Bu çalışma Richard Thomas Lösemi Fonu, Lady Tata Memorial Trust, Northwick Park Hastanesi Lösemi Araştırma Fonu ve Sağlık Araştırması Ulusal Enstitüsü (NIHR), Birleşik Krallık tarafından finanse edildi.

1. Iskelelerinin İskele imalatı ve Bio-fonksiyonlandırmalar <ol><li> Petri tabağı disk şeklinde poliüretan iskelesi (100-250 gözenek büyüklüğü mm, gözenekliliği% 90-95) imal etmek için, ardından bir polimer solüsyonu (Dioksan içinde 5wt%) hazırlayarak ısının neden olduğu faz ayırma işlemi kullanan 13 donmaya ve müteakip solvent yüceltme (Şekil 1A). </li><li> ECM proteinleri (Şekil 1B) ile kaplama öncesinde 0.5 x 0.5 x 0.5 mm küpler halinde iskele diski kesin. </li><li> Sonra 1 dk ve fosfat içine aktarmak için etanol (% 70) onları daldırarak Öncesi ıslak iskeleleri 20 dakika (PBS) tamponlu tuz. Daha sonra 3630 x g&#39;de 10 dakika boyunca santrifüj edilir ve protein çözeltisi ilave edin. </li><li> 20 dakika boyunca 1420 x g&#39;de protein çözeltisi içinde iskelesi santrifüjlenir. </li><li> Yüzey boşluklannın engeli ve iskelesi içine derin nüfuz tohumlanmış hücreleri imkan vermek üzere, 10 dakika boyunca 910 x g&#39;de bir kez daha iskeleleri santrifüjPBS içinde. </li><li> Etanol içinde 2 saat (% 70) için daldırma ile 230 v, 50 Hz, 0.14 A, UV lamba, az 8 dakika maruz kalma bir arada kullanarak iskeleleri sterilize edin. Bundan sonra,% 30 fetal bovin serumu (FBS) ve% 1 penisilin ve streptomisin (P / S) ile desteklenmiş Iscove modifiye Dulbecco Orta (IMDM) eklenmeden önce PBS ile iki kez iskelesi yıkanır. ° C ve% 5 CO2 kullanımdan önce 37 az 3 gün süreyle nemlendirilmiş bir inkübatörde iskelesi yerleştirin. Steril iskelesi bir 24 well-doku kültür plakaları (kuyu başına bir yapı iskelesi) yerleştirilir. </li></ol> 2. Mononükleer Hücre İzolasyon ve İskele Seeding <ol><li> Ficoll-Paque yoğunluk gradiyenti santrifüj (1500 rpm de 35 dakika) (Şekil 1C) kullanarak, kordon kanı veya lösemi kemik iliği örneklerinden, insan MNC ayıklayın. </li><li> IMDM in Pastör pipetiyle MNC% 30 FBS ve 1%, P / S içeren </li><li> Bir mikro-pipet kullanarak iskeleler üzerine MNC süspansiyon Tohum 100μl (2 × 10 6 hücre / iskelet). </li><li> İskeleler yerleşmeye hücreleri için 10 dakika boyunca% 5 CO2 altında 37 ° C&#39;de numaralı seribaşı iskeleleri inkübe edin. </li><li> 37 kuvöz% 30 FBS ve% 1 P / S ve yer kuyucuğu içeren IMDM 1.5 ml ile her yanı kadar kontör ° C ve deneme süresi için% 5 CO 2. </li><li> Numaralı seribaşı iskelelerinin tüm medya günlük değişime uğrarlar. </li></ol> 3 in situ Hücre Çoğalması ve Morfoloji olarak:. MTS, SEM ve Cytospins <ol><li> MTS deneyi: kültür bir un numaralı seribaşı ve temiz yeni bir 24 iyi plaka iki numaralı seribaşı iskeleler ve yerden çıkarın. Ortamın 1 ml ve 37 MTS çözeltisi ve 3 saat süre ile inkübe edilir ve 200 ul ekleme ° C&#39;de ve% 5 CO2. <ol><li> Bir mikroplak okuyucu kullanarak 490 nm&#39;de 96-plaka ve ölçü absorbans yer, üstteki 8 x 100 ul örnek alabilir. </li></ol></li><li> Th farklı zaman noktalarında: elektron mikroskobu (SEM)e kültür, medya MNC ile tohumlanan iskeleleri kaldırmak ve 4 40 dakika boyunca% 2.5 PBS-tamponlanmış glutaraldehit çözüm ° C düzeltmek, daha sonra PBS ile iki kez yıkayın. <ol><li> Susuz 4 saat için aseptik ortamda 10 dakika ve kuru etanol için bir aşamalı dizisi (25, 50, 70, 80, 90, 95, ve% 100), her bir destek mi. </li><li> Bölüm örnekleri ve daha sonra önceden SEM değerlendirilmesi için 2 dakika süre ile, bir argon atmosfer altın