Name: co-culture of living microbiome with microengineered human intestinal villi in a gut-on-a-chip microfluidic device
Uploaded: 2016-08-30T15:00:00
Description: Watch this Scientific Journal Video about Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device at JoVE.com

We thank Sri Kosuri (Wyss Institute at Harvard University) for providing the GFP-labeled E. coli strain. This work was supported by the Defense Advanced Research Projects Agency under Cooperative Agreement Number W911NF-12-2-0036, Food and Drug Administration under contract #HHSF223201310079C, and the Wyss Institute for Biologically Inspired Engineering at Harvard University. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies, either expressed or implied, of the Army Research Office, Army Research Laboratory, Food and Drug Administration, or the U.S. Government. The U.S. Government is authorized to reproduce and distribute reprints for Government purposes notwithstanding any copyright notation hereon.

1. Microfabbricazione di un dispositivo Gut-on-a-chip Nota: L&#39;intestino-on-a-chip è un dispositivo microfluidico fatta da, polimero siliconico gas-permeabile trasparente (polidimetilsilossano, PDMS), contenente due microcanali paralleli (1 mm larghezza lunghezza x 150 micron di altezza x 1 cm) separati da un flessibile poroso (10 micron di diametro dei pori, 25 micron di pori spaziatura a pori) PDMS membrana 5,9. Realizzare il (Figura 1A, a sinistra) gut-on-a-c...

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Capire interazioni ospite-microbiome è fondamentale per far progredire la medicina; tuttavia, tradizionali modelli di coltura cellulare eseguiti in un piatto di plastica o di una piastra ben statica non supportano la stalla co-coltura di cellule intestinali umane con microbi intestinali vivere per più di 1-2 giorni, perché le cellule microbiche soprattutto invadere le cellule di mammifero in vitro. La popolazione microbica overgrowing consuma rapidamente ossigeno e sostanze nutritive, in seguito la produzion...

L&#39;intestino umano ospita una gamma incredibilmente diversificata di specie microbiche (&lt;1.000 specie) e un numero enorme di cellule microbiche (10 volte di più rispetto alle cellule ospiti umane) e geni (100 volte più del genoma umano) 1. Questi microbiomes umane giocano un ruolo chiave nel metabolismo di sostanze nutritive e xenobiotici, che regola le risposte immunitarie, e mantenere l&#39;omeostasi intestinale 2. Non sorprende che, date queste diverse funzioni, l&#39;intestino microbiome commensale modula ampiamente salute e la malattia 3. Così, la comprensione del ruolo del budello microbioma e host-microbo interazioni sono di grande importanza per promuovere gastrointestinale (GI) di salute e di esplorare nuove terapie per i disturbi intestinali 4. Tuttavia, esistenti in modelli intestinali in vitro (ad esempio, le culture statiche) limita host-microbiome co-coltura per un breve periodo di tempo (&lt;1 giorno) perché le cellule microbiche invadere e compromettono barriera intestinaleFunzione 5. Modelli animali surrogati (ad esempio, di germi free 6 o tecniche di ingegneria genetica topi 7) sono, inoltre, non comunemente utilizzati per lo studio ospite-gut microbiome crosstalk perché la colonizzazione e il mantenimento stabile di microbioma intestinale umana sono difficili. Per superare queste difficoltà, abbiamo recentemente sviluppato un essere umano biomimetico &quot;Gut-on-a-Chip&quot; sistema microphysiological (Figura 1A, a sinistra) per emulare le interazioni ospite-microbioma intestinale che si verificano nel vivere intestino umano 5,8. Il Microdevice gut-on-a-chip contiene due canali microfluidica parallele separate da un flessibile, porosa, matrice extracellulare (ECM) membrana Rivestiti fiancheggiata da epiteliali intestinali umane cellule Caco-2, imitando il intestinale lume-capillari interfaccia tessuto (Figura 1A , a destra) 9. ciclici deformazioni ritmiche sotto vuoto spinto inducono deformazioni meccaniche fisiologiche che simulano i cambiamenti normalmente Induccati da peristalsi (Figura 1A, a destra). È interessante notare che, quando cellule Caco-2 sono coltivate nell&#39;intestino-on-a-chip per più di 100 ore, hanno spontaneamente forma tridimensionale (3D) villi intestinali con giunzioni strette, bordi pennello apicali, le cellule proliferative limitate a cripte basali, produzione di muco, una maggiore attività metabolizzare farmaci (ad esempio, citocromo P450 3A4, CYP3A4), e una maggiore glucosio ricaptazione 8. In questo microambiente &#39;privo di germi&#39;, è stato possibile co-cultura probiotico Lactobacillus GG rhamnosus o una formazione terapeutica di una miscela batterico probiotico con cellule epiteliali ospitante per due settimane 5,10. In questo studio, descriviamo il protocollo dettagliato per eseguire host-intestino microbiome co-coltura nel dispositivo gut-on-a-chip per un periodo prolungato. In aggiunta, discutiamo criticità e sfide potenziali da considerare per una vasta applicazione di questo host-microbiome co-coltura pROTOCOLLO.

