Diese Arbeit wurde von der Richard Thomas Leukämie-Fonds, die Lady Tata Memorial Trust, das Northwick Park Hospital Leukämieforschung Treuhandfonds und des National Institute of Health Research (NIHR), UK finanziert.

1. Scaffold Herstellung und Bio-Funktionalisierung von Gerüsten <ol><li> Um PU Gerüste (Porengröße 100-250 mm, Porosität 90-95%) in Form von Scheiben Petrischale herzustellen, verwenden die thermisch induzierte Phasentrennung 13 process durch Herstellen einer Polymer-Lösung (5 Gew.% in Dioxan), gefolgt von Einfrieren und anschließende Lösungsmittel Sublimation (Abbildung 1A). </li><li> Schneiden Sie das Gerüst Festplatte in Würfel von 0,5 x 0,5 x 0,5 mm vor der Beschichtung mit ECM-Proteine ​​(Abbildung 1B). </li><li> Vornässen die Gerüste durch Eintauchen in Ethanol (70%) für 1 min und danach in Phosphat-gepufferter Salzlösung (PBS) für 20 min. Dann für 10 min zentrifugieren bei 3630 xg und fügen Sie die Protein-Lösung. </li><li> Zentrifugieren Sie die Gerüste in der Protein-Lösung bei 1420 xg für 20 min. </li><li> Um die Oberflächenporen freizugeben, und damit die Zellen ausgesät, um tiefer in das Gerüst zu durchdringen, zentrifugieren die Gerüste noch einmal bei 910 xg für 10 minin PBS. </li><li> Sterilisieren Sie die Gerüste mit einer Kombination von 8 min Belichtung bei 230 V, 50 Hz, 0,14 A, UV-Lampe, mit Eintauchen für 2 h in Ethanol (70%). Danach wäscht die Gerüste zweimal in PBS vor der Zugabe Iscoves modifiziertem Dulbecco Medium (IMDM) mit 30% fötalem Rinderserum (FBS) und 1% Penicillin und Streptomycin (P / S) ergänzt. Platzieren der Gerüste in einem befeuchteten Inkubator für 3 Tage bei 37 ° C und 5% CO 2 vor der Verwendung. Sterile Gerüste werden in einer 24-Well-Gewebekulturplatten (ein Gerüst pro Vertiefung) angeordnet ist. </li></ol> 2. Mononukleären Zellen Isolierung und Gerüstbau Seeding <ol><li> Auszug menschlichen MNC aus Nabelschnurblut oder Knochenmark bestimmt Proben mit Ficoll-Paque Dichtegradientenzentrifugation (35 min bei 1500 rpm) (Abbildung 1C). </li><li> Resuspendieren MNC in IMDM, enthaltend 30% FBS und 1% P / S </li><li> Seed 100 &amp;mgr; von MNC-Suspension (2 x 10 6 Zellen / Gerüst) auf die Gerüste mit Hilfe eines Mikro-Pipette. </li><li> Inkubieren Sie die gesetzten Gerüste bei 37 ° C unter 5% CO 2 für 10 Minuten für die Zellen, in die Gerüste zu begleichen. </li><li> Füllen jeder Vertiefung mit 1,5 ml IMDM, enthaltend 30% FBS und 1% P / S und Ort Well-Platten im Inkubator bei 37 ° C und 5% CO 2 für die Dauer des Experiments. </li><li> Gesetzt Gerüste unterziehen täglich von allen Medien ändern. </li></ol> 3 In situ Cell Proliferation und Morphologie:. MTS, SEM und Cytospins <ol><li> MTS-Assay: Sofort von der Kultur ein un-entkernt und zwei gesetzten Gerüste und in einem sauberen neuen 24-Loch-Platte. 1 ml der Medien-und 200 ul der MTS-Lösung, Inkubation für 3 h bei 37 ° C und 5% CO 2. <ol><li> Nehmen Sie 8 x 100 ul-Proben aus dem Überstand; Platz in einer 96-Well-Platte und messen die Absorption bei 490 nm mit einem Mikroplatten-Reader. </li></ol></li><li> Die Rasterelektronenmikroskopie (REM): Zu verschiedenen Zeitpunkten von the Kultur, entfernen Sie die Gerüste mit MNC aus den Medien ausgesät, und fixieren mit 2,5% PBS-gepuffertem Glutaraldehyd-Lösung für 40 min bei 4 ° C, dann zweimal mit PBS waschen. <ol><li> Dehydratisierte die Gerüste in einer abgestuften Serie von Ethanol (25, 50, 70, 80, 90, 95 und 100%), jeweils für 10 min und trocken in einer aseptischen Umgebung für 4 Stunden. </li><li> § Proben und dann Sputter-Schicht sie mit Gold in einer Argon-Atmosphäre für 2 min vor dem REM-Auswertung, verwenden Sie eine Beschleunigung von 20 kV. </li></ol></li><li> Cytospins: Sammeln 