sputter-kaplama, bunların, 20 kV voltaj bir hızlanma kullanabilir. </li></ol></li><li> Cytospins: kültüründe farklı zaman noktalarında iskele gelen hücreleri aspire tarafından 2.0 x 10 4 hücre / slayt toplayın. <ol><li> 1500 rpm&#39;de 3 dakika için bir cam slayt üzerine hücreler santrifüjleyin. </li><li> Wright-Giemsa Modifiye Leke ve optik mikroskop kullanılarak gözlemlemek ile slaytlar Leke. Yumuşak Görüntüleme Sistemi fotoğraf çekmek için kullanılabilir F-görüntüleyebilirsiniz. </li><li> Mikroskop gözlem öncesinde slaytlar yıkayın. </li></ol></li><li> M<html> ultiphoton analizi: Daha fazla analiz kadar -20 etanol buharı (% 70) gece ve daha sonra kuru ve mağaza ° C ile farklı zaman noktalarında Fix iskeleleri. Önce multiphoton mikroskop 30 mikron kalınlığında slaytlara bölümünde iskele ve PBS ile ıslak görselleştirme için. <ol><li> Oda sıcaklığında 30 dakika süreyle% 10 fetal bovin serumu ile PBS içinde tohumlu iskelesi bloke eder. </li><li> Fare anti-insan CD71: ° C karanlıkta primer monoklonal antikorun a 1:50 seyreltme ile 4 azından iskelesi gece boyunca inkübe edilir. </li><li> PBS ile üç kez yıkanır örnekleri ve sekonder antikor ile lekesi: karanlık oda sıcaklığında 1 saat boyunca anti-fare Alexa Fluor 488. </li><li> PBS ile numuneler üç kere yıkayın ve bir su emisyon x60 objektif lens kullanarak bir konfokal mikroskop ile gözlemlemek. </li><li> Fluorofor Alexa Fluor 488 pulsatil lazerler 488 nm de heyecanlı. Volocity 5.3.2 yazılımı sonraki görüntü analizi için de kullanılabilir. </li></ol></li></ol>Hücresel Nüfus &quot;&gt; 4 jove_title. Sitometrik Akış Analizi <ol><li> Akış sitometresi (FC) hücresel analizinden önce tespiti için ilgili immünfloresan antikorlar ile ilgi hücreleri etiketleyin. Örnekleri arasında bir dizi tespiti için seçilen antikorlar göre hazırlanmaktadır. MNC algılaması için antikor kombinasyonları kullanılır: CD45-FITC/CD71-PE/CD235a-PE-Cy5. </li><li> Farklı zaman noktalarında ve santrifüj azından iskelesi gelen hücreleri aspire. 100 ul FC tamponu (PBS +% 0.1 sodyum azit) içinde 6 1x10 etrafında hücreleri bir hücre peleti eritilir ve her bir antikor floresans boyasının 10 uL ekleyebilir. 4 azından 30 dakika ° C&#39;de inkübe hücreleri, PBS ile iki kez yıka, sonuçta FC tampon içinde tekrar askıya. </li><li> Antikor ifade &quot;olumsuzluk&quot; kurmak ve akış sitometresi kontrolü ile tespit kanalları kalibre izotip kontrolü ile akış sitometresi lekeli içine örnek yükleyin. </li><li> Örnek lekeli w Oku<html> Üç farklı antikorları i ve en sonunda WinList yazılımı kullanılarak veri temsil eder. </li></ol> 5.. Temsilcisi Sonuçlar Eksojen büyüme faktörlerinin ilave edilmeden hematopoietik hücre büyümesi kinetik bir örnek, Şekil 2 &#39;de gösterilmiştir. Hematopoiesis heterojen, iki farklı hücre nedeniyle: normal ve anormal hücre hematopoetik gösterilmiştir. Şekil 2A, insan CBMNC hücre proliferasyonu kültüründe 28 gün sonra açıktır. Şekil 2B, insan primer lösemi hücre kullanılarak taklitçilik büyüme kinetikleri gösterir. Hücresel proliferasyon absorbans ilişkin hücresel metabolik aktivitesini ölçer MTS testi kullanılarak değerlendirilir. Her iki durumda da, hücrelerin çoğalması ve modeli kuruldu. Büyüme kinetiği farklılıklar gözlenir; kültür kurmak, normal hematopoetik hücreler daha hızlı lösemik hücrelerden daha. iskelelerinin _content &quot;&gt; eksojen büyüme faktörlerinin yokluğunda kültürün 28 gün, normal hücrelerin hematopoetik (Şekil 3A) ve lösemi hücre (Şekil 3B) tipik olarak daha sonra hücreler hasat morfolojisi. Orta bölümleri sonra SEM ile analiz edildi İskeleler kültürü kaldırılır ve kümeler halinde ve &quot;niş&quot; gibi yapılar (Şekil 3A &#39;&amp; B&#39;) kendilerini kurarak, iskele boyunca numaralı seribaşı hücrelerinin yayılmasını saptandı. Şekil C gösteren bir dereceye giremeyen içinde gözenek boyutu ve dağıtım iskelesi, bir kontrol olarak kullanılmıştır. 