<table border="0" cellpadding="0" cellspacing="0" width="1454">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:509px;">Dulbecco&#39;s Modified Eagle Medium (DMEM) containing 25 mM glucose and 25 mM HEPES</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:175px;">10564-011</td>
			<td style="width:544px;">Warm it up at 37&#176;C in a water bath.</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:509px;">Difco Lactobacilli MRS broth</td>
			<td style="width:227px;">BD</td>
			<td style="width:175px;">288120</td>
			<td>Run autoclave at 121&#176;C for 15 min.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:509px;">Poly(dimethylsiloxane)</td>
			<td style="width:227px;">Dow Corning</td>
			<td style="width:175px;">3097358-1004</td>
			<td>15:1 (w/w), PDMS : cureing agent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:509px;">Caco-2BBE human colorectal carcinoma line</td>
			<td style="width:227px;">Harvard Digestive Disease Center</td>
			<td style="width:175px;"></td>
			<td>Human colorectal adenocarcinoma&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:509px;">Heat-inactivated FBS</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:175px;">10082-147</td>
			<td>20% (v/v) in DMEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:509px;">Trypsin/EDTA solution (0.05%)</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:175px;">25300-054</td>
			<td>Warm it up at 37&#8451; in a water bath.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:509px;">Penicillin-streptomycin-glutamine</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:175px;">10378-016</td>
			<td>1/100 dilution in DMEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:509px;">4&#8242;,6-Diamidino-2-phenylindole dihydrochloride</td>
			<td style="width:227px;">Molecular Probes</td>
			<td style="width:175px;">D1306</td>
			<td>Nuclei staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:509px;">Phalloidin-CF647 conjugate (25 units/mL)</td>
			<td style="width:227px;">Biotium</td>
			<td style="width:175px;">00041</td>
			<td>F-actin staining</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:509px;">Flexcell FX-5000 tension system</td>
			<td style="width:227px;">Flexcell International Corporation</td>
			<td style="width:175px;">FX5K</td>
			<td>Peristalsis-like stretcing motion (10% cell strain, 0.15 Hz frequency)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:509px;">Inverted epifluorescence microscope</td>
			<td style="width:227px;">Zeiss</td>
			<td style="width:175px;">Axio Observer Z1</td>
			<td>Imaging, DIC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:509px;">Scanning confocal microscope</td>
			<td style="width:227px;">Leica</td>
			<td style="width:175px;">DMI6000</td>
			<td>Imaging, Fluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:509px;">UVO Cleaner</td>
			<td>Jelight Company Inc</td>
			<td style="width:175px;">342</td>
			<td>Surface activation of the gut-chip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Type I collagen&#160;</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:175px;">A10483-01</td>
			<td>Extracellular matrix component for cell culture into the chip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel</td>
			<td style="width:227px;">BD</td>
			<td style="width:175px;">354234</td>
			<td>Extracellular matrix component for cell culture into the chip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mL disposable syringe</td>
			<td style="width:227px;">BD</td>
			<td style="width:175px;">309628</td>
			<td>Cell and media injection stuff</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25G5/8 needle</td>
			<td style="width:227px;">BD</td>
			<td style="width:175px;">329651</td>
			<td>Cell and media injection stuff</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe pump</td>
			<td>Braintree Scientific Inc.</td>
			<td style="width:175px;">BS-8000</td>
			<td>Injection equipment into the chip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VSL#3</td>
			<td>Sigma-Tau Pharmaceuticals</td>
			<td>7-45749-01782-6</td>
			<td>A formulation of 8 different commensal gut microbes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reinforced Clostridial Medium</td>
			<td style="width:227px;">BD</td>
			<td style="width:175px;">218081</td>
			<td>Anaerobic bacteria culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GasPak EZ Anaerobe Container System with Indicator</td>
			<td style="width:227px;">BD</td>
			<td style="width:175px;">260001</td>
			<td>Anaerobic gas generating sachet&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4% paraformaldehyde</td>
			<td>Electron Microscopy Science</td>
			<td style="width:175px;">157-4-100</td>
			<td>Fixing the cells for staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:175px;">T8787</td>
			<td>Permeabilizing the cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:175px;">A7030</td>
			<td>Blocking agent for staining of the cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corona treater</td>
			<td>Electro-Technic Products</td>
			<td>BD-20AC</td>
			<td>Plasma generator for fabrication of the chip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:509px;">Steriflip&#160;</td>
			<td style="width:227px;">Millipore</td>
			<td style="width:175px;">SE1M003M00</td>
			<td>Degasing the complete culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:509px;">Disposable hemocytometer</td>
			<td style="width:227px;">iNCYTO</td>
			<td style="width:175px;">DHC-N01</td>
			<td>For manual cell counting</td>
		</tr>
	</tbody>
</table>

co-culture of living microbiome with microengineered human intestinal villi in a gut-on-a-chip microfluidic device

Qui, descriviamo un protocollo per eseguire a lungo termine co-coltura di multi-specie intestino microbioma umano con villi intestinali microengineered in un dispositivo umano gut-on-a-chip microphysiological. Noi ricapitolare i intestinale interfaccia di tessuto lume-capillare in un dispositivo di microfluidica, dove deformazioni meccaniche fisiologiche e flusso di taglio fluido sono costantemente applicati per imitare la peristalsi. Nel microcanali lume, epiteliali intestinali Caco-2 cellule umane sono coltivate in modo da formare un epitelio villi &#39;germ-free&#39; e rigenerare piccoli villi intestinali. cellule microbiche pre-coltivate vengono inoculati nel lato lume di stabilire un ecosistema host-microbo. Dopo che le cellule microbiche aderiscono alla superficie apicale dei villi, flusso di fluido e deformazioni meccaniche sono ripresi per produrre un microambiente stato stazionario in cui terreno fresco viene costantemente alimentato e batteri non legato (nonché scarti batteriche) sono continuamente rimossi. Dopo estesa co-coltura fgiorni o settimane rom, più microcolonie sono trovati per essere collocata in modo casuale tra i villi, ed entrambi microbica e cellule epiteliali rimangono vitali e funzionale per almeno una settimana in coltura. Il nostro protocollo co-coltura può essere adattato a fornire una piattaforma versatile per altri ecosistemi ospitanti-microbiome che si possono trovare in vari organi umani, che potrebbe agevolare studio in vitro del ruolo del microbioma umano nell&#39;orchestrare la salute e la malattia.

Per emulare il intestinale ecosistema host-microbioma umano in vitro, è necessarie per sviluppare un protocollo sperimentale per ricostituire la stabilità a lungo termine co-coltura di batteri intestinali e le cellule epiteliali intestinali umane in condizioni fisiologiche come la meccanica peristalsi-simili e flusso del fluido. Qui, utilizziamo un biomimetico Microdevice gut-on-a-chip (Figura 1A) per co-coltura cellule microbiche a diret...

Watch this Scientific Journal Video about Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device at JoVE.com

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

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Stabilire coculture basate su una singola cellula in modo deterministico con un chip microfluidico

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Generazione di condizioni ambientali dinamiche utilizzando un dispositivo microfluidico ad alta produttività

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Preparation and Structural Evaluation of Epithelial Cell Monolayers in a Physiologically Sized Microfluidic Culture Device

Preparazione e valutazione strutturale di monostrati di cellule epiteliali in un dispositivo di coltura microfluidica di dimensioni fisiologiche

In vitro microfluidic experimentation holds great potential to reveal many insights into the microphysiological phenomena occurring in conditions such as ...