Name: the ex vivo culture and pattern recognition receptor stimulation of mouse intestinal organoids
Uploaded: 2016-05-18T15:00:00
Description: Watch this Scientific Journal Video about The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids at JoVE.com

The authors would like to thank Dr. Sheryl Coutermarsh-Ott, Dylan McDaniel and Bettina Heid for technical discussions. The authors thank Dr. Nanda Nanthakumar for providing the Caco-2 cells. The authors also thank The Multicultural Academic Opportunities Program (MAOP) at Virginia Tech. This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Award K01DK092355 (to I.C.A.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

All forskning ble godkjent og gjennomført under Virginia Tech IACUC retningslinjer 1. Forbered R-Spondin1 Betinget Media Fra HEK293T-Rspo1 cellelinje <ol><li> Generering av HEK293T-Rspondin1 celler er blitt beskrevet tidligere 10. Seed HEK293T-Rspondin1 utsondrende celler ved 5-10% konfluens, ca 8 x 10 5 -1.7 x 10 6 celler, i en T-175-kolbe med 40 ml 1 x Dulbeccos modifiserte Eagle-medium (DMEM) + 10% føtalt bovint serum ( FBS) i vekstmediet, og inkuber ved 37 ° C + 5%<...

<ol>
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E., Giangreco, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+the+relevance+of+human+stem+cell-derived+organoid+models+for+epithelial+translational+medicine.">Concise review: the relevance of human stem cell-derived organoid models for epithelial translational medicine.</a> Stem Cells. 31 (3), 417-422 (2013).</li><li>Kaiko, G. E., Stappenbeck, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-microbe+interactions+shaping+the+gastrointestinal+environment.">Host-microbe interactions shaping the gastrointestinal environment.</a> Trends Immunol. 35 (11), 538-548 (2014).</li><li>Kim, K. 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Kulturen og vedlikehold av tarm organoids er en prosedyre som kan mestres av enhver person med tilstrekkelig vev kultur teknikk. Det er Raffinert i aging når sammenlignet med voksende celler i en mer konvensjonell monolag, men disse finesser er ikke vanskelig å overvinne. De kritiske trinnene i denne fremgangsmåten involverer å være i stand til å vokse organoids til en tilstrekkelig høy tetthet for optimal poding. Eksperimenter må skaleres ned med organoids som store seeding tettheter som kan vanligvis oppnås m...

Evnen til å høste og kultur primære organoids er blitt beskrevet for tynntarm, tykktarm, bukspyttkjertel, lever og hjerne og er spennende fremskritt relevante for forståelsen en mer fysiologisk fenomen representative for vev biologi 1-5. Den første fremgangsmåter som beskriver kulturen og vedlikehold av tynntarms organoids ble rapportert av Sato et al., Ut av laboratoriet til Hans Clevers 1. Forut for denne metoden, høsting og kultur av primære intestinale epitelceller viste seg å være begrenset og ineffektiv i å opprettholde epitelial cellevekst. Fremgangsmåter følger dissosiasjon av vev via inkubering med enzymer, så som kollagenase og dispase, som til slutt ville føre til utveksten av blandede primære fibroblastceller 6. Disse forholdene vil også bli tid begrenset i å opprettholde den epiteliale cellekultur. Minimal til ingen epitelcelle nisje vil danne, som epitelcellene ville komme inn på grunn av apoptoseav mangel på egnede vekstfaktorer eller tap av kontakt integritet, betegnet anokis 7. Ankomsten av den 3D-organoid kultursystem er tilveiebrakt en fremgangsmåte for å dyrknings primære tarmceller som inneholder et spektrum av tarmcelletyper i kultur vedvarende 1. Disse epiteliale organoids ha fordeler i forhold til cellelinjer er at de er sammensatt av flere differensierte celler, og bedre etterligner organet de er avledet fra in vivo 8. Prosessen til slutt &quot;vokse en mini gut i en skål&quot; har vist seg å være et verdifullt verktøy for å vurdere responsen tarmepitelet under forskjellige stimuli. Gransker samspillet mellom primærtarmceller med mikrobielle patogener forbundet molekylære mønstre (PAMPs) er relevant til feltet av immunologi som disse molekylære mønstre kan regulere ulike svar fra både vert og mikrobe 9. Ikke bare kan etterforskerne nå utforske disse interaksjoner med muse organoids, men dekan dyrkes fra mennesker som vel to. Denne teknologien har potensial til å dramatisk endre personlig medisin, og det er fristende å spekulere om fremskritt at denne teknikken vil gjøre mulig i nær fremtid. Det overordnede målet med denne metoden er å tilveiebringe en protokoll for kulturen, ekspansjon, og behandling av tarm organoids med en rekke stimuli. Slike stimuli kan til slutt være alt fra vaksiner, bakterielle PAMPs, live patogener, gastrointestinal (GI) og kreftbehandling. Isoleringen og kultur av mus tarm organoids har blitt tilpasset fra Sato et al. Selv om det er mindre avvik fra den opprinnelige metoden, at sluttproduktet blir organoid kulturen er fremdeles oppnås ved å følge denne protokoll. Denne metoden er fokusert på å beskrive en tilstrekkelig teknikk for riktig normalisering når du arbeider med ikke-homogene cellestrukturer, som må tas i betraktning når man gjennomfører en analyse basert på celle numbra.