28 gün sonra Multiphoton mikroskopi in situ 3D iskelesi içinde hücrelerinin dağılımını vurgulamak için kullanılan ve bu işaretleyici tarafından ifade (Şekil 4) iskele merkez bölümde eritroid adalar varlığını gösterdi erythroblasts pozitiftir. CD71 Bu olgun ve olgunlaşan hücreler du önemini ortaya koyuyorhalka eritropoez. Insan CBMNCs ve Şekil 5B anormal hematopoetik hücreler gösterir: Birincil lösemik hücrelerin Son olarak, hücrelerin önceden ekimi sitometrisi grafikleri Şekil 5A, normal hematopoetik hücreler temsil hematopoetik hücrelerin fenotipi farkı gösterir. CD235a düzeyleri + ve CD45 + sırasıyla lösemilerin hemoblastic doğasını vurgulayarak lösemik daha normal bir örnek daha yüksektir eritrosit ve lökositlerin karşılık. <img alt="Şekil 1" src="/files/ftp_upload/3654/3654fig1.jpg" /> Şekil 1. PU iskele imalatı ve bio-fonksiyonlandırmalar yer alan süreçlerin İllüstrasyon. A) poliüretan Dioksan (5wt%) içinde çözülür ve ısının neden olduğu faz ayırma işlemi ve daha sonra çözücü, süblimleştirme yolu ile iskelesi Safinia ark 13 tarafından tarif edildiği gibi, üretilir. B) İskele diski sonra int kesiliro küpleri 0.5 x 0.5 hücre dışı matriks (ECM) proteinleri ile santrifüj kaplı x 0,5 mm ve daha sonra. C) MNC insan umbilikal CB veya bir mikropipet ile yoğunluk gradiyenti santrifüj ve PU iskeleleri taneli (2x10 6 hücre / iskelet) ile BM aspirasyon ayıklanır. <img alt="Şekil 2" src="/files/ftp_upload/3654/3654fig2.jpg" /> . Şekil 2 Hücresel proliferasyon MTS deneyi ile ölçülmüştür: A) insan kordon kanı ÇUŞ kullanarak; insan birincil lösemik hücreleri kullanarak) B. Eksojen sitokinlerin ilave edilmeden poliüretan iskelesi tohumlanmış olduğunda kolon zamanla hücrelerinin büyümesini göstermektedir. <img alt="Şekil 3" src="/files/ftp_upload/3654/3654fig3.jpg" /> Şekil 3: iskelelerinin etrafında Hücre morfolojisi ve dağılımı, scanni cytospins kullanarakng elektron mikrografikleri. (AB) kültür 28 gün sonra PU iskeleleri toplanan A) kordon kanı mononükleer hücrelerinin Temsilcisi Wright-Giemsa lekeli cytospins, ve B) kemik aspire lösemik hücrelerin kültür 14 gün sonra PU iskeleleri toplanmıştır. Her ikisi de deneylerin sitokin serbest durumda gerçekleştirilmiştir. (A&#39;-B &#39;)) tohumlu kordon kan ÇUŞ ve B&#39;) 28 gün süreyle kültüre edildikten sonra lösemi hücre ve hiçbir hücreleri ile birlikte C) kontrolü iskelesi A poliüretan iskelesi SEM temsil eden merkezi bölümü. <img alt="Şekil 4" src="/files/ftp_upload/3654/3654fig4.jpg" /> Şekil 4. Eritroid belirteç CD71 ile kültür ve lekeli 28 gün sonra kordon kanı ÇUŞ ile PU iskele numaralı seribaşı ve Multiphoton mikrografı. <img alt="Şekil 5," src="/files/ftp_upload/3654/3654fig5.jpg" /> Şekil 5. Flow sitometri 3D nokta araziler CD45, CD71 ve CD235a yüzey ifade belirteçleri için boyandı. Elde edilen izotip kontrolü de nispi floresans yoğunluğunu ve karşılaştırma için verilmiştir. Panel A yukarıdaki belirteçler pozitif insan kordon kanı mononükleer hücreler gösterir; B Paneli insan birincil lösemik hücreleri gösterir.

