Gli autori desiderano ringraziare le seguenti fonti di finanziamento: Canadian Institutes of Health Research (CIHR), le scienze naturali e ingegneria Research Council (NSERC).

1. Sviluppo di LabView Programma <ol><li> Un controllore di flusso di massa (MKS M100B Mass-Flo Controller) è collegato alla linea di gas elio per garantire una portata gas precisa. Questa unità è controllata da una tensione di ingresso, compreso tra 0 e 5 V. Un circuito di controllo nel programma LabView determina la tensione richiesta in un dato momento. Interfacciamento tra il computer ed il controllore di flusso di massa è eseguita con un dispositivo di acquisizione dati (NI USB-6009), e un alimentatore 12 V fornisce alimentazione al controllore di flusso di massa. </li><li> Due slitte di posizionamento lineari sono usati per controllare il movimento della piastra da 6 pozzetti, uno per ciascuna direzione. Ogni gruppo è costituito da una serie MA15 Velmex Assemblea Unislide accoppiato con un motore passo-passo, ed entrambi i gruppi sono controllati da un unico Velmex VXM Stepping Motor Controller. Il controllore consente pareggiare manuale di ogni motore o accetta una stringa di comandi seriali esterni, la cui combinazione consente di movimento specifico patterns. Diversi modelli possono essere programmati nel programma LabView, richiedere all&#39;utente di fornire solo la velocità torcia desiderato così come le dimensioni di percorso. </li></ol> 2. Preparazione delle attrezzature <ol><li> Aprire sia la bombola di elio e il regolatore. </li><li> Accendere il controller del motore. </li><li> Aprire il programma LabView e verificare tutte le condizioni. </li><li> Assicurarsi che la piattaforma sia centrato. Per controllare, guardare in ogni fase del motore e verificare che ogni carrello non è vicino a entrambe le estremità palco. O hanno al centro del programma le carrozze o regolare manualmente utilizzando i comandi da jogging sul frontale del controller del motore. </li><li> Se questa è la prima corsa della giornata, si consiglia di eseguire il programma una volta senza le cellule, al fine di garantire il setup è corretto. </li></ol> 3. Preparazione del Height Gas Jet capillare Nozzle <ol><li> Rimuovere il capillare dal supporto e azzerare il quadrante altezza. </li><li> Posizionare la piastra da 6 pozzetti on la piattaforma. </li><li> Sostituire e abbassare il capillare getto di gas al fondo del pozzo fino a raggiungere il vetrino. Fissare la capillare nel supporto. </li><li> Utilizzando la manopola di altezza, sollevare il capillare 3 mm. Segnare questa altezza sulla base capillare. </li><li> Sollevare il capillare a un&#39;altezza di sicurezza e quindi rimuovere la piastra da 6 pozzetti. </li></ol> 4. Cella Permeabilization protocollo <ol><li> Culture linea cellulare aderente a confluenza sul coperchio vetro scivola in una piastra da 6 pozzetti utilizzando tecniche di coltura standard. Per le cellule HeLa, sementi 6 pozzetti con 2 x 10 5 cellule per pozzetto e incubare per 48 ore prima del trattamento. </li><li> Preparare appropriato volume di soluzione contenente il vettore desiderato. Per hrGFPII-1, una concentrazione di 50 ng / ml in PBS 1x fornisce un forte segnale fluorescente in cellule HeLa 24 ore dopo la trasfezione. Per gli studi di destrano, si raccomanda di 80 mg / ml di 10 kDa destrano fluorescente verde. D&#39;ora in poi, questa soluzione si farà riferimento ad uns &quot;vettore soluzione&quot;. </li><li> Rimuovere cellule di coltura da bene e sciacquare tre volte con PBS 1x. </li><li> Pipettare 1.370 ml di soluzione di vettore preparati in fase 4.2 nel bene. Ciò dovrebbe tradursi in una profondità liquida di circa 1,3 mm. </li><li> Posizionare il pozzetto contenente il vettore soluzione nel centro della piattaforma. Abbassare l&#39;ugello getto di gas al livello precedentemente contrassegnato indica un&#39;altezza di ugello 3 mm sopra la diapositiva. </li><li> Impostare portata elio nel programma LabView e selezionare il modello di movimento desiderata da utilizzare. Seleziona &quot;Esegui&quot; quando è pronto per iniziare il protocollo di permeabilizzazione. Ci sarà un periodo di ritardo iniziale in cui il controllore di flusso di massa si appresta a fornire la portata necessaria. Una volta portata appropriata è stato raggiunto, il programma LabView attiverà i motori passo-passo per spostare il bene nel modello permeabilizzazione desideri. Una volta che la piattaforma è fermato, attendere circa 30 sec e rimuovere la soluzione vettoriale.Nota: permeabilizzazione ottimale si verifica vicino ad una pressione dinamica di 100 a 200 Pa all&#39;uscita dell&#39;ugello, a seconda del diametro del capillare. </li><li> Lavare ben tre volte con PBS 1x. </li><li> Riempire bene con terreni di coltura fresco. </li><li> Ripetere i passaggi 4,3-4,6 per il numero desiderato di esperimenti. Nota: Assicurati di includere campioni di controllo che consistono di pozzi essere riempiti con la soluzione di vettore senza il getto di gas che viene eseguito su di loro. Lasciare la soluzione nei pozzetti di controllo per circa 1 minuto poi procedere alle fasi 4.5 e 4.6. </li><li> Se si esegue un esperimento di transfezione hrGFP, ritorno 6 pozzetti di incubatore per 24 ore per permettere il materiale transfettati da trascrivere dalle cellule. Se si esegue un esperimento di permeabilizzazione destrano, ritorno 6 pozzetti di incubatore per 15 min. </li></ol> 5. Imaging cellulare <ol><li> Se lo si desidera, subito prima di imaging, di contrasto con adeguate macchia dal vivo / morto per valutare la morte cellulare. </li><li> Celle di immagine con appropriate microscopio per studiare l&#39;efficacia di trasfezione / permeabilizzazione. </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>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Getto di gas inerte permeabilizzazione è una tecnica utile per la trasfezione di cellule aderenti. Offre la possibilità di trasferire biomolecole nelle cellule con l&#39;uso di forze meccaniche, che elimina la necessità di prodotti chimici potenzialmente dannosi o vettori virali. La tecnica potrebbe potenzialmente offrire ai ricercatori e medici un modo efficiente e relativamente semplice per transfettare con precisione le cellule. La selettività del permeabilizzazione è unico anche consentendo ai ricercatori di tr...

Con l&#39;evoluzione della biomedicina e la comprensione della meccanica delle cellule, la consegna di biomolecole nelle cellule è diventato di vitale importanza per molti settori di ricerca e le terapie mediche. Diverse tecniche sono state sviluppate per introdurre molecole estranee attraverso la membrana plasmatica e nel citoplasma cellulare. Questi possono essere generalmente classificati come: tecniche virale, chimici o meccanici. Tecniche virali possono trasfettare materiale genetico come RNA e DNA utilizzando vettori virali 1. Tecniche virali tendono ad avere alte efficienze in certe linee cellulari, tuttavia hanno il potenziale di reazioni immunitarie e infiammatorie 2. I metodi più comuni di transfezione chimici includono precipitazione con fosfato di calcio a 3, esotossine batteriche 4 e lipofezione 5. Queste tecniche hanno dimostrato di essere efficace, tuttavia, alcuni problemi sorgono come tossicità cellulare e non specificità. Inoltre, tecniche come phosphat calcioe precipitazione sono stati trovati per avere efficienze di trasfezione fino al 70%, ma solo in certe linee cellulari, raramente in cellule primarie 6. Questo è da notare come crescenti sforzi di ricerca sono stati messi in cellule primarie, soprattutto nel caso di progettazione di vari trattamenti per uso clinico e lo studio del funzionamento del DNA. Interruzione temporale della membrana cellulare attraverso stimoli meccanici è un&#39;alternativa per introdurre molecole estranee nelle cellule. Tecniche includono: microiniezione di molecole 7, elettroporazione usando un campo elettrico di distruggere la membrana 8,9, sonoporazione utilizzando onde ad ultrasuoni per distruggere la membrana cellulare 10-12, bombardamento di particelle come dimostrato dal &quot;gene gun&quot;, che spara particelle legate con geni nella cella 13, e, più recentemente, l&#39;applicazione di un fluido sforzo di taglio che ha dimostrato di permeabilize temporalmente cellule di mammifero 14,15. Sebbene meccanicometodi AL evitare alcuni dei problemi di cui sopra, sono in genere accompagnati da efficienze inferiori e configurazioni molto complesse e specializzate. Una torcia scarica a bagliore a pressione atmosferica (APGD-t) è stato sviluppato presso la McGill con l&#39;obiettivo iniziale di funzionalizzazione di superfici e staccando le cellule aderenti in vitro 16. Casualmente, si è scoperto che una macchia dal vivo / morto utilizzato era in qualche modo essere presa in dalle cellule senza essere aggiunto un agente permeabile. Inoltre, questo sembrava si sono verificati solo in aree ristrette dei pozzi che è accaduto a corrispondere con il percorso della torcia. Da studi sulle capacità di permeabilizzazione della APGD-t proseguito in studi di controllo, è stato trovato che il gas di trasporto inerte getto di plasma senza eccitazione era anche in grado di permeabilize cellule. Questo contraddice l&#39;ipotesi iniziale che le specie reattive create dal getto di plasma alterati temporaneamente la funzione della membrana e ha suggerito che semplicemente le meccanicheforze CAL erano sufficienti a causare permeabilizzazione cellulare. Da qui, gli studi continuarono nel nostro laboratorio per cercare di quantificare e caratterizzare l&#39;efficienza permeabilizzazione di un getto di gas inerte come pure un&#39;occhiata alle sue capacità di trasfezione 16. Attraverso questi studi, si è scoperto che micropori costituiscano effettivamente nella membrana plasmatica, e questi pori tendono a richiudere entro circa 5 sec 15. Abbiamo dimostrato l&#39;utilità della tecnica in vitro e in vivo nella membrana corioallantoidea di embrione di pollo.

<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

Esistono varie tecniche di trasfezione cellulare e questi possono essere suddivisi a tre grandi categorie: virale, chimici e meccanici. Questo protocollo descrive un metodo meccanico per permeabilize temporalmente cellule aderenti mediante un getto di gas inerte che può facilitare il trasferimento di normalmente macromolecole non permeabili nelle cellule. Crediamo che questa tecnica funziona impartendo forze di taglio sulla membrana plasmatica di cellule aderenti, causando la formazione temporanea di micropori. Una volta che questi pori sono creati, le cellule sono poi permeabile al materiale genetico e altre biomolecole. Le forze meccaniche coinvolte non corrono il rischio di cellule permanentemente danneggiare o staccano dal loro substrato. Esiste, quindi, una ristretta gamma di dinamica dei gas inerti dove la tecnica è efficace. Un getto di gas inerte è dimostrata efficace in permeabilizzante varie linee cellulari aderenti, compresi HeLa, HEK293 e cellule endoteliali dell&#39;aorta addominale umani. Questo protocollo è appropriato per la permeabilization di cellule aderenti sia in vitro che, come abbiamo dimostrato, in vivo, mostrando esso può essere usato per la ricerca e potenzialmente in future applicazioni cliniche. Essa ha anche il vantaggio di permeabilizzante cellule in modo spazialmente restrittiva, che potrebbe rivelarsi un valido strumento di ricerca.

Permeabilizzazione delle cellule HeLa con 10 kDa destrano fluorescente verde utilizzando un getto di gas elio con un diametro interno di 0,86 millimetri capillare è mostrato in Figura 1. Le cellule sono state contrastate immediatamente dopo permeabilizzazione con 2 microlitri / ml EthD-soluzione 1 (LIVE / DEAD Viabilità / citotossicità Kit) per visualizzarne la morte cellulare. Subito dopo di contrasto, vetrini sono stati montati e ripreso. Questa figura mostra risultati dell&#39;esecuzione del getto...

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

Questo protocollo descrive un metodo per la permeabilizzazione temporanea di cellule aderenti mediante un getto di gas inerte. Questa tecnica facilita il trasferimento di materiale genetico e biomolecole in cellule di mammifero aderenti mediante l&#39;utilizzo delle forze meccaniche di distruggere la membrana plasmatica.

Permeabilizzazione delle cellule Aderito utilizzo di un gas inerte Jet

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

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9.6K Views.  McGill University.  Questo protocollo descrive un metodo per la permeabilizzazione temporanea di cellule aderenti mediante un getto di gas inerte. Questa tecnica facilita il trasferimento di materiale genetico e biomolecole in cellule di mammifero aderenti mediante l'utilizzo delle forze meccaniche di distruggere la membrana plasmatica. 

Video: 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.

Caratterizzazione qualitativa della frazione acquosa da idrotermale Liquefazione di alghe Utilizzando 2D gascromatografia con tempo di volo spettrometria di massa

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

Ricerca

Bioingegneria

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.

Quantificare i microrganismi alle concentrazioni basse usando microscopia digitale oleografica (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.

Alta risoluzione Patterning utilizzando due modalità di idrodinamiche Jet: Drop on Demand e Near-field elettrofilatura

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.

Una nuova tecnica chirurgica come una Fondazione per In Vivo parziale ingegneria del fegato nel ratto

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.

Purificazione e analisi di un anticorpo monoclonale da cellule ovaio Cesster cinese utilizzando un sistema microbioreante automatizzato

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.

Screening e identificazione di piccoli peptidi mirati Fibroblast Growth Factor Receptor2 utilizzando una 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 ...

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 fabbricazione e il funzionamento di un sistema di micro-elettroporazione a flusso continuo con rilevamento di permeabilizzazione

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.

Formulazione e caratterizzazione di un sistema di trasporto della dopamina basato sugli esosomi

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

Sintesi di nanoparticelle lipidiche utilizzando le tecnologie dei miscelatori CIJ e 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.

Autore in primo piano: Migliorare la formazione di nanoparticelle lipidiche attraverso la miscelazione turbolenta in geometrie confinate

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.

Sintesi di nanoparticelle lipidiche mediante flusso turbolento in miscelatori a getto a impatto confinato

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