<table>
	<tbody>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11050</td>
			<td>(Section 1,3,6) Or equivalent brand</td>
		</tr>
		<tr>
			<td>Sorvall Legend XTR Centrifuge</td>
			<td>Thermo</td>
			<td></td>
			<td>(Section 1,3)</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>GE Healthcare</td>
			<td>Sh30243.01</td>
			<td>(Section 1,6) For Caco-2 and HEK293 Rspondin1 cells</td>
		</tr>
		<tr>
			<td>HEK293T-Rspondin1 secreting cell line</td>
			<td></td>
			<td></td>
			<td>(Section 1) Described and modified from Kim, K.A. et al. Lentiviral particles contained RSPO1(NM_138683) ORF cDNA cloned into a pReceiver-Lv105 backbone custom ordered and purchased from GeneCopoeia.&#160;</td>
		</tr>
		<tr>
			<td>50 ml conical tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td>(Section 1) Or equivalent brand</td>
		</tr>
		<tr>
			<td>T-175 Flask</td>
			<td>Corning</td>
			<td>431079</td>
			<td>(Section 1) Or equivalent brand&#160;</td>
		</tr>
		<tr>
			<td>Protein Matrix</td>
			<td>Corning</td>
			<td>356231</td>
			<td>(Section 2,3,4,5,6) Matrigel Growth Factor Reduced&#160;</td>
		</tr>
		<tr>
			<td>HyClone Dulbecco&#39;s (DPBS)</td>
			<td>GE Healthcare</td>
			<td>SH30264.01</td>
			<td>(Section 2,3)</td>
		</tr>
		<tr>
			<td>DMEM/F12&#160;</td>
			<td>Life Technologies</td>
			<td>12634-010</td>
			<td>(Section 2,3) Advanced DMEM/F12</td>
		</tr>
		<tr>
			<td>Corning 24 Well TC Plates</td>
			<td>Corning</td>
			<td>3524</td>
			<td>(Section 2)</td>
		</tr>
		<tr>
			<td>N2 Supplement 100x&#160;</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td>(Section 2)</td>
		</tr>
		<tr>
			<td>B27 without vitamin A 50x&#160;</td>
			<td>Life Technologies</td>
			<td>12587-010&#160;</td>
			<td>(Section 2)</td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Life Technologies</td>
			<td>15596-026</td>
			<td>(Section 2)</td>
		</tr>
		<tr>
			<td>Glutamine Supplement (Glutamax)</td>
			<td>Life Technologies</td>
			<td>35050-061</td>
			<td>(Section 2) Can Combine with Advanced DMEM/ F12</td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Life Technologies</td>
			<td>15630-080</td>
			<td>(Section 2) Can Combine with Advanced DMEM/ F12&#160;</td>
		</tr>
		<tr>
			<td>10ml Serological Pipet</td>
			<td>Falcon</td>
			<td>357551</td>
			<td>(Section 2) Or equivalent brand</td>
		</tr>
		<tr>
			<td>Murine Noggin</td>
			<td>Peprotech</td>
			<td>250-38</td>
			<td>(Section 2) Stock = 100 mg/ml</td>
		</tr>
		<tr>
			<td>N-Acetyl-L-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td>(Section 2) Stock = 1M</td>
		</tr>
		<tr>
			<td>Recombinant Mouse EGF</td>
			<td>Biolegend</td>
			<td>585608</td>
			<td>(Section 2) Stock = 500 mg/ml</td>
		</tr>
		<tr>
			<td>Rocker Variable</td>
			<td>Bioexpres</td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>dissecting scissors</td>
			<td></td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td></td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>glass slides</td>
			<td></td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>dissecting tweezers</td>
			<td></td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>25 ml Serological Pipet</td>
			<td>Falcon</td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>EDTA&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>SLBB9821</td>
			<td>(Section 3) 0.5M or alternative TC grade EDTA</td>
		</tr>
		<tr>
			<td>Sterile Petri Dish 100mm x 15mm</td>
			<td>Fisher</td>
			<td>FB0875712</td>
			<td>(Section 3) Or equal sized TC dish</td>
		</tr>
