Name: in vitro organoid culture of primary mouse colon tumors
Uploaded: 2013-05-17T12:00:00
Description: Watch this Scientific Journal Video about In vitro Organoid Culture of Primary Mouse Colon Tumors at JoVE.com

Denne undersøgelse blev støttet af tilskud til YMS fra National Institutes of Health (CA148828), The University of Michigan Gastrointestinal Peptid Center og Jeffrey A. Colby Colon Cancer Research og Tom Liu Memorial Funds fra University of Michigan Comprehensive Cancer Center.

1.. Colontumor Isolering og celledissociering <ol><li> Intestinal tumorer kan isoleres fra enhver sporadisk eller behandling-induceret tyktarmskræft model. Musene bør aflives med CO2. Kolon, opsamles derefter, skylles med koldt phosphatbufret saltvand (PBS) og åbnet på langs. Identificer regioner, der indeholder tumorer ved hjælp af et stereomikroskop, dissekere ud med en saks, og vask med kold PBS. </li><li> Inkuber tarm fragmenter indeholdende tumorer i EDTA chelation puffer (2 mM EDTA, 5,6 mmol / L ...

<ol>
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De eksperimentelle procedurer, der beskrives i denne protokol vil give mulighed for isolering og dyrkning af primære murine colontumorer. Protokollen er tilpasset fra banebrydende arbejde udført af Dr. Clevers gruppe 1,24,27. Vi optimeret fordøjelse tid og collagenase koncentration for at få et bedre udbytte af tumor organoids. De kritiske trin omfatter tumorcelle fordøjelse i enkelte celler, Matrigel resuspension, og selektiv kultur. Tumor-celle fordøjelse, for at opnå en effektiv adskillelse af colon...

De tarmepithelceller formere og slå over på en ekstraordinær sats, overgår alle andre væv i hvirveldyr kroppen 2,3. De delende celler, herunder tarm stamceller (ISC) og transit-forstærkende celler differentierer til enten sekretoriske (bæger, Paneth og enteroendocrine) celler eller enterocyterne 3. ISC er placeret i bunden af ​​krypten. Paneth celler bevæger sig ned til bunden af krypter og lever længe, ​​mens andre slægter migrere opad til villi 3,4. Her cellerne udsættes for tarmindholdet herunder mikrobiota og er kastet fra villus tip gennem en anoikis-induceret apoptotisk mekanisme. Selv tyktarmen mangler villi og Paneth celler, mekanismen for at opretholde homeostase ligner 4.. Wnt signalvejen har været impliceret i at spille en afgørende rolle i den intestinale spredning og ISC vedligeholdelse 4.. Sletning af the transskription faktor TCF4, en downstream effektor af Wnt signalering, fører til tab af tarm stamceller og efterfølgende opdeling af vævet 5.. Ligeledes transgen ekspression af Wnt inhibitor kr.1 reducerer epitelproliferation og udtømning sekretoriske celleafstamninger 6.. Omvendt overekspression af Wnt agonist R-spondin-1 inducerer potent og hurtige udbredelse af intestinale kryptceller 7. Betragtning af betydningen af Wnt signalering til tarm homeostase er Wnt pathway mutationer observeret hyppigere i tyktarmskræft 8. Tyktarmskræft er den tredje hyppigste årsag til kræftdødsfald i USA 9.. Overskydende kosten med rødt kød og alkohol, nedsat fysisk aktivitet, og nedarvede og somatiske mutationer anses for at være risikofaktorer for tyktarmskræft 10,11. Den adenomatøs polypose coli (APC)-gen, en nøgle Wnt signalering faktor, er muteret i et flertal af patienter med familiær, sporadisk og colitis-associeret tyktarmskræft 12,13. Mutationer af andre faktorer involveret i Wnt signalvejen herunder Axin2 og β-catenin er også observeret i tyktarmskræft 14,15. Men den præcise mekanisme og effektiv behandling for tyktarmskræft mangler stadig. For at lette efterforskningen af de molekylære mekanismer for tyktarmskræft, har humane coloncancer cellelinier, der repræsenterer de forskellige stadier af kræft progression fra en godartet til en aggressiv celletype blevet etableret 16-18. Mus coloncarcinomceller cellelinier med forskellige metastatiske egenskaber er også tilgængelige 19,20. Alligevel er de primære celler eller organoid kulturer foretrækkes frem transformerede cellelinier, fordi de nøje efterligner in vivo tilstand og generere mere fysiologisk relevant data 21.. De fleste kolon kræft afledte cellelinjer vokse som monolag fastgjort til plade eller som celle suspensioner, Lacking apikal-basolateral orientering og tætte forbindelser mellem cellerne. Også normale og tumor tarmepitelceller in vivo undergår en spontan form for apoptose betegnes anoikis, som de differentierede celler nå villus tips og kaste 22. Disse funktioner er vanskelige at rekapitulere i cellelinier, men er vigtige i den udviklingsmæssige proces for tyktarmskræft 23.. Disse funktioner er opretholdt i primære organoids. Desuden giver tumor organoid kulturer et effektivt system til at vurdere tumor autonome funktioner af gener i forhold til in vivo studier. Genetisk manipulation in vivo af tarmen er en tidskrævende proces hovedsagelig ved at skabe transgene og / eller knockout-mus ved hjælp af tarmen-specifikke drivere. Men tumor organoids er let modtagelig for virale medieret genetiske manipulationer, og dermed et godt redskab til at vurdere præcise molekylære mekanismer. Primære tarm tumor organoid kulturer HAVe blevet påvist at være en gennemførlig og kraftfuld teknik. Primær tarm cellekultur kan etablere funktionelle tarm organoids med krypt-villi struktur in vitro fra en enkelt voksen Lgr5 + stamceller 24.. Disse organoids kan transplanteres og indpodet i det beskadigede tyktarmsvæv til regenerering 25.. Yderligere tilpasning af de dyrkningsbetingelser havde gjort lignende epitelial organoids fra muse colon og humane tyndtarm og tyktarm gennemførlig 1.. For primær normal colon epitel kultur, naeringsoploesning samt vækstfaktorer, herunder EGF, Noggin, R-spondin og Wnt3a er afgørende, mens naeringsoploesning og EGF er tilstrækkelig til dyrkning primære muse colontumorceller organoids 1.. Her beskriver vi en detaljeret protokol for at isolere, kultur og generere colontumorceller organoids.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane Matrix</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td>5 mg/ml</td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Worthington</td>
			<td>LS004188</td>
			<td>375 U/mg</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td>1.8 U/mg</td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12634010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor (EGF), Murine, Natural</td>
			<td>Invitrogen</td>
			<td>53003-018</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
			<td>100 x</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>50 x</td>
		</tr>
		<tr>
			<td>Glutamax-I</td>
			<td>Gibco</td>
			<td>35050-079</td>
			<td>100x</td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma</td>
			<td>A9165-5G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Modified Eagle Medium</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PicoPureTM RNA Isolation Kit</td>
			<td>Invitrogen</td>
			<td>KIT0204</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