2,0 x 10 4 Zellen / Dia durch Absaugen der Zellen aus dem Schafott zu verschiedenen Zeitpunkten in der Kultur. <ol><li> Zentrifugation der Zellen auf einen Objektträger für 3 min bei 1500 Umdrehungen pro Minute. </li><li> Stain die Folien mit Wright-Giemsa-Färbung und beobachten Geändert durch Verwendung eines optischen Mikroskops. F-View Soft Imaging System verwendet werden, um Bilder zu machen sein. </li><li> Den Objektträger vor dem Mikroskop Beobachtung. </li></ol></li><li> M<html> ultiphoton Analyse: Fix Gerüste zu verschiedenen Zeitpunkten mit Ethanol Dampf (70%) und dann über Nacht trocken und bei -20 ° C bis zur weiteren Analyse. Vor der Visualisierung mit dem Mikroskop Multiphotonen Abschnitt das Gerüst in 30 Mikrometer dicke Folien und nass mit PBS. <ol><li> Sperren der geimpften Gerüste in PBS mit 10% fötalem Rinderserum für 30 min bei Raumtemperatur. </li><li> Inkubieren Sie die Gerüste über Nacht bei 4 ° C im Dunkeln mit einer 1:50-Verdünnung des primären monoklonalen Antikörper: anti-Maus menschlichen CD71. </li><li> Proben dreimal Waschen in PBS und Färbung mit dem sekundären Antikörper: Anti-Maus-Alexa Fluor 488 für 1 h bei Raumtemperatur im Dunkeln. </li><li> Waschen Proben dreimal mit PBS und beobachten mit einem konfokalen Mikroskop mit Hilfe eines Wasser-Emission x60 Objektiv. </li><li> Fluorophor Alexa Fluor 488 wird bei 488 nm durch den pulsierenden Laser angeregt. Endgeschwindigkeit, 5.3.2-Software kann für die nachfolgende Bildanalyse genutzt werden. </li></ol></li></ol>jove_title &quot;&gt; 4. Die durchflusszytometrische Analyse der Zellpopulation <ol><li> Bevor zelluläre Analyse im Durchflusszytometer (FC), beschriften Sie die Zellen von Interesse mit den entsprechenden Antikörpern Immunfluoreszenz für die Erkennung. Eine Anzahl von Proben werden gemäß den ausgewählten Antikörper zum Nachweis hergestellt. Für MNC-Erkennung folgende Kombination von Antikörpern verwendet werden: CD45-FITC/CD71-PE/CD235a-PE-Cy5. </li><li> Absaugen der Zellen von dem Gerüst zu verschiedenen Zeitpunkten und zentrifugiert. Lösen Sie ein Zellpellet von etwa 1x10 6 Zellen in 100 ul FC-Puffer (PBS + 0,1% Natriumazid) und mit 10 ul jeder Antikörper-Fluoreszenz-Farbstoff. Inkubieren der Zellen für 30 min bei 4 ° C, zweimal waschen mit PBS gewaschen und schließlich in FC-Puffer erneut zu suspendieren. </li><li> Legen Sie die Probe in das Durchflusszytometer befleckt mit dem Isotyp-Kontrolle, um die &quot;Negativität&quot; des Antikörpers Ausdruck festlegen und kalibrieren die Erkennung Kanäle mit dem Durchflusszytometer Kontrolle. </li><li> Lesen Sie die Probe befleckt w<html> it den drei verschiedenen Antikörpern und schließlich stellen die Daten bei der Nutzung des WinList Software. </li></ol> 5. Repräsentative Ergebnisse Ein Beispiel von hämatopoetischen Zellen Wachstumskinetik ohne die Zugabe von exogenen Wachstumsfaktoren in 2 gezeigt. Aufgrund der heterogenen Natur der Blutbildung, zwei verschiedene Zellen: normale und abnormale hämatopoetische Zellen dargestellt. In 2A ist die zelluläre Proliferation von humanen CBMNC nach 28 Tagen in Kultur zeigt. 2B zeigt die Wachstumskinetik in der Mimik mit primären humanen Leukämiezellen. Die Zellproliferation wird anhand des MTS-Assay, der zellulären metabolischen Aktivität in Bezug auf Absorption misst. In beiden Fällen vermehrten sich die Zellen und ihren Sitz in dem Modell. Unterschiede in der Wachstumskinetik werden beachtet; normalen hämatopoetischen Zellen gründen die Kultur schneller als die leukämischen Zellen. _content &quot;&gt; Die Morphologie der geernteten Zellen auch nach 28 Tagen Kultur in Abwesenheit von exogenen Wachstumsfaktoren typisch für die normale hämatopoetische Zellen (Abbildung 3A) und leukämischen Zellen (Abbildung 3B) war. Zentrale Teile der Gerüste wurden mittels SEM analysiert nach Die Gerüste wurden von der Kultur entfernt, und zeigte die Ausbreitung der ausgesäten Zellen in dem Gerüst, etablieren sich in Clustern und in &quot;Nischen-like&quot;-Strukturen (3A &#39;&amp; B&#39;). Abbildung C zeigt die Porengröße und-verteilung in einem ungesetzte Gerüst als Kontrolle verwendet. Multiphotonenmikroskopie nach 28 Tagen wurde verwendet, um die Verteilung der Zellen in dem 3D-Gerüst in situ zu markieren, und es zeigte das Vorhandensein von Erythrozyten-Inseln in mittleren Abschnitten des Gerüsts (4) durch die Expression des Markers CD71, die positiv ist in Erythroblasten. Dies beweist die Bedeutung der reifen und reifenden Zellen duRing Erythropoese. Schließlich zeigt Durchflusszytometrie Graphen der Zellen vor Seeder den Unterschied in der Phänotyp von hämatopoetischen Zellen, wobei 5A stellt normalen hämatopoetischen Zellen: menschliche CBMNCs und 5B zeigt abnormale hämatopoetische Zellen: primäre Leukämiezellen. Ebenen der CD235a + und CD45 + entspricht, Erythrozyten und Leukozyten jeweils höher sind in der normalen Probe als in der leukämischen Hervorhebung der hemoblastic Art der Leukämien. <img alt="1" src="/files/ftp_upload/3654/3654fig1.jpg" /> Abbildung 1. Illustration der Prozesse in der PU-Gerüst Herstellung und Bio-Funktionalisierung beteiligt. A) PU in Dioxan (5 Gew.%) gelöst und durch thermisch induzierte Phasentrennung und die anschließende Lösungsmittel Sublimation das Stützgerüst hergestellt wird, wie durch Safinia et al 13 beschrieben. B) Das Gerüst Scheibe wird dann geschnitten into Würfel von 0,5 x 0,5 x 0,5 mm und dann durch Zentrifugation mit extrazellulären Matrix (ECM) Proteinen beschichtet. C) MNC werden aus humanen Nabelschnur CB oder von BM Aspiration mittels Dichtegradientenzentrifugation und entkernt (2x10 6 Zellen / Gerüst) in der PU-Gerüste mit einer Mikropipette entnommen. <img alt="2" src="/files/ftp_upload/3654/3654fig2.jpg" /> . 2 Zellproliferation unter Verwendung des MTS-Assay: A) mit humanem Nabelschnurblut MNU, B) unter Verwendung von humanen primären Leukämiezellen. Die Spalten zeigen das Wachstum von Zellen im Laufe der Zeit, wenn in der PU-Gerüsten ohne die Zugabe von exogenen Zytokinen ausgesät. <img alt="Abbildung 3" src="/files/ftp_upload/3654/3654fig3.jpg" /> Abbildung 3: Cell Morphologie und Verteilung um die Verwendung von Gerüsten Zytospins, scanning elektronenmikroskopischen Aufnahmen. (AB) Vertreter Wright-Giemsa gefärbten Zytospins von A) Nabelschnurblut mononukleäre Zellen aus PU Gerüste nach 28 Tagen Kultur gesammelt, und B) Knochen aspiriert Leukämiezellen aus PU Gerüste nach 14 Tagen Kultur gesammelt. Beide Versuche wurden in Zytokin-freien Zustand durchgeführt. (A&#39;-B &#39;) Vertreter zentralen Abschnitte der PU-Gerüst REM-Aufnahmen von A&#39;) seeded Nabelschnurblut Multis und B &#39;) leukämischen Zellen, nachdem sie 28 Tage lang kultiviert, und C) Kontrolle Gerüst ohne Zellen. <img alt="Abbildung 4" src="/files/ftp_upload/3654/3654fig4.jpg" /> Abbildung 4. Multiphotonen Aufnahme eines PU-Gerüst entkernt mit Nabelschnurblut MNCs nach 28 d in Kultur und gebeizt mit der erythroiden Markers CD71. <img alt="Abbildung 5" src="/files/ftp_upload/3654/3654fig5.jpg" /> Abbildung 5. Durchflusszytometrie 3D Dot-Plots für CD45, CD71 und CD235a Oberflächenexpression Marker gefärbt. Die Isotyp-Kontrolle erhalten wird auch für den Vergleich der relativen Fluoreszenzintensität dargestellt. Feld A zeigt menschlichem Nabelschnurblut mononukleäre Zellen positiv für die oben genannten Marker; Tafel B zeigt primären humanen leukämischen Zellen.