		<tr>
			<td>1ml Syringe</td>
			<td>Becton Dickinson</td>
			<td>309659</td>
			<td>(Section 4)</td>
		</tr>
		<tr>
			<td>Precision Glide Needle</td>
			<td>Becton Dickinson</td>
			<td>305120</td>
			<td>(Section 4) 23G x 1 1/4 (0.6mm x 30mm)</td>
		</tr>
		<tr>
			<td>Flagellin from Bacillus subtilis</td>
			<td>Invivogen</td>
			<td>tlrl-bsfla&#160;</td>
			<td>(Section 5,6)</td>
		</tr>
		<tr>
			<td>Listeria monocytogenes</td>
			<td>ATCC</td>
			<td>19115</td>
			<td>(Section 5,6)&#160;(Murray&#160;et&#160;al.)&#160;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629-1EA</td>
			<td>(Section 5,6)Or equivalent brand</td>
		</tr>
		<tr>
			<td>BBL Brain Heart Infusion Agar</td>
			<td>Becton Dickinson</td>
			<td>211065</td>
			<td>(Section 5)</td>
		</tr>
		<tr>
			<td>Bacto Brain Heart Infusion</td>
			<td>Becton Dickinson</td>
			<td>237500</td>
			<td>(Section 5)</td>
		</tr>
		<tr>
			<td>Caco-2</td>
			<td>ATCC</td>
			<td>HTB-37</td>
			<td>(Section 6)</td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>gibco</td>
			<td>25200056</td>
			<td>(section 6)</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>A412-4</td>
			<td>(Section 6)</td>
		</tr>
		<tr>
			<td>SpectraMax M5</td>
			<td>Molecuar Devices</td>
			<td></td>
			<td>(Section 6)</td>
		</tr>
		<tr>
			<td>96 Well Assay Plate</td>
			<td>Corning</td>
			<td>3603</td>
			<td>(Section 6) Black Plate, Clear Bottom TC treated</td>
		</tr>
		<tr>
			<td>Nuclear Staining Dye</td>
			<td>Life Technologies</td>
			<td>H1399</td>
			<td>(section 6) Hoechst 33342</td>
		</tr>
		<tr>
			<td>T-75 Flask</td>
			<td>Corning</td>
			<td>430641</td>
			<td>(Section 6) Or equivalent brand</td>
		</tr>
		<tr>
			<td>15 ml conical tube</td>
			<td>Falcon</td>
			<td>352096</td>
			<td>(Section1,3) Or equivalent brand</td>
		</tr>
		<tr>
			<td>1.7 ml polypropylene tube</td>
			<td>Bioexpress</td>
			<td>C-3262-1</td>
			<td>Or equivalent brand</td>
		</tr>
		<tr>
			<td>Quick-RNA MiniPrep</td>
			<td>Zymo Research</td>
			<td>R1054</td>
			<td>Or equivalent brand</td>
		</tr>
		<tr>
			<td>TNF-alpha&#160;</td>
			<td>Applied Biosystems</td>
			<td>Mm 00443260_g1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>IL-6&#160;</td>
			<td>Applied Biosystems</td>
			<td>(Mm 00446190_m1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>IL-1beta</td>
			<td>Applied Biosystems</td>
			<td>Mm 00434228_m1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>IL-18</td>
			<td>Applied Biosystems</td>
			<td>Mm 00434225_m1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>18s</td>
			<td>Applied Biosystems</td>
			<td>Hs 99999901_s1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>7500 Fast Real Time PCR System</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nexus gradient Mastercycler</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Fast Universal PCR Master Mix</td>
			<td>Life Technologies</td>
			<td>4352042</td>
			<td></td>
		</tr>
		<tr>
			<td>High Capacity cDNA Reverse Transcription Kit</td>
			<td>Life Technologies/Applied Biosystems</td>
			<td>4368814</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Optical 96-Well Reaction Plate, 0.1 mL</td>
			<td>Life Technologies/Applied Biosystems</td>
			<td>4346907</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse R-Spondin 1 Protein</td>
			<td>R&#38;D Systems</td>
			<td>3474-RS-050</td>
			<td>500 ng/ml</td>
		</tr>
		<tr>
			<td>chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>C7559</td>
			<td></td>
		</tr>
	</tbody>
</table>