in vitro organoid culture of primary mouse colon tumors

Adskillige humane og murine coloncancer cellelinier er blevet etableret, er fysiologisk integritet tyktarmstumorer såsom flere cellelag, basal-apikal polaritet, evne til at skelne, og anoikis ikke vedligeholdes i tyktarmskræft afledte cellelinier. Den foreliggende undersøgelse viser en metode til dyrkning af primære muse colontumorceller organoids tilpasset fra Sato T et al. 1, som bevarer vigtige fysiologiske funktioner i colontumorer. Denne metode består af muse kolon tumorvæv indsamling, tilstødende normale colon epitel dissociation colontumorceller fordøjelse i enkelte celler, indlejring colontumorceller i Matrigel, og selektiv kultur baseret på princippet om, at tumorceller fastholde væksten på begrænsende næringsstof betingelser sammenlignet med normale epitelceller. Den primære tumor organoids hvis isoleret fra genetisk modificerede mus giver et meget nyttigt system til at vurdere tumor autonome funktion af specifikke gener. Desuden tumor organoids er modtagelig for genmanipulation af virus mediterede genlevering, og derfor signalveje involveret i tyktarmen tumorigenese kunne også grundigt undersøgt ved overekspression eller knockdown. Primær tumor organoids kultur giver en fysiologisk relevante og gennemførlige midler til at studere de mekanismer og terapeutiske modaliteter for colon tumorigenese.

Tidsforløbet for en colontumor organoid dannelse fra en tre-måneder gamle Apc min / + mus er vist i figur 1.. På dag 0, kan enkelte celler observeret flere timer efter udpladning (fig. 1A). På dag 1, overlevede colontumorceller epitelceller med refraktær kerner kunne observeres. På dag 3, fordoblet størrelsen af ​​celler. På dag 6, udvidet størrelsen af ​​organoid mere end ti gange og viste tegn på apoptose i midten. På dag 14 ville orgnoids vokse ...

Watch this Scientific Journal Video about In vitro Organoid Culture of Primary Mouse Colon Tumors at JoVE.com

In vitro Organoid Culture of Primary Mouse Colon Tumors

En simpel metode til at etablere den primære murine colontumor organoid beskrives. Denne metode udnytter den funktion, der colontumorceller overleve og vokse ind organoids i medier indeholder begrænsede vækstfaktorer, mens normal colon epitel ikke.

In vitro Organoid Kultur i Primary Mouse tyktarmstumorer

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

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In vitro Organoid Culture of Primary Mouse Colon Tumors

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Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Forskolininduceret Hævelse i Intestinal Organoids: An<em&gt; In vitro</em&gt; Assay for Vurdering Drug respons i patienter med cystisk fibrose

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

Dissektion og eksplantat kultur af Murine Allantois til In Vitro -analyse af allantois vedhæftet fil

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental ...