&#39;une contrainte de cisaillement de fluide qui a été montré pour perméabiliser des cellules de mammifères 14,15 temporellement. Bien que mécanicienméthodes al éviter certains des problèmes mentionnés ci-dessus, ils sont généralement accompagnés par une efficacité moindre et des configurations très complexes et spécialisés. Une torche à décharge luminescente à la pression atmosphérique (APGD-t) a été développé à l&#39;Université McGill dans le but initial de la fonctionnalisation de surfaces et de détacher les cellules adhérentes in vitro 16. Par un heureux hasard, il a été découvert qu&#39;une tache vivants / morts a été utilisé en quelque sorte d&#39;être pris en charge par les cellules sans agent de perméabilisation est ajouté. En outre, cela semblait n&#39;avoir eu lieu que dans les zones réglementées des puits qui ont eu lieu à correspondre avec le chemin de la torche. Enquête sur les capacités de perméabilisation de la APGD-t continué et dans les études de contrôle, il a été constaté que le support inerte jet de plasma sans excitation de gaz a également été en mesure de perméabiliser les cellules. Cela contredit l&#39;hypothèse initiale selon laquelle les espèces réactives créées par le jet de plasma perturbés temporairement la fonction de la membrane et a suggéré que simplement les mécaques forces étaient suffisantes pour provoquer la perméabilisation de la cellule. De là, les études poursuivies dans notre laboratoire pour tenter de quantifier et de caractériser l&#39;efficacité de perméabilisation d&#39;un jet de gaz inerte ainsi que regarder ses capacités de transfection 16. Grâce à ces études, il a été trouvé que des micropores font sous forme de fait dans la membrane plasmique, et ces pores ont tendance à refermer au sein d&#39;environ 5 sec 15. Nous avons démontré l&#39;utilité de la technique in vitro et in vivo dans la membrane chorioallantoïque d&#39;un embryon de poulet.

<table border="1">
	<tbody>
		<tr>
			<td>Vitality hrGFPII-1</td>
			<td>Agilent Technologies Canada</td>
			<td>240143-57</td>
			<td>Green fluorescent protein for transfection experiments on HeLa cells</td>
		</tr>
		<tr>
			<td>Dextran, AlexaFluor 488; 10,000 MW, Anionic, Fixable</td>
			<td>Life Technologies Inc.</td>
			<td>D22910</td>
			<td>dextran for permeabilization experiments</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Viability/Cytotoxicity Kit</td>
			<td>Life Technologies Inc.</td>
			<td>L3224</td>
			<td>for counterstaining of mammalian cells</td>
		</tr>
		<tr>
			<td>Prolong Gold Antifade Reagent</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td>for mounting slides when imaging</td>
		</tr>
		<tr>
			<td>Ultra High Purity Helium</td>
			<td>Praxair Canada Inc.</td>
			<td>HE 5.0UH-T</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyclone DMEM/ High Glucose Media - 500 ml</td>
			<td>Thermo Scientific</td>
			<td>SH30022.01</td>
			<td>for HeLa cells</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Invitrogen</td>
			<td>26140079</td>
			<td>For DMEM media</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin, liquid</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>For DMEM media</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS) 1x</td>
			<td></td>
			<td></td>
			<td>Produced in lab</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. List of required reagents</td>
		</tr>
		<tr>
			<td>6-Well Clear TC-Treated Microplates</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr>
			<td>#1 22 x 22 Coverslips</td>
			<td>Fisher</td>
			<td>12-542B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td></td>
			<td>WPI Sizing Information</td>
		</tr>
		<tr>
			<td>ID (mm): 1, 0.86, 0.68, 0.5 Order #: 1B200, 1B150, 1B120, 1B100</td>
		</tr>
		<tr>
			<td>Mass-Flo Controller</td>
			<td>MKS</td>
			<td>M100B01314CS1BV</td>
			<td>Mass flow controller, Series M100B, requires an external 12 V power supply</td>
		</tr>
		<tr>
			<td>USB-6009 DAQ Device</td>
			<td>National Instruments</td>
			<td>779026-01</td>
			<td></td>
		</tr>
		<tr>
			<td>UniSlide Assembly with stepping motor and controller</td>
			<td>Velmex</td>
			<td></td>
			<td>Two series MA15 UniSlide Assemblies, fitted with Vexta 1.8&#176; stepping motors provided by Velmex, and controlled by a VXM Stepping Motor Controller</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. List of required equipment</td>
		</tr>
	</tbody>
</table>

permeabilization of adhered cells using an inert gas jet

Diverses techniques de transfection cellulaire existent et peuvent être décomposés en trois grandes catégories: virale, chimiques et mécaniques. Ce protocole décrit un procédé mécanique pour perméabiliser les cellules adhérentes dans le temps au moyen d&#39;un jet de gaz inerte qui peut faciliter le transfert de macromolécules normalement non-perméables dans des cellules. Nous croyons que cette technique fonctionne en transmettant des forces de cisaillement sur la membrane plasmique des cellules adhérentes, ce qui entraîne la formation de micropores temporaire. Une fois que ces pores sont créés, puis les cellules sont perméables à du matériel génétique et d&#39;autres biomolécules. Les forces mécaniques impliquées ne courent le risque de cellules en permanence endommager ou de détachement de leur support. Il est, par conséquent, une gamme étroite de la dynamique des gaz inertes où la technique est efficace. Un jet de gaz inerte, s&#39;est avérée efficace pour la perméabilisation de diverses lignées cellulaires adhérentes, y compris les cellules HeLa, HEK293 et ​​des cellules endotheliales aortiques abdominaux humains. Ce protocole est approprié pour la permeabilization de cellules adhérentes à la fois in vitro et, comme nous l&#39;avons démontré, in vivo, montrant qu&#39;il peut être utilisé pour la recherche et potentiellement dans de futures applications cliniques. Il a aussi l&#39;avantage de perméabilisation des cellules d&#39;une manière restrictive l&#39;espace, ce qui pourrait s&#39;avérer un précieux outil de recherche.

La perméabilisation de cellules HeLa avec 10 kDa de dextran fluorescent vert à l&#39;aide d&#39;un jet de gaz d&#39;hélium avec un capillaire de diamètre intérieur de 0,86 mm est représenté sur la figure 1. Les cellules ont été contre immédiatement après perméabilisation avec 2 pl / ml EthD-1 solution (LIVE / DEAD viabilité / cytotoxicité Kit) pour visualiser la mort cellulaire. Immédiatement après une contre-coloration, lamelles ont été montées et imagés. Cette figure montre les ré...

Watch this Scientific Journal Video about Permeabilization of Adhered Cells Using an Inert Gas Jet at JoVE.com

Permeabilization of Adhered Cells Using an Inert Gas Jet

Ce protocole décrit un procédé pour la perméabilisation temporaire de cellules adhérentes au moyen d&#39;un jet de gaz inerte. Cette technique facilite le transfert de matériel génétique et de biomolécules dans des cellules de mammifères adhérentes par l&#39;utilisation des forces mécaniques pour rompre la membrane de plasma.

Perméabilisation des cellules adhérant à l&#39;aide d&#39;un gaz inerte Jet

Various cell transfection techniques exist and these can be broken down to three broad categories: viral, chemical and mechanical. This protocol describes ...

permeabilization-of-adhered-cells-using-an-inert-gas-jet

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9.6K vues.  McGill University.  Ce protocole décrit un procédé pour la perméabilisation temporaire de cellules adhérentes au moyen d'un jet de gaz inerte. Cette technique facilite le transfert de matériel génétique et de biomolécules dans des cellules de mammifères adhérentes par l'utilisation des forces mécaniques pour rompre la membrane de plasma. 

Vidéo: Permeabilization of Adhered Cells Using an Inert Gas Jet

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

Caractérisation qualitative de la fraction aqueuse de hydrothermale Liquéfaction d&#39;algues Utilisation 2D chromatographie en phase gazeuse avec le temps de vol Spectrométrie de masse

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

Recherche

Bio-ingénierie

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy (DHM)

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in medical diagnostics, and for life detection by robotic missions to other planets and moons of the solar system. Currently, sparse bacterial samples are counted by culture plating or epifluorescence microscopy. Culture plates require long incubation times (days to weeks), and epifluorescence microscopy requires extensive staining and concentration of the sample. Here, we demonstrate how to use off-axis digital holographic microscopy (DHM) to enumerate bacteria in very dilute cultures (100-104 cells/mL). First, the construction of the custom DHM is discussed, along with detailed instructions on building a low-cost instrument. The principles of holography are discussed, and a statistical model is used to estimate how long videos should be to detect cells, based on the optical performance characteristics of the instrument and the concentration of the bacterial solution (Table 2). Video detection of cells at 105, 104, 103, and 100 cells/mL is demonstrated in real time using un-reconstructed holograms. Reconstruction of amplitude and phase images is demonstrated using an open-source software package.

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM

Digital holographic microscopy (DHM) is a volumetric technique that allows imaging samples 50-100X thicker than brightfield microscopy at comparable resolution, with focusing performed post-processing. Here DHM is used for identifying, counting, and tracking microorganisms at very low densities and compared with optical density measurements, plate count, and direct count.

Quantification des microorganismes à des Concentrations faibles en utilisant la microscopie holographique numérique (DHM)

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in ...

High-resolution Patterning Using Two Modes of Electrohydrodynamic Jet: Drop on Demand and Near-field Electrospinning

Electrohydrodynamic (EHD) jet printing has drawn attention in various fields because it can be used as a high-resolution and low-cost direct patterning tool. EHD printing uses a fluidic supplier to maintain the extruded meniscus by pushing the ink out of the nozzle tip. The electric field is then used to pull the meniscus down to the substrate to produce high-resolution patterns. Two modes of EHD printing have been used for fine patterning: continuous near-field electrospinning (NFES) and dot-based drop-on-demand (DOD) EHD printing. According to the printing modes, the requirements for the printing equipment and ink viscosity will differ. Even though two different modes can be implemented with a single EHD printer, the realization methods significantly differ in terms of ink, fluidic system, and driving voltage. Consequently, without a proper understanding of the jetting requirements and limitations, it is difficult to obtain the desired results. The purpose of this paper is to present a guideline so that inexperienced researchers can reduce the trial and error efforts to use the EHD jet for their specific research and development purposes. To demonstrate the fine-patterning implementation, we use Ag nanoparticle ink for the conductive patterning in the protocol. In addition, we also present the generalized printing guidelines that can be used for other types of ink for various fine-patterning applications.

Here, we present a protocol to produce high-resolution conductive patterns using electrohydrodynamic (EHD) jet printing. The protocol includes two modes of EHD jet printing: the continuous near-field electrospinning (NFES) and the dot-based drop-on-demand (DOD) EHD printing.

Haute résolution en utilisant deux Modes de Jet électrohydrodynamique le Patterning : Drop on Demand et contigu électrofilage

Electrohydrodynamic (EHD) jet printing has drawn attention in various fields because it can be used as a high-resolution and low-cost direct patterning ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Une nouvelle Technique chirurgicale comme fondement pour In Vivo partielle génie hépatique chez le Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Purification et analyse d'un anticorps monoclonal à partir de cellules ovaires de hamster chinois à l'aide d'un système automatisé de microbioréacteur

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various biological activities including cell proliferation, survival, migration and differentiation. Several aberrations in the FGFR signaling pathway, owing to mutations or gene amplification events, have been identified in various types of cancers. Hence, recent research has focused on developing strategies involving therapeutic targeting of FGFRs. Current FGFR inhibitors at various stages of pre-clinical and clinical development include either small molecule inhibitors of tyrosine kinases or monoclonal antibodies, with only a few peptide- based inhibitors in the pipeline. Here, we provide a protocol using phage display technology to screen small peptides as antagonists of FGFR2. Briefly, a library of phage-displayed peptides was incubated in a plate coated with FGFR2. Subsequently, unbound phage was washed off by TBST (TBS + 0.1% [v/v] Tween-20), and bound phage was eluted with 0.2 M glycine-HCl buffer (pH 2.2). The eluted phage was further amplified and used as input for the next round of biopanning. Following three rounds of biopanning, the peptide sequences of individual phage clones were identified by DNA sequencing. Finally, the screened peptides were synthesized and analyzed for affinity and biological activity.

Herein, we present a detailed protocol for screening small peptides that bind to FGFR2 using a phage display peptide library. We further analyze the affinity of the selected peptides toward FGFR2 in vitro and its ability to suppress cell proliferation.

Dépistage et identification des petits peptides ciblant le récepteur du facteur de croissance du fibroblaste2 à l'aide d'une bibliothèque de peptides d'affichage de phage

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various ...

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

La fabrication et l’exploitation d’un système de micro-électroporation à flux continu avec détection de perméabilisation

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...

Formulating and Characterizing an Exosome-based Dopamine Carrier System

Exosomes between 40 and 200 nm in size constitute the smallest subgroup of extracellular vesicles. These bioactive vesicles secreted by cells play an active role in intercellular cargo and communication. Exosomes are mostly found in body fluids such as plasma, cerebrospinal fluid, urine, saliva, amniotic fluid, colostrum, breast milk, joint fluid, semen, and pleural acid. Considering the size of exosomes, it is thought that they may play an important role in central nervous system diseases because they can pass through the blood-brain barrier (BBB). Hence, this study aimed to develop an exosome-based nanocarrier system by encapsulating dopamine into exosomes isolated from Wharton's jelly mesenchymal stem cells (WJ-MSCs). Exosomes that passed the characterization process were incubated with dopamine. The dopamine-loaded exosomes were recharacterized at the end of incubation. Dopamine-loaded exosomes were investigated in drug release and cytotoxicity assays. The results showed that dopamine could be successfully encapsulated within the exosomes and that the dopamine-loaded exosomes did not affect fibroblast viability.

Here, we aimed to obtain a formulation by dopamine loading of the isolated exosomes of stem cells from Wharton's jelly mesenchymal stem cells. Exosome isolation and characterization, drug loading into the resulting exosomes, and the cytotoxic activity of the developed formulation are described in this protocol.

Formulation et caractérisation d’un système de transport de dopamine à base d’exosomes

Exosomes between 40 and 200 nm in size constitute the smallest subgroup of extracellular vesicles. These bioactive vesicles secreted by cells play an ...

Synthèse de nanoparticules lipidiques à l’aide des technologies de mixage CIJ et μMIVM

Synthesizing Lipid Nanoparticles by Turbulent Flow in Confined Impinging Jet Mixers

Lipid nanoparticles (LNPs) have demonstrated their enormous potential as therapeutic delivery vehicles, as evidenced by the approval and global usage of two COVID-19 messenger RNA (mRNA) vaccines. On a small scale, LNPs are often made using microfluidics; however, the limitations of these devices preclude their use on a large scale. The COVID-19 vaccines are manufactured in large quantities using confined impinging jet (CIJ) turbulent mixers. CIJ technology enables production at a laboratory scale with the confidence that it can be scaled to production volumes. The key concepts in CIJ mixing are that the mixing length and time scale are determined by the turbulence intensity in the mixing cavity and that the nanoparticle formation occurs away from walls, eliminating the problem of deposition on surfaces and fouling. This work demonstrates the process of making LNPs using confined impinging jet mixer technology with two geometries: the two-jet CIJ and the four-jet multi-inlet vortex mixer (MIVM). The advantages and disadvantages of each mixing geometry are discussed. In these geometries, LNPs are formed by rapid mixing of an organic solvent stream (usually ethanol containing the ionizable lipids, co-lipids, and stabilizing PEG-lipids) with an aqueous anti-solvent stream (aqueous buffer containing RNA or DNA). The operating parameters for the CIJ and MIVM mixers are presented to prepare reproducible LNPs with controlled size, zeta potential, stability, and transfection effectiveness. The differences between LNPs made with poor mixing (pipetting solutions) compared to CIJ mixing are also presented.

Pleins feux sur l’auteur : Améliorer la formation de nanoparticules lipidiques grâce à un mélange turbulent dans des géométries confinées

A detailed protocol for synthesizing lipid nanoparticles (LNPs) using confined impinging jet (CIJ) mixer technologies, including a two-jet CIJ and a four-jet multi-inlet vortex mixer (&#181;MIVM), is demonstrated. The CIJ mixers generate reproducible, turbulent micro-mixing environments, resulting in the production of monodisperse LNPs.

Synthèse de nanoparticules lipidiques par écoulement turbulent dans des mélangeurs à jet d’impact confinés

Lipid nanoparticles (LNPs) have demonstrated their enormous potential as therapeutic delivery vehicles, as evidenced by the approval and global usage of ...