Name: ex vivo mimicry of normal and abnormal human hematopoiesis
Uploaded: 2012-04-10T12:00:00
Description: Watch this Scientific Journal Video about Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis at JoVE.com

Dette arbeidet ble finansiert av Richard Thomas Leukemi Fund, Lady Tata Memorial Trust, den Northwick Park Hospital Leukemi Research Trust Fund og National Institute of Health Research (NIHR), Storbritannia.

1. Stillas Produksjon og Bio-funksjonalisering av Stillaser <ol><li> For å dikte PU stillasene (porestørrelse 100-250 mm, porøsitet 90-95%) i form av petriskål disker, bruke termisk induserte faseseparasjon 13 prosessen med å utarbeide en polymer løsning (5wt% i Dioxan) etterfulgt av frost og påfølgende løsemiddel sublimering (figur 1A). </li><li> Skjær stillaset disken i terninger på 0,5 x 0,5 x 0,5 mm før belegg med ECM proteiner (Figur 1B). </li><li> Pre-våt...

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Den ex vivo 3D kultur systemet presenteres her gjør oss i stand til å etablere en 3D biomimicry av hematopoiesis som sammenfatter den opprinnelige BM arkitektur og cellulær fenotype uavhengig av eksogene cytokiner. 3D-modellen gir struktur og mikromiljøet som gjør at normale og unormale blodkreft cellene til å spre seg i forhold som ligner dem som møtte in vivo. Valget av polymere stillaset materialet representerte et avgjørende skritt i biomimicry design. Viktige f...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue No</td>
		</tr>
		<tr>
			<td>Dioxan</td>
			<td>Invitrogen</td>
			<td>D20,186-3</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190-094</td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Invitrogen</td>
			<td>12440-053</td>
		</tr>
		<tr>
			<td>Ficoll-Paque</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
		</tr>
		<tr>
			<td>MTS</td>
			<td>Promega</td>
			<td>G3580</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Fluka Biochemika</td>
			<td>49624</td>
		</tr>
		<tr>
			<td>Wright-Giemsa</td>
			<td>Sigma-Aldrich</td>
			<td>WG32</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10108-165</td>
		</tr>
		<tr>
			<td>CD71</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-32272</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A11001</td>
		</tr>
		<tr>
			<td>CD45-FITC</td>
			<td>BD Pharmigen</td>
			<td>74895</td>
		</tr>
		<tr>
			<td>CD71-PE</td>
			<td>BD Pharmigen</td>
			<td>555537</td>
		</tr>
		<tr>
			<td>CD235a-PE-Cy5</td>
			<td>BD Pharmigen</td>
			<td>555570</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S-8032</td>
		</tr>
	</tbody>
</table>

ex vivo mimicry of normal and abnormal human hematopoiesis

Blodkreft stamceller krever et unikt mikromiljøet for å opprettholde blod celle formasjon 1; benmarg (BM) er en kompleks tredimensjonal (3D) vev der hematopoiesis er regulert av romlig organisert cellulære microenvironments kalt nisjer 2-4. Organiseringen av BM nisjer er kritisk for funksjon eller dysfunksjon av normal eller ondartet BM 5. Derfor en bedre forståelse av in vivo mikromiljøet med en ex vivo parodiering ville hjelpe oss belyse de molekylære, cellulære og microenvironmental determinanter for leukemogenesis 6. Foreløpig er hematopoetiske celler dyrket in vitro i to-dimensjonale (2D) vevskulturstudier kolber / brønn-plater 7 krever enten co-kulturen med allogenic eller xenogenic stromale celler eller tillegg av eksogene cytokiner 8. Disse forholdene er kunstig og skiller seg fra in vivo </em&gt; mikromiljøet i at de mangler 3D cellulære nisjer og eksponere cellene til unormalt høye cytokin konsentrasjoner som kan resultere i differensiering og tap av pluripotency 9,10. Heri presenterer vi en ny 3D beinmarg kultur system som simulerer in vivo 3D vekst miljøet og støtter multilineage hematopoiesis i fravær av eksogene vekstfaktorer. Den svært porøse Stillaset brukes i dette systemet laget av polyuretan (PU), forenkler høy tetthet cellevekst over en høyere spesifikk overflate enn den konvensjonelle monolayer kulturen i 2D 11. Vårt arbeid har vist at denne modellen støttes veksten av menneskelig ledningen blod (CB) mononukleære celler (MNC) 12 og primære leukemic celler i fravær av eksogene cytokiner. Denne romanen 3D mimikk gir en levedyktig plattform for utvikling av en menneskelig eksperimentell modell for å studere hematopoiesis og å utforske nye behandlinger for leukemi.

Watch this Scientific Journal Video about Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis at JoVE.com

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

En 3D kultur system for hematopoiesis beskrives ved hjelp av menneskelig navlestrengsblod og leukemic benmargceller. Metoden er basert på bruk av en porøs syntetisk polyuretan stillas belagt med ekstracellulære matrix proteiner. Dette stillaset er tilpasningsdyktige for å imøtekomme et bredt spekter av celler.

Ex vivo Herming av normale og unormale Menneskelig hematopoiesis

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional ...

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Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

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