<ol>
	<li>Orkin, S., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoiesis:+an+evolving+paradigm+for+stem+cell+biology.">Hematopoiesis: an evolving paradigm for stem cell biology.</a> Cell. 132, 631-644 (2008).</li><li>Spradling, A., Drummond-Barbosa, D., Kai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+find+their+niche.">Stem cells find their niche.</a> Nature. 414, 98-104 (2001).</li><li>Panoskaltsis, N., Mantalaris, A., Wu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+a+mimicry+of+bone+marrow+tissue+ex+vivo.">Engineering a mimicry of bone marrow tissue ex vivo.</a> J Biosci. Bioeng. 100, 28-35 (2005).</li><li>Lo Celso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-animal+tracking+of+individual+haematopoietic+stem/progenitor+cells+in+their+niche.">Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche.</a> Nature. 457, 92-96 (2009).</li><li>Mantalaris, A., Bourne, P., Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+human+osteoclasts+in+a+three-dimensional+bone+marrow+culture+system.">Production of human osteoclasts in a three-dimensional bone marrow culture system.</a> Biochem. Eng. J. 20, 189-196 (2004).</li><li>Placzek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+bioprocessing:+fundamentals+and+principles.">Stem cell bioprocessing: fundamentals and principles.</a> J. R. Soc. Interface. 6, 209-232 (2009).</li><li>Dexter, T., Testa, N., Prescott, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Cell Biology. 14, 387-405 (1976).</li><li>Piacibello, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+growth+factor+requirement+of+primitive+cord+blood+hematopoietic+stem+cell+for+self-renewal+and+amplification+vs+proliferation+and+differentiation.">Differential growth factor requirement of primitive cord blood hematopoietic stem cell for self-renewal and amplification vs proliferation and differentiation.</a> Leukemia. 12, 718-727 (1998).</li><li>Yoshida, T., Takagi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+processing+engineering+for+ex+vivo+expansion+of+hematopoietic+cells:+a+review.">Cell processing engineering for ex vivo expansion of hematopoietic cells: a review.</a> Biochemical Engineering Journal. 20, 99-106 (2004).</li><li>Lim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intelligent+bioprocessing+for+haemotopoietic+cell+cultures+using+monitoring+and+design+of+experiments.">Intelligent bioprocessing for haemotopoietic cell cultures using monitoring and design of experiments.</a> Biotechnol. Adv. 25, 353-368 (2007).</li><li>Mortera-Blanco, T., Mantalaris, A., Bismarck, A., Panoskaltsis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+a+three-dimensional+scaffold+for+ex+vivo+biomimicry+of+human+acute+myeloid+leukaemia.">The development of a three-dimensional scaffold for ex vivo biomimicry of human acute myeloid leukaemia.</a> Biomaterials. 31, 2243-2251 (2010).</li><li>Mortera-Blanco, T., Mantalaris, A., Bismarck, A., Aqel, N., Panoskaltsis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+cytokine-free+expansion+of+cord+blood+mononuclear+cells+in+three-dimensional+scaffolds.">Long-term cytokine-free expansion of cord blood mononuclear cells in three-dimensional scaffolds.</a> Biomaterials. 32, 9263-9270 (2011).</li><li>Safinia, L., Datan, N., Hohse, M., Mantalaris, A., Bismarck, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+methodology+for+the+effective+surface+modification+of+porous+polymer+scaffolds.">Towards a methodology for the effective surface modification of porous polymer scaffolds.</a> Biomaterials. 26, 7537-7547 (2005).</li></ol>

