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. mikrofabrikation af en Gut-on-a-chip-enhed Bemærk: gut-on-a-chip er en mikrofluidanordning lavet af gennemsigtigt, gaspermeabelt siliconepolymer (polydimethylsiloxan, PDMS), der indeholder to parallelle mikrokanaler (1 mm bredde x 150 um højde x 1 cm længde) adskilt af en fleksibel porøs (10 um i porediameter, 25 um i afstand pore pore) PDMS membranen 5,9. Fabrikere tarmen-on-a-chip (figur 1A, venstre) efter de angivne trin. <ol><li> Mikrofabrikation Proc...

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Forståelse host-microbiome interaktioner er afgørende for at fremme medicin; imidlertid har traditionelle cellekulturmodeller udført i en plastik skål eller en statisk brønds plade ikke understøtter den stabile co-dyrkning af humane tarmceller med levende gut mikrober i mere end 1-2 dage, fordi mikrobielle celler meste overgrow de mammale celler in vitro. Den tilgroning mikrobielle population hurtigt forbruger ilt og næringsstoffer, efterfølgende at producere stor mængde af metaboliske affald (f.ek...

Den humane tarm huser et fantastisk forskelligartet array af mikrobielle arter (&lt;1.000 arter) og en enorm række af mikrobielle celler (10 gange mere end de humane værtsceller) og gener (100 gange mere end det humane genom) 1. Disse humane microbiomes spiller en central rolle i metabolizing næringsstoffer og xenobiotika, regulering immunresponser, og opretholde intestinal homeostase 2. Ikke overraskende, givet disse forskellige funktioner, den kommensale tarm microbiome modulerer udstrakt sundhed og sygdom 3. Således at forstå betydningen af tarmen microbiome og vært-mikrobe interaktioner er af stor betydning for at fremme gastrointestinal (GI) sundhed og udforske nye lægemidler til tarmlidelser 4. Men de eksisterende in vitro tarmen modeller (f.eks, statiske kulturer) begrænse host-microbiome co-kultur til en kort periode (&lt;1 dag), fordi mikrobielle celler gror og kompromittere tarmbarrierenfunktion 5. Surrogat dyremodeller (f.eks kim-fri 6 eller genetisk manipulerede mus 7) også ikke almindeligt anvendt til at studere vært-gut microbiome krydstale fordi kolonisering og stabil opretholdelse af menneskets tarme microbiome er vanskelige. For at overvinde disse udfordringer, vi for nylig udviklet en biomimetisk menneske &quot;Gut-on-a-chip&quot; microphysiological (figur 1A, venstre) at efterligne vært-gut microbiome samspil, der opstår i levende menneske tarm 5,8. Tarmen-on-a-chip microdevice indeholder to parallelle mikrofluidkanaler adskilt af en fleksibel, porøs, ekstracellulær matrix (ECM) overtrukne membran foret med human intestinal epitel Caco-2 celler, der efterligner den intestinale lumen-kapillære væv interface (figur 1A , højre) 9. Vakuum-drevet cykliske rytmiske deformationer fremkalde fysiologiske mekaniske deformationer, der efterligner ændringer normalt induced ved peristaltik (figur 1A, højre). Interessant, når Caco-2-celler dyrkes i tarmen-on-a-chip til mere end 100 timer, de spontant danner tredimensionale (3D) intestinal villi med tight junctions, apikale børste grænser, proliferative celler begrænset til basale krypter, slimproduktion, øget stofmetaboliserende aktivitet (fx cytochrom P450 3A4, CYP3A4), og øget glucose reuptake 8. I denne &quot;kim-fri&quot; mikromiljø, var det muligt at co-kultur den probiotiske Lactobacillus rhamnosus GG eller et terapeutisk dannelse af en probiotisk bakterie blanding med vært epitelceller i op til to uger 5,10. I denne undersøgelse beskriver vi detaljeret protokol til at udføre host-gut microbiome co-kultur i tarmen-on-a-chip enhed i en længere periode. Desuden diskuterer vi kritiske spørgsmål og potentielle udfordringer, der skal i betragtning til en bred anvendelse af denne vært-microbiome co-kultur protokol.

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			<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>
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		<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>
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		<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>
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		<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>
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		<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

Her beskriver vi en protokol til at udføre langsigtede co-kultur af flere arter menneskelige tarm microbiome med microengineered intestinale villi i en menneskelig gut-on-a-chip microphysiological enhed. Vi rekapitulere den intestinale lumen-kapillære væv grænseflade i en mikrovæskeanordning, hvor fysiologiske mekaniske deformationer og flow væske shear konstant anvendes til at efterligne peristaltikken. I lumen mikrokanalplade, humane intestinale epiteliale Caco-2 celler dyrkes for at danne en &quot;kim-fri&quot; villus epithelium og regenerere tyndtarmen villi. Pre-dyrkede mikrobielle celler podes i lumen side at etablere en vært-mikrobe økosystem. Efter mikrobielle celler klæbe til den apikale overflade af villi, er fluidstrømning og mekaniske deformationer genoptaget for at fremstille en stabil mikromiljø, hvori frisk dyrkningsmedium konstant tilføres og ubundne bakterier (samt bakterielle affald) fjernes kontinuerligt. Efter forlænget co-kultur from dage til uger, findes flere mikrokolonier at være tilfældigt placeret mellem villi, og begge mikrobielle og epitelceller forbliver levedygtige og funktionelle i mindst en uge i kultur. Vores co-kultur protokol kan tilpasses til at tilvejebringe en alsidig platform for andre værtsplanter-microbiome økosystemer, der kan findes i forskellige humane organer, der kan lette in vitro-undersøgelse af den rolle af humant microbiome for tilrettelæggelsen sundhed og sygdom.

At efterligne det humane intestinale vært-microbiome økosystem in vitro, er det nødvendigt at udvikle en eksperimentel protokol til at rekonstituere den stabile langsigtede co-kultur af tarmbakterier og humane intestinale epitelceller under fysiologiske betingelser, såsom peristaltik-lignende mekanik og fluidstrømning. Her anvender vi en biomimetisk gut-on-a-chip microdevice (Figur 1A) til at co-kultur levende mikrobielle celler i dire...

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

Vi beskriver en in vitro-protokol til at co-kultur gut microbiome og intestinal villi i en længere periode ved anvendelse af en human gut-on-a-chip microphysiological system.

Co-kultur af Living microbiome med Microengineered Menneskelig Intestinal Villi i en Gut-on-a-Chip mikrofluidanordning

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

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

Generering af dynamiske miljøforhold ved hjælp af en mikrofluidisk højoverførselsenhed

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

Forberedelse og strukturel evaluering af epitelcellemonolag i en fysiologisk størrelse mikrofluidisk kulturanordning

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