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. microfabrication av en Gut-on-a-chip enhet Merk: gut-på-en-brikke er et mikrofluid enhet laget av gjennomsiktig gass-permeabel silikonpolymer (polydimetylsiloksan, PDMS), inneholdende to parallelle mikrokanaler (1 mm bredde x 150 pm høyde x 1 cm lengde) adskilt av en fleksibel porøs (10 mikrometer i pore diameter, 25 mikrometer i avstand pore å pore) PDMS membran 5,9. Dikte gut-on-a-chip (figur 1A, venstre) å følge trinnene. <ol><li> Microfabrication Pr...

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Forstå verts mikrobiomer interaksjoner er avgjørende for å fremme medisin; imidlertid ikke tradisjonelle cellekultur modeller utført i en plastskål eller en statisk brønn plate støtter ikke stabil co-kultur av humane tarmceller med levende gut mikrober i mer enn 1-2 dager fordi mikrobielle celler hovedsakelig overgrow pattedyrceller in vitro. Den gjengroing mikrobiell befolkning forbruker raskt oksygen og næringsstoffer, deretter produsere overdreven bruk av metabolske avfall (f.eks, organiske ...

Den menneskelige tarmen havner en utrolig variert utvalg av mikrobielle arter (&lt;1000 arter) og en enorm rekke mikrobielle celler (10 ganger mer enn de menneskelige vertsceller) og gener (100 ganger mer enn det menneskelige genom) 1. Disse menneskelige microbiomes spille en nøkkelrolle i metabolizing næringsstoffer og xenobiotics, regulerer immunresponser, og opprettholde intestinal homeostase to. Ikke overraskende, gitt disse ulike funksjoner, den commensal gut mikrobiomer modulerer omfattende helse og sykdom 3. Dermed forstå rollen av tarmen mikrobiomer og vert-mikrobe interaksjoner er av stor betydning for å fremme gastrointestinal (GI) helse og utforske nye behandlingsformer for tarmsykdommer 4. Men eksisterende in vitro tarmen modeller (f.eks statiske kulturer) begrense host-mikrobiomer co-kultur til en kort periode (&lt;1 dag) fordi mikrobielle celler overgrow og kompromiss intestinal barrierefunksjon 5. Surrogat dyremodeller (for eksempel bakteriefritt 6 eller genmodifisert mus 7) er heller ikke vanlig å studere host-gut mikrobiomer crosstalk fordi kolonisering og stabil vedlikehold av menneskelige tarmen mikrobiomer er vanskelig. For å overvinne disse utfordringene, vi nylig utviklet en biomimetic menneskelige &quot;Gut-on-a-chip&quot; microphysiological system (figur 1A, venstre) for å emulere host-gut mikrobiomer interaksjoner som oppstår i levende menneske tarmen 5,8. Tarmen-on-a-chip microdevice inneholder to parallelle microfluidic kanaler adskilt av en fleksibel, porøs, ekstracellulære matrix (ECM) belagte membran omgitt av menneskets tarm epitel Caco-2 celler, etterligne intestinal lumen-kapillær vev-grensesnitt (figur 1A , høyre) 9. Vakuum-drevet sykliske rytmiske deformasjoner tale fysiologiske mekaniske deformasjoner som etterligner endringer normalt induced av peristaltikk (figur 1A, høyre). Interessant, da Caco-2 celler dyrkes i tarmen-on-a-chip for mer enn 100 timer, de spontant danne tredimensjonale (3D) tarmtottene med tett veikryss, apikale pensel grenser, proliferative celler begrenset til basale krypter, slimproduksjon, økt metaboliserende aktivitet (f.eks cytokrom P450 3A4, CYP3A4), og forbedret glukose reuptake 8. I denne &quot;bakteriefritt&quot; mikromiljøet, var det mulig å co-kulturen den probiotiske Lactobacillus rhamnosus GG eller en terapeutisk dannelsen av en probiotiske bakterier blanding med verts epitelceller i opptil to uker 5,10. I denne studien, beskriver vi detaljert protokoll for å utføre vert-gut mikrobiomer ko-kultur i tarm-på-en-brikke anordning for en lengre periode. I tillegg diskuterer vi kritiske spørsmål og potensielle utfordringer for å bli vurdert for en bred anvendelse av denne host-mikrobiomer co-kultur protocol.

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		<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

Her beskriver vi en protokoll for å utføre langsiktig co-kultur av multi-art menneskelige tarmen mikrobiomer med mikro-oppbygd tarmtottene i en menneskelig gut-on-a-chip microphysiological enhet. Vi rekapitulere tarm lumen-kapillære vev-grensesnitt i en microfluidic enhet, der fysiologiske mekaniske deformasjoner og væske skjærstrømmen blir stadig søkt å etterligne peristaltikk. I lumen microchannel, humane intestinale epitelceller Caco-2-celler dyrkes for å danne en &quot;bakterie-fri&quot; villus epitel og regenerere liten tarmtottene. Pre-dyrkede mikrobielle celler inokuleres inn i hulrommet side for å etablere en vert-mikrobe økosystem. Etter at mikrobielle celler holder seg til den apikale overflate av villi, blir fluidstrømningen og mekaniske deformasjoner gjenopptatt for å fremstille en stabil tilstand mikromiljø i hvilket friskt kulturmedium blir stadig tilføres og ubundet bakterier (så vel som bakterielle avfall) fjernes kontinuerlig. Etter lengre co-kultur fROM dager til uker, blir flere mikrokoloniene funnet å være tilfeldig plassert mellom villi, og både mikrobielle og epitelceller forblir levedyktig og funksjonell i minst en uke i kultur. Vår co-kulturen protokollen kan tilpasses for å gi en allsidig plattform for andre verts mikrobiomer økosystemer som finnes i ulike organer, som kan legge til rette for in vitro studie av hvilken rolle menneskelige mikrobiomer i orkestre helse og sykdom.

For å etterligne den menneskelige tarm host-mikrobiomer økosystem in vitro, er det nødvendig å utvikle en forsøksprotokoll for å rekonstituere den stabile langtids ko-kultur av tarmbakterier og humane intestinale epitelceller under fysiologiske betingelser slik som peristaltikk lignende mekanikk og fluidstrømmen. Her benytter vi et biomimetic gut-on-a-chip microdevice (Figur 1A) til co-kultur levende mikrobielle celler i direkte kont...

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-protokoll for å ko-kultur gut mikrobiomer og tarmtottene i en lengre periode ved hjelp av et humant tarm-på-en-brikke microphysiological system.

Co-kultur of Living mikrobiomer med mikro-oppbygd Menneskelig tarmtotter i en Gut-on-a-Chip mikrofluid Device

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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Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Etablering single-Cell basert co-kulturer i en deterministisk måte med en Mikrovæskebasert chip

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

Generering av dynamiske miljøforhold ved hjelp av en mikrofluidisk enhet med høy gjennomstrømning

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 strukturell evaluering av 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 ...