Wir haben nichts zu offenbaren.

Die Ex-vivo-3D-Kultur hier vorgestellte System ermöglicht es uns, eine 3D-biomimicry der Blutbildung feststellen, dass der ursprüngliche BM rekapituliert Architektur und zelluläre Phänotyp unabhängig von exogenen Zytokinen. Das 3D-Modell stellt die Struktur und die Mikroumgebung, die normale und abnormale hämatopoetische Zellen unter Bedingungen ähnlich denen in vivo angetroffen vermehren können. Die Auswahl der polymeren Trägermaterial zu vertreten ein entscheiden...

<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

Hämatopoetischen Stammzellen erfordern eine einzigartige Mikroumgebung um die Bildung roter Blutkörperchen ein zu unterhalten; das Knochenmark (BM) ist eine komplexe dreidimensionale (3D) Gewebe, bei Hämatopoese wird durch räumlich organisiert zelluläre Mikroumgebung reguliert bezeichnet Nischen 2-4. Die Organisation der BM Nischen ist entscheidend für die Funktion oder Dysfunktion des normalen oder malignen BM 5. Deshalb ein besseres Verständnis für die in-vivo-Mikroumgebung mit einem Ex-vivo-Mimikry würde uns helfen, Aufklärung der molekularen, zellulären und Determinanten der Mikroumgebung Leukämogenese 6. Derzeit werden hämatopoetischer Zellen in vitro in einem zweidimensionalen (2D) Gewebekulturflaschen / Well-Platten 7 erfordern entweder Co-Kultur mit allogenen oder xenogenen Stromazellen oder Zugabe von exogenen Zytokinen 8 kultiviert. Diese Bedingungen sind künstliche und unterscheiden sich von der in vivo </em&gt; Mikroumgebung, dass sie nicht über die 3D-zellulären Nischen und entlarven die Zellen auf abnorm hohe Zytokin-Konzentrationen, die zur Differenzierung und zum Verlust der Pluripotenz 9,10 führen kann. Hier präsentieren wir eine neuartige 3D-Knochenmark-Kultur-System, das die 3D simuliert in vivo das Wachstum und Umwelt unterstützt multilineage Hämatopoese in Abwesenheit von exogenen Wachstumsfaktoren. Die hoch porösen Gerüst in diesem System aus Polyurethan (PU) verwendet, ermöglicht eine hohe Dichte Zellwachstum über eine höhere spezifische Oberfläche als die herkömmliche 2D-Monolayer-Kultur in 11. Unsere Studien zeigen, dass dieses Modell das Wachstum von menschlichem Nabelschnurblut (CB) mononukleäre Zellen (MNC) 12 und primären leukämischen Zellen in Abwesenheit von exogenen Zytokinen unterstützt. Diese neuartige 3D-Mimikry bietet eine tragfähige Plattform für die Entwicklung eines menschlichen experimentelles Modell zur Blutbildung zu studieren und neuartige Behandlungsmöglichkeiten gegen Leukämie zu erforschen.