<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>

Biz ifşa hiçbir şey yok.

Burada sunulan ex vivo 3D kültür sistemi bize hematopoiesis 3D biyomimikri kurmaya olanak veren özetlediği BM özgün mimarisi ve eksojen sitokinler bağımsız hücre fenotipi. 3D modeli yapısı ve normal ve anormal hücre hematopoetik in vivo olarak karşılaşılan benzer koşullarda prolifere sağlayan mikro sağlar. Polimerik iskelesi malzeme seçimi biyomimikri tasarım kritik bir adımı temsil ediyordu. Biyomalzeme Önemli avantajları gerekli mimari, yapısal ...

<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

Kemik iliği (Kİ) mekansal organize hücresel mikro tarafından düzenlenir karmaşık üç boyutlu (3D) doku neyin hematopoez olduğunu nişler 2-4 denir; hematopoetik kök hücreler kan hücresi oluşumu 1 sürdürebilmek için benzersiz bir mikro gerektirir. BM nişler organizasyon normal veya malign BM 5 işlev veya işlev bozukluğu için önemlidir. Bu nedenle, bir ex vivo taklitçilik kullanılarak in vivo mikro çevresinde daha iyi anlaşılmasını us lökomogenezisin 6 arasında, hücresel ve moleküler microenvironmental belirleyicilerini açıklamak konusunda yardımcı olur. Şu anda, hematopoetik hücreleri ya allojenik ya ksenojenik stromal hücreleri ile birlikte kültür ya da egzojen sitokinlerin 8 ilavesiyle gerektiren (2B) iki boyutlu bir doku kültür şişeleri / kuyu-plakaları 7 in vitro kültürlenmiştir. Bu şartlar, yapay ve in vivo olarak farklılık </em&gt; bunlar 3D hücresel nişler yoksun ve pluripotency 9,10 farklılaşma ve kaybına neden olabilir anormal yüksek sitokin konsantrasyonları için hücrelerde açığa çıkarırlar içinde mikro. Burada, in vivo ortamda 3D büyümesini uyaran ve eksojen büyüme faktörlerinin yokluğunda multilinaj hemopoeziste destekler yeni bir 3D kemik iliği kültürü sistemi sunmaktadır. Poliüretan (poliüretan) yapılmış, bu sistemde kullanılan çok gözenekli yapı iskelesi, 2B 11, geleneksel tek tabakalı kültürden daha yüksek bir spesifik yüzey alanı boyunca yüksek yoğunluklu hücre büyümesini kolaylaştırır. Bizim bu iş modeli insan kanı kordonu (kesici) mononükleer hücreler (MNC) eksojen sitokinlerin yokluğunda 12 ve birincil lösemi hücre büyümesini desteklenen belirtmiştir. Bu yeni 3D taklit hematopoezisi incelemek ve lösemi için yeni tedavilerin keşfetmek için deneysel insan modelinin geliştirilmesi için uygun bir platform sağlar.

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

Hematopoez için 3D kültür sistemi, insan kordon kanı ve lösemiye kemik iliği hücreleri kullanılarak anlatılmıştır. Metodu hücre dışı matris proteinlerine ile kaplanmış bir emici sentetik poliüretan iskele kullanımına dayanmaktadır. Bu yapı iskelesi hücreleri geniş bir uyum sağlamak için uyarlanabilir.

Normal ve Anormal İnsan Hematopoezin ex vivo Mimicry

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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Research

JoVE Journal

Bioengineering

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

15.4K Görüntüleme.  Imperial College London. Hematopoez için 3D kültür sistemi, insan kordon kanı ve lösemiye kemik iliği hücreleri kullanılarak anlatılmıştır. Metodu hücre dışı matris proteinlerine ile kaplanmış bir emici sentetik poliüretan iskele kullanımına dayanmaktadır. Bu yapı iskelesi hücreleri geniş bir uyum sağlamak için uyarlanabilir.

Video: Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

Yüksek Çözünürlüklü 3D Görüntüleme Ex-Vivo Biyolojik örnekler Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

Biyomühendislik

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.

Üç Boyutlu İnsan Cilt Model Yeniden İnşa: Aracı Normal Cilt ve Melanom İlerleme Eğitim

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

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.

Devirli Basınç Biyoreaktör Tasarım Ex vivo</emAort Kalp Vanalar> Eğitim

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.

Için Mekanik-adaptif Polimer Nanokompozitler bir çevre kontrollü Mikrogerilim Test<em&gt; Ex vivo</em&gt; Karakterizasyonu

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.

Plasenta karşısında Ksenobiyotik ve Nanomalzemelerin Ulaştırma Hızı belirlenmesi kullanarak<em&gt; Ex vivo</em&gt; İnsan Plasental Perfüzyon Modeli

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.

Doğallığı Kollajen Tellerinin Görüntüleme<em&gt; In vivo</em&gt; Ve<em&gt; Ex vivo</em&gt; Kafesli Kollajen Mimetik Peptidlerinin Fotoğraf-tetiklenen Hybridization yoluyla

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

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential to establish an ex vivo model that can evaluate SIM performance accurately, for developing high-performance SIMs. In our previous study, we developed a new ex vivo model that can be used to evaluate the performance of various SIMs in detail by applying constant tension to the specimen&#39;s ends. We also confirmed that the proposed new ex vivo model allows accurate submucosal elevation height (SEH) measurement under uniform conditions and detailed comparisons of the performances of various types of SIMs. Here, we describe the new ex vivo model and explain the detailed setup methodology of this model. Since all parts of the new model were easy to obtain, the setup of the new model could be completed quickly. SEH of various SIMs could be measured more accurately by using the new model. The critical factor that determines SIM performance can be identified using the new model. SIM development speed will drastically increase after the factor has been identified.

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

We developed a new ex vivo model that applies constant tension to the porcine gastric specimen. This development made it possible to evaluate the performance (the height and duration of the submucosal elevation) of various SIMs accurately.&#160; The detailed setup methodology of this new model is explained.

Endoskopik Submucosal enjeksiyon malzemesi performans değerlendirilmesi için yeni bir Ex Vivo Model

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential ...

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.

Optik Diffüz Korelasyon Spektroskopisi ile İnsan Beyninin Serebral Kan Akışı Nadayalı Dinlenme Durumu Fonksiyonel Bağlantısı

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.

Kardiyak Trabeculae Ex Vivo'nun Eşzamanlı Brightfield, Floresan ve Optik Tutarlılık Tomografik Görüntülemesi

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).

Çok Fonksiyonlu Bir Sistem Kullanarak İnsan Kanseri ve Normal Hücrelerde Evet İlişkili Proteinin Tamamen Optik Mekanobiyoloji Sorgulaması

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