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.

Al forskning blev godkendt og gennemført under Virginia Tech IACUC retningslinjer 1. Klargør R-Spondin1 konditionerede medier fra HEK293T-Rspo1 cellelinje <ol><li> Generering af HEK293T-Rspondin1 celler er tidligere blevet beskrevet 10. Seed HEK293T-Rspondin1 secernerende celler ved 5-10% konfluens, ca. 8 x 10 5 -1.7 x 10 6 celler, i en T-175 kolbe med 40 ml 1x Dulbeccos Modified Eagle Medium (DMEM) + 10% kalvefosterserum ( FBS) som vækstmedierne, og inkuberes ved 37 ° C + ...

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Kulturen og vedligeholdelse af intestinale organoids er en procedure, der kan beherskes af enhver person med tilstrækkelig vævskultur teknik. Der er finesser i passage sammenlignet med voksende celler i en mere konventionel monolag, men disse finesser er ikke vanskelige at overvinde. De kritiske trin i denne fremgangsmåde involverer at kunne dyrke organoids til en høj nok densitet for optimal såning. Eksperimenter skal skaleres ned med organoids som store seeding tætheder, der kan ofte opnås med cellelinjer er ik...

Evnen til at høste og kultur primære organoids er blevet beskrevet til tyndtarmen, colon, pancreas, lever og hjerne og er spændende forskud germane at forstå en flere fysiologisk repræsentativt fænomener for vævs biologi 1-5. De første fremgangsmåder beskriver kulturen og vedligeholdelse af små intestinale organoids blev rapporteret af Sato et al. Ud af laboratoriet af Hans Clevers 1. Forud for denne fremgangsmåde, høst og kultur af primære intestinale epitelceller viste sig at være begrænsede og ineffektive i at opretholde epitelcellevækst. Fremgangsmåder inkluderet dissociation af vævet via inkubering med enzymer, såsom collagenase og dispase, som i sidste ende vil føre til udvæksten af sammenblandede primære fibroblastceller 6. Disse betingelser vil også blive tid begrænset til at opretholde den epithelcellekultur. Minimal til ingen epitelcelle niche ville danne, som epitelcellerne ville træde apoptose grundmanglen på egnede vækstfaktorer eller tab af kontakt integritet, betegnes anokis 7. Fremkomsten af 3D-organoide dyrkningssystem har tilvejebragt en fremgangsmåde til kultur primære intestinale celler indeholdende et spektrum af intestinale celletyper i vedvarende kultur 1. Disse epiteliale organoids har fordele i forhold cellelinier er, at de er sammensat af flere differentierede celler, og bedre efterligner organ, de er afledt af in vivo 8. Processen til i sidste ende &quot;vokse en mini gut i et fad&quot; har vist sig at være et værdifuldt redskab til at vurdere svaret fra tarmepitel under forskellige stimuli. Undersøgelse af samspillet af primære tarmceller med mikrobielle patogen forbundet molekylære mønstre (PAMPs) er relevant for området for immunologi, da disse molekylære mønstre kan regulere forskellige reaktioner fra både vært og mikrobe 9. Ikke alene kan efterforskerne nu udforske disse interaktioner med mus organoids, men dekan dyrkes fra mennesker samt to. Denne teknologi har potentiale til dramatisk ændre personlig medicin og det er fristende at spekulere over fremskridt, at denne teknik vil gøre det muligt i den nærmeste fremtid. Det overordnede mål med denne metode er at give en protokol for kulturen, ekspansion, og behandling af intestinale organoids med en række forskellige stimuli. Sådanne stimuli kan i sidste ende spænder fra vacciner, bakterielle PAMPs, levende patogener, gastrointestinale (GI) og kræftmedicin. Den isolering og dyrkning af mus tarm organoids er blevet tilpasset fra Sato et al. Selv om der er mindre afvigelser fra den oprindelige metode, slutproduktet bliver organoide kultur stadig opnås, når følger denne protokol. Denne metode er fokuseret på at beskrive en passende teknik til ordentlig normalisering, når der arbejdes med ikke-homogene cellestrukturer, som skal tages i betragtning, når der udføres en analyse baseret 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 intestinale organoids er et værdifuldt modelsystem, der har potentiale til at signifikant indvirkning inden for mucosal immunologi. Men kompleksiteten af ​​de organoide vækstkarakteristika bære betydelige forbehold for investigator. Specifikt er vækstmønstre enkelte organoide er meget varierende og skabe en heterogen population af epitelceller i kultur. Med sådanne forbehold, fælles vævskultur praksis kan ikke blot anvendes på organoide system på grund af kompleksiteten af ​​den cellulære struktur. Tælle og plating udelukkende baseret på celle nummer, som er fælles for individuelt adskilte celler, såsom cellelinjer, er ikke en pålidelig metode til organoids medmindre nogle normalisering teknik anvendes. Normalisering af det samlede proteinindhold er lavet kompleks på grund af beboeren protein matrix. bør tages Disse egenskaber med hensyn til celle nummer, form og celletype i betragtning, når de evaluerer udskilt contelte fra organoide masse. Denne protokol er blevet genereret at skitsere en enkel procedure til kultur og behandle små tarm organoids med mikrobielle patogener og patogen forbundet molekylære mønstre (PAMPs). Det understreger også normaliseringsreglerne teknikker, der skal anvendes, når protein analyse udføres efter sådan en udfordring.

Når du følger denne protokol til at dyrke tarm organoids vil karakteristiske sfære formede organoids være til stede efter høst. Tilføjelsen af ​​R-spondin1 konditionerede medier dagligt vil indlede vækst og spirende af organoids. Væksten af organoids er vist i figur 1A - F, og repræsenterer intestinale organoids på dag 1, 2, 4, 5, 6 og dag 14. Figur 1F repræsenterer de ikke-homogene vækstkarakteristika af...

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 protokol til at høste, vedligeholde og behandle mus små tarm organoids med patogen forbundet molekylære mønstre (PAMPs) og Listeria monocytogenes beskrevet, samt fokus på genekspression og ordentlig normalisering teknikker til protein.

Det<em&gt; Ex vivo</em&gt; Kultur og Pattern Recognition Receptor Stimulering af Mouse Intestinal 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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