the ex vivo culture and pattern recognition receptor stimulation of mouse intestinal organoids

Primære intestinal organoids er en verdifull modellsystem som har potensial til å betydelig påvirke feltet av slimhinne immunologi. Men kompleksiteten i de organoid vekstegenskaper bære betydelige begrensninger for etterforsker. Spesifikt vekstmønster for hver individuell organoid er svært variabel og skape en heterogen populasjon av epitel-celler i kultur. Med slike betingelser, felles vevskultur praksis, kan ikke bare brukes på organoid systemet på grunn av kompleksiteten av cellestrukturen. Opptelling og Plating basert utelukkende på cellenummer, som er felles for individuelt separerte celler, slik som cellelinjer, er ikke en pålitelig metode for organoids med mindre noe normalisering teknikk er anvendt. Normalisering til totalt proteininnhold er gjort komplekse på grunn av den fastboende proteinmatriks. Disse egenskapene når det gjelder cellenummer, form og celletype bør tas i betraktning ved vurdering av utskilt contelt fra organoid masse. Denne protokollen er blitt generert for å skissere en enkel prosedyre til kultur og behandle tynntarms organoids med mikrobielle patogener og patogen assosiert molekylære mønstre (PAMPs). Det understreker også normaliserings teknikker som skal brukes når protein analyse gjennomføres etter en slik utfordring.

Når du følger denne protokollen for å dyrke tarm organoids, vil karakteristiske sfære formet organoids være tilstede etter høsting. Tilsetting av R-spondin1 klimaanlegg media daglig vil starte vekst og spirende av organoids. Veksten av organoids er vist i Figur 1A - F, og er representative for tarm organoids på dagene 1, 2, 4, 5, 6 og dag 14. Figur 1F viser de ikke-homogene vekstkarakteristika av organoids på dag...

Watch this Scientific Journal Video about The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids at JoVE.com

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Her er en protokoll for å høste, vedlikeholde og behandle mus tynntarms organoids med patogen forbindelse molekylmønstre (PAMPs) og Listeria monocytogenes er beskrevet, så vel som vekt på genekspresjon og riktig normaliserings teknikker for protein.

De<em&gt; Ex Vivo</em&gt; Kultur og mønstergjenkjenning Receptor Stimulering av Mouse Tarm Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

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Immunology and Infection

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

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