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

Ein 3D-Kultur für Hämatopoese wird unter Verwendung von humanem Nabelschnurblut und leukämischen Knochenmarkszellen. Verfahren basiert auf der Verwendung eines porösen, synthetischen Polyurethan Gerüst mit Proteinen der extrazellulären Matrix beschichtet ist. Dieses Gerüst ist anpassbar an eine Vielzahl von Zellen aufnehmen.

Ex-vivo-Mimikry von normalen und anormalen menschlichen Hämatopoese

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

ex-vivo-mimicry-of-normal-and-abnormal-human-hematopoiesis

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Bioengineering

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

15.4K Aufrufe.  Imperial College London. Ein 3D-Kultur für Hämatopoese wird unter Verwendung von humanem Nabelschnurblut und leukämischen Knochenmarkszellen. Verfahren basiert auf der Verwendung eines porösen, synthetischen Polyurethan Gerüst mit Proteinen der extrazellulären Matrix beschichtet ist. Dieses Gerüst ist anpassbar an eine Vielzahl von Zellen aufnehmen.

Serie: Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

Die dreidimensionalen humanen Haut Rekonstruieren Modell: ein Tool für normale Haut und Melanom Progression Study

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Forschung

Biotechnik

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Entwurf eines zyklischen Druck Bioreaktor für die Ex Vivo Studium der Aorten-Herzklappen

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

Umweltfreundliche kontrollierten Microtensile Testing von mechanisch-adaptive Polymer-Nanokomposite für<em&gt; Ex vivo</em&gt; Charakterisierung

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Bestimmung der Transport von Xenobiotika und Nanomaterialien über die Plazenta mit dem<em&gt; Ex vivo</em&gt; Menschliche Plazentaperfusion Modell

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

Imaging denaturierten Kollagen-Stränge<em&gt; In-vivo-</em&gt; Und<em&gt; Ex-vivo-</em&gt; Über die Photo-ausgelöst Hybridisierung von Caged Collagen mimetischen Peptiden

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Cerebral Blood Flow-Based Resting State Functional Connectivity of the Human Brain using Optical Diffuse Correlation Spectroscopy

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a key hemodynamic parameter related to cerebral oxygen supply. Resting state low frequency fluctuations based on oxygenation contrast have been shown to provide correlations between functionally connected regions. The presented protocol uses optical diffuse correlation spectroscopy (DCS) to assess blood flow-based resting state functional connectivity (RSFC) in the human brain. Results of CBF-based RSFC in human frontal cortex indicate that intra-regional RSFC is significantly higher in the left and right cortices compared to inter-regional RSFC in both cortices. This protocol should be of interest to researchers who employ multi-modal imaging techniques to study human brain function, especially in the pediatric population.

This protocol demonstrates how to measure resting state functional connectivity in the human prefrontal cortex using a custom-made diffuse correlation spectroscopy instrument. The report also discuss practical aspects of the experiment as well as detailed steps for analyzing the data.

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a ...

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric&#160;deformation. Our understanding of the internal relationships between these events has been limited because we can neither &#39;see&#39; inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue&#39;s geometric&#160;changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 &#181;m by 540 &#181;m) and the length of a typical trabecula (&#62;2000 &#181;m). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric&#160;data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

Simultane Hellfeld-, Fluoreszenz- und optische Kohärenztomographische Bildgebung kontrahierender Herztrabekel Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and ...

All-optical Mechanobiology Interrogation of Yes-associated Protein in Human Cancer and Normal Cells using a Multi-functional System

Long-term multi-functional imaging and analysis of live cells require streamlined, functional coordination of various hardware and software platforms. However, manual control of various equipment produced by different manufacturers is labor-intensive and time-consuming, potentially decreasing the accuracy, reproducibility, and quality of acquired data. Therefore, an all-in-one and user-programmable system that enables automatic, multi-functional, and long-term image acquisition and is compatible with most fluorescent microscopy platforms can benefit the scientific community. This paper introduces the complete operating protocols of utilizing a novel integrated software system that consists of (1) a home-built software program, titled "Automatic Multi-functional Integration Program (AMFIP)," which enables automatic multi-channel imaging acquisition, and (2) a suite of quantitative imaging analysis and cell traction computation packages. 
This integrated system is applied to reveal the previously unknown relationship between the spatial-temporal distribution of mechano-sensitive Yes-associated protein (YAP) and the cell mechanics, including cell spreading and traction, in CRISPR/Cas9-engineered human normal cells (B2B) and lung cancer cells (PC9). Leveraging this system's capability of multi-channel control and readout, the result shows: (1) B2B normal cells and PC9 cancer cells show a distinct relationship between YAP expression, traction, and cell dynamics during cell spreading and migration processes; and (2) PC9 cancer cells apply noticeable peri-nuclear forces on substrates. In summary, this paper presents a detailed stepwise protocol on how to utilize an integrated user-programmable system that enables automatic multi-functional imaging and analysis to elucidate YAP mechano-sensitivity. These tools open the possibility for detailed explorations of multifaceted signaling dynamics in the context of cell physiology and pathology.

This paper presents a detailed stepwise protocol on how to utilize an integrated multi-functional and user-programmable system that enables automatic multi-channel imaging and mechanobiological analysis to elucidate the mechano-sensitivity of Yes-associated protein (YAP).

Rein optische Mechanobiologie Befragung von Yes-assoziiertem Protein in menschlichen Krebs- und normalen Zellen mit einem multifunktionalen System

Long-term multi-functional imaging and analysis of live cells require streamlined, functional coordination of various hardware and software platforms. ...

Ein neuartiger 3D-gedruckter Bioreaktor für die ex vivo-Kultur von Blutgefäßen in der Perfusion

Ex Vivo Perfusion Culture of Large Blood Vessels in a 3D Printed Bioreactor

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious surgical and pharmacological interventions to prevent and treat vascular disease are urgently needed. In part, the shortage of translational models limits the understanding of the cellular and molecular processes involved in vascular disease. Ex vivo perfusion culture bioreactors provide an ideal platform for the study of large animal vessels (including humans) in a controlled dynamic environment, combining the ease of in vitro culture and the complexity of the live tissue. Most bioreactors are, however, custom manufactured and therefore difficult to adopt, limiting the reproducibility of the results. This paper presents a 3D printed system that can be easily produced and applied in any biological lab, and provides a detailed protocol for its setup, enabling users&#39; operation. This innovative and reproducible ex vivo perfusion culture system enables the culture of blood vessels for up to 7 days in physiological conditions. We expect that adopting a standardized perfusion bioreactor will support a better understanding of physiological and pathological processes in large animal blood vessels and accelerate the discovery of new therapeutics.

Autor im Rampenlicht: EasyFlow - ein wirtschaftlicher und anpassungsfähiger Perfusionsbioreaktor für die Kultur großer Blutgefäße 

This protocol presents the setup and operation of a newly developed, 3D printed bioreactor for the ex vivo culture of blood vessels in perfusion. The system is designed to be easily adopted by other users, practical, affordable, and adaptable to different experimental applications, such as basic biology and pharmacological studies.

Ex Vivo Perfusionskultur großer Blutgefäße in einem 3D-gedruckten Bioreaktor

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious ...