Gli autori desiderano ringraziare il Dott. Peiman Hemmati (Dipartimento di Medicina dell&#39;Università del Wisconsin-Madison) per offrire generosamente la MSC H1, e il dottor Tim Hacker, il Dr. Gouqing Song e la signora Jill Koch della University of Wisconsin Cardiovascolare Fisiologia Fondo centrale per l&#39;esecuzione di interventi chirurgici del mouse. Questo lavoro è stato sostenuto dal National Science Foundation attraverso una borsa di studio Graduate Research a Brian Freeman e NIH R21 HL089679.

1. Donatore di cellule Transfection <ol><li> Raccolta delle cellule staminali mesenchimali (MSC, derivate da cellule staminali embrionali H1 gentilmente donato dal Dott. Peiman Hematti, in alternativa, qualsiasi tipo di cellula di qualsiasi specie ipotizzato di fondere in vivo potrebbe essere impiegato) quando il 70 - 80% confluenti con 1X tripsina (Mediatech, Manassas VA) per 5 min. Inattivare tripsina con α-MEM terreno completo (antibiotico libero, Invitrogen, Carlsbad CA) 18. Centrifugare a 300 xg per 5 min. </li><li> Con attenzione aspirare il surnatante e risospendere pellet in 1 ml di PBS 1X e contare le cellule utilizzando un emocitometro. </li><li> Trasferire 1,5 x 10 6 cellule in una provetta da 1,5 ml per centrifuga. Centrifugare a 300 xg per 5 min. </li><li> Con attenzione aspirare il surnatante. Risospendere il pellet in 300 ml di R Buffer (Sistema Transfection Neon, Invitrogen) e 6 mg (2 mg / 5,0 x 10 5 cellule) del P231 pCMVe-betaAc-STOP-luc (Addgene, Cambridge, MA). Mettere 3 ml di tampone E (InvitrOgen) nella porta elettroporazione docking per il protocollo del produttore (Sistema Transfection Neon, Invitrogen). </li><li> Trasferimento cellule plasmide soluzione ad una punta 100 ul pipetta Neon e electroporate con una durata dell&#39;impulso di 20 ms e una grandezza di 1500 volt. Luogo elettroporate cellule in una provetta da 15 ml conica contenente 9,7 ml di α-MEM terreno completo. </li><li> Ripetere il passo 1,5 altre due volte e cellule transfettate piscina per ottenere un volume totale di 10 ml. Aggiungere i 10 ml di sospensione cellulare (1,5 x 10 6 cellule) in un pallone T175 contenente 10 ml di α-MEM terreno completo. La vitalità cellulare dopo l&#39;elettroporazione è circa il 30%, per produrre circa 4,5 x 10 5 cellule vitali per T175. </li><li> Cambia α-MEM completo di media 24 ore dopo la trasfezione. </li><li> Vendemmia transfettate cellule quando 70-80% confluenti (~ 2 - 3 giorni dopo l&#39;elettroporazione). Eseguire il conteggio delle cellule utilizzando un emocitometro e risospendere le cellule ad una concentrazione di 1,0 x 10 6cells/50 ml di α-MEM terreno completo. Ridurre al minimo le cellule tempo trascorso in sospensione per ridurre la morte cellulare prima dell&#39;iniezione. </li></ol> 2. Iniezione intramiocardico <ol><li> Indurre l&#39;anestesia da isoflurano (Phoenix Pharmaceuticals, Inc., San Giuseppe, MO) su topi transgenici ingegnerizzati per esprimere costitutivamente Cre ricombinasi in ogni cellula (B6.C-Tg (CMV-CRE) 1Cgn / J, Jackson Laboratory, Bar Harbor, ME). </li><li> Rimuovere i capelli dalla regione toracica utilizzando tosatrici capelli o di rimozione chimica. </li><li> Intubare con un catetere 18 gauge (Becton Dickinson &amp; Co, Franklin Lakes NJ) e posto sul ventilatore del mouse a 120-130 respiri al minuto con una gittata sistolica di 150 microlitri. </li><li> Fai incisione laterale attraverso il quarto spazio intercostale, producendo così una toracotomia. </li><li> Visualizzazione del cuore, fare due iniezioni di 25 ml di sospensione cellulare transfettate con una siringa da 1 ml (Temuro Medical Corporation, Somerset, NJ) e 28 gauge (Bectsu Dickinson &amp; Co, Franklin Lakes NJ). Per facilitare l&#39;iniezione intramiocardico ed evitare danni eccessivi per l&#39;organo, piegare la testa di spillo a 90 gradi. </li><li> Dopo l&#39;iniezione, usare suture assorbibili (ad esempio, Vicryl) per chiudere le costole e strati muscolari. Sutura della pelle chiusi con nylon 4-0 o seta. </li><li> Lasciare il mouse per recuperare da anestesia e estubare. </li><li> Gruppi di controllo dovrebbe includere i topi Cre ricevere iniezioni unico mezzo, i topi Cre ricevendo la stessa concentrazione di cellule untransfected e topi wild-type ricevere cellule trasfettate (in alternativa, i topi Cre ricevendo cellule transfettate non incline a fondere). </li></ol> 3. Imaging Biophotonic <ol><li> Da cinque a quindici minuti prima di imaging, intraperitoneale (IP) iniettare 10 L per grammo di peso corporeo del mouse di 15 mg / ml di D-Luciferin (Caliper Life Sciences, Hopkinton, MA). </li><li> Indurre l&#39;anestesia sui topi via isoflurano al 4% per l&#39;induzione unod 1 - 2% per la manutenzione. </li><li> Topi posto in posizione supina nella casella di imaging con un facciale che l&#39;amministrazione 1 - 2% isoflurano per l&#39;anestesia di mantenimento (Imaging System Xenogen Biophotonic, Hopkinton, MA). Diversi i topi possono essere esposte contemporaneamente. Immagine falsa controllo del mouse con i topi sperimentali per facilitare il confronto del segnale luminescente. </li><li> Utilizzando immagini software Living (Xenogen), impostare il tempo di esposizione appropriato (tipicamente 60 sec, vedi Risultati). Imposta area di immagine per adattarla topi e mantenere zona coerenti in tutta l&#39;imaging per evitare modifiche nella sensibilità. Impostare l&#39;altezza soggetto a 4,5 cm. </li><li> Acquisire intensità del segnale di luminescenza corrispondente al mouse o topi all&#39;interno del campo di visualizzare e salvare i file di immagine non modificato. Elaborare le immagini per rimuovere il segnale di fondo che corrisponde a un controllo o un mouse non manipolata. Valori di intensità di sopra di fondo corrispondono a cellule fuse all&#39;interno dell&#39;animale. Analisi intensità può essere effettuato utilizzando software di immagine vivente (Xenogen) o aprire l&#39;immagine source software di analisi. Typically, una regione di interesse (ROI) è stato selezionato per confrontare i dati di intensità tra gli animali ed esperimenti. </li></ol> 4. Rappresentante Risultati Per determinare la sensibilità del sistema Biophotonic Imaging Xenogen, una linea cellulare che esprime costitutivamente luciferasi (231-LUC-D3H1, Xenogen) è stato consegnato al miocardio di topi C57/Bl6 (Jackson Laboratory). Cellule sono state iniettate a concentrazioni di 1 x 10 6, 1 x 10 3, oppure 1 x 10 1 celle. Sei ore dopo la consegna delle cellule, i topi sono stati iniettati per via intraperitoneale con luciferina e ripreso con il sistema Xenogen. Un segnale specifico è stato possibile rilevare con 1.000 cellule (2 su 6 topi ripreso, Figura 2), ma il rilevamento era più affidabile con 10.000 celle (6 di 6 topi ripreso). È importante sottolineare che questo studio ha anche servito per stabilire una correlazione approssimativa tra numero di cellule che esprimono luciferasi e l&#39;intensità del segnale. <p class="jove_content"&gt; Per dimostrare l&#39;utilità del protocollo descritto nel rilevamento e monitoraggio fusione cellulare, cellule staminali mesenchimali sono state trasfettate con il loxP-Stop-loxP-luciferasi plasmide (Addgene) e consegnato al miocardio di Cre-topi che esprimono. Circa una settimana dopo la consegna delle cellule, i topi sono stati fotografato utilizzando il sistema Xenogen senza D-luciferina iniezione. Come previsto, senza il substrato enzimatico, nessun segnale di intensità è stato rilevato (Figura 3). Successivamente, D-luciferina è stato iniettato per via intraperitoneale e un segnale corrispondente alla intensità luciferasi, e quindi la fusione cellulare è stato rilevato in due dei quattro topi testati. Un segnale simile è stata rilevata una settimana dopo (Figura 3), suggerendo MSC-coupled prodotti di fusione può essere mantenuto in vivo. In questo caso, lo studio è stato interrotto per consentire la valutazione del cuore e dei tessuti circostanti, ma si potrebbe immaginare a lungo termine analisi e di imaging più frequenti per monitorare l&#39;, la proliferazione di manutenzione unod forse la migrazione dei prodotti di fusione nei topi. <img alt="Figura 1" src="/files/ftp_upload/3581/3581fig1.jpg" /> Figura 1. Schematica della tecnica per rilevare la fusione cellulare in vivo. Se la fusione tra Cre-esprimono cellule di topo e cellule trapiantate che esprimono un plasmide floxed luciferasi si verifica, luciferasi sarà espressa. Luciferasi può essere rilevato, iniettando il substrato enzimatico, D-luciferina, nel mouse e allora immagini con il mouse utilizzando un Biophotonic Imaging System Xenogen (Adattato da 19) <img alt="Figura 2" src="/files/ftp_upload/3581/3581fig2.jpg" /> Figura 2. Sensibilità di rilevazione di luciferasi cellule che esprimono nel tessuto cardiaco con l&#39;imaging Biophotonic. Una linea cellulare che esprime costitutivamente luciferasi (231-LUC-D3H1, Xenogen) è stato consegnato allo spazio intramiocardico di C57/Bl6 topi a vari numeri di cellulare totale. Immagini rappresentative di topi receiving 1 x 10 6, 1 x 10 3 e 1 x 10 1 celle (da sinistra a destra) sono indicati, l&#39;imaging è stato condotto circa 6 ore dopo l&#39;iniezione. <img alt="Figura 3" src="/files/ftp_upload/3581/3581fig3.jpg" /> Figura 3. Nella quantificazione della luminescenza Vivo indicativo di fusione cellulare. Cellule staminali mesenchimali sono state trasfettate con il loxP-Stop-loxP-luciferasi plasmide e consegnato al miocardio di Cre-topi che esprimono. Circa una settimana e due settimane dopo la consegna delle cellule, i topi sono stati Cre fotografato utilizzando il Biophotonic Imaging System Xenogen per misurare l&#39;intensità di luminescenza indicativo di fusione cellulare. (A) Sovrapposizione della fotografia e l&#39;intensità di luminescenza di finzione e topi 1-4 (da sinistra a destra) giorni 17 dopo la consegna delle cellule. (B) Intensità di luminescenza di finzione e topi 1-4 (da sinistra a destra) giorni 17 dopo la consegna delle cellule. Una regione di interesse è stato selezionato (giallo) corrispondente al sito di iniezione e l intensitàevels sono stati determinati utilizzando ImageJ (source) del software 20. (C) intensità di luminescenza è stata normalizzata alla stessa regione di interesse sulla farsa del mouse per tutte le condizioni sperimentali. A una settimana, i topi 3 e 4 hanno mostrato segnale positivo luminescente che suggerisce la fusione spontanea di una cellula del mouse e MSC trapiantate. Il segnale continuato a mouse 3 a due settimane. Per determinare organo-specifiche localizzazione del segnale corrispondente al mouse 3, la cavità toracica è stato esposto e organi primari asportato e immagine. (D) Sovrapposizione della fotografia e l&#39;intensità di luminescenza del mouse 3. Localizzazione nota del segnale di intensità nel piccolo intestino. (E), intensità di luminescenza del mouse 3. (F) Sovrapposizione della fotografia e l&#39;intensità di luminescenza di falsa mouse. (G) L&#39;intensità di luminescenza di falsa mouse.

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Nessun conflitto di interessi dichiarati.

Il metodo qui descritto consente, per la prima volta, l&#39;identificazione e l&#39;analisi temporale discreto di fusione cellulare negli organismi, compresi gli animali di piccole dimensioni. L&#39;approccio combina Cre-loxP ricombinazione con conseguente analisi delle immagini Biophotonic. L&#39;approccio è suscettibile di monitoraggio non solo cellula-cellula fusion, ma anche la fusione virus-cellula e quindi potrebbe rivelarsi utile per il monitoraggio delle infezioni virali. Analisi delle immagini è rapida ed è ...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Neon Transfection di sistema </td><td> Invitrogen, Carlsbad, CA </td><td> MPK5000 </td><td></td></tr><tr><td> Neon 100 Kit microlitri </td><td> Invitrogen, Carlsbad, CA </td><td> MPK10025 </td><td> Contiene R ed E Buffer </td></tr><tr><td> a-MEM polvere </td><td> Invitrogen, Carlsbad, CA </td><td> 12000-022 </td><td></td></tr><tr><td> Siero fetale bovino (FBS) </td><td> Hyclone, Logan UT </td><td> SH30070.03 </td><td></td></tr><tr><td> B6.C-Tg (CMV-CRE) 1Cgn / J </td><td> Jackson Laboratory, Bar Harbor, ME </td><td> 006054 </td><td></td></tr><tr><td> Tripsina 10X </td><td> Fisher Scientific, Forest Lawn, NJ </td&gt; <td> MT-25-054-Cl </td><td></td></tr><tr><td> L-Glutammina </td><td> Fisher Scientific, Forest Lawn, NJ </td><td> 25030-081 </td><td></td></tr><tr><td> D-Luciferin </td><td> Caliper Life Sciences, Hopkinton, MA </td><td> 122796 </td><td></td></tr><tr><td> Xenogen Imaging System Biophotonic </td><td> Caliper Life Sciences, Hopkinton, MA </td><td> IVIS Spectrum </td><td></td></tr><tr><td> Sodio Biocarbonate </td><td> Sigma Aldrich, St. Louis, MO </td><td> S6014-500G </td><td></td></tr><tr><td> Aminoacidi non essenziali </td><td> Invitrogen, Carlsbad, CA </td><td> 11140-050 </td><td></td></tr></tbody></table>

a cre-lox p recombination approach for the detection of cell fusion in vivo

La capacità di due o più cellule dello stesso tipo di fusibile è stato utilizzato nei metazoi nel corso dell&#39;evoluzione per formare molti organi complessi, inclusi i muscoli scheletrici, ossa e placenta. Studi contemporanei dimostrano la fusione di cellule dello stesso tipo conferisce funzioni avanzate. Per esempio, quando le cellule del trofoblasto della placenta fondono per formare il sinciziotrofoblasto, il sinciziotrofoblasto è più in grado di trasportare le sostanze nutritive e gli ormoni attraverso la barriera materno-fetale di trophoblasts non condensato 1-4. Studi più recenti dimostrano la fusione di cellule di tipo diverso possono indirizzare il destino della cellula. Il &quot;ritorno&quot; o la modifica del destino delle cellule di fusione era una volta pensato per essere limitata a sistemi di colture cellulari. Ma l&#39;avvento del trapianto di cellule staminali hanno portato alla scoperta da noi e altri che le cellule staminali possono fondersi con le cellule somatiche in vivo e che la fusione facilita la differenziazione delle cellule staminali 5-7. Così, la fusione cellulare è un processo regolato capable di promuovere la sopravvivenza cellulare e della differenziazione, e quindi potrebbe essere di fondamentale importanza per lo sviluppo, la riparazione dei tessuti e anche la patogenesi della malattia. Limitare lo studio della fusione cellulare, è la mancanza di tecnologie adeguate a 1) identificare accuratamente prodotti di fusione e di 2) prodotti di fusione traccia nel tempo. Qui vi presentiamo un nuovo approccio per affrontare sia le limitazioni attraverso l&#39;induzione di bioluminescenza su fusione (Figura 1). Bioluminescenza è possibile rilevare con alta sensibilità in vivo 8-15 Utilizziamo un costrutto che codifica per la luciferasi lucciola (Fotino pyralis) gene collocato adiacente al un codone di stop affiancato da sequenze loxP. Quando le cellule che esprimono questo gene fondono con cellule che esprimono la proteina Cre ricombinasi, i siti loxP sono spaccati e il segnale di stop è escisse che permette la trascrizione della luciferasi. Poiché il segnale è induciBLE, l&#39;incidenza di falsi positivi segnali è molto bassa. A differenza dei metodi esistenti che utilizzano il Cre / loxP sistema 16, 17, abbiamo incorporato un segnale &quot;vivente&quot; di rilevazione e quindi permettere per la prima volta l&#39;opportunità di monitorare la cinetica di fusione cellulare in vivo. Per dimostrare l&#39;approccio, i topi che esprimono ubiquitariamente Cre ricombinasi servito come destinatari di cellule staminali transfettate con un costrutto per esprimere a valle luciferasi di un codone di stop floxed. Cellule staminali sono state trapiantate tramite iniezione intramiocardico e dopo il trapianto di analisi d&#39;immagine intravitale è stata condotta per monitorare la presenza di prodotti di fusione nel cuore e tessuti circostanti nel corso del tempo. Questo approccio potrebbe essere adattato per analizzare la fusione cellulare in qualsiasi tipo di tessuto in qualsiasi stadio di sviluppo, la malattia o la riparazione dei tessuti adulti.

Watch this Scientific Journal Video about A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo at JoVE.com

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

Un metodo per tenere traccia fusione cellulare in organismi viventi nel corso del tempo è descritto. L&#39;approccio utilizza Cre-<em&gt; LoxP</emRicombinazione&gt; per indurre l&#39;espressione luciferasi sulla fusione cellulare. Il segnale luminescente generato può essere rilevato negli organismi viventi con sistemi di imaging Biophotonic con una sensibilità di rilevazione di circa 1.000 cellule nei tessuti periferici.

Un Cre-Lox P approccio ricombinazione per la rilevazione di fusione cellulare In Vivo</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

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Research

JoVE Journal

Bioengineering

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

18.7K Views.  University of Wisconsin-Madison. Un metodo per tenere traccia fusione cellulare in organismi viventi nel corso del tempo è descritto. L'approccio utilizza Cre- LoxP per indurre l'espressione luciferasi sulla fusione cellulare. Il segnale luminescente generato può essere rilevato negli organismi viventi con sistemi di imaging Biophotonic con una sensibilità di rilevazione di circa 1.000 cellule nei tessuti periferici.

Video: A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

Biomolecolare Detection impiega il sensore di immagine interferometrica riflettanza (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Ricerca

Bioingegneria

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

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

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

Fabbricazione di elettrochimica-DNA biosensori per il rilevamento Reagentless di acidi nucleici, proteine ​​e piccole molecole

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

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

La creazione di substrati Patterned bidimensionali per confinamento di proteine ​​e cellule

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Semplici dispositivi microfluidici per<em&gt; In vivo</em&gt; Imaging di<em&gt; C. elegans</em&gt;,<em&gt; Drosophila</em&gt; E Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

Microiniezione Wound Assay e<em&gt; In vivo</em&gt; Localizzazione di Epidermal Wound risposta Giornalisti a<em&gt; Drosophila</em&gt; Embrioni.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Una pelle piena Difetto modello per valutare Vascolarizzazione di Biomateriali<em&gt; In Vivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Una tecnica senza etichetta per l&#39;Imaging spazio-temporale delle secrezioni monocellulari

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

Bioreattori fibra cava per<em&gt; In Vivo</em&gt; -come Mammiferi Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparazione del 3D collagene Gel e microcanali per lo studio delle interazioni 3D<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

Riconoscimento visivo delle più acidi nucleici in una matrice capillare

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...